Measurement of guinea pig eosinophil major basic protein by radioimmunoassay

hfolecular Immunology, Vol. 16, pp. 71 l-719. Pergamon Press Ltd. 1979. Printed in Great Britain.

MEASUREMENT OF GUINEA PIG EOSINOPHIL MAJOR BASIC PROTEIN BY RADIOIMMUNOASSAY* DONALD L. WASSOM,? DAVID A. LOEGERING

and GERALD

J. GLEICH

Departments of Medicine and Immunology, Mayo Clinic and Mayo Foundation, Rochester, MN, U.S.A. (Received 10 August 1978) Abstract-Major basic protein (MBP)$ was measured by radioimmunoassay (RIA) using 1311-MBP,rabbit anti-MBP, and burro anti-rabbit IgG. Two critical features of the assay were: (1) alkylation of the MBP with iodoacetamide prior to radioiodination and (2) inclusion of another basic protein, either protamine or histone, in the phosphate buffer. Freshly isolated non-alkylated MBP was immunologically deficient when compared to alkylated or reduced and alkylated MBP, but its reactivity could be restored by reduction with dithiothreitol and alkylation. Polymerized MBP also showed reduced immunoreactivity which could be regenerated by reduction and alkylation. MBP activity was rapidly destroyed at 56”C, but it could be iyophilized, and it survived treatment with 6 M guanidinium hydrochloride (GuHCI). After initial reduction and alkylation in the absence of dissociating solvents, subsequent reduction and alkylation in 6 M GuHCl renders the protein unreactive. MBP levels in normal guinea pig serum were less than 5 ng/ml and were not elevated in sera from guinea pigs parasitized with Trichinellu spiralis and having peripheral blood eosinophilia. Muscle extracts from Trichinella infected animals showed significantly higher levels of MBP activity than normal controls. MBP was measurable in extracts of untreated eosinophils, but reduction and alkylation of these extracts increased MBP activity several fold. Extracts of purified eosinophils averaged 1.13 x IO- 3 ntz MBP per cell. The RIA oermits detection of MBP in bodv fluids and tissues at levels as low as 2 ng/ml. The KIA is-useful in assessing increased or decreased levels of MBP activity in samples from experimental animals when compared to samples from controls.

INTRODUCTION

has been associated with allergic and parasitic diseases for many years, yet the specific functions of eosinophils in such interactions are not fully known. Mahmoud has used a mono-specific antieosinophil serum to implicate the eosinophil as an important component of acquired immunity to Schistosoma mansoni in mice (Mahmoud et al., 19750, b) and Grove obtained similar results studying infections with Trichinella spiralis (Grove et al., 1977). Additionally, Butterworth has demonstrated antibody dependent killing of S. mansoni schistosomules by human eosinophils in vitro (Butterworth et al., 1975, 1977). Eosinophil products have been shown to modulate release of histamine (Hubscher. 1975a,b; Jones & Kay, 1976; Zeiger et al., 1976) and to inactivate slow reacting substance of anaphylaxis (Wasserman et al., 1975) and platelet The eosinophil

*Supported in part by grants from the National Institutes of Allergy and Jnfectious Diseases, AI 9728, AI 11483, AI 07047 and from the Mayo Foundation. tAddress for correspondence: Donald L. Wassom, Allergic Diseases Research Laboratory, Mayo Clinic, Rochester, MN 55901, U.S.A 1 Abbreviations used: Major basic protein, MBP; dithiothreitol, DTT; bovine serum albumin, BSA; iodoacetamide, IA; sodium dodecyl sulfate, SDS; ethylenediamine tetraacetate, EDTA; fetal calf serum, FCS; Hank’s balanced salt solution, BSS; Dulbecco’s phosphate buffered saline. PBS: guanidinium hvdrochloride. GuHCI: polyacrylamide gel ele&opho&sis, PAGE; intradeimal, IDi phosphate-protamine-fetal calf serum buffer, PPF; counts per minute, counts/min; radioimmunoassay, RIA. M.1 M.M. 1619~~

activating factor (Kater et al., 1976) and may possibly play a role in modulating the allergic response (Hubscher, 1977). Additionally, large numbers of eosinophils accumulate in the rat uterus during estrus and the eosinophil may play a role in rat fertility (Rytomaa, 1960; Tchernitchin et al., 1974). A major basic protein (MBP), localized within the core of the eosinophil granule, has been isolated from eosinophils of guinea pigs, man and rats (Gleich et al., 1973, 1974, 1976; Lewis et al., 1976a, 1978) and is estimated to account for approximately 25% of all protein present within the cell. The protein is composed of 13% arginine, has an isoelectric point greater than ten and has a mol. wt of 11,000 daltons. It contains two disulfide bonds and two free sulfhydryl groups and readily forms polymers which are multiples of the 11,000 mol. wt sub-unit. The monomer form of the MBP can be stabilized either by alkylation of freshly isolated MBP or by reduction and alkylation of the MBP. The MBP is not a histone and is distinct from the Charcot-Leyden crystal protein. Major basic protein possesses weak anti-bacterial activity and it does not contract guinea-pig ileum. Recently, the authors, in collaboration with Butterworth and David have found that the major basic protein is cytotoxic for schistosomules of S. mansoni, and may be responsible for the observed antibody dependent damage to these parasites by purified eosinophils. To better assess the importance of MBP as an effector of eosinophil function, a method for accurately measuring the content of MBP in biological samples was needed. This report outlines a sensitive radioimmunoassay which allows detection of as little as 2 ng of MBP/ml.

711

712

DONALD

L. WASSOM.

DAVID

A. LOEGERING

MATERIALS AND METHODS

Guinea pigs Dunkin-Hartley, randomly bred guinea pigs obtained from Bio-Lab Corp.. St. Paul, MN, were used for all studies. Mutrrials Protamine sulfate, bovine serum albumin (BSA), Triton X100, dithiothreitol (DTT) and iodoacetamide (IA) were obtained from Sigma Chemical Co.. St. Louis, MO. Sodium dodecyl sulfate (SDS), polyacrylamide, Tween-80, sucrose, ethvlenediamine tetraacetate (EDTA), and potassium iodide were purchased from Fisher Scientific Co., Pittsburgh, PA. Fetal calf serum (FCS). Hank’s balanced salt solution (BSS) (10 x ) and Dulbeccos phosphate buffered saline (PBS) (IO x ) were obtained from Grand Island Biological, Grand Island, NY. i3i1 was obtained from New England Nuclear, Boston. MA: Senhadex G-50 was from Pharmacia Fine Chemicals, Inc.: Piscataway, NY; guanidinium hydrochloride (GuCHI) was froml-leico, Delaware Water Gap, PA; and metrizamide from Nygaard and Co. A/S, Oslo. Phloxine B and methylene blue were purchased from Allied Chemical, National Aniline Div.. New York, NY. PuriJicurion

ofguinea pig eosinophil MBP

The method for isolating MBP from guinea pig eosinophils has been described elsewhere (Gleich et ul., 1973, 1974). Briefly. eosinophil-rich cell suspensions were obtained from the peritoneal cavities of guinea pigs repeatedly lavaged with 50 ml of normal saline. Cells were concentrated by centrifugation at 400g for IO min, and erythrocytes lysed by adding 9 ml distilled water to 3 ml cell suspension in 0. I5 M NaCI, mixing for 20 set and reconstituting isotonicity with 1 ml IO x PBS. The remaining cells were washed twice in cold fresh 0.34 M sucrose by centrifugation at 400~ for IO min and they were

and GERALD

J. GLEICH

resuspended in 10 ml fresh 0.34 M sucrose. The cells, which become fragile to lysis during the sucrose washes, were disrupted in a Tenbroeck tissue grinder in an ice bath (Kontes Glass Co., Evanston, IL) by ten passes of the pestle. Disrupted cells were centrifuged for IO min at 4OOg and the opalescent supernatant was transferred to another tube and centrifuged at lO.OOOg for 20 min (J-21 centrifuge Beckman Instruments. Spinco Division. Palo Alto. CA). The brownish-green pellet, containing the eosinophil granules, was solubilized in I ml 0.01 M HCI and centrifuged at 40.000 g for 5 min. The supernatant, containing the MBP. was passed over a Sephadex G-50 fine column ( I .Z x 45 cm) equilibrated with 0.025 ,2/1acetate buffer-O. I5 M NaCI, pH 4.3. The second peak off the column contained the MBP. Freshly isolated MBP readily polymerizes but it can be stabilized by alkylating its two free sullhydryl groups with iodoacetamide as follows: EDTA at a final concentration of 0.002 M and dithiothreitol (DTT) at a 60-fold molar excess above free sullhydryl groups in the MBP were added to the MBP at pH 8.0 and incubated for 40 min at room temp. Iodoacetamide at a l20-fold molar excess was then added. and the mixture incubated in the dark for 20 min. The reduced and alkylated sample was transferred to a 3500 dalton cutoff dialysis casing (Spectrum Medical Industries Inc., Los Angeles, CA) and dialyzed overnight vs 0. I5 M NaCl. All MBP used as standards and for labelling with 13i1 were similarly reduced and alkylated. The concentration of MBP was determined by its absorbance at 277 nm: El ,,,, = 26.3. The purity of the reduced and alkylated MBP used for standards and for radiolabelling has been assessed by several methods. Electrophoresis in SDS-polyacrylamide or urea acetic acid polydcrylamide gels shows only a single protein band. Preliminary amino acid sequencing studies showed that only a single amino acid was cleaved during Edman degradation. Additionally, the purified MBP gives only a single precipitin band when tested with specific antibody by

Fig. I. Reaction of identity between purified, reduced and‘alkylated MBP standard and MBP present in crude eosinophil extracts. Rabbit antiserum to the MBP (center well) was reacted with dilutions of the MBP standard (well Nos. 2 and 5) and with dilutions of a crude eosinophil extract which had been reduced and alkylated (well Nos. 1. 4 and 6). In immunodiffusion experiments it is essential to use agarose as the supporting gel. If agar IS used, precipitin bands do not form, presumably because the MBP binds to acidic groups on the agar.

713

RIA for Eosinophil Major Basic Protein immunoelectophoresis. Analysis of purified, reduced and alkylated MBP by the Ouchterlony procedure likewise shows only a single precipitin band when reacted with specific antibody and shows a reaction of identity with MBP present in crude eosinophil extracts. Figure 1 shows a reaction of identity between the precipitin bands formed between the purified MBP standard and a crude eosinophil extract which was reduced in 0.0075 A4 DTT and alkylated with a 2-fold molar excess of IA. Non-reduced and alkylated MBP reacts with specific antibody as will be shown later but fails to form precipitin bands in gel immunodiffusion experiments, presumably due to polymerization and subsequent failure to diffuse through the gel.

1Ot

Production of antiserum

Rabbit antiserum to the MBP of guinea pig eosinophil granules, as described by Lewis et al. (1976b), was used for all studies. When this antiserum was reacted with a crude eosinophil extract, only a single band formed as shown by immun~I~trophoresis and by Ouchterlony immun~iffusion (Fig. 1). No precipitin bands were formed when the antiserum was reacted with extracts of eosinophil-poor peritoneal exudate ceils.

t lO-3

iO-4

Antlbody

16"

G6

dilution

The radioimmunoassay

Fig. 2. Antiserum titration curve of rabbit antiserum to Reduced and alkylated MBP was radioiodinated with 1311 guinea pig eosinophil MBP. 0.1 ng 1311-MBP in 0.6 ml PPF by a modification of the procedure described by McConahey buffer was reacted at 4°C with 0.1 ml of antiserum at increasing and Dixon (1966). Approximately 600 pCi of *3‘I, diluted in dilutions. The next day, 0.1 ml of normal rabbit serumdiluted 200 ~1 of 0.5 M Na,HPO,-KH,PO, buffer, pH 7.5, was l/20 in PPF buffer and 0.1 ml of burro anti-rabbit IeG added to 5 ml plastic tubes on ice followed by 10 pig of were added, and tubes were mixed and then incubated for reduced and alkylated MBP. Chloramine T, 25 pi of 1 mg/ml two hours at 4°C. Counts of 1311-MBP in the precipitate were in 0.5 ,&.f phosphate buffer, was added and mixed determined after cent~fugation at 25006. The percentage of inte~ittently for 3 min. Sodium metabi~lfito, 25 pl of counts in the precipitate is plotted on the ordinate and the 1 mg/ml, was added and the mixture incubated for another 2 reciprocal log of the ant&mm dilution plotted on the min after which 50 ~1of 1% KI was added and the contents of abscissa. the tube thoroughly mixed. Next, 0.1 MNa,HPO,-KH,PO, buffer, pH 7.4, containing I mg/ml protamine sulfate and fluids collected. The cells, after centrifugation at 400 g for 5 0.5% fetal calf serum (PPF buffer) was added and the min, were resuspended in 10 ml Hank’s BSS, layered over 8 contents transferred to a 3500 dalton cut-off dialysis casing ml 22y0 metrizamide and centrifuged at 400 g for 30 min. and dialyzed against four changes of 0.15 M NaCl. In Cells at the interface contained less than 20% eosinophils preliminary experiments, attempts were made to separate free (usually < 10%) while cells in the pellet contained >80% l3lX from 1311-MBPby passing the mixture over a Sephadex eosinophils (often >950/,). After counting total cells and total G-25 column. This proved unsatisfactofy as large amounts of eosinophils, volumes were adjusted to contain 10’ total *311-MBP bound to the column and could not be eiuted. leukocytes per ml. Erythrocytes were lysed by exposure to Aliquots of 13’I-MBP were frozen and 1 &ml working stock distilled water as described above. 1 ml of cell sus~nsion (10’ stored at 4°C. Specific activity averaged about 32 &i per pg leukocytes) in Hank’s BSS wascentrifuged at 400g for 5 min, and more than 98% of counts were precipitated by 10% the BSS discarded and the cells resuspended in PPF buffer tungstic acid. As shown in Fig. 2, maximum precipitabitity of containing 0.1% Triton X-100. The cells were subjected to a *3’1-MBP by antibody was found to be 83% when 1 ng ‘311rapid freeze-thaw procedure by alternating freezing in dry MBP was reacted with increasing concentrations of ice-acetone followed by thawing in a 37°C water bath. After antiserum. Unless otherwise indicated, the radioimten freeze-thaw cycles the suspension was centrifuged at munoassay (RIA) was performed by adding 0.1 ml of a 10m4 40,OOOgfor 20 min at 4°C. The supematant fluid was assayed dilution of rabbit anti-MBP in PPF buffer, 0.5 ml PPF buffer by RIA for MBP activity. For some experiments the 40,OOOg and 0.1 ml of inhibitor to 10 x 75 glass tubes. The contents were supernatant was reduced in 0.04 M or 0.0075 M DTT and thoroughly mixed and were incubated at 37°C for 30 min and at alkyiated in 0.08 M or 0.015 M IA prior to assay. 4°C for 15 min followed by additon of 1 ng 1311-MBPdiluted Tissues extracted for MBP were removed from freshly in 0.1 ml PPF buffer. The tubes were covered and incubated killed animals and placed in cold Hank’s BSS. The tissues overnight at 4°C. Immune complexes were pr~ipitat~ by were blotted dry, weighed, and approximately OS-I.0 gm the ad&tion of 0.1 ml of a 1:20 dilution of normal rabbit thoroughly _ . chopped using a McIlwain tissue chopper serum (NRSl in PPF buffer, and 0.1 ml of burro anti-rabbit IgG. ‘l&es’were mixed, incubated at 4°C for 2 hr and (Mickle Laboratories Engineering Co., Gomshall, Su&ey, England). The ChoDDed tissue was washed in Hank’s BSS. centrifuged for 20 min at 4°C and 2500 g. The supematants sus-prided in 1 mi_PPF buffer-O.l% Triton X-100 and were discarded, the tubes drained and the precipitate counted in a Nuclear-Chicago gamma scintillation counter (G. D. freezethawed as described above for cells. Supernatants Searle and Co., Des Plaines, II). Controlscontaining 100% of were assayed for MBP activity by RIA. the 1311-MBP added to each tube were counted for a sufficient time to accumulate 10,000 counts or more; assay tubes were counted for an equal time and expressed as a percentage of total counts bound. extraction of M3P from cells and titisues

Peritoneal cavities of guinea pigs were lavaged with 50 ml sterile, nonpyrogenic 0.15 M NaCI and the eosinophil-rich

RESULTS

Initially we tested rabbit antibody to the guinea pig eosinophil MBP for its ability to bind 1311-MBP in different buffer systems. We wished to select a buffer which allowed optimal binding of antibody to the

714

DONALD L. WASSOM, DAVID A. LOEGERING and GERALD J. GLEICH

ng MBP Fig. 3. Binding of i3iI-MBP to specific antibody in different buffer systems. Inhibition curves were produced by incubating increasing amounts of unlabeled MBP standard with 1 ng i3iI-MBP, specific rabbit anti-guinea pig MBP and the following buffers: 0.1 M Na,HPO,-KH,PO,, pH 7.4 (AL---A); 0.1 M Na,HPO,-KH,PO,, pH 7.4,0.5x heat inactivated FCS (A-A); 0.1 M Na,HPO,-KH,PO,, pH 7.4, 1 mg/ml protamine sulfate (M); and 0.1 M Na,HPO,-KH,PO,, pH 7.4, 1 mg/ml protamine sulfate, 0.5% heat inactivated FCS (O---+). Trap values indicate non-specific levels of free 13’1-MBP bound in the precipitate in the absence of specific antibody. MBP and minimized the non-specific trapping of free

13iI-MBP during the double antibody precipitation of antibody-bound MBP. We found that several buffer systems could be used as long as a polycationic protein such as protamine or histone was included in the medium. In the absence of these cationic proteins, binding curves lacked sensitivity. Addition of fetal calf serum further enhanced sensitivity and was essential to keep non-specific trapping of free i3iI-MBP below 10%. Figure 3 illustrates an experiment showing the requirement for protamine and FCS in the assay system. In these experiments, binding of i311-MBP was assayed in the presence of increasing amounts of unlabeled MBP. Protamine greatly enhances the sensitivity of the assay and FCS is a necessary component if traps are to be kept acceptably low. As protamine works equally as well as histone in this system, we prepared our buffer to contain 0.1 M Na,HPO,-KH,PO,, pH 7.4, 1 mg/ml protamine sulfate and 0.5% heat inactivated FCS (PPF buffer). Sodium azide at a final concentration of 0.1% was added to inhibit microbial growth and the buffer was stored at 4°C. The RIA established for myelin basic protein (Cohen et al., 1975) used an overnight pre-incubation with protein standard or sample prior to addition of the radiolabelled protein. We found that we could obtain acceptable results in the MBP assay by using a 30 min pre-incubation at 37°C followed by 15 min at 4°C prior to addition of l3 ‘I-MBP. We also tested various incubation times after addition of i3iI-MBP and found that little sensitivity was gained by incubation times in excess of 18-24 hr. Using the 45 min pre-incubation, we were able to complete an assay within 24 hr. The tendency of MBP to bind nonspecifically to glass, foreign proteins, or other negatively charged moieties makes assay of the protein

difficult. It is important, therefore, to keep all reagents on ice and to work quickly. Addition of 1 mg/ml protamine to the assay buffer is essential for measurement of small quantities of MBP. The protamine, being present in marked excess over MBP, does not interfere with the assay and yet presumably competes for and occupies binding sites on glass and proteins that might otherwise be occupied by MBP. Additionally, we found that the double-antibody precipitation of antibody-bound MBP must be conducted at 4°C to minimize non-specific trapping of free 13*1-MBP. For example, incubation for 2 hr at room temp as opposed to 4°C results in a doubling of the value for the traps. Figure 3 also shows a typical standard curve for this assay obtained by adding increasing amounts of unlabeled MBP standard to 1 ng i3iI-MBP and incubation in PPF buffer in the presence of specific rabbit anti-guinea pig MBP. Having established conditions for the assay, we compared immunoreactivity of freshly isolated MBP with MBP that had been subjected to chemical alteration. It has been shown previously that the MBP readily polymerizes unless free sulfhydryl groups are alkylated (Gleich et al., 1974). To see if polymerized MBP was reactive in the RIA, and to assess the effect of reduction and alkylation and alkylation alone, we tested the reactivity of the following preparations: (1) freshly isolated MBP, (2) MBP stored under nitrogen at 4°C for one day, (3) MBP through which oxygen had been bubbled at room temp for one day, (4) freshly isolated MBP alkylated with iodoacetamide and (5) freshly isolated MBP reduced with DTT and then alkylated with iodoacetamide. SDS polyacrylamide gels of the above preparations showed a single band except for the sample through which oxygen had been bubbled which showed bonds corresponding to polymers of the 11,000 mol. wt sub-unit (Gleich et al.,

715

RIA for Eosinophil Major Basic Protein

6Or

I Oo

1

1

I

I

1

I

2

3

4

5

ng MBP Fig. 4. Comparisons of inhibition curves obtained by adding increasing amounts of alkylated (O--+), reduced and alkylated (M) or non-alkylated (‘-----A) MBP to 1 ng i3tI-MBP, specific rabbit anti-guinea pig MBP and PPF buffer.

1974).Of the five samples tested, only the alkylated or the reduced and alkylated preparations of MBP were potent inhibitors in the RIA. Non-alkylated MBP assayed immediately after isolation was over lo-fold less active in the assay than MBP which had been alkylated. Similarly, MBP stored under nitrogen and the polymerized MBP were less active. Figure 4 compares inhibition curves produced by an alkylated, a reduced and alkylated, and a non-alkylated sample

of MBP. Alkylation or reduction and alkylation greatly enhanced antigenic activity as measured by the RIA. Non-alkylated preparations of MBP, even if polymerized, regain full activity in the RIA if reduced and alkylated at a later time. Because reduction in DTT followed by alkylation with iodoacetamide in the absence of a diss~iating solvent stabilizes the MBP, we determine the effect of reduction and alkylation in the presence of 6 M GuHCl. MBP was reduced and alkylated in the absence of GuHCI, dialyzed vs 0.1 M ammonium bicarbonate and lyophilized. The lyophilized, reduced and alkylated MBP was reconstituted in 6 M GuHCl and a 60-fold molar excess of DTT over MBP free sulfhydryl groups was added (Konigsberg, 1972). After incubation at 25°C for 18 hr, IA at 120-fold molar excess was added and the incubation continued in the dark for an additional 20 min. After dialysis vs 0.15 M NaCl, the MBP was tested for reactivity in the RIA. Figure 5 reveals that MBP reduced and alkylated in the presence of 6 M GuHCl is completely inactive. In contrast, incubation of reduced and alkylated MBP in 6 M GuHCl but without further reduction and alkylation results in only a 2-fold loss of immunoreactivity. Presumably, when reduction and alkylation is conducted in the absence of 6 M GuHCl, only free sulfhydryls and freely accessible disulfide bonds are reduced and alkylated. When MBP is equilibrated with 6 M GuHCl and again reduced and alkylated, immunoreactivity is lost. This implies that the disulfide bonds are important in the maintenance of its antigenic configuration. To assess theeffect of temperature on stability of the MBP, we incubated alkylated MBP ai different

40 P 8 Q $ S 4 2 w

30

20

I

Oo

I

I

1

2

3

4

5

ng MBP Fig. 5. Comparison of inhibition curves obtained by adding increasing amounts of reduced and alkylated MBP (O----+); reduced and alkylated MBP, dialyzed vs 0.1 M ammonium bicarbonate, lyophii~~, r~onstitut~d in 6 M GuHCl and incubated for 18 hr at 25°C (M); and reduced and alkylated MBP dialyzed, vs 0.1 &f ammonium bicarbonate, lyophilized, reconstituted in 6 M GuHCl, further reduced with DTT for 18 hr at 25°C and alkylated with IA (W---¤).

0

IO

20

30

40

50

ng MBP

Fig. 6. Comparison of inhibition curves produced by reduced and alkylated MBP incubated for 4 hr at different tem~ratures; 4°C (O----O); 25°C (A-A); 37°C (m---m); 56°C (M). After incubation, increasing amounts of reduced and alkylated MBP were added to samples containing 1 ng i3’I-MBP, specific rabbit anti-guinea pig MBP and PPF buffer.

DONALD L. WASSOM, DAVID A. LOEGERING and GERALD J. GLEICH

716

Table

1. Measurement

of MBP in eosinophil y0 Eosinophils

Experiment

____

Sample

____

1

No. tested

____

mean

Fresh extract eosinophil-rich

7.5

12,983 + 1.973

Fresh extract eosinophil-rich

92.8 + 2.5

9,041 + 898

1.3

118 f

92.8 k 2.5

eosinophil-poor

103,000

6.0 + 1.3

for 4 hr and then measured activity by RIA. Figure 6 shows that incubation at 56°C completely destroyed activity while temperatures at 37°C or lower had little effect. Figure 6 is shown with an expanded scale for MBP concentrations to illustrate that the loss of activity after heating was evident even at high concentrations of MBP. We further found that reduced and alkylated MBP was

8.45 x 10m3 f 4.6 x 10-3 2.33 x 10m3 k 1.5

9.86 x 10 -A & 0.12 x IO_’ 1.98 x lO-3 i 0.48 x 10-i

42

i

32,200

1.08 x 10 Z + 0.33 x IO Z 2.42 x IO-” + 0.78 10 J

1,230 + 314

* IO’ cells, either highly enriched for eosinophils or comparatively eosinophil-poor. succession in a solution of PPF buffer-O.l% Triton X-100. Supernatants of extracts after centrifugation at 40,000 g.

temperatures

13”,,

& 46,588

2,670 &-1,230

6.0 i

Reduced and alkylated extract (0.0075 M DTT) eosinophil-rich

78,338

40-

s 2 P

30-

$-

20-

-1.6 -

,

,

,

,

,

,

(

40

5.0

-.a

-.6

-.4

-.2

0

.2

.4

.6

0.4

05

-1.8

-1.6

-2.ot O0

I.0

21)

0

0.1

0.2

31)

ng MBP

PI eorlnophll

0.3 extract

95”;

28”,, 54””

67”,, 72” (,

stable for months at 4°C when stored in PPF buffer at concentrations of 100 pg/ml or higher. The above experiments were conducted to establish conditions for the optimal performance of the RIA and to determine the effects of various conditions on reactivity of the MBP in this system. Next, we sought to measure the content of MBP in body fluids and tissues of normal guinea pigs. We partially purified

x IO-

76:”

were freeze-thawed ten times in quick were assayed for MBP activity by RIA

50-

b

C.V.

68”,

14.5 & 4.5

eosinophil-poor

ng MBP activity Eos_il mean + sem

473 + 93

88.5 + 7.5

eosinophil-poor

2

*sem

.14.5 * 4.5

Reduced and alkylated extract (0.04 M DTT) eosinophil-rich

extracts”

ng MBP activity ml mean +sem

88.5 f

eosinophil-poor

freeze-thaw

Log,,

MBP

-1.4

-1.2

Loqo

pl

concsntratlon -1.0

coslnophil

-.6

-.6

-4

extract

Fig. 7. Comparison of the reduced and alkylated MBP standard with freshly isolated eosinophil extract by RIA. (a) Inhibition curves obtained when increasing amounts of MBP standard (*A) and increasing amounts of untreated eosinophil extract (O---O) are measured by RIA. (b) Logit-log regression lines for ). The correlation coefficients for MBP MBP standard (M) and untreated eosinophil extract (W standard inhibition regression line were -0.977 and for the eosinophil extract -0.999. Comparison of the log&-log regression lines revealed that the null hypothesis of a common slope could not be rejected (F,,., = + 0.78:NS).

717

RIA for Eosinophil Major Basic Protein

eosinophifs from peritoneal Iavage fluids as described above and measured MBP in freeze-thaw extracts of leucocytes either enriched or deficient in eosinophils. Both the eosinophil-rich and eosinophil-poor extracts were made from 10’ total leucocytes suspended in PPF buffer containing &lo/, Triton X-100. The suspensions were freeze-thawed ten times in quick succession and supernatants were assayed for MBP activity after centrifugatinn at 40,000 g. Results of seven ex~riments are shown in Table 1. ~osinophiI-rich

extracts contained much more MBP activity than extracts from eosinophil-poor cell populations. Although each of the seven experiments was conducted with different cell populations, there is reasonably good agreement in values for the amount of MBP extracted per eosinophil from the eosinophil-rich cell fractions; freshly isolated extracts yielded an average of 1.13 x IO- 3 ng per eosinophif with a coefficient of variation of 30%. Figure 7 shows the inhibition curves and the logit-log transfo~ed regression lines obtained when samples of the freshly isolated eosinophil extract and the reduced and alkylated MBP standard are compared. An analysis of covariance performed on the pair of lines shown in Fig. 7(b) failed to distinguish any significant difference in their slopes. The similar inhibition curves for the two preparations indicate that the reduced and alkylated MBP may be used as a standard in measuring non-reduced and alkylated MBP in biologic samples. In addition to assaying the freshly prepared extract, each extract was reduced and alkylated to see if this procedure increased MBP activity. Extracts were reduced using either 0.04 M or 0.0075 M DTT followed by alkylation with a Z-fold molar excess of IA as described in Materials and Methods. Samples reduced in 0.04 M DTT and alkylated were diaiized vs NaCI prior to assay whereas samples reduced with DTT at 0.0075 iLf and alkylated were diluted for assay immediately without dialysis, Reduction and alkylation of extracts resulted in 3- to 20-fold increases in MBP activity. The two samples reduced with 0.04 M DTT showed poor agreement in values for ng MBP per eosinophil. The five samples reduced at the lower concentration of DTT also showed considerable variability (C.V.67%). Several possible explanations for the difference in

variability between the fresh and the reduced and alkylated samples can be offered. First, the freshly isolated extract is assayed immediately after preparation whereas the reduction and alkylation process takes time which may allow proteolysis or other degradations to take place. Even after reduction and alkylation, preparations continue to lose activity with time indicating that degradation of the MBP is occurring. Secondly, the reduction and alkylation itself is conducted in an empirical manner on such samples. It is not known beforehand how much contarninat~i~~ protein or MBP is present in each sample and the concentration of DTT may be more appropriate for some samples than for others. It must be kept in mind that each experiment involves different cell populations from different animals containing slightly different ratios of eosinophils and. conta~nating cells. Examination of Table t reveals that the ratio of contaminating cells to eosinophils alters the recovery of MBP from extracted eosinophils, less MBP per eosinophil being measured in the eosinophil-poor WBC extracts. Having established that MBP could be measured in extracts of cell populations containing eosinophils, we tested extracts from tissues of normal guinea pigs and compared them with extracts from tissues of animals infected with the nematode Trjch~~~~lff spiralis. Infected guinea pigs developed eosinophil~a during the second week of infection with peak levels of eosinophils occurring in blood and tissue one or two weeks later. Seven guinea pigs were each fed 750 infective larvae of T. spiralis on day 1, and on days 18, 21, 22 and 23, an infected animal and a control were exsan~~nated by cardiac puncture and the diaphragm removed for MBP extraction. On day 25, three infected animals and two controls were treated similarly. Infected muscle tissues, fixed in formalin and stained with chromotrope ZR, showed small infiltrates of eosinophils in the muscle but no eosinophils were seen in contact with encysting larvae. Control animals contained no muscle larvae. Diaphragm extracts were prepared as described in Materials and Methods, and MBP activity in the supernatant was then assayed by RIA. Results are given in Table 2. Eos~nophil counts in infected animals were elevated above those of controls, and signi~cantly more MBP activity was

Table 2. Measurements of MBP in muscle extracts and serum from guinea pigs infected with Trjc~jne~lu spiralis” Na. animals

tested Experi- Days post ment infection

Total eosinophil count (blood)

T. sp. infected

Control

T. sp. infected

Control

ng MBP activity -gm tissue T. sp. infected Control

1

18

1

1

440

24

11.6

I I 1

1 I 1

-

-

13.5

2

21 22 23 25 25

3 5

2 5

269 24 171 37 1255_+354 86kO.O 666-1227’ 35+16

1S.I 21.3 23.8k3.7 21.5*1.9”

P

7.2 12.5 20.7b < 0,023 15.2 8.Oj,O.9 8.8jIl.l <0.007

ng MBP activity -_---ml serum T. sp. infected Control P 4.8 4.3 4.2

4.2 4.2 3.7

3.0

2.9

NS NS

“Animals were fed infective larvae of T. ~pir~~j~andkilled at specific intervals. Total peripheral blood eosinophil counts were performed and diaphragms were removed, weighed, and extracts made by repeated freeze-thawing in PPF buffer containing 0.1% Triton X-100. MBP activity, as measured by RIA, was compared to activity present in extracts from normal controls. Sera from infected animals and controis were also tested for MBP activity by RIA. ’ Eosinophils were present in uninfected muscle tissue. ‘Mean of four values; one animal negative for muscle larvae at assay.

718

DONALD

L. WASSOM,

DAVID A. LOEGERING

extracted from infected tissues. In the single case. 22 days post infection, where MBP activity in the normal control exceeded that of the infected guinea pig. eosinophils were seen in muscle sections of both animals. Why eosinophils were present in the muscle of this uninfected control is not known. Of the other controls examined. all six diaphragm sections were devoid of eosinophils. We repeated the experiment a second time but examined all animals 25 days post infection rather than at timed intervals. Five animals were infected with 1000 larvae of T. .spirnli.sand they along with controls were killed by exsanguination. The diaphragms were extracted and extracts measured as before. Results of the second experiment are shown in Table 2. Once again, significantly more MBP activity is seen in the tissues extracted from infected guinea pigs. As total eosinophil counts from guinea pigs infected with T. $~~~~~~~ range many fold higher than controls, we determined whether or not MBP levels in serum were elevated in such animals. Blood was allowed to clot at room temperature and fresh serum placed on ice until assayed. Levels of MBP activity in sera from animals with eosinophila were not significantly elevated above those of controls and in both cases ranged between 2 and 5 ng MBPjml (Table 2). Reduction and alkylation of guinea pig serum does not increase MBP activity in such samples. DlSCUSSlON

The cationic nature of the MBP and its possession of two free sulfhydryl groups impart properties to the MBP that make its measurement by RIA difficult. The protein binds to giass and plastic and will precipitate spontaneously with acidic proteins. If free sulfhydryl groups are not alkylated, it is IO-fold less reactive in the RIA than when stabilized by alkylation. We have described an assay in which these difficulties are minimized, and which provides a means for detecting the protein in biologic fluids and extracts of tissue. We found that addition of protamine and FCS to the assay buffer compensated for difficulties presumably attributable to the cationic charge of the MBP and that the MBP is stable for months at 4°C if alkylated with i~oacetamide. The advantages of the RIA over the previously described complement fixation assay (Lewis et al.. 1976b) are the increased sensitivity and the ease of analyzing large numbers of samples. We first used this assay to evaluate the effect of chemical or physical alterations on ~mmunoreactivity of the protein. We found that it was rapidly inactivated at 56°C and that it must be reduced and alkylated or alkylated in order to achieve optimal activity. The reduced activity of unalkylated preparations was seen even in the absence of detectable polyn~erizat~on, but such samples regain original activity if reduced and alkylated at a later time. We suspect that the non-alkylated MBP may be reacting with FCS proteins in the assay buffer and losing reactivity with antibody. When MBP is reduced in DTT and then alkylated with IA. the molecule is stabilized and very reactive in the RIA. However, if the reduced and alkylated MBP is equilibrated with 6 M GuHCl and again reduced and alkylated, all activity is lost. This suggests that a

and GERALD

_) GL.t:I( f-1

disulfide bond(s) may not have been acccssibie for reduction during the initial DTT treatrn~l~t but was subsequently exposed and reduced during the second reduction in 6 A4 GuHCI. It is aiso of interest that reduced and alkylated MBP can bc denatured in 6 bI GuHCl and yet will retain its antigenic configuration with only a 2-fold loss in activity when GuHCl is removed by dialysis. The RIA, as described. is a convenient tool for assessing the effect ofchemical and physical alterations on MBP activity and may be used in a similar manner to explore other aspects of MBP structure. Using the RIA as descri bed. MBPcan be detected in biologic fluids or tissue extracts when present in concentrations of 2 ng;ml or greater. It is probable, however. that a small portion of the activity observed in the RIA. when conducted in the presence of high concentrations of ~ont~nlinat~ilg proteins, is due to non-specific binding of ‘“‘I-MBP to substances other than specific antibody. For example. in preliminary tests we found that if MBP is added to undiluted serum and assayed, about 90”,, of the MBP added is recovered. It must be assumed that lO”,, of the labeled MBP is bound to other proteins when it iii added to bv other such samples. If i311-MBP is adsorbed proteins and is therefore no longer accessible to antibody, falsely elevated values of MBP activity may be obtained. This problem can be partially solved by establishing standard curves with the MBP standards diluted in a normal pool of the biologic fluid to be tested. We have performed such experiments by diluting MBP standards in serum fi-om guinea pigs rendered eosinopenic by cortisone treatment. The slopes of inhibition curves performed in guinea pig serum are very similar to those ofcurves prepared with MBP diluted in PPF buffer and serve little purpose except to move normal baselines back to zero. As long as adequate controls are run with each assay, significant differences in MBP activity between can be samples controls and experimental reproducibly and reliably demonstrated. When sera from normal and T~ichinelin-infected guinea pigs were assayed, we found that levels of activity were low in both groups and that while mean values for the Tri~i?it?c,i(u-infected animals were higher, the difference was not statistically signi~cant. In this instance we cannot be certain that the activity measured in controls represents baseline levels of serum MBP; it is possible that the activity observed was due entirely to non-specific factors. ft is clear, however, that eosinophilia. due to T. .~~i~~i/i,s infection, does not significantly elevate serum levels ofdetectable MBP in the guinea pig. As expected. when eosinophilrich and eosinophil-poor WBC populations were extracted and assayed for MBP activity, we found significantly more activity in extracts from purified eos~nophils. F~lrthermore. this rictiuity could be measured in a dose-dependent fashion and the dose response curves of such samples paralleled those of the purified MBP standards. Also, reduced and alkylated eosinophil extracts show precipitin bands of identity with MBP standards when tested by the Ouchtcrlony procedure. The measurement of MBP in diaphragm extracts of normal and TrichineNLI-int~cted guinea pigs illustrates an example where activjity in parasitized tissue exceeded that found in normal controls. Because

RIA for Eosinophil activity is seen in extracts of normal tissue, however, we can only measure the absolute amount of MBP present in parasitized muscle if we assume that the activity in normal muscle extract is non-specific. Alternatively, the RIA can be used to detect differences between experimental samples and controls. The ability to measure levels of MBP in biologic specimens may be of value if it is found that increased or decreased levels are associated with other clinical findings. Venge et al., (1977~) have described a RIA for a human eosinophil cationic protein and have correlated serum levels of this protein with levels of eosinophils in the peripheral blood. Patients with eosinophilia usually show elevated levels of cationic protein; however, abnormally low levels of cationic protein were measured in serum from patients with asthma (Venge et al., 1977b). The protein assayed by Venge and characterized by Olsson et al., (1974,1977) differs from the eosinophil MBP in size and amino acid composition and may represent another cationic molecule localized within eosinophil granules. Using the guinea pig model described in this report, we have recently established a radioimmunoassay for the human eosinophil MBP* and have begun testing human sera for MBP activity. Preliminary tests show greatly elevated values of MBP activity in sera from some patients with eosinophilia, and this activity has been shown to be due to high concentrations of MBP which can be isolated chromatographically from other serum proteins. The ability to measure MBP both in purified form and in body fluids of guinea pigs and man provides another tool which may be of value in defining a functional role for the eosinophil. MBP

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Major

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719

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