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Detection of eosinophils using an eosinophil peroxidase assay. Its use as an assay for eosinophil differentiation factors
Journal oflmrnunologicalMethods, 83 (1985) 209-215
209
Elsevier JIM03641
Detection of Eosinophils Using an Eosinophil Peroxidase Assay. Its Use as an Assay for Eosinophil Differentiation Factors Malcolm Strath, David J. Warren and Colin J. Sanderson The National Institute for Medical Research, The Ridgewav, Mill ttill, London N W 7 IAA, U.K.
(Received 16 January 1985, accepted 18 June 1985)
A colorimetric assay for peroxidase has been applied to the detection of eosinophils in bone marrow cultures and tissue cell suspensions. The substrate solution consists of 0.1 mM o-phenylenediamine in 0.05 M Tris-HCl buffer pH 8.0 containing 0.1% Triton and 1 mM hydrogen peroxide. The method is shown to be an easy and reproducable method of detecting eosinophils, with insignificant interference from neutrophils. Key words: eosinophil peroxidase - eosinophil differentiation activity
Introduction T h e use of a p e r o x i d a s e assay for the i d e n t i f i c a t i o n of g r a n u l o c y t e s in b l o o d s m e a r s and tissues was suggested b y G r a h a m (1919). H u m a n eosinophils are k n o w n to c o n t a i n relatively large a m o u n t s of p e r o x i d a s e (West et al., 1975; W e v e r et al., 1982), which is different in structure a n d p r o p e r t i e s from the m y e l o p e r o x i d a s e of n e u t r o p h i l s ( K l e b a n o f f et al., 1980; W e v e r et al., 1982). A s this e n z y m e is easy to q u a n t i f y we have investigated the p o s s i b i l i t y that m o u s e eosinophils could be a s s a y e d directly b y assaying for EPO. Both e o s i n o p h i l s a n d n e u t r o p h i l s from mice stain with b e n z i d i n e i n d i c a t i n g the presence of p e r o x i d a s e in b o t h cell types. However, using o - p h e n y l e n e d i a m i n e as h y d r o g e n d o n o r n e u t r o p h i l s show no signific a n t reactivity. Thus eosinophils in p e r i t o n e a l exudate, spleen a n d b o n e m a r r o w cell s u s p e n s i o n s could be d e t e c t e d in the presence of large n u m b e r s of neutrophils. These assays are facilitated b y the use of c o l o r i m e t e r s d e s i g n e d to m e a s u r e a b s o r p t i o n directly on s a m p l e s in microplates. T h e d e v e l o p m e n t of techniques for p r o d u c i n g eosinophils in l o n g - t e r m b o n e m a r r o w cultures (Strath a n d S a n d e r s o n , 1985a) p r o v i d e s a source of e o s i n o p h i i s for
Abbreviations: AMT, 3-amino-I, 2, 4-triazole; EDA, eosinophil differentiation activity; EDF, eosinophil differentiation factor; EPO, eosinophil peroxidase; Tris, tris(hydroxymethyl) methylamine.
0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
210 analytical work as well an assay for lymphokines controlling eosinophil development. In our initial studies aimed at identifying these lymphokines, an assay was developed for assessing the number of eosinophils produced in bone marrow cultures in microplates. This involved differential counts on stained preparations and total counts on a Coulter Counter (Sanderson et al., 1985). This time-consuming assay has been improved using the EPO assay to detect eosinophils in bone marrow cultures. This gives a reliable nonsubjective assay suitable for testing large numbers of T clone and T hybrid supernatants or column fractions during purification procedures.
Materials and Methods
Substrate The substrate solution consists of 0.1 mM o-phenylenediamine dihydrochloride (OPD) (Sigma, Poole) in 0.05 M Tris-HC1 containing Triton X-100 (Sigma) and 1 m M hydrogen peroxide (BDH, Poole). The O P D is made up as a 5 mM stock solution in water, stored at - 7 0 ° C and diluted 1 : 50 into buffer immediately before use. Except where stated otherwise, all incubations were carried out at pH 8.0 in the presence of 0.1% Triton X-100.
Eosinophil peroxidase (EPO) assay Substrate solution (100 ttl) was added to cells or cultures in microtitre plate wells, the plates left at room temperature for 30 min before stopping the reaction by the addition of 50/tl of 4 M suiphuric acid. The absorbance was then determined at 492 nm using a Titertek Multiskan (Flow Labs., Irvine, Scotland). The blanks used were either, (a) substrate solution incubated with medium for the cell titrations or (b) wells lacking eosinophil differentiation activity (EDA) in the case of bone marrow cultures. Once the reaction is stopped the colour is stable for several hours in the dark. Different coocentrations of the EPO inhibitor 3-amino-I, 2, 4-triazole (AMT, Ralph N. Emanuel, Alperton, Middx.) (Cramer et al., 1984) were added to the substrate solution before addition to aliquots of 4 X 104 eosinophils in 50 /tl of medium/well. Potassium cyanide was incorporated into the substrate solution at 1.0, 5.0 and 10 mM final concentration and the peroxidase activity of 2 X 104 eosinophils and neutrophils determined.
Eosinophil and neutrophil titration Quadruplicate aliquots of purified eosinophiis and neutrophils from mice (Lopez et al., 1981), and cells from bone marrow cultures (Strath and Sanderson, 1985a), were added to microtitre plate wells, made up to 50 ~tl with medium (bicarbonate-free RPMI-1640 containing 20 mM Hepes and 5% newborn calf serum), and the EPO activity determined.
211
Eosinophil differentiation activity assay Femoral bone marrow was obtained from Mesocestoides corti parasitised BALB/c. nimr mice (Strath and Sanderson, 1985b), and a single cell suspension prepared in bicarbonate-free RPMI-1640 containing 20 mM Hepes and 5% newborn calf serum. After washing, the cells were resuspended in RPMI-1640 containing bicarbonate, 10 mM Hepes, 2 mM glutamine, 1 mM sodium pyruvate, 7.5 x 10 s M monothioglycerol, 10 ~ M hydrocortisone sodium succinate and 15% foetal calf sera. Aliquots of the cells were then added to 96-well round bottom microtitre plates (Nunc, Denmark) to give 100 #1 cells/well. Mitogen-stimulated spleen supernatant (MSSS) known to contain eosinophil differentiation activity was added (10 yI) to each well (Strath and Sanderson, 1985a). After 6 days at 37°C, the medium was removed and substrate solution added to determine EPO activity.
Tissue eosinophils Peritoneal exudate and bone marrow from normal and M. corti-infected mice were obtained in bicarbonate-free RPMI-1640 containing 20 mM Hepes and 5% foetal calf serum. Cytocentrifuge preparations were methanol-fixed and stained with Giemsa for differential counts. Total cell numbers were determined on a Coulter Counter. Aliquots of the cells in 50 ktl medium were then tested for EPO activity, the same number of cells/well independent of the degree of eosinophilia. Results and Discussion
Optimum conditions for detecting EPO Different numbers of eosinophils, made up to a constant total cell number with neutrophils, were tested at pH 7.0, 8.0 and 9.0. Fig. 1 shows that p H 8.0 is optimum
2.0
A 492
10 L"
10 s
Eosinophils/well
Fig. t. Increasing numbers of eosinophils were made up to 2 x 1 0 s total cells with neutrophils, and assayed at pH 7.0 (A), pH 8.0 (I), pH 9.0 (O). The EPO was measured as the absorbance at 492 nm, with the colorimeter blanked against the substrate solution. Analysis of variance indicated that the data at pH 8.0 was significantly higher than that at pH 9.0 (at 5% level) and pH 7.0 (at 1% level).
212 20-
(A)
20
/'~-- . . . .
A4~
/
10-
/
/~x~: (B)
/
i ~// 103
10~
10~
10]
10L
10~'
Cells/well
Fig. 2. (A) EPO activity of enriched peritoneal eosinophils ( O ) and neutrophils (O). Each point represents the mean of 4 replicate wells +_ 1 SD. Most errors are smaller than the symbols used. The eosinophil preparation contained 77% eosinophils, 8% neutrophils and 15% mononuclear cells. The neutrophil preparation contained 86% neutrophils, 10% eosinophils and 4% mononuclear cells. Thus the activity in neutrophils can be attributed to contaminating eosinophils. (B) EPO activity of cultured eosinophils (D) and neutrophils (l). Values represent means from 4 duplicate wells. The errors are smaller than the symbols used. The eosinophil preparation contained 91% eosinophils, 2% neutrophils and 7% mononuclear cells. The neutrophil preparation contained 84% neutrophils and 16% mononuclear cells.
and that neutrophils have little peroxidase detected by this assay. It was found that 0.1% Triton X-100 permits maximal EPO activity, higher concentrations being inhibitory and lower concentrations presumably not sufficient to lyse the cells and allow release of enzyme (data not shown). In all subsequent experiments the assay was carried out at pH 8.0 in the presence of 0.1% Triton X-100.
Comparison of eosinophil and neutrophil peroxidase acti~fitv Fig. 2A shows the assay of increasing numbers of peritoneal eosinophils purified from M. corti-infected mice. It can be seen that the assay is sensitive enough to detect 103 eosinophils with maximum activity at 104 eosinophils. The activity in the neutrophils can be accounted for by the presence in the cell preparation of 10% eosinophils. This can be compared with Fig. 2B which shows the assay of eosinophils and neutrophils obtained from bone marrow cultures. It can be seen from Fig. 2B that the neutrophil peroxidase contributes little to the total peroxidase activity, thus allowing the differentiation of eosinophil and neutrophil peroxidase without the addition of specific inhibitors.
Inhibition of EPO Fig. 3A shows the effect of different concentrations of A M T on the EPO activity of 4.02 X 104 eosinophils. Maximum activity is obtained in the absence of AMT, but addition of the inhibitor results in a dramatic decrease of enzyme activity. Fig. 3B shows the effect of cyanide on eosinophil and myeloperoxidase, and it can be seen that the small amount of myeloperoxidase is decreased, while the eosinophil peroxidase is relatively resistant to cyanide inhibition. This pattern of sensitivity to enzyme inhibitors is similar to that found in human eosinophils (Yam et al., 1971; Cramer et al., 1984).
213
20- I
20] (B)
(A)
A4~
A492
I0
r
~
10 20 30 mM AMT
40
o
lO
50
lOO
mM KCN
Fig. 3. (A) The effect of different concentrations of AMT on the EPO activity of 4 x 104 eosinophils. EPO activity measured as the absorbance at 492 nm. (B) The inhibition of peroxidase activity by cyanide on 2 × 104 eosinophils (O) and neutrophils (e). Values represent means _+ 1 SD of 4 duplicate wells. Most errors are smaller than the symbols used.
Eosinophil differentiation actiuity assay The demonstration t h a t t h e a s s a y f o r E P O w o u l d d e t e c t e o s i n o p h i l s b u t n o t neutrophils provides a simple method for assessing the development of eosinophils in b o n e m a r r o w m i c r o c u l t u r e s . T h i s is i l l u s t r a t e d i n Fig. 4 w h e r e t h e e f f e c t o f i n i t i a l b o n e m a r r o w i n o c u l u m o n t h e a m o u n t o f E P O o b t a i n e d a f t e r 6 d a y s i n c u b a t i o n is s h o w n . P l a t e a u levels o f E P O w e r e o b t a i n e d w i t h 5 x 10 4 c e l l s / w e l l .
Detection of eosinophils in tissues Fig. 5 A s h o w s t h a t e o s i n o p h i l s c a n b e d e t e c t e d in p e r i t o n e a l e x u d a t e a n d b o n e m a r r o w . T h e s e d a t a s h o w E P O a c t i v i t y in cells f r o m p a r a s i t i s e d a n d n o r m a l a n i m a l s .
2.0
A492
1.0
#
+1
(1)(2)(3)(4) Fig. 4. The effect of incubating different numbers of bone marrow cells in the presence of eosinophil differentiation activity on the EPO activity after 6 days incubation. Values represent means _+ 1 S D of 4 duplicate wells. (1) = 5 × 103 cells/well; (2) = 104 cells/well: (3) = 5 x 104 cells/well; (4) = 105 cells/well. The colorimeter was blanked against wells containing bone marrow cells but lacking EDA.
214 20
A4~2 10
10 ?
10 ~
Total cells/well
10 s
102
10 ? Eosinophils
10 ~
10 s
/ well
Fig. 5. (A) EPO activity from normal and parasitised mouse peritoneal exudate and bone marrow plotted against the total cell number per well. Peritoneal exudate cells from normal (e) and parasitised (O} animals: bone marrow from normal (h,) and parasitised (zx) animals. Values represent means + 1 SD from 4 duplicate wells. Most errors are smaller than the symbols used. (B) EPO activity plotted against the calculated number of eosinophils/well. Legend as for Fig. 5A,
It can be seen that in each case the EPO activity was higher in cells from parasitised animals, reflecting the higher proportion of eosinophils in these tissues. The peritoneal exudate from parasitised mice contains 85% eosinophils, compared to 5% in normal mice. The bone marrow preparation from parasitised mice contained 35% eosinophils, compared to 6% from normal mice. However, when the EPO activity is plotted against the calculated number of eosinophils present in the wells (Fig. 5B), very little difference can be seen between the different tissues, showing the EPO activity is the same for the same number of eosinophils independent of the tissue of origin. Erythrocytes arising by erythropoiesis in microwell bone marrow cultures incubated in the presence of 10% fresh normal mouse serum were negative in the EPO assay (data not shown). Triton X-100-induced lysis of the erythrocytes in whole blood and subsequent release of their contents obscured the EPO reaction of circulating eosinophils. The small number of erythrocytes found in peritoneal exudate and bone marrow caused no interference in the EPO assay of these tissues.
Conclusions The use of this assay to quantify the relative number of eosinophils could be limited if the amount of EPO were to differ significantly in eosinophils from different sources. In practise this has not been a problem in the mouse. Firstly, eosinophils from different tissues showed a similar level of EPO when plotted against the actual number of eosinophils (Fig. 5B). Secondly, a comparison of peritoneal exudate eosinophils with eosinophils grown in culture also showed similar levels of EPO (Strath and Sanderson, 1985a). However, as a precautionary measure, we routinely count the eosinophils in each system before relying on EPO activity. In this way we could detect changes in EPO activity per cell.
215 T h e m a i n a d v a n t a g e s of the E P O assay s y s t e m is to assess the r e l a t i v e n u m b e r of e o s i n o p h i l s f r o m s i m i l a r s o u r c e s w h e n large n u m b e r s o f s a m p l e s are i n v o l v e d . F o r e x a m p l e , for f o l l o w i n g t h e d e v e l o p m e n t o f e o s i n o p h i l s in the tissues ( b u t not the b l o o d ) of p a r a s i t i s e d mice, in assays for e o s i n o p h i l d i f f e r e n t i a t i o n f a c t o r s ( S a n d e r s o n a n d Strath, 1985; S a n d e r s o n et al., 1985; W a r r e n a n d S a n d e r s o n , 1985) o r for a s s e s s i n g the r e l a t i v e n u m b e r o f e o s i n o p h i l p r e c u r s o r s in the b o n e m a r r o w d u r i n g the d e v e l o p m e n t o f e o s i n o p h i l i a ( S t r a t h a n d S a n d e r s o n , 1985b).
References Cramer, R., M.R. Soranzo. R. Menegazzi, A. Pitotti, G. Zabucchi and P. Patriarca, 1984, J. lmmunol. Methods 70, 119. Graham, G.S., 1919, J. Med. Res. 19, 15. Klebanoff, S.J., E.C. Jong and W.R. Henderson, 1980, in: The Eosinophil in Health and Disease, ed. A. Mahmoud (Grune and Stratton, London) p. 99. Lopez, A.F., M. Strath and C.J. Sanderson, 1981, Immunology 43, 779. Sanderson, C.J. and M. Strath, 1985, Immunology, 54, 275. Sanderson, C.J., D.J. Warren and M. Strath, 1985, J. Exp. Med. 162, in press. Strath, M. and C.J. Sanderson, 1985a, J. Cell Science 74, in press. Strath, M. and C.J. Sanderson, 1985b, Exp. Hematol., in press. Warren, D.J. and Sanderson, C.J., 1985, Immunology 54, 615. West, B.C., N.A. Gelb and R.S. Rosenthak 1975, Am. J. Pathol. 81, 575. Wever, R., M.N. Hamers, C.J. De Graaf, R.S. Weening and D. Roos, 1982, Adv. Exp. Med. Biol. 141, 501. Yam, L.T., C.Y. Li and W.H. Crosby, 1971, Am. J. Clin. Pathol. 55, 283.
209
Elsevier JIM03641
Detection of Eosinophils Using an Eosinophil Peroxidase Assay. Its Use as an Assay for Eosinophil Differentiation Factors Malcolm Strath, David J. Warren and Colin J. Sanderson The National Institute for Medical Research, The Ridgewav, Mill ttill, London N W 7 IAA, U.K.
(Received 16 January 1985, accepted 18 June 1985)
A colorimetric assay for peroxidase has been applied to the detection of eosinophils in bone marrow cultures and tissue cell suspensions. The substrate solution consists of 0.1 mM o-phenylenediamine in 0.05 M Tris-HCl buffer pH 8.0 containing 0.1% Triton and 1 mM hydrogen peroxide. The method is shown to be an easy and reproducable method of detecting eosinophils, with insignificant interference from neutrophils. Key words: eosinophil peroxidase - eosinophil differentiation activity
Introduction T h e use of a p e r o x i d a s e assay for the i d e n t i f i c a t i o n of g r a n u l o c y t e s in b l o o d s m e a r s and tissues was suggested b y G r a h a m (1919). H u m a n eosinophils are k n o w n to c o n t a i n relatively large a m o u n t s of p e r o x i d a s e (West et al., 1975; W e v e r et al., 1982), which is different in structure a n d p r o p e r t i e s from the m y e l o p e r o x i d a s e of n e u t r o p h i l s ( K l e b a n o f f et al., 1980; W e v e r et al., 1982). A s this e n z y m e is easy to q u a n t i f y we have investigated the p o s s i b i l i t y that m o u s e eosinophils could be a s s a y e d directly b y assaying for EPO. Both e o s i n o p h i l s a n d n e u t r o p h i l s from mice stain with b e n z i d i n e i n d i c a t i n g the presence of p e r o x i d a s e in b o t h cell types. However, using o - p h e n y l e n e d i a m i n e as h y d r o g e n d o n o r n e u t r o p h i l s show no signific a n t reactivity. Thus eosinophils in p e r i t o n e a l exudate, spleen a n d b o n e m a r r o w cell s u s p e n s i o n s could be d e t e c t e d in the presence of large n u m b e r s of neutrophils. These assays are facilitated b y the use of c o l o r i m e t e r s d e s i g n e d to m e a s u r e a b s o r p t i o n directly on s a m p l e s in microplates. T h e d e v e l o p m e n t of techniques for p r o d u c i n g eosinophils in l o n g - t e r m b o n e m a r r o w cultures (Strath a n d S a n d e r s o n , 1985a) p r o v i d e s a source of e o s i n o p h i i s for
Abbreviations: AMT, 3-amino-I, 2, 4-triazole; EDA, eosinophil differentiation activity; EDF, eosinophil differentiation factor; EPO, eosinophil peroxidase; Tris, tris(hydroxymethyl) methylamine.
0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
210 analytical work as well an assay for lymphokines controlling eosinophil development. In our initial studies aimed at identifying these lymphokines, an assay was developed for assessing the number of eosinophils produced in bone marrow cultures in microplates. This involved differential counts on stained preparations and total counts on a Coulter Counter (Sanderson et al., 1985). This time-consuming assay has been improved using the EPO assay to detect eosinophils in bone marrow cultures. This gives a reliable nonsubjective assay suitable for testing large numbers of T clone and T hybrid supernatants or column fractions during purification procedures.
Materials and Methods
Substrate The substrate solution consists of 0.1 mM o-phenylenediamine dihydrochloride (OPD) (Sigma, Poole) in 0.05 M Tris-HC1 containing Triton X-100 (Sigma) and 1 m M hydrogen peroxide (BDH, Poole). The O P D is made up as a 5 mM stock solution in water, stored at - 7 0 ° C and diluted 1 : 50 into buffer immediately before use. Except where stated otherwise, all incubations were carried out at pH 8.0 in the presence of 0.1% Triton X-100.
Eosinophil peroxidase (EPO) assay Substrate solution (100 ttl) was added to cells or cultures in microtitre plate wells, the plates left at room temperature for 30 min before stopping the reaction by the addition of 50/tl of 4 M suiphuric acid. The absorbance was then determined at 492 nm using a Titertek Multiskan (Flow Labs., Irvine, Scotland). The blanks used were either, (a) substrate solution incubated with medium for the cell titrations or (b) wells lacking eosinophil differentiation activity (EDA) in the case of bone marrow cultures. Once the reaction is stopped the colour is stable for several hours in the dark. Different coocentrations of the EPO inhibitor 3-amino-I, 2, 4-triazole (AMT, Ralph N. Emanuel, Alperton, Middx.) (Cramer et al., 1984) were added to the substrate solution before addition to aliquots of 4 X 104 eosinophils in 50 /tl of medium/well. Potassium cyanide was incorporated into the substrate solution at 1.0, 5.0 and 10 mM final concentration and the peroxidase activity of 2 X 104 eosinophils and neutrophils determined.
Eosinophil and neutrophil titration Quadruplicate aliquots of purified eosinophiis and neutrophils from mice (Lopez et al., 1981), and cells from bone marrow cultures (Strath and Sanderson, 1985a), were added to microtitre plate wells, made up to 50 ~tl with medium (bicarbonate-free RPMI-1640 containing 20 mM Hepes and 5% newborn calf serum), and the EPO activity determined.
211
Eosinophil differentiation activity assay Femoral bone marrow was obtained from Mesocestoides corti parasitised BALB/c. nimr mice (Strath and Sanderson, 1985b), and a single cell suspension prepared in bicarbonate-free RPMI-1640 containing 20 mM Hepes and 5% newborn calf serum. After washing, the cells were resuspended in RPMI-1640 containing bicarbonate, 10 mM Hepes, 2 mM glutamine, 1 mM sodium pyruvate, 7.5 x 10 s M monothioglycerol, 10 ~ M hydrocortisone sodium succinate and 15% foetal calf sera. Aliquots of the cells were then added to 96-well round bottom microtitre plates (Nunc, Denmark) to give 100 #1 cells/well. Mitogen-stimulated spleen supernatant (MSSS) known to contain eosinophil differentiation activity was added (10 yI) to each well (Strath and Sanderson, 1985a). After 6 days at 37°C, the medium was removed and substrate solution added to determine EPO activity.
Tissue eosinophils Peritoneal exudate and bone marrow from normal and M. corti-infected mice were obtained in bicarbonate-free RPMI-1640 containing 20 mM Hepes and 5% foetal calf serum. Cytocentrifuge preparations were methanol-fixed and stained with Giemsa for differential counts. Total cell numbers were determined on a Coulter Counter. Aliquots of the cells in 50 ktl medium were then tested for EPO activity, the same number of cells/well independent of the degree of eosinophilia. Results and Discussion
Optimum conditions for detecting EPO Different numbers of eosinophils, made up to a constant total cell number with neutrophils, were tested at pH 7.0, 8.0 and 9.0. Fig. 1 shows that p H 8.0 is optimum
2.0
A 492
10 L"
10 s
Eosinophils/well
Fig. t. Increasing numbers of eosinophils were made up to 2 x 1 0 s total cells with neutrophils, and assayed at pH 7.0 (A), pH 8.0 (I), pH 9.0 (O). The EPO was measured as the absorbance at 492 nm, with the colorimeter blanked against the substrate solution. Analysis of variance indicated that the data at pH 8.0 was significantly higher than that at pH 9.0 (at 5% level) and pH 7.0 (at 1% level).
212 20-
(A)
20
/'~-- . . . .
A4~
/
10-
/
/~x~: (B)
/
i ~// 103
10~
10~
10]
10L
10~'
Cells/well
Fig. 2. (A) EPO activity of enriched peritoneal eosinophils ( O ) and neutrophils (O). Each point represents the mean of 4 replicate wells +_ 1 SD. Most errors are smaller than the symbols used. The eosinophil preparation contained 77% eosinophils, 8% neutrophils and 15% mononuclear cells. The neutrophil preparation contained 86% neutrophils, 10% eosinophils and 4% mononuclear cells. Thus the activity in neutrophils can be attributed to contaminating eosinophils. (B) EPO activity of cultured eosinophils (D) and neutrophils (l). Values represent means from 4 duplicate wells. The errors are smaller than the symbols used. The eosinophil preparation contained 91% eosinophils, 2% neutrophils and 7% mononuclear cells. The neutrophil preparation contained 84% neutrophils and 16% mononuclear cells.
and that neutrophils have little peroxidase detected by this assay. It was found that 0.1% Triton X-100 permits maximal EPO activity, higher concentrations being inhibitory and lower concentrations presumably not sufficient to lyse the cells and allow release of enzyme (data not shown). In all subsequent experiments the assay was carried out at pH 8.0 in the presence of 0.1% Triton X-100.
Comparison of eosinophil and neutrophil peroxidase acti~fitv Fig. 2A shows the assay of increasing numbers of peritoneal eosinophils purified from M. corti-infected mice. It can be seen that the assay is sensitive enough to detect 103 eosinophils with maximum activity at 104 eosinophils. The activity in the neutrophils can be accounted for by the presence in the cell preparation of 10% eosinophils. This can be compared with Fig. 2B which shows the assay of eosinophils and neutrophils obtained from bone marrow cultures. It can be seen from Fig. 2B that the neutrophil peroxidase contributes little to the total peroxidase activity, thus allowing the differentiation of eosinophil and neutrophil peroxidase without the addition of specific inhibitors.
Inhibition of EPO Fig. 3A shows the effect of different concentrations of A M T on the EPO activity of 4.02 X 104 eosinophils. Maximum activity is obtained in the absence of AMT, but addition of the inhibitor results in a dramatic decrease of enzyme activity. Fig. 3B shows the effect of cyanide on eosinophil and myeloperoxidase, and it can be seen that the small amount of myeloperoxidase is decreased, while the eosinophil peroxidase is relatively resistant to cyanide inhibition. This pattern of sensitivity to enzyme inhibitors is similar to that found in human eosinophils (Yam et al., 1971; Cramer et al., 1984).
213
20- I
20] (B)
(A)
A4~
A492
I0
r
~
10 20 30 mM AMT
40
o
lO
50
lOO
mM KCN
Fig. 3. (A) The effect of different concentrations of AMT on the EPO activity of 4 x 104 eosinophils. EPO activity measured as the absorbance at 492 nm. (B) The inhibition of peroxidase activity by cyanide on 2 × 104 eosinophils (O) and neutrophils (e). Values represent means _+ 1 SD of 4 duplicate wells. Most errors are smaller than the symbols used.
Eosinophil differentiation actiuity assay The demonstration t h a t t h e a s s a y f o r E P O w o u l d d e t e c t e o s i n o p h i l s b u t n o t neutrophils provides a simple method for assessing the development of eosinophils in b o n e m a r r o w m i c r o c u l t u r e s . T h i s is i l l u s t r a t e d i n Fig. 4 w h e r e t h e e f f e c t o f i n i t i a l b o n e m a r r o w i n o c u l u m o n t h e a m o u n t o f E P O o b t a i n e d a f t e r 6 d a y s i n c u b a t i o n is s h o w n . P l a t e a u levels o f E P O w e r e o b t a i n e d w i t h 5 x 10 4 c e l l s / w e l l .
Detection of eosinophils in tissues Fig. 5 A s h o w s t h a t e o s i n o p h i l s c a n b e d e t e c t e d in p e r i t o n e a l e x u d a t e a n d b o n e m a r r o w . T h e s e d a t a s h o w E P O a c t i v i t y in cells f r o m p a r a s i t i s e d a n d n o r m a l a n i m a l s .
2.0
A492
1.0
#
+1
(1)(2)(3)(4) Fig. 4. The effect of incubating different numbers of bone marrow cells in the presence of eosinophil differentiation activity on the EPO activity after 6 days incubation. Values represent means _+ 1 S D of 4 duplicate wells. (1) = 5 × 103 cells/well; (2) = 104 cells/well: (3) = 5 x 104 cells/well; (4) = 105 cells/well. The colorimeter was blanked against wells containing bone marrow cells but lacking EDA.
214 20
A4~2 10
10 ?
10 ~
Total cells/well
10 s
102
10 ? Eosinophils
10 ~
10 s
/ well
Fig. 5. (A) EPO activity from normal and parasitised mouse peritoneal exudate and bone marrow plotted against the total cell number per well. Peritoneal exudate cells from normal (e) and parasitised (O} animals: bone marrow from normal (h,) and parasitised (zx) animals. Values represent means + 1 SD from 4 duplicate wells. Most errors are smaller than the symbols used. (B) EPO activity plotted against the calculated number of eosinophils/well. Legend as for Fig. 5A,
It can be seen that in each case the EPO activity was higher in cells from parasitised animals, reflecting the higher proportion of eosinophils in these tissues. The peritoneal exudate from parasitised mice contains 85% eosinophils, compared to 5% in normal mice. The bone marrow preparation from parasitised mice contained 35% eosinophils, compared to 6% from normal mice. However, when the EPO activity is plotted against the calculated number of eosinophils present in the wells (Fig. 5B), very little difference can be seen between the different tissues, showing the EPO activity is the same for the same number of eosinophils independent of the tissue of origin. Erythrocytes arising by erythropoiesis in microwell bone marrow cultures incubated in the presence of 10% fresh normal mouse serum were negative in the EPO assay (data not shown). Triton X-100-induced lysis of the erythrocytes in whole blood and subsequent release of their contents obscured the EPO reaction of circulating eosinophils. The small number of erythrocytes found in peritoneal exudate and bone marrow caused no interference in the EPO assay of these tissues.
Conclusions The use of this assay to quantify the relative number of eosinophils could be limited if the amount of EPO were to differ significantly in eosinophils from different sources. In practise this has not been a problem in the mouse. Firstly, eosinophils from different tissues showed a similar level of EPO when plotted against the actual number of eosinophils (Fig. 5B). Secondly, a comparison of peritoneal exudate eosinophils with eosinophils grown in culture also showed similar levels of EPO (Strath and Sanderson, 1985a). However, as a precautionary measure, we routinely count the eosinophils in each system before relying on EPO activity. In this way we could detect changes in EPO activity per cell.
215 T h e m a i n a d v a n t a g e s of the E P O assay s y s t e m is to assess the r e l a t i v e n u m b e r of e o s i n o p h i l s f r o m s i m i l a r s o u r c e s w h e n large n u m b e r s o f s a m p l e s are i n v o l v e d . F o r e x a m p l e , for f o l l o w i n g t h e d e v e l o p m e n t o f e o s i n o p h i l s in the tissues ( b u t not the b l o o d ) of p a r a s i t i s e d mice, in assays for e o s i n o p h i l d i f f e r e n t i a t i o n f a c t o r s ( S a n d e r s o n a n d Strath, 1985; S a n d e r s o n et al., 1985; W a r r e n a n d S a n d e r s o n , 1985) o r for a s s e s s i n g the r e l a t i v e n u m b e r o f e o s i n o p h i l p r e c u r s o r s in the b o n e m a r r o w d u r i n g the d e v e l o p m e n t o f e o s i n o p h i l i a ( S t r a t h a n d S a n d e r s o n , 1985b).
References Cramer, R., M.R. Soranzo. R. Menegazzi, A. Pitotti, G. Zabucchi and P. Patriarca, 1984, J. lmmunol. Methods 70, 119. Graham, G.S., 1919, J. Med. Res. 19, 15. Klebanoff, S.J., E.C. Jong and W.R. Henderson, 1980, in: The Eosinophil in Health and Disease, ed. A. Mahmoud (Grune and Stratton, London) p. 99. Lopez, A.F., M. Strath and C.J. Sanderson, 1981, Immunology 43, 779. Sanderson, C.J. and M. Strath, 1985, Immunology, 54, 275. Sanderson, C.J., D.J. Warren and M. Strath, 1985, J. Exp. Med. 162, in press. Strath, M. and C.J. Sanderson, 1985a, J. Cell Science 74, in press. Strath, M. and C.J. Sanderson, 1985b, Exp. Hematol., in press. Warren, D.J. and Sanderson, C.J., 1985, Immunology 54, 615. West, B.C., N.A. Gelb and R.S. Rosenthak 1975, Am. J. Pathol. 81, 575. Wever, R., M.N. Hamers, C.J. De Graaf, R.S. Weening and D. Roos, 1982, Adv. Exp. Med. Biol. 141, 501. Yam, L.T., C.Y. Li and W.H. Crosby, 1971, Am. J. Clin. Pathol. 55, 283.