Journal of Immunological Methods, 131 (1990) 25-32 Elsevier
25
JIM 05613
A cell-ELISA for the quantification of adherent murine macrophages and the surface expression of antigens Peter H. Nibbering, Joke S. Van de Gevel and R a l p h Van Furth Department of Infectious Diseases, University Hospital, Leiden, The Netherlands (Received 17 November 1989, revised received 9 February 1990, accepted 20 March 1990)
The present study was performed in order to establish whether a cell-ELISA could be used to determine the expression of antigens by adherent murine peritoneal macrophages and also quantify the numbers of such macrophages. Accurate determination of the number of adherent macrophages proved to be possible with a cell-ELISA designed to assess complement receptor type III (CRIII) expression. Expression of CRIII was considerably more sensitive than determination of the cell-protein or DNA content as a measure of the number of adherent macrophages. For the calculation of the expression of CRIII, Ia antigen, and antigen F4/80 by resident and activated macrophages, use was made of the linear part of the curve obtained when the numbers of macrophages were plotted against the absorbance values for each of the antigens. The values for CRIII expression did not differ significantly between resident macrophages, macrophages activated with recombinant interferon-y (rIFN-7) and macrophages activated with BCG/PPD. IFN-7-activated and BCG/PPD-activated macrophages expressed Ia antigen significantly more intensely than did resident peritoneal macrophages. In contrast the activated macrophages expressed F4/80 significantly less intensely than resident peritoneal macrophages. Key words: Macrophage activation; Cell-surface antigen; ELISA, quantitative cell-
Introduction
Quantification of the expression of cell-surface antigens, e.g., antigen F4/80 and Ia antigen (Nus-
Correspondence to: R. Van Furth, Department of Infectious Diseases, University Hospital Leiden, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Abbreviations: ELISA, enzyme-linked immunosorbent assay; i.p., intraperitoneal; BCG, Mycobacterium boris strain Bacillus Calmette-Gurrin; PPD, purified protein derivative of BCG; PBS, phosphate-buffered saline; CRIII, complement receptor type III; rIFN-y, recombinant interferon-'/; BSA, bovine serum albumin; TMB, 3,3',5,5'-tetramethylbenzidine; EDTA, ethylene diaminotetraacetic acid, disodium salt.
senzweig et al., 1981; Ezekowitz and Gordon, 1982), and of other proteins, e.g., p47b and p71/73 (MacKay and Russell, 1986, 1987) can be important for the evaluation of macrophage activation. Expression of antigens on macrophages is usually determined in large numbers of suspended cells by flow cytometry or a radioimmunobinding assay. These methods are sensitive, easy to perform, and rapid, but, flow cytometry cannot be used to determine the expression of antigens by adherent macrophages and radioimmunoassays require specific conditions for the handling of radioactivity. These drawbacks can be avoided by using the recently developed quantitative enzyme immunocytochemical method (Nibbering et al., 1985,
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
26 1987), but this method is time-consuming and certainly not suitable for studies with large numbers of cells. The cell-ELISA has been used to determine the presence of an antigen on cells, e.g., Ia antigen on cells of a murine macrophage-like cell line (Fairchild and Moorhead, 1985; Le Moal et al., 1989). This approach offers a useful alternative for determination of the expression of antigens by adherent macrophages if the results are related to the number of macrophages under study. The finding that expression of the complement receptor type Ill (CRIII) by adherent murine peritoneal macrophages is proportional to the number of cells (Hamilton et al., 1987) has made it possible to determine the number of adherent macrophages with a cell-ELISA. The purpose of the present study was to design a cell-ELISA to quantify both the expression of antigens by adherent murine peritoneal macrophages and the number of macrophages present.
Materials and methods
Isolation of cells 6-week-old female specific pathogen free C57BL6 mice were purchased from IFFA Credo (Saint Germaine-sur-L'Abresle, France). Peritoneal ceils were harvested by lavage with 2 ml phosphate-buffered saline (PBS, pH 7.4) containing 50 U / m l heparin as described (Van Furth and Cohn, 1968). Cells were collected from (a) untreated mice; (b) from mice 18 h after an intraperitoneal (i.p.) injection of 1 × 104 U recombinant rat interferon-v (rlFN-~/; a gift from Dr. P.H. Van der Meide, Primate Center TNO, Department of Virology, Rijswijk, The Netherlands); and (c) from mice 21 days after intravenous injection of 5 x 106 viable Mycobacterium boris (strain BCG, a gift from Dr. W.H. De Jong, National Institute of Public Health and Environmental Hygiene, RIVM, Bilthoven, The Netherlands). The latter had received an i.p. booster of 50 /tg purified protein derivative (PPD, from Dr. W.H. De Jong, RIVM) on day 19. The rlFN-y preparation contained less than 4 pg endotoxin per 104 U rlFN-y, as quantified with the Limulus assay.
Cell-ELISA To obtain a population of adherent macrophages, 100 /tl suspensions of peritoneal cells in Medium 199 (Microbiological Associates, Walkersville, MD, U.S.A.) supplemented with 10% fetal calf serum (Flow Laboratories, Irvine, U.K.), 1000 U / m l sodium penicillin G (Gist-Brocades, Delft, The Netherlands), and 50/~g/ml gentamycin (Gist-Brocades) were incubated at various concentrations ranging from 1 × 106 to 1 × 102 macrophages per well, in Nunclon microwell plates (Intermed, Roskilde, Denmark) for 2 h at 37 o C. Cell-free control wells received 100 /~1 medium without cells. The non-adherent cells were removed by three washes with PBS using an Immunowash (Intermed). Next, adherent cells were fixed with 0.05% glutaraldehyde in PBS at 4°C for 30 min, washed with saline, and covered with 10% heat-inactivated newborn calf serum in PBS. After removal of the serum, the cells were incubated for 60 min at room temperature with optimal concentrations of one of the following monoclonal antibodies diluted in PBS: M1/70 (an antibody against CD l l b antigen, i.e., the a chain of the complement receptor type III, CRIII (Belier et al., 1982)), F4/80 (an antibody directed to a macrophage-specific antigen (Austyn and Gordon, 1981), the cell line was a gift of Dr. S. Gordon, Sir William Dunn School of Pathology, Oxford, U.K.), and M5/114 (an antibody recognizing I A b'd'q I E d'k ) (Bhattacharya et al., 1981). These three monoclonal antibodies are of the rat IgG2b subclass. As controls, PBS or an antibody against the Thy-1 antigen (antibody 59AD2.2; rat IgG2a subclass (Ledbetter and Herzenberg, 1979)) and an antibody recognizing the B220 antigen (antibody RA3.6B2; rat IgM class (Coffman, 1982)) were used. After three washes with saline, the cells were incubated twice for 15 min each at room temperature with an optimal concentration of peroxidaseconjugated rabbit anti-rat Ig (Dakopatts, Copenhagen, Denmark) in PBS supplemented with 5% normal mouse serum. Excess conjugate was removed by two washes with saline containing 0.05% Tween 20 followed by two washes with saline alone. All reagents were adjusted to the temperature intended for incubation. Binding of monoclonal antibodies to the cells was detected with 0.1 mg/ml 3,3',5,5'-tetramethylbenzidine
27 (TMB, Miles Laboratories, Slough, U.K.) and 0.06% hydrogen peroxide in 0.1 M sodium acetate citric acid buffer (pH 6.0) for 30 rain at room temperature in the dark (Goka and Farthing, 1987). The supernatant was then transferred to a microwell plate provided with 25 #1 1 M sulfuric acid to develop the peroxidase reaction product. Absorbance by the final reaction product at 450 nm was read on a Titertek Multiscan plus (EFLAB, Helsinki, Finland). The observed absorbance was corrected for the non-specific binding of antibodies and the auto-oxidation of TMB by subtracting the absorbance of macrophages exposed to PBS from the observed absorbance, and the result was called specific absorbance. All experiments were performed in triplicate and the specific absorbances expressed as means + SD.
Measurement of adherent cell-protein After completion of the cell-ELISA, the proteins in the wells were solubilized by adding 100 /xl 0.5% w / v sodium dodecylsulfate in 0.2 N sodium hydroxide. Next, the plates were sealed and incubated at 85 o C for 60 rain, after which the content of the wells was transferred to test tubes and the amount of protein determined (Markwell et al., 1978) using serial dilutions of bovine serum albumin (BSA) as standards. The protein value was corrected for residual serum protein adhering to the well surface by subtracting the mean of the values obtained for cell-free wells containing culture medium with serum from the total value for wells containing cells. All experiments were performed in quadruplicate and the results expressed as means _ SD. Determination of DNA content The D N A content of the cells attached to the wells was determined after removal of the immunoperoxidase reaction product. The ceils were washed with PBS and incubated with 60 /~1 of a solution containing 0.06 m g / m l propidium iodide (Sigma Chemical Co., St. Louis, MO, U.S.A.) and 0.13% v / v carbon black (fluorescence quencher solution; Wild-Leitz, Wetzlar, F.R.G.) in 4.6% w / v E D T A (pH 7.0) for 30 min at room temperature. Propidium iodide fluorescence was measured with a computer-assisted Patimed microscope photometer (Wild-Leitz) provided with a mounting
frame for microtiter trays. Scanning of the microwells and data processing were performed with the Patimed Software package (Wild-Leitz). In each plate the fluorescence intensity of individual microwells was corrected for the background fluorescence measured in cell-free wells and expressed as a percentage of the maximum corrected fluorescence value. All experiments were performed six times and the results presented as median and range.
Results
Determination of the number of macrophages attached to microwells Experiments were performed to establish whether virtually all macrophages adhere to the plastic surface of the wells during the incubation period. For this purpose, microwells were plated with a suspension containing between 1 x 105 to 5 x 103 resident macrophages. After 2 h of incubation the supernatant was removed and the adherent cells were washed twice with 1 0 0 / d PBS. After collection of the supernatant and the washing fluids containing all non-adherent cells, the number of non-adherent viable macrophages was determined with a Btirker hemocytometer and differential counts performed in cytocentrifuge preparations fixed in methanol and stained with Giemsa stain. The results showed that 2.3% (range 1.2-4.3%; n = 4) of the viable macrophages added to the wells did not attach to the plastic. To determine the number of macrophages adhering to the wells, the cell protein content of wells plated with various numbers of macrophages was measured. G o o d correlation was found between the numbers of macrophage added and cell protein values over the range 1 x 10 4 to 5 x 105 macrophages per well (least squares regression r = 0.99, P < 0.001) (Fig. 1A). In wells offered less than 1 × 10 4 macrophages the amount of cell protein was below the limit of detection, i.e., 10 /tg protein/ml. The number of adherent macrophages was also determined by measuring the D N A content of the cells in the wells. A good correlation was found between the D N A content and the number of macrophages added over the range 2 x 10 5 to
28 B
A
c
1.5"
100"
~300" t.
200"
~ 1.0"
~ 100"
"~ 0.5"
D.
g~
o-~i
0
_
10 2
10 3
,
,
,
10 4
10 5
10 6
Number of macrophages per well
Z
o.~1
,
0
10 2
, 10 3
10 4
, l0 s
10 ~
Number of macrophages per well
~ 0.0" tO ~
10 a
10 4
lO s
i0 s
Number of macrophages per well
Fig. 1. Relationship between the n u m b e r s of resident macrophages added to the microwells and the a m o u n t of cell-protein (A), the D N A content of the adherent cells (B), and the expression of CRIII by the adherent macrophages, as assessed by a cell-ELISA (C). A: resident peritoneal macrophages were placed at concentrations up to l x 1 0 6 macrophages per well and fixed with 0.05% glutaraldehyde. After disruption of the cells and solubilization of the adherent proteins with 0.5% sodium dodecylsulphate in 0.2 N sodium hydroxide, the a m o u n t of protein in the wells was determined according to Markwell et al. (1978). The observed total protein values were corrected for protein measured in cell-free wells. The results are expressed as the m e a n and standard deviation for four experiments in each of which the mean was based on 3 - 4 wells. B: resident peritoneal macrophages were plated at concentrations up to 5 x 105 macrophages per well, fixed with 0.05% glutaraldehyde, and after removal of immunoperoxidase reaction product were stained with 0.06 m g / m l propidium iodide. Fluorescence was measured with a computer-assisted Patimed microscope photometer. The total fluorescence values were corrected for background fluorescence measured in cell-free wells. The corrected results are expressed as the median and range of the relative fluorescence values of 3 - 4 experiments. C: resident peritoneal macrophages were plated at concentrations up to 2 × 105 macrophages per well, fixed with 0.05% glutaraldehyde, and after a cell-ELISA for CRIII had been performed the absorbance by the final reaction product was determined at 450 nm. In each experiment absorbances were corrected for the mean absorbance of macrophages exposed to PBS. The results are expressed as the m e a n and standard deviation of five experiments in each of which the m e a n was based on at least three wells.
5 x 103 macrophages per well (least squares regression analysis r = 0.99; P < 0.001) (Fig. 1B). For the D N A assay the lower limit of detection was 1-5 x 103 macrophages per well. Similar relationships between the D N A content and the number of added macrophages were found for r l F N ,/-activated and B C G / P P D - a c t i v a t e d macrophages. For both kinds of activated macrophage the curve was linear from 2 × 105 to 5 × 103 macrophages added to each well (r = 0.98; P < 0.001). To find out whether the number of adherent macrophages per well could also be determined with a cell-ELISA, the relationship between the number of resident macrophages added to the wells and the expression of CRIII was analysed. The results showed a linear relationship between the expression of CRIII and the number of added macrophages for the range between 5 × 10 4 and 5 X 10 2 macrophages per well (r = 0.95; P < 0.001) (Fig. 1C). When more than 5 x 104 macrophages were added, the specific absorbances reflecting CRIII expression leveled off. For the range between 5 x 10 4 and 5 x 10 2 macrophages added
per well we found a linear relationship between the C R III expression and the number of rlFN-7activated macrophages ( r = 0.97; P < 0.001) and also of B C G / P P D - a c t i v a t e d macrophages ( r = 0.96; P < 0.001).
Characteristics of the cell-ELISA The optimal concentrations of the immunological reagents were determined by checkerboard analysis using wells that had been plated with a suspension of 5 x 104 macrophages (results not shown). The non-specific absorbances assessed in wells with or without cells and exposed to PBS instead of monoclonal antibody were low (Table I). Binding of monoclonal antibodies to the surface of microwells without cells did not occur (Table I). To evaluate the specificity of the cell-ELISA, the binding to peritoneal macrophages of antibodies detecting antigens on macrophages and antibodies against antigens on lymphocytes were quantified. The results showed that only antibodies recognizing antigens on macrophages bound to adherent peritoneal macrophages. To find out whether the
29 1.5 Complement receptor I!1
"7.
ia antigen
F4/80 antigen
4 1.0
r
e~
°m
g
0.5
e~ o~
0.0 0
7
14
28
0
7
14
28
56
0
7
14
28
56
Days of storage Fig. 2. Expression of CRIII, la antigen, and antigen F4/80 by stored BCG/PPD-activated peritoneal macrophages as determined on various days during a period of 2 months. Peritoneal macrophages from mice infected i.v. with 5 x 106 BCG and boosted i.p. with 50 /.tg PPD, were incubated for 2 h in microwells, fixed with 0.05% gltttaraldehyde, washed with PBS, and stored in PBS containing 0.2 m g / m l gelatin. Expression of an antigen by these cells was determined with a cell-ELISA on various days over a period of two months. The results are expressed as the mean and standard deviation of triplicate measurements. Two-way analysis of variance of the specific absorbances showed no significant decrease in the values during the storage period (P > 0.01).
specific absorbances determined on different days could be compared, we quantified the expression of antigens by glutaraldehyde-fixed B C G / P P D activated peritoneal macrophages stored at 4 ° C for 2 months. Under these conditions, the expression of the antigens under study does not change (Nibbering et al., 1985); no significant differences were observed in the specific absorbances reflecting the expression of various antigens by stored
macrophages during the storage period (two way analysis of variance P > 0.1; Fig. 2).
Expression of antigens by resident and activated macrophages For
this study,
the relationship between
the
numbers of added macrophages and the specific a b s o r b a n c e reflecting e x p r e s s i o n of the Ia a n t i g e n ( F i g . 3A) o r a n t i g e n F 4 / 8 0
(Fig. 3B) was first
TABLE I SPECIFIC ABSORBANCE VALUES REFLECTING THE EXPRESSION OF CRIII, Ia ANTIGEN, AND ANTIGEN F4/80 BY RESIDENT AND ACTIVATED PERITONEAL MACROPHAGES a All results are expressed as mean and standard deviation, n = number of experiments. Peritoneal
n
PBS b
macrophages Resident rIFN-y-activated BCG/PPD-activated None
5 3 4 4
0.04 + 0.03 0.09 + 0.04 0.11 + 0.08 0.08 + 0.03
Specific absorbance CRIII
Ia antigen
Antigen F4/80
0.92 + 0.19 0.94 :k 0.08 0.89:1:0.22 0
0.19 _+0.06 0.46 5:0.05 0.50 + 0.13 0
0.68:1:0.21 0.36 + 0.13 0.16 5:0.14 0
a Determined with a ceI1-ELISA. For the assessment of CRIII expression, microwells were plated with 2.5 x 1 0 4 macrophages, and for the determination of the expression of the Ia antigen or antigen F4/80, 5 × 104 macrophages were plated. b Non-specific absorbance was determined in wells with or without macrophages exposed to PBS.
30 A 1.0
U
1.0"
"
Q
el
e~
t-
0.5" 01
¢m
e~
e~
o.o- ~ -
,
I
0 10 z 10 3 10 4 l0 s 10 6 N u m b e r o f m a c r o p h a g e s per well
0.0.-it 0
I
10 5
i
10 3
10 4
l0 s
10 6
N u m b e r o f m a c r o p h a g e s per well
Fig. 3. Relationship between the number of rlFN-~,-activated peritoneal macrophages and the expression of la antigen (A) and between the numbers of resident macrophage and the expression of antigen F4/80 (B). A: peritoneal macrophages were isolated from mice that bad been injected intraperitoneally with 104 U rlFN-'y 18 h earlier. Expression of la antigen by various numbers of added activated macrophages was determined with a cell-ELISA. The results are expressed as the mean and standard deviation of the specific absorbances of three experiments. Over the range 1 x 103 to 1 x 105 macrophages per well, good correlation was found between the numbers of macrophages and the expression of la antigen (least squares regression analysis r = 0.98; P < 0.001). B: expression of antigen F4/80 by various numbers of resident peritoneal macrophages was determined with a celI-ELISA and the results expressed as for A. A linear relationship between the number of macrophages and the expression of antigen F4/80 was found over the range 5 x 104 to 1 x 103 macrophages per well (least squares regression analysis r = 0.95; P < 0.001).
determined. Expression of the various antigens by resident, rlFN-~,-activated, and B C G / P P D activated macrophages was calculated from the linear slope of these curves; the results are summarized in Table I. CRIII expression by the three kinds of macrophage did not differ significantly. Ia antigens were significantly more (Mann-Whitney U test: P
25
JIM 05613
A cell-ELISA for the quantification of adherent murine macrophages and the surface expression of antigens Peter H. Nibbering, Joke S. Van de Gevel and R a l p h Van Furth Department of Infectious Diseases, University Hospital, Leiden, The Netherlands (Received 17 November 1989, revised received 9 February 1990, accepted 20 March 1990)
The present study was performed in order to establish whether a cell-ELISA could be used to determine the expression of antigens by adherent murine peritoneal macrophages and also quantify the numbers of such macrophages. Accurate determination of the number of adherent macrophages proved to be possible with a cell-ELISA designed to assess complement receptor type III (CRIII) expression. Expression of CRIII was considerably more sensitive than determination of the cell-protein or DNA content as a measure of the number of adherent macrophages. For the calculation of the expression of CRIII, Ia antigen, and antigen F4/80 by resident and activated macrophages, use was made of the linear part of the curve obtained when the numbers of macrophages were plotted against the absorbance values for each of the antigens. The values for CRIII expression did not differ significantly between resident macrophages, macrophages activated with recombinant interferon-y (rIFN-7) and macrophages activated with BCG/PPD. IFN-7-activated and BCG/PPD-activated macrophages expressed Ia antigen significantly more intensely than did resident peritoneal macrophages. In contrast the activated macrophages expressed F4/80 significantly less intensely than resident peritoneal macrophages. Key words: Macrophage activation; Cell-surface antigen; ELISA, quantitative cell-
Introduction
Quantification of the expression of cell-surface antigens, e.g., antigen F4/80 and Ia antigen (Nus-
Correspondence to: R. Van Furth, Department of Infectious Diseases, University Hospital Leiden, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Abbreviations: ELISA, enzyme-linked immunosorbent assay; i.p., intraperitoneal; BCG, Mycobacterium boris strain Bacillus Calmette-Gurrin; PPD, purified protein derivative of BCG; PBS, phosphate-buffered saline; CRIII, complement receptor type III; rIFN-y, recombinant interferon-'/; BSA, bovine serum albumin; TMB, 3,3',5,5'-tetramethylbenzidine; EDTA, ethylene diaminotetraacetic acid, disodium salt.
senzweig et al., 1981; Ezekowitz and Gordon, 1982), and of other proteins, e.g., p47b and p71/73 (MacKay and Russell, 1986, 1987) can be important for the evaluation of macrophage activation. Expression of antigens on macrophages is usually determined in large numbers of suspended cells by flow cytometry or a radioimmunobinding assay. These methods are sensitive, easy to perform, and rapid, but, flow cytometry cannot be used to determine the expression of antigens by adherent macrophages and radioimmunoassays require specific conditions for the handling of radioactivity. These drawbacks can be avoided by using the recently developed quantitative enzyme immunocytochemical method (Nibbering et al., 1985,
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
26 1987), but this method is time-consuming and certainly not suitable for studies with large numbers of cells. The cell-ELISA has been used to determine the presence of an antigen on cells, e.g., Ia antigen on cells of a murine macrophage-like cell line (Fairchild and Moorhead, 1985; Le Moal et al., 1989). This approach offers a useful alternative for determination of the expression of antigens by adherent macrophages if the results are related to the number of macrophages under study. The finding that expression of the complement receptor type Ill (CRIII) by adherent murine peritoneal macrophages is proportional to the number of cells (Hamilton et al., 1987) has made it possible to determine the number of adherent macrophages with a cell-ELISA. The purpose of the present study was to design a cell-ELISA to quantify both the expression of antigens by adherent murine peritoneal macrophages and the number of macrophages present.
Materials and methods
Isolation of cells 6-week-old female specific pathogen free C57BL6 mice were purchased from IFFA Credo (Saint Germaine-sur-L'Abresle, France). Peritoneal ceils were harvested by lavage with 2 ml phosphate-buffered saline (PBS, pH 7.4) containing 50 U / m l heparin as described (Van Furth and Cohn, 1968). Cells were collected from (a) untreated mice; (b) from mice 18 h after an intraperitoneal (i.p.) injection of 1 × 104 U recombinant rat interferon-v (rlFN-~/; a gift from Dr. P.H. Van der Meide, Primate Center TNO, Department of Virology, Rijswijk, The Netherlands); and (c) from mice 21 days after intravenous injection of 5 x 106 viable Mycobacterium boris (strain BCG, a gift from Dr. W.H. De Jong, National Institute of Public Health and Environmental Hygiene, RIVM, Bilthoven, The Netherlands). The latter had received an i.p. booster of 50 /tg purified protein derivative (PPD, from Dr. W.H. De Jong, RIVM) on day 19. The rlFN-y preparation contained less than 4 pg endotoxin per 104 U rlFN-y, as quantified with the Limulus assay.
Cell-ELISA To obtain a population of adherent macrophages, 100 /tl suspensions of peritoneal cells in Medium 199 (Microbiological Associates, Walkersville, MD, U.S.A.) supplemented with 10% fetal calf serum (Flow Laboratories, Irvine, U.K.), 1000 U / m l sodium penicillin G (Gist-Brocades, Delft, The Netherlands), and 50/~g/ml gentamycin (Gist-Brocades) were incubated at various concentrations ranging from 1 × 106 to 1 × 102 macrophages per well, in Nunclon microwell plates (Intermed, Roskilde, Denmark) for 2 h at 37 o C. Cell-free control wells received 100 /~1 medium without cells. The non-adherent cells were removed by three washes with PBS using an Immunowash (Intermed). Next, adherent cells were fixed with 0.05% glutaraldehyde in PBS at 4°C for 30 min, washed with saline, and covered with 10% heat-inactivated newborn calf serum in PBS. After removal of the serum, the cells were incubated for 60 min at room temperature with optimal concentrations of one of the following monoclonal antibodies diluted in PBS: M1/70 (an antibody against CD l l b antigen, i.e., the a chain of the complement receptor type III, CRIII (Belier et al., 1982)), F4/80 (an antibody directed to a macrophage-specific antigen (Austyn and Gordon, 1981), the cell line was a gift of Dr. S. Gordon, Sir William Dunn School of Pathology, Oxford, U.K.), and M5/114 (an antibody recognizing I A b'd'q I E d'k ) (Bhattacharya et al., 1981). These three monoclonal antibodies are of the rat IgG2b subclass. As controls, PBS or an antibody against the Thy-1 antigen (antibody 59AD2.2; rat IgG2a subclass (Ledbetter and Herzenberg, 1979)) and an antibody recognizing the B220 antigen (antibody RA3.6B2; rat IgM class (Coffman, 1982)) were used. After three washes with saline, the cells were incubated twice for 15 min each at room temperature with an optimal concentration of peroxidaseconjugated rabbit anti-rat Ig (Dakopatts, Copenhagen, Denmark) in PBS supplemented with 5% normal mouse serum. Excess conjugate was removed by two washes with saline containing 0.05% Tween 20 followed by two washes with saline alone. All reagents were adjusted to the temperature intended for incubation. Binding of monoclonal antibodies to the cells was detected with 0.1 mg/ml 3,3',5,5'-tetramethylbenzidine
27 (TMB, Miles Laboratories, Slough, U.K.) and 0.06% hydrogen peroxide in 0.1 M sodium acetate citric acid buffer (pH 6.0) for 30 rain at room temperature in the dark (Goka and Farthing, 1987). The supernatant was then transferred to a microwell plate provided with 25 #1 1 M sulfuric acid to develop the peroxidase reaction product. Absorbance by the final reaction product at 450 nm was read on a Titertek Multiscan plus (EFLAB, Helsinki, Finland). The observed absorbance was corrected for the non-specific binding of antibodies and the auto-oxidation of TMB by subtracting the absorbance of macrophages exposed to PBS from the observed absorbance, and the result was called specific absorbance. All experiments were performed in triplicate and the specific absorbances expressed as means + SD.
Measurement of adherent cell-protein After completion of the cell-ELISA, the proteins in the wells were solubilized by adding 100 /xl 0.5% w / v sodium dodecylsulfate in 0.2 N sodium hydroxide. Next, the plates were sealed and incubated at 85 o C for 60 rain, after which the content of the wells was transferred to test tubes and the amount of protein determined (Markwell et al., 1978) using serial dilutions of bovine serum albumin (BSA) as standards. The protein value was corrected for residual serum protein adhering to the well surface by subtracting the mean of the values obtained for cell-free wells containing culture medium with serum from the total value for wells containing cells. All experiments were performed in quadruplicate and the results expressed as means _ SD. Determination of DNA content The D N A content of the cells attached to the wells was determined after removal of the immunoperoxidase reaction product. The ceils were washed with PBS and incubated with 60 /~1 of a solution containing 0.06 m g / m l propidium iodide (Sigma Chemical Co., St. Louis, MO, U.S.A.) and 0.13% v / v carbon black (fluorescence quencher solution; Wild-Leitz, Wetzlar, F.R.G.) in 4.6% w / v E D T A (pH 7.0) for 30 min at room temperature. Propidium iodide fluorescence was measured with a computer-assisted Patimed microscope photometer (Wild-Leitz) provided with a mounting
frame for microtiter trays. Scanning of the microwells and data processing were performed with the Patimed Software package (Wild-Leitz). In each plate the fluorescence intensity of individual microwells was corrected for the background fluorescence measured in cell-free wells and expressed as a percentage of the maximum corrected fluorescence value. All experiments were performed six times and the results presented as median and range.
Results
Determination of the number of macrophages attached to microwells Experiments were performed to establish whether virtually all macrophages adhere to the plastic surface of the wells during the incubation period. For this purpose, microwells were plated with a suspension containing between 1 x 105 to 5 x 103 resident macrophages. After 2 h of incubation the supernatant was removed and the adherent cells were washed twice with 1 0 0 / d PBS. After collection of the supernatant and the washing fluids containing all non-adherent cells, the number of non-adherent viable macrophages was determined with a Btirker hemocytometer and differential counts performed in cytocentrifuge preparations fixed in methanol and stained with Giemsa stain. The results showed that 2.3% (range 1.2-4.3%; n = 4) of the viable macrophages added to the wells did not attach to the plastic. To determine the number of macrophages adhering to the wells, the cell protein content of wells plated with various numbers of macrophages was measured. G o o d correlation was found between the numbers of macrophage added and cell protein values over the range 1 x 10 4 to 5 x 105 macrophages per well (least squares regression r = 0.99, P < 0.001) (Fig. 1A). In wells offered less than 1 × 10 4 macrophages the amount of cell protein was below the limit of detection, i.e., 10 /tg protein/ml. The number of adherent macrophages was also determined by measuring the D N A content of the cells in the wells. A good correlation was found between the D N A content and the number of macrophages added over the range 2 x 10 5 to
28 B
A
c
1.5"
100"
~300" t.
200"
~ 1.0"
~ 100"
"~ 0.5"
D.
g~
o-~i
0
_
10 2
10 3
,
,
,
10 4
10 5
10 6
Number of macrophages per well
Z
o.~1
,
0
10 2
, 10 3
10 4
, l0 s
10 ~
Number of macrophages per well
~ 0.0" tO ~
10 a
10 4
lO s
i0 s
Number of macrophages per well
Fig. 1. Relationship between the n u m b e r s of resident macrophages added to the microwells and the a m o u n t of cell-protein (A), the D N A content of the adherent cells (B), and the expression of CRIII by the adherent macrophages, as assessed by a cell-ELISA (C). A: resident peritoneal macrophages were placed at concentrations up to l x 1 0 6 macrophages per well and fixed with 0.05% glutaraldehyde. After disruption of the cells and solubilization of the adherent proteins with 0.5% sodium dodecylsulphate in 0.2 N sodium hydroxide, the a m o u n t of protein in the wells was determined according to Markwell et al. (1978). The observed total protein values were corrected for protein measured in cell-free wells. The results are expressed as the m e a n and standard deviation for four experiments in each of which the mean was based on 3 - 4 wells. B: resident peritoneal macrophages were plated at concentrations up to 5 x 105 macrophages per well, fixed with 0.05% glutaraldehyde, and after removal of immunoperoxidase reaction product were stained with 0.06 m g / m l propidium iodide. Fluorescence was measured with a computer-assisted Patimed microscope photometer. The total fluorescence values were corrected for background fluorescence measured in cell-free wells. The corrected results are expressed as the median and range of the relative fluorescence values of 3 - 4 experiments. C: resident peritoneal macrophages were plated at concentrations up to 2 × 105 macrophages per well, fixed with 0.05% glutaraldehyde, and after a cell-ELISA for CRIII had been performed the absorbance by the final reaction product was determined at 450 nm. In each experiment absorbances were corrected for the mean absorbance of macrophages exposed to PBS. The results are expressed as the m e a n and standard deviation of five experiments in each of which the m e a n was based on at least three wells.
5 x 103 macrophages per well (least squares regression analysis r = 0.99; P < 0.001) (Fig. 1B). For the D N A assay the lower limit of detection was 1-5 x 103 macrophages per well. Similar relationships between the D N A content and the number of added macrophages were found for r l F N ,/-activated and B C G / P P D - a c t i v a t e d macrophages. For both kinds of activated macrophage the curve was linear from 2 × 105 to 5 × 103 macrophages added to each well (r = 0.98; P < 0.001). To find out whether the number of adherent macrophages per well could also be determined with a cell-ELISA, the relationship between the number of resident macrophages added to the wells and the expression of CRIII was analysed. The results showed a linear relationship between the expression of CRIII and the number of added macrophages for the range between 5 × 10 4 and 5 X 10 2 macrophages per well (r = 0.95; P < 0.001) (Fig. 1C). When more than 5 x 104 macrophages were added, the specific absorbances reflecting CRIII expression leveled off. For the range between 5 x 10 4 and 5 x 10 2 macrophages added
per well we found a linear relationship between the C R III expression and the number of rlFN-7activated macrophages ( r = 0.97; P < 0.001) and also of B C G / P P D - a c t i v a t e d macrophages ( r = 0.96; P < 0.001).
Characteristics of the cell-ELISA The optimal concentrations of the immunological reagents were determined by checkerboard analysis using wells that had been plated with a suspension of 5 x 104 macrophages (results not shown). The non-specific absorbances assessed in wells with or without cells and exposed to PBS instead of monoclonal antibody were low (Table I). Binding of monoclonal antibodies to the surface of microwells without cells did not occur (Table I). To evaluate the specificity of the cell-ELISA, the binding to peritoneal macrophages of antibodies detecting antigens on macrophages and antibodies against antigens on lymphocytes were quantified. The results showed that only antibodies recognizing antigens on macrophages bound to adherent peritoneal macrophages. To find out whether the
29 1.5 Complement receptor I!1
"7.
ia antigen
F4/80 antigen
4 1.0
r
e~
°m
g
0.5
e~ o~
0.0 0
7
14
28
0
7
14
28
56
0
7
14
28
56
Days of storage Fig. 2. Expression of CRIII, la antigen, and antigen F4/80 by stored BCG/PPD-activated peritoneal macrophages as determined on various days during a period of 2 months. Peritoneal macrophages from mice infected i.v. with 5 x 106 BCG and boosted i.p. with 50 /.tg PPD, were incubated for 2 h in microwells, fixed with 0.05% gltttaraldehyde, washed with PBS, and stored in PBS containing 0.2 m g / m l gelatin. Expression of an antigen by these cells was determined with a cell-ELISA on various days over a period of two months. The results are expressed as the mean and standard deviation of triplicate measurements. Two-way analysis of variance of the specific absorbances showed no significant decrease in the values during the storage period (P > 0.01).
specific absorbances determined on different days could be compared, we quantified the expression of antigens by glutaraldehyde-fixed B C G / P P D activated peritoneal macrophages stored at 4 ° C for 2 months. Under these conditions, the expression of the antigens under study does not change (Nibbering et al., 1985); no significant differences were observed in the specific absorbances reflecting the expression of various antigens by stored
macrophages during the storage period (two way analysis of variance P > 0.1; Fig. 2).
Expression of antigens by resident and activated macrophages For
this study,
the relationship between
the
numbers of added macrophages and the specific a b s o r b a n c e reflecting e x p r e s s i o n of the Ia a n t i g e n ( F i g . 3A) o r a n t i g e n F 4 / 8 0
(Fig. 3B) was first
TABLE I SPECIFIC ABSORBANCE VALUES REFLECTING THE EXPRESSION OF CRIII, Ia ANTIGEN, AND ANTIGEN F4/80 BY RESIDENT AND ACTIVATED PERITONEAL MACROPHAGES a All results are expressed as mean and standard deviation, n = number of experiments. Peritoneal
n
PBS b
macrophages Resident rIFN-y-activated BCG/PPD-activated None
5 3 4 4
0.04 + 0.03 0.09 + 0.04 0.11 + 0.08 0.08 + 0.03
Specific absorbance CRIII
Ia antigen
Antigen F4/80
0.92 + 0.19 0.94 :k 0.08 0.89:1:0.22 0
0.19 _+0.06 0.46 5:0.05 0.50 + 0.13 0
0.68:1:0.21 0.36 + 0.13 0.16 5:0.14 0
a Determined with a ceI1-ELISA. For the assessment of CRIII expression, microwells were plated with 2.5 x 1 0 4 macrophages, and for the determination of the expression of the Ia antigen or antigen F4/80, 5 × 104 macrophages were plated. b Non-specific absorbance was determined in wells with or without macrophages exposed to PBS.
30 A 1.0
U
1.0"
"
Q
el
e~
t-
0.5" 01
¢m
e~
e~
o.o- ~ -
,
I
0 10 z 10 3 10 4 l0 s 10 6 N u m b e r o f m a c r o p h a g e s per well
0.0.-it 0
I
10 5
i
10 3
10 4
l0 s
10 6
N u m b e r o f m a c r o p h a g e s per well
Fig. 3. Relationship between the number of rlFN-~,-activated peritoneal macrophages and the expression of la antigen (A) and between the numbers of resident macrophage and the expression of antigen F4/80 (B). A: peritoneal macrophages were isolated from mice that bad been injected intraperitoneally with 104 U rlFN-'y 18 h earlier. Expression of la antigen by various numbers of added activated macrophages was determined with a cell-ELISA. The results are expressed as the mean and standard deviation of the specific absorbances of three experiments. Over the range 1 x 103 to 1 x 105 macrophages per well, good correlation was found between the numbers of macrophages and the expression of la antigen (least squares regression analysis r = 0.98; P < 0.001). B: expression of antigen F4/80 by various numbers of resident peritoneal macrophages was determined with a celI-ELISA and the results expressed as for A. A linear relationship between the number of macrophages and the expression of antigen F4/80 was found over the range 5 x 104 to 1 x 103 macrophages per well (least squares regression analysis r = 0.95; P < 0.001).
determined. Expression of the various antigens by resident, rlFN-~,-activated, and B C G / P P D activated macrophages was calculated from the linear slope of these curves; the results are summarized in Table I. CRIII expression by the three kinds of macrophage did not differ significantly. Ia antigens were significantly more (Mann-Whitney U test: P
