[7] Immunopharmacological methods to study murine allogeneic and syngeneic pregnancy

[7]

Immunopharmacological Methods to Study Murine Allogeneic and Syngeneic Pregnancy Marfa Elena Sales and Enri S. Borda

Introduction The bidirectional interaction between the neuroendorine and immune systems has been largely documented. We have specifically demonstrated that alloimmunization triggers a regulatory mechanism that modifies/3-adrenergic receptor expression on uterine membranes (1). The activation of/3-adrenergic receptors mediates smooth muscle relaxation in humans, as well as in experimental animals (2, 3) and, for this reason; it plays an important role in the maintenance of uterine quiescence during pregnancy and in the prevention of premature labor (4). We have also shown that antibodies produced by alloimmunization can elicit biological effects through the activation of postsynaptic/3-adrenoceptors. In this way alloantibodies inhibit the spontaneous motility of the murine oviductal tract (5). During pregnancy, the maternal production of alloimmune antibodies against paternal antigens has been largely documented (6). In the mouse, maternal immune response to paternal antigens is restricted to certain strain combinations and it is probably associated with an immune response gene located in or close to the H-2 locus (7). Thus, we have developed experimental procedures to show that allogeneic pregnancy immunoglobulin G (IgG) interacts with C3H uterine/3-adrenoceptors decreasing the spontaneous motility of IgG and increasing cAMP production. Moreover, allogeneic pregnancy IgG fixates on uterine tissue and interferes with the binding of a specific /3-adrenergic radioligand behaving as a noncompetitive inhibitor.

Animals Virgin female inbred mice from Comisi6n Nacional de Energfa At6mica are used throughout the study. All animals are 60-90 days old. BALB/c mice (H-2 d) and C3H mice (H-2 k) are chosen for experimental procedures and their H-2 haplotypes are checked by microcytotoxicity testing using monospecific alloimmune sera as described previously (8). Only females exhibiting a 4-day estrous cycle are selected and the stage 102

Methods in Neurosciences, Volume 24

Copyright 9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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IMMUNOPHARMACOLOGY AND ALLOGENEIC PREGNANCY Syngeneic Pregnancy

(~ Balb/c

x

103

AIIogeneic Pregnancy

~ Balb/c

~) Balb/c

x

0" C3H

AIIog-Preg IgG

Syng-Preg IgG

agglutination

J

C3H red blood cells

+ / -

IFI

"

C3H uterine slices

F[c. 1 Development of allogenic pregnancy (Allog-Preg) model (BALB/c x C3H) from which allogenic pregnancy IgG is obtained. Syngeneic pregnancy (Syng-Preg) (BALB/c x BALB/c) is the immunologic control of the model.

of the estrous cycle is determined on the basis of the cellular composition of vaginal fluid, which is examined daily at the same time. In order to obtain an allogeneic pregnancy female BALB/c mice are mated with C3H male mice (Fig. 1). The mating is done in proestrus since ovulation normally occurs at the beginning of the late estrus (9). The day on which vaginal plug is found is designated as Day 1 of allogeneic pregnancy. To obtain

104

I GENERALMETHODS syngeneic pregnant animals BALB/c female mice are caged in proestrus with BALB/c males and pregnancy is detected following the same procedure described above. The control group (nonpregnant animals) is composed of BALB/c and/or C3H female mice in diestrus. Animals are kept in a room with controlled temperature and illumination (14 hr light and 10 hr dark), fed Purina mouse chow, and allowed unrestricted access to water.

Production of Antibodies

Purification of Immunoglobulins Sera from pregnant and nonpregnant female mice are obtained by ocular bleeding. Whole blood is left at 37~ for 30 min; it is centrifuged at 1500 rpm for 10 min to remove blood cells and then centrifuged, for a second time, at 4000 rpm for 15 min at 4~ to separate denaturated proteins. Immunoglobulins are precipitated according to a previously reported procedure (10). An equal volume of 70% (w/v) saturated ammonium sulfate solution (freshly made, pH 7) is slowly mixed with the serum. After 2 hr the precipitated protein is centrifuged at 5000 rpm for 30 min. The pellet is dissolved (in deionized water) in one-half of the initial volume of serum and 1 volume of 33% (w/v) ammonium sulfate is added. This is immediately centrifuged again and the supernatant discarded. The pellet is dissolved again and the last step is repeated five times. Finally the pellet is dissolved in one-half volume of deionized water and dialyzed against 15-30 volumes of 0.01 M phosphate buffer solution, pH 8, at 4~ until the ammonium sulfate is totally eliminated (negative reaction of dialysis bath with barium chloride).

Purification of Immunoglobulin G Fraction Immunoglobulin G fraction is purified by ion-exchange chromatography in DEAE-cellulose (11). About 1 g of resin is needed for 100 mg of precipitated immunoglobulins. The Ig's are loaded onto a column which has been washed and equilibrated with the same buffer. It is important that the conductivity of effluent buffer be the same as that of the initial one. The IgG fraction is eluted with 0.02 M phosphate buffer solution, pH 8. Fractions that correspond to the first peak of absorbance at 280 nm are pooled and concentrated by ultrafiltration (Minicon B 15 concentrator, Amicon, Danvers, MA). Protein concentration is determined by the method of Lowry (12).

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E v a l u a t i o n o f T i t e r for A n t i b o d i e s

Direct Hemagglutination Test This technique is useful for mice because their red blood cells have a significant number of antigens that belong to the major H system and are involved in humoral immune response during allogeneic pregnancy (13). We use this procedure to evaluate the titer of antibodies, directed to paternal antigens, present in allogeneic pregnant animals (BALB/c x C3H). C3H erythrocytes are obtained from nonpregnant female mice and washed with phosphate-buffered saline (PBS) until hemolysis residues are no longer present. Then the erythrocytes are suspended in a 1.5% solution made up in a 50% normal C3H serum (heated to 56~ for 30 min to inactivate complement proteins) in PBS. Allogeneic pregnancy IgG is diluted in a 2% (v/v) dextran (molecular weight 70,000) solution in PBS from 1"40 to 1" 1280. Equal volumes (25 txl) of the red cell suspension and allogeneic pregnancy IgG dilutions are incubated in microtiter plates of round-bottom wells for 2 hr at 37~ and then overnight at 4~ Negative controls were one volume each of red blood cells and dextran solution, red blood cells and PBS, and red blood cells and IgG purified from syngeneic pregnant animals (BALB/c x BALB/c) or nonpregnant animals (BALB/c or C3H) diluted in dextran. We also include positive controls like IgG obtained from BALB/c female mice immunized with C3H lymphoid cells in the assay. The results are read macroscopicaUy: agglutinated red cells appear as a carpet spread over the entire bottom of the well, whereas nonagglutinated cells form a small tight button. Positive and negative controls are essential and doubtful results can be checked microscopically after the content of the well is placed gently onto a glass slide with a Pasteur pipette.

Indirect Immunofluorescence We use this assay to determine if allogeneic pregnancy IgG is able to recognize and bind to antigenic structures from paternal origin present in C3H uterine tissues, which is also used to evidence the biological effects of these antibodies. Uterine tissue from nonpregnant C3H female mice in diestrus is snapfrozen (after removing fat and peritoneal structures) by placing it in the side wall of a flask previously stored at -20~ The flask contains absolute ethanol at -70~ Tissue freezes rapidly and is stored at -20~ (14).

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GENERAL METHODS

Sections from unfixed tissues are snap-frozen onto the microtome chuck by applying the smallest amount of water, with a syringe and needle, around its base to effect attachment by freezing. Sections 7/zm thick are transferred to acid-washed slides kept at room temperature by bringing the slides close to the knife surface so that the sections become attached and firmly bound to the glass. Dry the sections thoroughly with an electric fan for 10 min. Tissue slices are incubated with allogeneic pregnancy IgG diluted in PBS from 1 : 10 to 1 : 1280 (v/v). Each dilution is applied over uterine-duplicated sections and incubated for 30 min in a wet chamber at room temperature. Then slices are washed twice with PBS for 10 min. Sections are stained with fluorescein-labeled goat anti-mouse IgG diluted 1:20 in PBS for 30 min in a dark wet chamber at room temperature. Then two or three washes with PBS for 1 hr are done. Finally the sections are mounted with a glycerin: PBS (9:1) (v/v) solution and sealed. Syngeneic pregnancy IgG and nonpregnant IgG are used as controls. Readings are carried out in an epi-illuminated microscope. S t u d i e s o f in Vitro U t e r i n e M o t i l i t y In order to investigate if uterine motility is altered in allogeneic pregnancy in comparison with syngeneic pregnancy, we have studied spontaneous uterine motility in an isolated organ bath system. Animals are killed by cervical dislocation in different stages of allogeneic and syngeneic pregnancy as well as in diestrus (control females). The entire uterus is immediately removed, trimmed of fat and peritoneal structures, and placed in gassed (95% 02 and 5% CO2, v/v) Krebs-Ringer-bicarbonate (KRB) solution (15):

Compound Na + K+ Ca 2+ Mg2+ C1HCO3 PO~SO~Glucose

Concentration (mM) 145.00 6.02 2.40 1.34 126.00 25.00 1.20 1.33 5.50

When uteri from pregnant mice are used, fetuses and placentas are carefully separated. Uterine horns are opened longitudinally and 0.5-cm-long strips

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from each one are cut between the ovarian and cervical regions. These strips are mounted in 15-ml organ baths which contain KRB solution at 37~ pH 7.4, and are gassed with 95% O2 and 5% CO2. One end of the strip is attached to a fixed hook and the other is connected to a force transducer coupled to a recording oscillograph. After a resting tension of 500 mg is applied to the preparation by means of a micrometric device, the murine uterus contracts isometrically. Tissues are allowed to equilibrate for 20 min until they present regular spontaneous contractions. The magnitude of each contraction is measured in milligrams. Tension control values are obtained by calculating the mean amplitude of all contractions recorded after the isolation period. Uterine spontaneous activity is stable during the whole experimental period (60 min) (15). In this system we evaluate the inhibition of uterine motility with cumulative concentration response curves of/3-adrenergic agonists like L-(--)-isoproterenol, L-(-)-epinephrine, or L-(-)-norepinephrine. It is also possible to block/3-adrenergic effects on uterine motility by using/3-adrenergic antagonists like propranolol or butoxamine. In the experiments of in vitro uterine motility, tissue is incubated 20 min with the/3-antagonist when necessary. For concentration-response curves, the tissue is incubated with each concentration of adrenergic agonist or IgG. In our experiments, the final concentration of agonists in the bath ranged from 10-~~ to 10 -6 m.

Uterine Membrane Preparations To prepare purified membranes, uteri from allogeneic pregnant, syngeneic pregnant, and nonpregnant mice (C3H or BALB/c in diestrus) are mixed in four volumes of ice-cold buffer containing 0.25 M sucrose, 5 mM Tris-HCl, and 1 mM MgC12, pH 7.4, and are homogenized with Polytron PT 20 at low, medium, and high speed for 30 sec in 15-sec intervals; this procedure is repeated twice. The homogenates are filtered through four layers of gauze and spin at 700 g for 15 min. The supernatants are stored at 0~ The pellets are homogenized again with one-half volume of the upper buffer and centrifuged at 10,000 g for 15 rain; the mitochondrial fraction is discarded. Finally supernatants are combined and centrifuged at 40,000 g for 30 min. The pellets are resuspended in 2-3 ml of 50 mM Tris-HCl, 10 mM MgCI2 (pH 7.4) and are stored at -70~ until they are used. Membrane protein concentration is measured using a standard curve of bovine albumin (fraction V) (12). We found 5-nucleotidase activity in our preparation, indicating the presence of the microsomal fraction (16).

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GENERAL METHODS

R a d i o l i g a n d B i n d i n g to U t e r i n e / 3 - A d r e n e r g i c R e c e p t o r s

Saturation Assay Murine uterine /3-adrenergic receptor is characterized using the tritiated /3-antagonist dihydropalprenolol ([3H]DHA) and purified uterine membranes from nonpregnant female mice (BALB/c or C3H). Aliquots of the membrane fraction (200/xg protein) are incubated (in duplicate) with shaking in 150/zl assay buffer (50 mM Tris-HC1, 10 mM MgC12, pH 7.4), containing increasing concentrations of the radioligand (0.3 to 3 nM), for 20 min at 37~ Incubation is stopped via rapid vacuum filtration with 2 ml of ice-cold buffer through Whatman (Clifton, NJ) GF/C glass fiber filters. The filters are immediately rinsed with 10 ml of ice-cold buffer, dried, and added to 10 ml of Tritontoluene-based scintillation fluid [30% Triton X- 100 in 4 g ofPPO (2,5-diphenyloxazole) and 50 mg of POPOP {1,4-bis[2-(5-phenyl-2-oxazolyl]benzene} per liter of toluene]. Radioactivity is measured in a liquid scintillation/3 counter. Nonspecific binding is determined by filtering duplicated aliquots of membranes incubated with 10/xM of the/3-antagonist propranolol (DL isomer), which should not exceed 10% of specific binding. Specific binding is calculated by subtracting duplicated observations of nonspecific binding assessed separately each time the assay is run. To characterize/3-adrenergic receptor in mice uterine tissue, we have chosen the ideal protein concentration, temperature, and incubation time for binding assays, from kinetics experiments (Fig. 2). In order to obtain the amount of radioligand bound in femtomoles (fmol) from data obtained in counts per minute (cpm), we consider the efficiency ('0) of the counter in the equation fmol [3H]DHA =

[3H]DHA bound (cpm) x 1012fmol/mol (cpm/dpm) x 2.22 x 1012dpm/Ci x SA (Ci/mmol)'

where SA is the specific activity of the radioligand and dpm is disintegrations per minute. Femtomoles are then normalized per milligram of protein in each assay. The equilibrium dissociation constant (Kd) and the maximal number of binding sites (Bmax) are calculated by Scatchard analysis of the saturation curves (17): Bound/Free: Bmax(1/Kd) - 1/Kd (Bound).

Competition Binding Experiments In order to determine the subtype of/3-adrenergic receptor which is predominant in murine uteri, competition assays are done using 200/xg of uterine

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IMMUNOPHARMACOLOGY

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PREGNANCY

._r 100

2o.6 0

A

o

E a z

"~ 40

o so

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,< la -r" m.., "~

B

m 50 E

30-

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0

o

~

~

,~

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C

~ :tz

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Temperature (~

;I;

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"-"

;

'

~E -,-_~ 20 ~o 10 0 0

9

I

I

L

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Time (rain)

FIG. 2 [3H]DHA binding to uterine membranes. (A) Membrane protein concentration dependence of [3H]DHA binding. (B) Effect of temperature on [3H]DHA specific binding. Uterine membranes (2 mg/ml) were incubated with 1.5 nM of [3H]DHA for 20 min at different temperatures. (C) Time course of the association reaction (e) and the dissociation reaction (9 For the association reaction, uterine membranes (2 mg/ml) and 1.5 nM of [3H]DHA were incubated at 37~ for different times and the specific binding was determined in the presence of 10 /xM propranolol. For the dissociation reaction, at 20 min 10/xM propranolol was added and the specific binding was determined at the different times shown.

membrane per tube and increasing concentrations of adrenergic agonists (L-isoproterenol, [.-epinephrine, and L-norepinephrine prepared in 0.1% ascorbic acid to prevent drug oxidation) in the presence of 1.2 nM [3H]DHA. Table I shows the Ka values for the agonists that were calculated using the Cheng and Prussoff equation. The order of potency of the agonists (isoproterenol > epinephrine > norepinephrine) indicates that/32-adrenoceptors are predominant in murine uterine tissue. In this type of experiment we can also evaluate the ability of allogeneic pregnancy IgG to interact with/3-adrenergic receptors present in C3H uteri. Uterine membranes are incubated with increasing concentrations of alloge-

110

I GENERAL METHODS TABLE I Inhibition of [3H]DHA Binding by fl-Agonists to Control Uteri Adrenergic agent

Kda (/zM)

(-)-Isoproterenol (-)-Epinephrine (-)-Norepinephrine

5.4 26.5 92.3

The Kd for the interaction of competing ligands is calculated using the equation of Cheng and Prussoff: Kd = IC50/1 + [L]/Kd, where IC50is the competing ligand concentration which half-maximally inhibits the specific binding of the radioligand at a concentration (L). IC50 values were obtained from competition experiments performed in duplicate at several concentrations of each agent.

neic pregnancy IgG, syngeneic pregnancy IgG, and control IgG (from nonpregnant animals) for 30 min at 30~ in buffer binding. After incubation time is completed, membranes are centrifuged at 40,000 g for 30 min at 4~ The pellet is resuspended in the same buffer and used in competition assays that follow the same procedure described above. Results are expressed as percent of the ratio of [3H]DHA specifically bound in the presence of IgG/[3H]DHA specifically bound in the absence of IgG (Fig. 3).

Cyclic AMP Assay In order to evaluate whether the activation of uterine fl-adrenergic receptors produced by agonists or antibodies was transducted to the intracellular compartment, we measure cAMP production by a radioimmunoassay-like procedure. Uteri from allogeneic and syngeneic pregnant mice and nonpregnant mice are immediately removed after sacrifice and the fetuses and placentas are separated from uterine tissue as well as uterine implantation zones. Uteri are weighed and left with spontaneous activity in 1 ml of KRB with 1 mM 3-isobutyl-l-methylxanthine (MIX) (an inhibitor of phosphodiesterase activity) gassed with 5% CO2 and 95% O2 (v/v) with shaking at 37~ for 30 min. When agonists or antagonists of fl-adrenoceptors are used to modify cAMP production, they are added 3 and 15 min before incubation time is over, respectively. Tissues are then homogenized with an Ultraturrax T 18/10 disperser at maximal speed for 30 sec, three times, in 15-sec intervals, in

[7]

IMMUNOPHARMACOLOGY

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111

100

~=

75

"0 C

h~ .-r. a ..r ~

25

0 9

I 8

I 7

I 6

m 5

IgG (-Log M)

FIG. 3 Inhibition of binding of [3H]DHA by increasing concentrations of allogeneic pregnancy IgG (O), control IgG (9 and syngeneic pregnancy IgG (11). C3H uterine membranes were preincubated with different concentrations of IgG for 30 min at 30~ and then with 1.2 nM of [3H]DHA at 37~ for 15 min. Control binding of 100% refers to the radioactivity bound to uterine membranes in the absence of IgG. Means +_ SEM of five independent experiments are plotted.

2 ml of absolute ethanol, and centrifuged at 2500 g for 15 min at 4~ The supernatants are collected and pellets are homogenized in 1 ml of ethanol: water (2 : 1, v/v) and centrifuged. Supernatants are combined and evaporated at 55~ under a nitrogen stream. cAMP residues are dissolved in 2 ml of assay buffer (50 mM Tris-HC1, 8 mM theophylline, 6 mM 2-mercaptoethanol, 0.45 mM EDTA, pH 7.4) and stored at -20~ until the assay is carried out. Aliquots of 100 ~1 are taken for the nucleotide determination using the procedure outlined in Table II.

Protocol 1. In glass tubes add 100 kd of standard solutions, samples, or buffer. 2. Add 50 ~1 of tritiated radioactive tracer diluted in assay buffer. 3. Vortex the tubes.

112

I GENERAL METHODS

TABLE II Procedure for cAMP Assay ,,

,

Parameter Total counts

Bo

Nonspecific binding

Standard curve c

Tube

Assay buffer (/zl)

cAMP (/zl)

[3H]cAMP a (/zl)

1 2 3 4 5 6 7 8 9 10

350 350 350 100 100 100 150 150 150 --

----~ ~ ~ ~ ~ 100 100 100

50 50 50 50 50 50 50 50 50 50 50 50

11

~

12

~

Protein kinase b (/xl) m

m

m

5O 5O 5O

50 50 50 i

a [3H]cAMP (specific activity, 31 Ci/mmol), 7 fmol/tube. b Protein kinase was purified following Brown's method (18). c Standard solutions of cAMP: 20, 10, 5, 2.5, 1.25, 0.625, 0.315, and 0.156 pmol/100/~1.

4. 5. 6. 7. 8. 9. 10. 11.

12.

Add 50/zl of protein kinase diluted (1:7) in assay buffer. Vortex the tubes. Incubate for 90 min at 4~ Rapidly add 200/zl of charcoal-albumin solution (except total counts tubes) (5% charcoal and 1% albumin in assay buffer) to each tube. Vortex the tubes. Incubate for 10 min at 0~ in ice-water. Centrifuge at 2000 g for 15 min at 4~ Decant the supernatant from each tube in a vial containing 10 ml of scintillation cocktail. Touch the rim of the test tube onto the surface of the scintillation fluid to "draw-off" the last drop from each tube. Determine the amount of radioactivity present in a/3 counter.

Mathematical Calculation of Results The concentration of cAMP in unknown samples may be calculated mathematically after constructing the calibration curve as follows: 1. Determine the nonspecific binding in counts per minute for the assay by averaging the cpm for tubes 7 to 9.

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2. Determine Bo (the cpm bound in the absence of unlabeled cAMP) by averaging cpm for tubes 4 to 6 and subtracting the nonspecific binding. 3. Determine B (the cpm bound in the presence of standard or unknown cAMP) by averaging the cpm for the remaining triplicated tubes and subtracting the nonsoecific binding. 4. Calculate B/Bo for each level of standard cAMP and for the unknowns. 5. Calculate logit B/Bo (ln B/Bo/1 - B/Bo) for the standard curve and for the unknowns. 6. Calculate log c (c is picomoles of unlabeled cAMP in each tube of standard curve). 7. Plot logit B/Bo against log c on logit-log paper. 8. From the logit B/Bo values for the unknown samples read the number of log c of inactive cAMP from the standard curve. 9. Calculate c from log c. The within-assay (intrassay) variation is estimated for different amounts of cAMP and the mean is 9.3%. The interassay variation for 43 duplicated determinations is 13.6%.

Acknowledgment This work has been supported by Grant BID-PID 0352 from the National Research Council from Argentina CONICET.

References 1. M. E. Sales, L. Sterin-Borda, and E. S. Borda, Int. J. Immunopharmacol. 8, 947 (1989). 2. G. Berg, R. G. G. Anderson, and G. Ryden, Am. J. Obstet. Gynecol. 151, 392 (1985). 3. E. M. M. Chow and J. M. Marshall, Eur. J. Pharmacol. 68, 1377 (1981). 4. M. H. Litime, G. Pointis, M. Breuiller, D. Cabrol, and F. Ferre, J. Clin. Endocrinol. Metab. 68, 1 (1989). 5. E. S. Borda, A. M. Genaro, G. A. Cremaschi, M. E. Sales, and L. Sterin-Borda, Eur. J. Pharmacol. 100, 195 (1984). 6. C. Cunningham, D. A. Power, A. Innes, T. Lind, and G. R. D. Catto, Hum. lmmunol. 19, 716 (1987). 7. N. Kaliss, in "Immunology of Reproduction" (K. Bratanov, ed.), p. 495. Bulgarian Academic of Sciences, Sofia, 1973. 8. G. A. Cremaschi, L. Sterin-Borda, A. M. Genaro, E. Borda, and M. Braun, J. Immunol. 133, 2681 (1984).

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I GENERAL METHODS

10. 11. 12. 13.

14.

15. 16. 17. 18.

T. Nicol, D. L. J. Bilbey, L. M. Charles, J. L. Codingley, and B. VernonRoberts, J. Endocrinol. 30, 277 (1964). R. A. Margni, in "Inmunologia e Inmunoquimica" (Panamericana, ed.), p. 481. Buenos Aires, Argentina, 1982. R. A. Margni, in "Inmunologia e Inmunoquimica" (Panamericana, ed.), p. 673. Buenos Aires, Argentina, 1982. O. H. Lowry, N. J. Rosenbrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). K. I. Welsh and J. R. Batchelor, in "Handbook of Experimental Immunology" (D. M. Weir, ed.), Vol. 2, p. 35.6. Blackwell Scientific Publications, London, 1978. G. D. Johnson, E. J. Holborow, and J. Dorling, in "Handbook of Experimental Immunology" (D. M. Weir, ed.), Vol. 1, p. 15.1. Blackwell Scientific Publications, London, 1978. E. S. Borda, J. Sauvage, L. Sterin-Borda, M. F. Gimeno, and A. L. Gimeno, Eur. J. Pharmacol. 56, 61 (1979). R. W. Lerner, G. D. Lopaschuk, and P. M. Olley. Can. J. Physiol. Pharmacol. 68, 1574 (1990). G. Scatchard, Ann. N.Y. Acad. Sci. 51, 660 (1949). B. L. Brown, J. D. M. Albano, R. P. Ekins, and A. M. Sgherzi. Biochem. J. 121~ 561 (1971).