A rapid and sensitive method allowing photometric determination of erythrophagocytosis by mononuclear phagocytes

Journal of Immunological Methods, 82 (1985) 141-153

141

Elsevier JIM 03583

A Rapid and Sensitive Method Allowing Photometric Determination of Erythrophagocytosis by Mononuclear Phagocytes T.W.

Jungi

Institute for Clinical and Experimental Cancer Research, University of Berne, Tiefenau- Hospital, CH- 3004 Berne, Switzerland

(Received 15 February 1985, accepted 26 April 1985)

A spectrometric assay for assessing erythrophagocytosis by mononuclear phagocytes is described. It is based on the haemoglobin-catalyzed conversion of a benzidine derivative into a coloured product in the presence of H202 (pseudoperoxidase activity). The assay is set up in microtitre plates, and following an uptake phase and removal of non-ingested erythrocytes, pseudoperoxidase activity is measured in detergent lysates of phagocytes, using an ELISA reader photometer. Various detergents and substrates were evaluated. SDS was found to be the most suitable detergent. Diaminobenzidine (in PBS, pH 7.4) was the substrate of choice for enumerating ingested erythrocytes in a range from 104 to 5-8 x 105 sheep erythrocytes. Ortho-tolidine (in acetate buffer, pH 5.5) could be used in a range from 2 x 103 to 2 × 103 sheep erythrocytes. The results obtained with human peripheral blood monocytes or monocyte-derived macrophages and IgG-sensitized sheep erythrocytes correlated well with those obtained using 51Cr-labelled, IgG-sensitized erythrocytes. Key words: mononuclear phagocyte function - phagocytosis assay - peroxidase - hemoglobin enzymatic activity- erythrophagocytosis

Introduction T h e d e t e r m i n a t i o n of particle ingestion b y m o n o n u c l e a r p h a g o c y t e s in vitro is an i m p o r t a n t f u n c t i o n a l p a r a m e t e r of these cells. P h a g o c y t o s i s assays using o p s o n i z e d e r y t h r o c y t e s have f o u n d w i d e s p r e a d a c c e p t a n c e a n d the o p s o n i s a t i o n p r o c e d u r e s used i n c l u d e sensitization with I g G a n t i b o d i e s (Michl et al., 1979), sensitization with a n t i b o d i e s a n d c o m p l e m e n t (Bianco et al., 1975); C3 a c t i v a t e d b y the alternative p a t h w a y ( C z o p a n d A u s t e n , 1980), t r e a t m e n t of gelatinized particles with fibronection (Bevilacqua et al., 1981), t a n n i c acid t r e a t m e n t (Biegel a n d R a b i n o v i t c h , 1983), o r a c o m b i n a t i o n of these p r o c e d u r e s ( W r i g h t et al., 1983). The m e t h o d s used for q u a n t i t a t i o n have i n c l u d e d m i c r o s c o p i c e x a m i n a t i o n of mon01ayer cells or cells in suspension, r a d i o m e t r i c e v a l u a t i o n of the u p t a k e of i s o t o p i c a l l y labelled cells (Newm a n et al., 1980) a n d s p e c t r o s c o p i c m e a s u r e m e n t of h a e m o g l o b i n in cell lysates 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

142 (Cooper et al., 1984). The spectroscopic method described in this report has increased sensitivity and utilizes a pseudoperoxidase reaction catalyzed by haemoglobin (Naumann, 1964). The procedure allows rapid and accurate determination of erythrophagocytosis by microtitre plate monolayers with > 30-fold range of phagocytic indices between < 0.1 > 20, depending on the phagocyte number per monolayer and the substrate used.

Materials and Methods

Chemicals Media and additives to culture media were obtained from Seromed, Basle. Bovine serum albumin was purchased from Organon Technika, Turnhout, as a 30% solution (Boseral S). Hydroxyethylpiperazone ethane sulphonic acid (Hepes) and o-tolidine were bought from Fluka, Buchs, and diaminobenzidine (DAB) was from Sigma, St. Louis, MO. SlCr (CEA, France) was obtained through Medipro, Teufen, as sodium chromate in saline, 300 mCi/mg specific activity. Affinity-purified rabbit anti-sheep erythrocyte IgG antibodies (anti-E S antibodies) were from Cordis (Miami, FL). All other reagents were purchased from Merck (Darmstadt). Phagocytes Monocytes were isolated from 50 ml heparinized venous blood, drawn from healthy volunteers, or from the leukocyte buffy coats of whole blood donations in CPD-A, respectively, by Ficoll-Hypaque centrifugation (Boyum, 1968) and repeated washing. The washed mononuclear cells were resuspended at 1.5-2.5 x 106 cells/ml in minimal essential medium (MEM) which contained 0.5% albumin and which was adjusted to pH 7.3 with 1 M Hepes, pH 8.0. This medium (MHA) was used for phagocyte adherence to microtitre plates (Nunc, Roskilde) and for the phagocytosis test. Each well received 100 ~tl cell suspension, and after an adherence phase of 45 min at 37°C, non-adherent cells were washed off with phosphate-buffered saline (0.01 M, pH 7.4) (PBS), using a Nunc Immunowash washing device for microtitre plates, and 100/~1 fresh medium then added. In some experiments, mon0cytes were isolated under sterile conditions and in the presence of low EDTA concentrations (0.3 mM) (Johnson et al., 1977). These cells were used for establishing 8-22-day cultures in teflon bags exactly as described (Andreesen et al., 1983). During this time, monocytes differentiated to mature macrophages. The latter were harvested after chilling the bags for 1 h at 4°C, spun at 200 x g for 8 min, washed once in PBS, resuspended in MHA and placed in microtitre wells (2-4 x 104 macrophages/well). Medium was replaced after 1-2 h of adherence at 37°C, and the phagocytosis test was initiated. Phagocytosis test To microtitre wells containing adherent phagocytes, and 100 ffl MHA, 50/~1 of a 1% solution of sheep erythrocytes (E S) in PBS was added. These had been sensitized with anti-E s IgG antibodies of various subagglutinating concentrations according to

143 standard procedures (Kabat and Mayer, 1961), resulting in E s opsonized by 140 to 1.8 )< 104 IgG molecules per cell (Jungi and Barandun, 1985). Erythrocytes with different antibody densities were tested in triplicate. In some experiments, E s had been labelled with 51Cr prior to sensitization. Packed E ~ were mixed with an equal volume of sodium chromate, 1 mCi/ml, incubated for 40 min at 37°C and washed twice with PBS. The microtitre plates were incubated for 90 min in a humidified chamber at 37°C. Then, non-adherent E ~ were removed with the Nunc Immunowash and adherent non-ingested cells were lysed by a hypotonic shock (PBS: H 2 0 = 2:7, 30 s). In radiometric tests, shocked monolayers were then incubated in isotonic PBS for a further 5 min prior to detergent lysis and radiometric evaluation of the lysates. In spectroscopic tests, PBS was removed, 100 ~tl SDS were added as described in the Results section and complete mixing was assured by repeated pipetting with an Eppendorf yellow tip micropipette. Finally, the substrate (200/~1) was added, and plates were read within 45 rain for extinction at the indicated wave length. The number of erythrocytes ingested per monolayer, as determined spectroscopically or radiometrically, was related to the number of nuclei per monolayer, using cetavlon-amido black for lysing a quadruplicate set of monolayers and counting the stained nuclei microscopically (Nakagawara and Nathan, 1983). The number of E ~ per nucleus was taken as the phagocytic index (PI). The spectroscopic determination of the number of erythrocytes ingested per monolayer was performed with a calibration curve over a suitable E ~ range as described in the Results section.

Spectroscopic determinations Microtitre plates containing a pseudoperoxidase-catalyzed product were read for extinction at 405 nm, 450 nm or 488 nm in a Dynatech MicroELISA Reader, Model AM 115. In some experiments, the content of the well was diluted, transferred to quartz cuvettes and read in a Zeiss spectrophotometer, Model PM QIII containing a monochromator M4 QIII and a tungsten lamp, either at a given wavelength or over a range from 325 nm to 800 nm.

Other procedures The procedures used for lysing phagocyte monolayers and for the preparation of the pseudoperoxidase substrates (DAB) and o-tolidine, are described in the Results section.

Results

Detergent lysis of mononuclearphagocyte monolayers A large number of detergents, tested at different p H values and different ionic strengths were investigated for their ability to achieve complete lysis of phagocyte monolayers without producing turbidity. These included SDS, Triton X-100, Tween 20, saponin, cetavlon, and 3 commercial products lacking information of their composition. The only satisfactory detergent which was compatible with the sub-

144 strates used in the p s e u d o p e r o x i d a s e reaction was SDS. One h u n d r e d /tl S D S c o m p l e t e l y lysed m o n o l a y e r s of freshly e x p l a n t e d m o n o c y t e s d o w n to a c o n c e n t r a tion of 0.1% regardless o f the ionic stength. It could be used in the n e u t r a l p H range d o w n to p H 5, b u t was f o u n d to be i n c o m p a t i b l e with high c o n c e n t r a t i o n s of acetate (see below). F o r the lysis of m o n o l a y e r s of c u l t u r e d m a c r o p h a g e s , 100 # l of 0.3% S D S were used, a n d a clear lysate was o b t a i n e d with m o n o l a y e r s not exceeding 3 x 104 cells. W i t h higher cell n u m b e r s , a slight t u r b i d i t y o c c u r r e d which, however, d i s a p p e a r e d after a d d i t i o n of 1 5 0 - 2 0 0 #l of p e r o x i d a s e s u b s t r a t e (see below).

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145

Spectroscopic assessment of the reaction product of the haemoglobin-catalyzed conoersion of diaminobenzidine (DAB) P r e l i m i n a r y e x p e r i m e n t s revealed that the a d d i t i o n of D A B / H 2 0 z to S D S - l y s e d p h a g o c y t e s which h a d ingested E ~ led to f o r m a t i o n of a green c o l o u r which later t u r n e d b r o w n while c o n t r o l lysates (with m a c r o p h a g e s that h a d n o t ingested E s) d i d n o t change the colour. Systematic e v a l u a t i o n of the a b s o r b a n c e over a w a v e l e n g t h range b e t w e e n 325 n m a n d 800 n M showed that SDS-lysed, D A B / H 2 0 2 - t r e a t e d E ~ e x h i b i t e d strong a b s o r b a n c e in the n e a r - U V r a n g e (Fig. 1A). U s i n g 0.013% H 2 0 2 , the p e a k at 350 n m a p p r o a c h e d its m a x i m a l height after 9 h, a n d a second, b r o a d p e a k d e v e l o p e d in the visible range ( m a x i m a l a b s o r b a n c e at 470 nm). T h e a d d i t i o n of h u m a n m o n o c y t e s or m a c r o p h a g e s to the e r y t h r o c y t e lysate d i d not c h a n g e the characteristic a b s o r b a n c e pattern. D A B alone or D A B / H 2 0 2 a l o n e showed high

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Erythrocytes / Well (xlO -3) Fig. 2. Extinction at 360 nm of a reaction mixture of DAB, SDS and SDS-lysed E s plotted as a function of the E s number added to triplicate wells. Final SDS concentrations were 0.125%. Final DAB concentrations were 0.104 mg/ml in panel A and 0.052 (e), 0,104 (v), 0.173 (&), 0.346 (I) mg/ml, respectively, in panel B. Final H202 concentrations were 0.02 (O), 0.063 (v), 0.10% (A) respectively, in panel A and 0.063% in panel B. Readings were performed at appropriate dilutions using a control sample without E s, and extinctions of the undiluted samples were calculated.

146 absorbance at 325 nm, which fell sharply towards higher wave lengths (Fig. 1B). U p o n prolonged incubation with or without mononuclear phagocytes, there was a shift in the absorbance towards higher wavelengths.

Establishment of optimal conditions for spectroscopic enumeration of SDS-lysed erythrocytes In experiments with graded numbers of E ~, the optimal conditions for spectroscopic enumeration of lysed E S were determined. It was first found that increasing concentrations of SDS progressively inhibited the generation of a product absorbing in the range of 350 nm. However, a final concentration up to 0.1% was found to be acceptable, and lower SDS concentrations did not increase the sensitivity. In other tests, the concentrations of H202 were varied, and absorbance was measured at both 360 nm and 470 nm. Variation of H202 between 0.035% and 0.3% did not lead to a significant change in the absorbance at either wave length (Fig. 2A, and data not shown), but as expected, higher H202 concentrations accelerated the development of a coloured product. Using 0.1% assured that the colour had reached an intensity close to maximum within 15-30 min and did not require stopping prior to photometric analysis. When the reaction was performed with different DAB concentrations, ranging from 0.05 to 0.35 m g / m l , different effects were observed at 360 nm and 470 nm. Readings performed at 350 nm revealed linear dose-response relationships over a broad erythrocyte range, the slope of the curve being similar for all DAB concentrations tested (Fig. 2B). The curves then leveled off for higher E S numbers, the plateau levels being proportional to the amount of DAB used. If readings were performed in the visible range, sigmoid curves were obtained, the (useful) steep quasi-linear part of the curve being shifted to the higher E s range if DAB was used at higher concentrations. DAB concentrations exceeding 0.5 m g / m l could not be used since a brown reaction product was generated spontaneously, i.e., in the absence of added H202, E ~, SDS and phagocytes. These and other experiments established the conditions defined in Table I which were used for spectroscopic assessment of erythrophagocytosis with DAB as a substrate.

Spectrometric and radiometric assessment of erythrophagocytosis by cultured human macrophages Macrophages cultured for 15 or 22 days in vitro were seeded in microtitre wells and then given E ~ sensitized with various amounts of anti-E ~ antibodies. After 90 min of incubation and after removal of non-ingested cells by washing and hypotonic lysis, monolayers were lysed with 100 ~1 SDS, followed by the addition of 200 ~1 of D A B / H 2 0 2 . A control set of monolayer wells received various known amounts of E ~ and were then treated appropriately to establish calibration curve. Cetavlonamidoblack was added to a triplicate well for microscopic enumeration of nuclei per monolayer (Nakagawara and Nathan, 1983). Extinctions read at 450 nm ( a n d / o r at 360 nm) were then used for calculating phagocytic indices (PI) by dividing the number of ingested erythrocytes by the number of nuclei per monolayer (Fig. 3). Ingestion depended on the amount of opsonic antibodies, and a sigmoid curve was obtained as in other phagocytosis systems (Jungi and Barandun, 1985).

Composition 0.1% SDS in PBS, p H 7.4 0.3% SDS in PBS, p H 7.4 0.1% SDS in sodium phosphate buffer, pH 5.5

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P R O C E D U R E F O R S P E C T R O M E T R I C E N U M E R A T I O N OF I N G E S T E D E ~ IN P H A G O C Y T E M O N O L A Y E R S

TABLE I

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In other experiments, phagocytosis by day 8 macrophages was assessed in parallel by spectrometry and by radiometry, using 51Cr-labelled E S in the latter assay. Fig. 4 shows that there was a close correlation between the 2 measuring methods, matching the correlation obtained when 2 individual quadruplicate runs of the radiometric test were performed. The lower limit of both assays was 10 4 E ~, the upper limit of the spectrometric test as determined by the flattening of the calibration curve, was 0.5-0.8 x 106 E s.

Use of o-tolidine as a substrate for enumerating SDS-lysed erythrocytes o-Tolidine was found to be an appropriate alternative substrate for enumerating ingested E s particularly when used below p H 6 and in the presence of acetate. Since low pH and high acetate concentrations interfered with the solubility of SDS lysates, the conditions defined in Table I were found to be optimal for enumerating erythrocytes in such lysates. The product exhibited a high absorbance, peaking at 620 nm and at 380 nm, within 5 min after addition of the substrate. The plates had to be read within 45 rain since a blue precipitate formed in the wells containing high haemoglobin concentrations upon prolonged storage. The calibration curves obtained had a sigmoid shape and could be used for counting E s numbers between 2 x 103 and 200 x 103, thus falling into a range particularly suited for assessing E s ingested by monolayers of freshly explanted monocytes.

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S p e c t r o m e t r i c d e t e r m i n a t i o n o f m o n o c y t e erythrophagocytosis using o-tolidine

M o n o l a y e r s o f freshly i s o l a t e d m o n o c y t e s were allowed to ingest E s sensitized w i t h various a m o u n t s of antibodies, a n d the n u m b e r of ingested cells was d e t e r m i n e d s p e c t r o m e t r i c a U y with either D A B o r o-tolidine as a substrate. Fig. 5 shows t h a t o-tolidine is m o r e sensitive t h a n D A B a n d covers the w h o l e r a n g e of p h a g o c y t i c indices o c c u r r i n g in this test system. I n a n e x p e r i m e n t in which s p e c t r o m e t r i c a n d

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Antibody Molecules / Erythrocyte Fig. 5. Spectroscopic determination of phagocytic indices (means + SD of 4), plotted as a function of anti-E s antibody molecules per target cell. Phagocytes were freshly isolated monocytes; substrates were D A B / H 2 0 2 (A) and o-tolidine/H202 (B). - . . . . . denotes the level below which no significantly elevated extinction was obtained when compared with control wells (unsensitized E ~ or no E ~ added).

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Fig. 6. Correlation of phagocytic indices (means of 4) as determined radiometrically (51Cr-assay) or spectroscopically (o-tolidine assay). Phagocytes were freshly isolated monocytes, target cells were E s opsonized with various a m o u n t s of anti-E s antibodies. Regression lines for the functions y = a x + b a n d x = a ' y + b' ( ), the correlation coefficient (r 2) and the theoretically expected regression line ( . . . . . . ) are indicated.

151 radiometric assessment of phagocytosis were compared, a good correlation of the 2 methods was obtained (Fig. 6) although radiometry was less sensitive than spectrometry.

Discussion

Determination of phagocytosis of opsonized erythrocytes is widely used for quantitating the ingestive capacity of phagocytes for 2 reasons. First, a clear discrimination between non-ingested, attached and ingested particles is possible. Second, background phagocytosis (ingestion of non-opsonized erythrocytes) is virtually nil. In most reports, phagocytosis has been quantified either microscopically (Bianco et al., 1975; Michl et al., 1979; Czop and Austen, 1980; Newman et al., 1980; Wright et al., 1983) or radiometrically, using 5~Cr-labelled erythrocytes (Newman et al., 1980; Cooper et al., 1984). More recently, a spectrometric screening method has been introduced, allowing the measurement of the absorbance of haemoglobin at 412 nm in a photometer for reading microtitre plates, thus circumventing microscopy and the use of radioactive material (Cooper et al., 1984). Here we describe a spectrometric method which is greater than 30-fold more sensitive than the measurement of the absorbance of Hb. It is based on the pseudoperoxidase activity of haemoglobin (Naumann, 1964), catalyzing the conversion of a substrate to a coloured product in the presence of H202. Several peroxidase substrates have been evaluated, and the results obtained with 2 substrates covering different but overlapping ranges of sensitivity are presented. The procedures were adapted to their use in combination with SDS lysates of phagocytes, since it was found that this type of detergent lysis was most reliable for ensuring the complete release of haemoglobin from ingested erythrocytes. DAB as a substrate is suitable for enumerating ingested sheep erythrocytes between 10 4 and 5-8 x 105 per well. Within this working range, quasi-linear calibration curves were obtained, particularly when readings were performed in the near-UV range. The presence of mononuclear phagocytes in the lysates did not significantly influence the shape of the calibration curve if readings were performed within 1 h of addition of the substrate. Prolonged storage of the samples prior to reading led to an increased monocyte-derived absorption. Thus, spectrometry using DAB is suited for measuring phagocytosis of sheep erythrocytes by monocyte-derived macrophages in microtitre wells. If 3 X 10 4 macrophages per monolayer are used, phagocytic indices between > 20 and 0.3 can be determined. (The use of lower cell numbers per monolayer is also possible but it was found that the loss of adherent cells during the washing steps is minimized when cell numbers close to confluency are used.) The method correlates well with the radiometric method and is simila~ in sensitivity but has the advantage that no radioisotopes have to be used. o-Tolidine as a substrate is at least 4 times more sensitive than DAB, thus allowing E s enumeration between 2 x 103 and 1.5-2 x 10 4 cells. The increased sensitivity when compared with the radiometric test makes it well suited for erythrophagocytosis studies involving peripheral blood monocytes. When 4 x 10 4

152

monocytes per well are used, phagocytic indices between 0.05 and 4 can be accurately determined. The method matches the sensitivity of the microscopic examination (a phagocytic index of 0.05 is equal to 1 erythrocyte per 200 phagocytes) but results are available immediately after photometry and do not require time-consuming microscopy. Calibration curves established from monolayers with known E s numbers are of sigmoid shape, and since the presence of phagocytes in the lysate quenches the absorption by 10-20%, these wells must contain the same number of phagocytes as the experimental wells. The low level of endogenous peroxidase in freshly isolated monocytes does not influence the results since wells lacking erythrocytes yield the same extinction regardless of the presence or absence Of monocytes. It is assumed that longer incubation times are required for the generation of tolidine blue catalyzed by endogenous peroxidase than that which is required with haemoglobin as a peroxidase. Among the other peroxidase substrates evaluated, only tetramethylbenzidine (TMB) was found to be suitable for the erythrophagocytosis test. This substrate, which is said to be less carcinogenic than other benzidine derivatives (Holland et al., 1974), was found to have similar properties as o-tolidine, but unlike the latter, TMB had to be dissolved in ethanol and further diluted in sodium phosphate buffer (0.1 M pH 5.5) to give optimal results (S. Hafner and T.W. Jungi, unpublished data). o-Phenylenediamine, a substrate often used in ELISA assays, was lower in sensitivity than DAB. From the literature, it may be inferred that 3-amino-9-ethyl-carbazole is not suited for staining E S (Tubbs et al., 1979) and benzidine is no more sensitive than o-tolidine and TMP (Holland et al., 1974). It appears that the procedure described satisfies the criteria for a screening test in investigations of phagocytosis, using opsonized erythrocytes as a model, since it is rapid, quantitative and sensitive. It can also be used with tanned erythrocytes and, using indicator phagocytes, it may be used to probe phagocytosis of erythrocytes using clinical samples (e.g., autoimmune haemolytic anaemias) without further modification of the erythrocytes.

Acknowledgements This work was supported by the Central Laboratory of the Swiss Red Cross, Blood Transfusion Service, Berne, the Stanley Thomas Johnson Foundation and the Swiss National Science Fund (Grant No. 3.827.0.83). The author thanks Mrs. M. BrEi6 for her expert technical assistance and Dr. A. Morell and Dr. W. Riesen for critical reading of the manuscript.

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