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Delayed hypersensitivity and migration inhibition in two lines of mice genetically selected for high or low responsiveness to phytohemagglutinin
CELLULAR
IMMUNOLOGY
77,249-265
(1983)
Delayed Hypersensitivity and Migration Inhibition in Two Lines of Mice Genetically Selected for High or Low Responsiveness to Phytohemagglutinin S. GAUTHIER-RAHMAN,* S. EL ROUBY,*” M. LIACOPOULOS-BRIOT,* C. STIFFEL,? C. DECREUSEFOND,t AND P. LIACOPOULOS* *Institut d’lnzmuno-Biologie (Inserm U20, CNRS LA 143), H6pital Broussais. 96 rue Didot, 75674 Paris, C6dex 14, and TLaboratoire d’lmmuno-Gbhique (Inserm lJl25, CNRS ER 701, Institut Curie, 25 rue d’Ulm, 75005, Paris, France Received November 12, 1982; accepted December 30, I982 Delayed-type hypersensitivity (DTH) and cell migration inhibition (Ml) were studied in two lines of mice genetically selected for the high (Hi/PHA) or low (Lo/PHA) in vitro response of their lymphoid cells to phytochemaaglutinin (PHA). A rapid photoelectric procedure for reading cell migrations enabled the study of MI over a wide range (10 log) of antigen concentrations in vitro. Hi/PHA mice required immunization with a 10 times higher dose of ovalbumin (OVA) in Freund’s complete adjuvant (FCA) than Lo/PHA mice for a comparable response in DTH (footpad swelling) and MI oftheir induced peritoneal exudate cells (PEC). Lo/PHA spleen showed marked bizonal MI on Day 5 after immunization with low doses (0.1 and 0.5 pg) of OVA in FCA, one peak being obtained in presence of in vitro concentrations of IO-’ or lo-’ &ml OVA and another peak at 1 or IO &ml, whereas Hi/PHA spleen showed stimulation of migration. In contrast, MI in Lo/PHA spleen failed to persist beyond Day 19, whereas it appeared progressively in Hi/PHA spleen, being maximal by Day 27. Low-zone inhibition in Hi/PHA spleen and PEC was lacking or poor even after immunization with higher doses of OVA in FCA. The implications of these findings are discussed.
INTRODUCTION Genetic selection for the intensity of the response of mice lymphoid cells to phytohemagglutinin (PHA) has led to the development of two lines of mice characterized by either a high (Hi/PHA) or a low (Lo/PHA) response (1). The marked difference, 20-fold, in their in vitro response to this T-cell mitogen was shown to be accompanied by differences as marked in the development of T-cell-mediated immune reactions both in vitro and in vivo. Thus the mixed-lymphocyte reaction (MLR) (2) and graftversus-host reactivity (GVHR) (3) were shown to be much stronger in the Hi/PHA line. It was therefore of interest to investigate whether another parameter of T-cell reactivity, the development of delayed-type hypersensitivity (DTH) and its in vitro correlate, cell migration inhibition (MI) (4, 5), had also been affected during the course of selection, and, if so, in the same sense as the response to alloantigens and mitogen. ’ French Government Scholarship holder. This work was supported by CRL Inserm 821042. 249 0008-8749183 $3.00 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
250
GAUTHIER-RAHMAN
ET
AL.
The development in this laboratory of a rapid photoelectric procedure for reading cell migrations from capillary tubes (6) and, later, from agarose drops (7) enabled us to study the effect of a wide range (10 log) of antigen concentrations on the migration of cells of induced peritoneal exudates (PEC) or spleen (SC) from mice immunized with different doses of ovalbumin (OVA) in Freund’s complete adjuvant (FCA) and to follow the kinetics of this phenomenon. As is well known, DTH to simple protein antigens may not be easy to assessin mice by the footpad assay, due to the occurrence of anaphylactic reactions after local challenge with antigen (8- 10). However, a recent method involving the use of heat-aggregated antigen for elicitation of DTH (10) enabled us to establish a notable difference in the response of the Hi/PHA and Lo/ PHA lines, which correlated with that observed in vitro in the development of migration inhibition of their PEC and SC. The main finding was that the Hi/PHA line needs to be immunized with a dose of antigen 10 times higher in order to develop a reaction similar to that of the Lo/PHA line. MATERIALS
AND
METHODS
Mice These experiments were performed in mice of the Hi/PHA and Lo/PHA lines belonging to F22 to F24 generations from our own breeding colonies and in outbred Swiss albino mice purchased from Iffa Credo (69210J’Arbresle, France). It is recalled that the Hi/PHA and Lo/PHA lines were developed from a starting parental population of out-bred Swiss albino mice provided by different breeders. Antigen Salt-free Grade V crystallized and lyophilized Sigma Chemical Company.
ovalbumin
(OVA) was obtained from
Immunization For migration inhibition experiments and serum antibody determination, ovalbumin in a dose of 0.1, 0.5, 5, 50, or 500 pg/mouse, comprised in 0.1 ml of sterile saline, was emulsified with an equal volume of Freund’s complete adjuvant (Difco) and injected id at six sites, three on each side of the shaved dot-sum. For DTH experiments, the antigen dose per mouse was comprised in 25 ~1 of saline emulsified with an equal volume of FCA, and the emulsion mixture was injected subcutaneously on either side of the base of the tail. Delayed-Type
Hypersensitivity
The footpad assay was performed according to the method of Titus and Chiller (10) with minor modifications. Briefly, aggregated ovalbumin (AOVA) was prepared by heating a 2% solution of OVA in saline at 70°C for 1 hr, followed by slow cooling. The precipitate was washed twice and then made to the original volume in saline. DTH was elicited in groups of 5 to 8 mice on Day 8 after immunization as above with different doses of OVA. The suspension of AOVA (30 ~1) was injected subcutaneously in the right hind footpad with a 27-gauge needle, and the same volume of saline was injected into the left hind footpad. Footpad thickness was measured at 4, 24,48, 72, and 96 hr after challenge under ether anaesthesia using an “Oditest” skin-
DELAYED
HYPERSENSITIVITY
AND
MIGRATION
INHIBITION
251
thickness gauge (Kroplin, GMBH, 6490 Schluechtern, 1, W. Germany). The extent of swelling was calculated by subtracting the thickness of the saline-injected footpad from that of the antigen-injected footpad. Cell Migration
Inhibition
Experiments using the capillary-tube technique with peritoneal exudate cells and photoelectric readings were begun as described elsewhere (6). When the photoelectric procedure for reading migration from agarose microdroplets was developed, migration of spleen cells was studied using this latter method (7). (I) Peritoneal exudate cells. It was found by prior experiment that large numbers of PEC could be obtained from one mouse after ip injection of Freund’s incomplete adjuvant (FIA) (Difco). Groups of mice immunized with different doses of OVA in FCA were sacrificed by cervical section on Days 5, 12, 19, 60, or later, after immunization and 4 days after ip injection of 0.5 ml FIA. The peritoneal cavity was washed out with a total of 20 ml of cold 199 medium supplemented with antibiotics and 1% heat-inactivated fetal bovine serum (FBS). The cell suspension was washed twice and the number of viable cells ascertained by trypan blue exclusion. At the same time, the number of phagocytic cells in the exudate was determined by counting the cells containing oil droplets. In general, 70-75% of the cells contained oil droplets and were considered to be macrophages. About 30 to 60 X 1O6 cells could be obtained from one mouse by this method. Much fewer PEC were obtained from Lo/PHA mice as compared to Swiss or Hi/PHA mice. Inhibition of migration of PEC from individual mice was studied in many cases in the two latter strains, but pools of two or more mice had to be used in Lo/PHA mice. A cell concentration of 18-20 X 106/ ml was found suitable for photoelectric reading using the capillary-tube technique. About 4 ml of suspension was required for the 80 capillaries needed for each experiment. The cell suspension was drawn up into capillary tubes of O.l-mm internal diameter. One end of the capillary tube was sealed by flaming. After centrifugation the capillaries were cut just below the cell-liquid interface. The end containing the cell pellet was fixed individually in the well of a sterilin migration plate with a dab of sterile silicone grease, and the well was filled immediately with 0.45 ml of medium with or without antigen. Six to eight capillaries were mounted per concentration of antigen, with 8 to 16 controls (medium without antigen). Migrations were read at 18 hr generally and also at 48 hr in some cases. (2) Spleen cells. The spleen was excised after the peritoneal exudate had been removed and minced in a drop-by-drop stream of cold 199 medium with 1% FBS. The cell suspension (20 ml vol) was passed through a stainless-steel sieve (60 pm), the cells were washed, and cell viability was ascertained by trypan blue exclusion. In a few experiments, red cells were lysed with 0.83% cold NH&l. Cell suspensions and pellets were maintained in an ice bath. Inhibition of migration of SC suspensions from individual spleens was studied with the agarose microdroplet technique. In many cases migration inhibitions of PEC and SC from the same animal were studied in parallel. Antigen concentrations OVA concentrations of 10e5, 10m4, 10m3, lo-‘, IO-‘, 1, 10, 100, and 250 &ml were prepared in 199 medium containing 20% heat-inactivated FBS by the adjunction
252
GAUTHIER-RAHMAN
ET
AL.
of an appropriate concentration of OVA in sterile saline in l/ 100 of the final volume. Concentrations of 1, 2.5, 5, and 10 mg/ml were obtained by adding weighed dry OVA to medium. Antigen concentrations were prepared prior to sacrifice of the animals and maintained in an ice bath. The Agarose Microdroplet bition
Technique for Photoelectric
Readings of Migration
Inhi-
This technique was a modification of the usual technique ( 11, 12) and is described in detail elsewhere (7). Briefly, spleen cell suspensions of 4 X lO’/ml were prepared in 0.2% seaplaque agarose (final) in 199 medium supplemented with antibiotics and 15% FBS. The suspension was drawn up into a 50-~1 Hamilton syringe fitted with an automatic dispenser. A sterilin migration plate was previously prepared by greasing the rims of its wells and cooling or an LKB plate was held at strict horizontal on an ice slurry. Four l-p1 microdroplets were dispensed per well of the sterilin plate. After a uniform gelation time of 6 min the wells were filled with 0.45 ml of medium without antigen (controls) or with one of the 11 different concentrations of OVA prepared and covered with round 22-mm coverslips. The plates were then placed together with the LKB table at strict horizontal at 37°C. Eight microdroplets per concentration and 16 controls were usually studied. In addition, 8-12 microdroplets were allowed to dry before the wells were filled with control medium (no migration). These served as background controls. Migrations were read in general at 18 hr. Photoelectric Readings of Cell Migrations The migrating cells are viewed under dark-field illumination. According to the technique used, the glass stub of the cut capillary or the agarose microdroplet is hidden by a rectangular or a circular black mask placed in the ocular. The light reflected by the migrating cells is directed to the photosensitive surface of the Polaroid camera of an apparatus for automatic microphotography (Nachet, Model N 8901, Levallois-Perret, France) and the migration may, if desired, be photographed. The light diffracted is measured as electrical potential by the electronic exposure meter of the apparatus, which is connected to a voltmeter of sufficient sensitivity. The voltage, in volts, corresponds logarithmically to the amount of light diffracted by the migration in lumens. Inhibition is indicated by a fall in voltage. Thus, % inhibition
=
LC - Lo”* x loo L”
where L’ is the mean luminosity of migrating cells in control medium (no antigen) and Lo”* the mean luminosity of migration at a given antigen concentration. The luminosity, in lumens, is equal to the antilog of the observed voltage (in volts) minus the background luminosity. Migration inhibition of 20% or more was considered significant. Serum Antibody Determination Blood samples were obtained by retro-orbital puncture from groups of 5-6 mice on Days 7, 11, 19, 27, and 60 after immunization with 0.5, 5, 50, or 500 pg of OVA in FCA as described above. Anti-ovalbumin titers of individual set-awere determined
DELAYED
HYPERSENSITIVITY
by passive hemagglutination bent assay) (13). Cyclophosphamide
AND MIGRATION
and also by micro-ELISA
253
INHIBITION
(enzyme-linked
immunosor-
(Cy) Pretreatment
Mice were given an ip injection of 100 mg/kg Cy 72 hr prior to immunization with 0.5, 5, or 50 pugOVA emulsified in 0.1 ml FCA. Migration inhibition of their PEC was studied on Day 5. RESULTS Delayed-Type
Hypersensitivity
The results of the footpad assay in mice of the two strains performed on Day 8 after immunization with 0.5, 5, or 50 pg in FCA are presented in Fig. 1. It can be seen that Lo/PHA mice show stronger reactions than Hi/PHA after immunization with lower doses of OVA, 0.5 and 5 pg. After immunization with 50 pg OVA, Hi/PHA mice show the stronger reactions, those in Lo/PHA being depressed. Optimal immunization for DTH was obtained in Lo/PHA after 5 pg OVA in FCA. Furthermore, footpad swelling was found to be more persistent in Lo/PHA mice. Maximal DTH was observed at 24 hr, but some swelling was still present at 48 and 72 hr. These late reactions were always higher in Lo/PHA mice whatever the immunizing dose of antigen (Fig. 1). However, there was great variation in the DTH reactions of individual mice and the differences observed between the two lines were at the limit of significance.
Immunizing
c! ,o
50.
; E 0
40.
.-,” =u
30
Dose:
LO ..I.. H,
pg
#
t
t
0.5
5
50
_
4
24
40
Hours
72
96
after
4
24
48
72
96
4
24
48
72
challenge
FIG. 1. Delayed-type hypersensitivity response of Hi/PHA and Lo/PHA mice. Eight days after immunization with 0.5, 5, or 50 pg OVA in FCA, mice were injected with 30 ~1 of heat-aggregated OVA (AOVA) in the right footpad and the same volume of physiological saline in the left footpad. The difference between the swelling of the right and left footpad is shown j, SEM at various times after the challenge. Each curve was obtained with the mean readings for 5-8 mice.
254
GAUTHIER-RAHMAN
Migration Inhibition Antigen
ET
AL.
in Induced PEC and the Efect of the Immunizing
Dose of
On Day 5 after immunization of Swiss, Hi/PHA, or Lo/PHA mice with 0.1, 0.5, 5,50, or 500 Kg of OVA in FCA, MI was present in PEC. In accordance with classical data, it was generally observed in the presence of a high concentration of OVA in vitro, 1 to 5 or even 10 mg/ml. However, in some circumstances MI was observed in a zone of a much lower antigen concentration. The results obtained on Day 5 in the presence of high concentrations of OVA in vitro are summarized in Fig. 2. A marked effect of the immunizing dose of OVA can be seen, particularly in Hi/PHA mice. MI on Day 5 is quasi-maximal (30-40%) in
50
40
: .w .n ..c
30
C
20
ix!
10
0
-10
-20
-30
lmmunizir dose
50
500
(P9
FIG. 2. Effect of the immunizing dose of antigen on the development of migration inhibition (MI) in induced peritoneal exudate cells of Hi/PI&A, Lo/PHA, and Swiss mice. MI was studied by the capillarytube technique with photoelectric readings (as described under Methods) on Day 5 after immunization with different doses of OVA in FCA and in the presence of in vitro concentrations of OVA ranging from lo-’ &ml to 10 mg/ml. The means of the greatest MI, generally observed in the high zone of antigen concentration (250 fig to 10 m&ml), are shown with SEM. The figures near the tops of the columns indicate the number of experiments.
DELAYED
HYPERSENSITIVITY
AND
MIGRATION
INHIBITION
255
Swiss or Lo/PHA mice after immunization with as little as 0.5 pg OVA, whereas in Hi/PHA mice this is obtained only after immunization with 50 pg. After the largest immunizing dose, 500 pg, MI is diminished in both Hi/PHA and Lo/PHA mice, this effect being most marked in Lo/PHA mice, where MI is replaced by stimulation and least in Swiss mice, where mean MI is still 32.5%. Similarly, after the lowest immunizing dose, 0.1 pg, only Swiss mice show significant MI (27%). MI of PEC on Day 5 was often seen as a sharp peak obtained in the presence of a narrow range of high in vitro concentrations of antigen, l-5 mg/ml, particularly after immunization with nonoptional doses of OVA. After optimal immunization, in Lo/PHA MI was spread over a much wider range of antigen concentrations, with the appearance of a second peak at very low antigen concentrations, 1O-‘-1O-3 pg/ml. In contrast, in Hi/PHA mice MI on Day 5 remained a single peak in the high zone of antigen concentration even after optimal immunization (Fig. 3). The response of PEC of Swiss mice was similar to that of Lo/PHA (data not shown). Kinetics of Development
of MI in Induced PEC
The results of sequential study on Days 5, 12, 19, 30, and 60 after immunization with 0.5 or 500 pg OVA in FCA are summarized in Table 1. MI increased somewhat from Day 5 to Day 12, whatever the immunizing dose. This effect was only marked in Hi/PHA mice, where it was present after both high and low immunizing doses, and in Lo/PHA mice only after the higher dose. After immunization with a low dose, 0.5 pg OVA, mean MI in Hi/PHA mice was less than in Lo/PHA at all times studied (Table 1). On the contrary, after immunization with a high dose, 500 pg, mean MI was higher in Hi/PHA on Days 5, 12, and 60. Lo/PHA mice, showing no MI on Day 5 after immunization with 500 pg, developed a mean MI (2 1.35%) only half that of Hi/PHA (42.25%) by Day 12, which increased
40 I
Log,,
Cont.
Ova.
p9
I ml
FIG. 3. Lack of early low-zone inhibition in induced peritoneal exudate cells of Hi/PHA mice. The curves show the mean MI observed at different antigen concentrations in 3 experiments for each line of mice performed on Day 5 after immunization with 5 pg OVA in FCA.
256
GAUTHIER-RAHMAN
ET AL.
TABLE 1 Kinetics of Migration Inhibition in Induced Peritoneal Exudate Cells of Three Lines of Mice after Immunization with a Low or a High Dose of Antigen in FCA Immunizing dose of OVA (rg) 0.5
Days after immunization
Percentage inhibition observed in PEC Expt. No.
5
I 2 3
12
1 2 3
19
1 2 3
30 60 500
5
12
1 2
19
I 2 3
60
1 2 3
120
1
Lo/PHA
Hi/PHA 18.6 (I mg) 46.0 (10 mg) 19.0 (10 mg) Mean 27.83 f 9.08 27.6 (10 mg) 35.5 (5 mg) 43.0 (2.5 mg) Mean 35.33 f 4.47 7.7 (5 mg) 32.2 (1 mg) 54.5 (2.5 mg) Mean 31.46 + 13.51 28.4 (10 mg) 25.5 (5 mg) 33.8 (5 mg) 9.9 (2.5 mg) 21.5 (10 mg) Mean 2 I .70 f 6.90 42.3(1 ccg) 42.2 (1 d Mean 42.25 k 0.05 33.3 (10 rg)
32.2(1 4 20.9 (I mg) 4.1 (10 a9 Mean 19.06 f 8.16 ND
66.0 (10 mg) 38.4 (2.5 mg) 38.6 (IO mg) Mean 44.83 + 5.84 43.6 (1 mg) 47.0 (10 mg) 51.6 (1 mg) Mean 47.40 + 2.31 53.2 (2.5 mg) 12.9 (2.5 mg)
Swiss 44.5 (10 mg) 46.2 (10 mg) 33.8 (5 mg) Mean 41.5 f 3.88 30.6 (2.5 mg)
ND”
Mean 33.00 + 20.20 ND 41.6 (10 mg) 44.6 (5 mg) 44.5 (10 mg) -35.2 -40.0
(2.5 mg) (5 mg)
Mean -37.60 + 2.4 20.9 (1 4 21.8 (1 /.a) Mean 21.35 + 0.45 34.5 (10 pg) 42.8 (1 mg) Mean 38.65 f 4. I5 6.35 (100 pg) 4.65 (1 mg) Mean 5.50 f 0.85 ND
30.9 (2.5 mg) 35.5 (2.5 mg) Mean 33.2 + 2.30 44.2 (0. I fig) 39.5 (5 mg) 26.1 (5 mg) 48.5 (100 pg) Mean 38.03 + 6.50 54.0 (100 pg) 33.8 (I mg) 16.2 (100 /.& Mean 34.50 f 10.92 44.5 (2.5 mg)
No@. The maximal MI in each experiment is noted with, in parentheses, the antigen concentration in vitro where it was obtained. Means are indicated with standard error. Negative figures indicate stimulation percentage. ’ ND, not done.
to 38.65% on Day 19. By Day 60, however, Lo/PHA no longer showed significant MI as compared to Hi/PHA, where MI was still present in two out of three experiments (Table 1). Swiss mice appeared to retain practically unchanged MI from Day 5 to Day 60 after immunization with 0.5 or 500 pg, the titers after the latter dose being somewhat lower (Table 1). Bizonal Inhibition
in PEC
MI in PEC of Lo/PHA and Swiss mice was frequently bizonal with a high zone at 100 pg to 10 mg/ml OVA in vitro and a low zone around lo-‘- lop3 &ml (Fig.
DELAYED
HYPERSENSITIVITY
AND MIGRATION
INHIBITION
257
3). After immunization with 0.5 pg OVA, a small peak in the low zone was seen in Lo/PHA mice in two of three experiments on Day 5, one of three on Day 12, and one of two on Day 19. Hi/PHA mice did not show the low-zone peak on Day 5, but small low-zone peaks appeared on Days 12 and 19 in half the experiments if culture was prolonged to 48 hr. Low-zone inhibition was more marked in PEC of all three lines of mice at times later than Day 5 and after immunization of Hi/PHA with higher doses. In Lo/PHA mice, the low-zone peak could become as high as the other one (Fig. 4). Bizonal inhibition of PEC was observed in Swiss mice as late as 10 months after immunization with 50 pg OVA in FCA (data not shown). After immunization with 500 pg OVA, MI in Hi/PHA was shifted to the left on Days 12 and 19, the major peaks being at 1 or 10 pg/ml and 10m4 to 10e2 pg/ml with little or no inhibition in the usual high zone (around 1 mg/ml) (Table 1, Fig. 5). In Lo/PHA mice, under these circumstances, inhibition in the high zone still occurred (Fig. 4) together with an additional peak at 1 or 10 pg/ml, or was observed over a wide range of Ag concentrations. Efect of Cyclophosphamide Treatment of Lo/PHA or Hi/PHA mice with 200 mg/kg Cy ip 72 hr prior to immunization with 0.5, 5, or 50 pg OVA in FCA was without notable effect on MI of induced PEC on Day 5. Migration
Inhibition
in Spleen and the Efect of the Immunizing
Dose of Antigen
Except for some early experiments where pools of 2-3 spleens were employed, individual spleens were studied using the agarose microdroplet technique with photoelectric readings. OVA concentrations from 10m5 pg/ml to 5 mg/ml were used. 50.
X,
:p
a? ;--
2”
. . ..-I
/
10.
0
l-‘:
:
r
:
* -3
-10
L....
-2
-1
0
1
.
2
: :
:.
5 f
‘j 3 : b
4 ‘8 i
-20.
-30. L-lo
Cone.
Owe.
pglml
FIG. 4. Marked bizonal inhibition in induced peritoneal exudate cells and spleen of a Lo/PHA mouse on Day 19 after immunization with 500 pg OVA in FCA.
258
GAUTHIER-RAHMAN
ET AL. -
PE s.” sp,..n
50 I
40. 30.
20.
10 0 10.
. -4
-3
-2
-,
0
1
2
\' .
.v
3
4
-20
-30.
%
Cow.
Ova.
pa/ml
v\
.
FIG. 5. Bizonal inhibition in induced peritoneal exudate cells and spleen of a Hi/PHA mouse on Day 12 after immunization with SOOjtg OVA in FCA.
On Day 5 after immunization with low antigen doses, 0.1 or 0.5 pg OVA, in FCA MI of 35-40s was observed in Lo/PHA spleen, whereas this was absent in Hi/PHA (Figs. 6 and 7, Table 2). However, MI on Day 5 appeared in Hi/PHA spleen after immunization with 5 or 50 pg OVA (Fig. 7). MI in Lo/PHA spleen was quasi-maximal
LO%0
Cont.
Ova.
pglml
FIG. 6. Comparison of migration inhibition in the spleen of Hi/PHA and Lo/PHA mice on Day 5 after immunization with a low dose of OVA, 0.5 rg in FCA. Ml was studied in individual spleens by the agarose microdroplet technique with photoelectric readings (see Methods). The means of MI observed in four spleens of each line of mice at the different in vitro antigen concentrations of OVA are shown with the SEM. Marked bizonal inhibition is observed in Lo/PHA and only stimulation in Hi/PHA spleen.
DELAYED
HYPERSENSITIVITY
AND MIGRATION
A
I3
50 :
..P r -c o\
259
INHIBITION
I
I
HI
40. 30. 20
10. 0, -10 -2oL lmmunislng
Dose:
0.1
0.5
5
50
500
0.1
0.5
5
50
500
(U9) FIG. 7. Effect of the immunizing dose of antigen on the development of migration inhibition in spleens of Hi/PHA and Lo/PHA mice. Mice were sacrificed on Day 5 aher immunization with different doses of OVA in FCA. MI was studied in each experiment in the presence of in vitro concentrations of 10e5 to 2500 &ml. The means of the greatest MI observed in the low zone of antigen concentration, 10-3-10-2 &ml (A), and those of the high zone, l- 10 &ml (B), are shown. The figures near the tops of the columns represent the number of experiments.
after immunization with the lowest dose, 0.1 pg, whereas in Hi/PHA 5 pg appeared to be optimal. MI was markedly reduced in Lo/PHA spleen after immunization with 50 pg and less so in Hi/PHA. After 500 pg, MI was markedly reduced in both lines (Fig. 7). Bizonal Inhibition
in Spleen
Migration inhibition on Day 5 after immunization with low doses was found to be markedly bizonal in Lo/PHA spleens with one peak in the presence of OVA concentrations of 10e2 and 10e3 pg/ml and another peak generally at 1- 10 &ml, in some cases at 100 Clg/ml (Table 2, Figs. 6 and 7). In contrast, MI in Hi/PHA spleen was observed in the high zone of Ag concentration with but little inhibition in the low zone even after optimal immunization (Fig. 7). Kinetics of Migration
Inhibition
in Spleen
Sequential study was performed on Days 5, 12, 19, 27, and 40 or 60 after immunization with 0.5 or 500 fig OVA in FCA. The evolution of MI in spleen after immunization with 0.5 pg is shown in Fig. 8 and Table 2. Bizonal inhibition was maximal (about 40%) on Day 5 in Lo/PHA spleen. Both peaks decreased thereafter progressively on Days 12 and 19 and were insignificant by Day 27. On the contrary,
1 2 3 4 Mean SEM
1 2 3 4 Mean SEM
1 2 3 Mean SEM
12
19
27
(lo-’ (10-r ( lO-2 (10-Z
(10-r ( lO-3 (IO-’ (lo-3
(10-r (lo-3 (lo-3 ( lO-3
pg) rg) rg) pg)
pg) pg) pg) jJg)
pg) pg, 10-r rg) rg) pg)
34.4 ( 1om3 rg) 10.8 ( lO-3 pg) -20.8 (IO-’ pg) 8.13 kl3.05
0 16.6 27.5 37.5 20.40 k8.02
3.5 6.0 -6.5 -13.3 -2.57 k4.29
-41.2 7.1 -20.0 -24.0 -19.77 +10.17
A
Hi/PHA
(1 (1 (1 (1
Pia rg) rd d
-11.0 (10-r 26.7 (IO-’ 7.85 k18.85
Mean SEM
14.83 k21.15
1 2
Mean SEM rg) pg)
52.7 (10m2 pg) 12.2 (lo-2 pg) -20.4 ( 10m2 pg)
/*g) pg) pg) pg)
1 2 3
(lo-2 (lo-3 ( 1om2 ( lO-2
(250 (100 (100 (10
pg) pg) pg) pg)
5.70 kl9.50
1 2 3 Mean SEM
A
Swiss
rg)
B
The figures
rg) rid 15.7 (100 rg) 17.66 k3.62
24.7 (1 12.6 (1
52.5 (100 rg)
-0.20 k31.90
are
-32.1 (1 j& pool 36 31.7 (10 rg) pool 36
in parentheses.
33.2 (IO-’ pg) 9.1 (lo-3 pg) 12.7 (10m3 pg) 18.33 k7.50
48.5 (lO-3
-1.20 k27.8
-29.0 (IO-* fig) 26.6 ( 10m3 pg)
in FCA
in vitro where it was found
25.2 (1 pg) -13.8 (1 fig)
12.90 k5.02
14.8 (10 rg) 20.5 (100 rg) 3.4 (100 Pd
1
Mean SEM
1 2
Expt.
0.5 pg OVA
IO.2 (1 PI9 37.2 (100 rg) 6.4 (100 pg) 10.8 (100 pgg) 16.15 k7.08
37.7 70.2 34.1 18.0 40.00 210.93
B
of
with
in spleens
(IO-‘, lO-2 rg) ( 10-3, lO-2 pg) (lo-), 1om2 /Jg) (lo-‘, 10-r rg)
A
Lo/PHA
observed
Immunization
10.2 7.1 20.0 45.6 20.72 k8.73
24.0 54.6 41.0 27.8 36.85 f6.94
inhibition
Low-Dose
1 2 3 4 Mean SEM
1 2 3 4 Mean SEM
Expt.
Percentage
in Spleen after
(1 pg, 10 pg) (1 4 (10 pg) (1 pg, 10 pg)
(1 fig) (1 fig, 10 a) (10 /a) (10 jtg)
B
Inhibition
57.0 (10 rg) 1.65 (1 rg) 37.0 (1 @cg, 10 pg) 31.88 kl6.18
29.1 24.40 k5.10
27.5
31.7 9.3
-15.85 42.1 17.4 21.1 16.18 fll.98
-68.5 -23.32 + 16.62
-6
-26.9 7.1
of Migration
2
Nofe. The inhibition observed in the low zone (A) or high zone (B) is noted with the antigen concentration for individual spleens except where pools are indicated. Negative figures indicate percentage stimulation.
I 2 3 4 Mean SEM
Expt.
5
Days after immunization
Kinetics
TABLE
DELAYED
50 .-: 5 .I c
40.
30. * 20.
10.
O-
-10 -
-20.
-30 -
HYPERSENSITIVITY
AND MIGRATION
261
INHIBITION
4 4 I 4 1 5
12
19 Days
27 after
5 lmmunizat
12
19
27
ion
FIG.8. Kinetics of migration inhibition in Hi/PHA and Lo/PHA spleen after immunization with a low antigen dose, 0.5 pg OVA in FCA. Mice were sacrificed on Days 5, 12, 19, and 27 after immunization and MI was studied in individual spleens as under Fig. 7. The means of the greatest MI observed in the low zone, 10-3-10-2 pg/ml (A), and in the high zone, I-10 pdrnl (B), of in vitro Ag concentrations are shown with the SEM. The figures near the tops of the columns represent the number of spleens. Lo/PHA spleens show maximal bizonal inhibition on Day 5, which declines rapidly thereafter. In contrast, Hi/PHA spleens, after initial enhancement of migration, develop increasing MI particularly in the high zone, which is maximal by Day 27.
after initial stimulation on Day 5 (approx -20%) MI appeared in Hi/PHA spleen from Day 12 onward and increased progressively until Day 27 (3 1.88%). It was observed only in the high zone at 1 or 10 pg/ml except on Day 19, when a small peak (20.4%) was also present in the low zone (Fig. 8). MI was no longer observed in Hi/PHA spleen by Day 40. Swiss spleen was found to be like Lo/PHA spleen in that it responded to low immunizing doses of OVA, 0.1 and 0.5 pg, with marked bizonal inhibition on Day 5, but only in one of two experiments, and was like Hi/PHA spleen in that mean
262
GAUTHIER-RAHMAN
ET
AL.
MI appeared to increase with time, to 33 and 50% on Day 12 after immunization with 0.1 and 0.5 Kg, respectively (Table 2). After an immunizing dose of 500 pg OVA (Table 3), MI in both Lo/PHA and Swiss spleen was insignificant on Days 5 and 12 with presence of stimulation on Day 12, whereas in Hi/PHA spleen, bizonal MI in a low degree was present on Days 5 and 12. By Day 19, bizonal inhibition was observed in Lo/PHA and Swiss spleen, whereas only a low-zone peak was retained in Hi/PHA spleen. Practically unchanged bizonal MI of 33% was still present in Swiss spleen on Day 60, by which time Lo/ PHA and Hi/PHA spleen no longer showed significant MI (Table 3). Serum Antibody On Day 5, after ovalbumin antibody immunization with titers were found Hi/PHA mice.
immunization with 0.5, 50, or 500 pg OVA in FCA, no antiwas detected by passive hemagglutination or by ELISA. After 0.5 or 500 pg OVA in FCA, Day 12, 19, and 27 serum antibody to be very similar by both techniques in Lo/PHA and
DISCUSSION Earlier studies performed in the two lines of mice genetically selected for their low or high response to phytohemagglutinin, a T-cell mitogen, showed a marked difference to exist between them in reactions involving the T-cell response to cell-associated Ag, namely mixed-lymphocyte culture (2) and graft versus host (3). The Hi/PHA line was shown to have the much greater T-cell reactivity. The present study utilizing a soluble protein Ag, OVA in FCA, brings to light differences in the behavior of the two lines in reactions of DTH and MI. These differences are related to the optimal immunizing dose of Ag, to the dose response in vitro in MI of cells sensitized in vivo, and to the kinetics of MI in spleen and PEC of immunized mice. The use of a rapid photoelectric procedure for reading cell migration (6, 7) permitted the study of MI over a wide range (10 log) of Ag concentrations in vitro and led to the observation of the phenomenon of bizonal inhibition. The optimal immunizing dose of Ag was found to be 10 times higher in the case of Hi/PHA mice, both for the in vivo footpad assay of DTH (Fig. 1) and for in vitro MI of spleen and PEC (Figs. 2 and 7). Early (Day 5) marked low-zone inhibition in presence of in vitro concentrations of 10e3 to 10m2 &ml OVA, with a second peak at 1 to 100 lg/ml, was a characteristic feature of Lo/PHA spleen after immunization with low Ag doses of 0.1 or 0.5 pg, but was found not to persist, decreasing rapidly by Days 12 and 19. On the contrary in Hi/PHA spleens, after initial stimulation, MI progressively appeared and became maximal by Day 27, the major or single peak always being in the high zone, 1 to 10 pg/ml (Fig. 8, Table 2). Similarly, early lowzone inhibition was lacking in PEC of optimally immunized Hi/PHA mice (Fig. 3). Bizonal inhibition was also observed in Swiss PEC and spleen. It suggests the existence of two cell populations responding to very different in vitro concentrations of Ag, 2 to 4 log apart. The lack of marked bizonal inhibition in Hi/PHA spleen and PEC at early times after immunization would suggest that cells responding to very low doses of Ag with liberation of MIF are either lacking or too few in number or prevented from expressing themselves in this line of mice. Even at times later than Day 5 and after immunization with larger Ag doses, low-zone inhibition, although present in Hi/PHA spleen and PEC, was not as high as in Lo/PHA (Figs. 4-7, Table
DELAYED
HYPERSENSITIVITY
AND
MIGRATION
INHIBITION
264
GAUTHIER-RAHMAN
ET
AL.
3). That antibody was responsible for the phenomena observed is unlikely, since no circulating antibody was found on Day 5 after immunization with 0.5 or 500 )~g OVA in FCA, and the antibody titers by passive hemagglutination at later times were very similar in the two lines. The lack of persistance of bizonal inhibition in Lo/PHA spleen could be due to failure of multiplication of the cells concerned, their migration out of the spleen, or the development of suppressive phenomena. It has been shown in the case of DTH that two cell populations are involved, one of which is replicating as shown by its sensitivity to vinblastine (14). On the other hand, the progressive increase of MI in Hi/PHA spleen after low-dose immunization could be due to slow multiplication of a rare cell population and/or the progressive removal of a suppressive effect. The response in MI of Swiss mice was superior to that of both lines. Swiss PEC responded on Day 5 after immunization with very low (0.1 pg) or very high (500 pg) doses of Ag, whereas the dose-response range of the two lines was much more restricted, Hi/PHA failing to respond after low and Lo/PHA after high immunizing doses. Persistance of MI in PEC and spleen was also much longer in Swiss mice (Tables 1 and 3). It would appear that the adequate presence or expression of both cell populations, responding respectively to high or low Ag doses in vitro, was necessary for the better development of MI seen in Swiss mice. Parallel study of MI in individual spleens and PEC showed that the two did not appear or disappear simultaneously. Thus, the distribution or expression of cells responding in MI was not homogenous in the different lymphoid cell populations of the same mouse, particularly at early or later times (Day 60) after immunization (compare Tables 1 and 3). MI in the spleen appeared to be a more transient phenomenon than in PEC. The time after immunization when MI appeared in the spleen and its duration were related both to genetic character and the immunizing dose of Ag (Tables 1 and 3). Hanington ( 15) found that MI in the spleen after cancerization appeared around Day 11 and became maximal by 3 weeks, long after the rejection of ip-injected allogenic tumor cells. Our data would suggest that this does not necessarily preclude the role of cells responding in MI in the rejection of the tumor allograft. The significance of bizonal migration inhibition observed herein in both spleen and PEC remains to be established. Two types of MIF have been isolated in vitro ( 16, 17) with different molecular weights and isoelectric points. Could the two types of MIF be produced by two different cell populations responding in vitro to very different Ag concentrations? Cells producing MIF after antigenic or mitogenic stimulation have generally been found to be of Lyt l+ phenotype (18-20). However, in some cases, such as immunization with active influenza virus or DNFB, involvement in DTH of T cells of different phenotype has been observed (2 1, 22). The possibility that the poor low-zone inhibition in Hi/PHA mice is due to the relative lack of cells of a particular Lyt phenotype is presently being explored. Moreover, the greater Tcell reactivity of the Hi/PHA line was demonstrated in response to cell-associated Ags in MLC and GVH. MIF production during MLC was found to be by both Lyt l+ and Lyt 2+ cells (23). It is possible that the Hi/PHA line would be a better producer of MIF in MLC than the Lo/PHA, and this study is under way. Bizonal inhibition has not hitherto been described. However, MI at low in vitro concentrations of OVA was observed by us in PEC of guinea pigs after treatment with Corynebacterium parvum (24). MI of leukocytes from some human subjects was observed at extremely low concentrations of PPD (5 X 10e7 &ml) by McCoy et al.
DELAYED
HYPERSENSITIVITY
AND MIGRATION
INHIBITION
265
(25) and also by us (unpublished data). Our experiments would suggest that this type of response could be related to genetic character. Finally, migration stimulation, sometimes of marked degree, seen in Hi/PHA spleen, particularly after immunization with small doses of Ag (Table 2, Figs. 6-8), would suggest the presence of a migration stimulation factor. Such a factor has been shown to be produced by certain human T-cell subsets (26, 27) and also in mice (27, 28). Suppressor T cells of the Lyt 2+,2 phenotype were found to be involved (28). Although no suppressor cells sensitive to cyclophosphamide were found in our experiments, suppressor T cells producing migration stimulation factor may be present with higher frequency in Hi/PHA mice. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 1I. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
Stiffel, C., Liacopoulos-Briot, M., Decreusefond, C., and Lambert, F., Eur. J. Immunol. 7, 291, 1977. Liacopoulos-Briot, M., Stiffel, C., Lambert, F., and Decreusefond, C., Cell. Immunol. 44, 29, 1979. Liacopoulos-Briot, M., Stiffel, C., Lambett, F., and Decreusefond, C., Cell. Immunol. 62, 448, 1981. Bloom, B. R., and Bennett, B., Science 153, 102, 1966. David, J. R., Al-Askari, S., Lawrence, H. S., and Thomas, L., J. Immunol. 93, 264, 1964. Gauthier-Rahman, S., and Morlat, J. L., J. Immunol. Methods 23, 7, 1978. Gauthier-Rahman, S., Morlat, J. L., Leca, G., and Bouin, M., J. Immunol. Methods 53, 7, 1982. Crowle, A. J., Advan. Immunol. 20, 197, 1975. Clark, C., Azar, M. M., Gleason, D. F., Cell. Immunol. 26, 228, 1976. Titus, R. G., and Chiller, J. N., J. Immunol. Methods 45, 65, 1981. Harrington, J. I., and Statsny, P., J. Immunol. 110, 752, 1973. Adelman, N., Cohen, S., Yoshida, T., J. Immunol. 121, 209, 1978. Vos, J. G., Buerkamp, J., Buys, J., and Steerenberg, P. A., Stand. J. Immunol. 12, 289, 1970. Lagrange, P. H., and Mackaness, G. B., J. Exp. Med. 141, 82, 1975. Harrington, J. T., Cell. Immunol. 24, 195, 1976. Remold, H. G., and Mednis, A. L., J. Immunol. 118, 2015, 1977. Remold, H. G., and Mednis, A. D., J. Immunol. 122, 1920, 1979. Kuhner, A. L., Cantor, H., and David, J. R., J. Immunol. 125, I 117, 1980. Adelman, N. E., Ksaizek, J., Yoshida, T., and Cohen, S., J. Immunol. 124, 825, 1980. Chensue, S. W., Boros, D. L., and David, C. S., J. Exp. Med. 151, 1398, 1980. Leung, K. N., and Ada, G. L., Stand. J. Immunol. 1980, 12, 48 I. Vadas, M. A., Miller, J. F.-A. P., Whitelaw, A. M., and Gamble, J. R., Immunogenetics 4, 137, 1977. Newman, W., Gordon, S., Hammerling, U., Senik, A., and Bloom, B. R., J. Immunol. 120,927, 1978. Gauthier-Rahman, S., Immunology 42, 99, 198 1. McCoy, J. L., Dean, J. H., and Herberman, R. B., J. Immunol. Methods 15, 355, 1977. Aaskov, J. O., and Anthony, H. M., Aust. J. Exp. Biol. Med. Sci. 54, 527, 1976. Fox, R. A., and Rajaraman, K., Cell. Immunol. 47, 69, 1979. Fox, R. A., and Rajaraman, K., Cell. Immunol. 59, 448, 1981.
IMMUNOLOGY
77,249-265
(1983)
Delayed Hypersensitivity and Migration Inhibition in Two Lines of Mice Genetically Selected for High or Low Responsiveness to Phytohemagglutinin S. GAUTHIER-RAHMAN,* S. EL ROUBY,*” M. LIACOPOULOS-BRIOT,* C. STIFFEL,? C. DECREUSEFOND,t AND P. LIACOPOULOS* *Institut d’lnzmuno-Biologie (Inserm U20, CNRS LA 143), H6pital Broussais. 96 rue Didot, 75674 Paris, C6dex 14, and TLaboratoire d’lmmuno-Gbhique (Inserm lJl25, CNRS ER 701, Institut Curie, 25 rue d’Ulm, 75005, Paris, France Received November 12, 1982; accepted December 30, I982 Delayed-type hypersensitivity (DTH) and cell migration inhibition (Ml) were studied in two lines of mice genetically selected for the high (Hi/PHA) or low (Lo/PHA) in vitro response of their lymphoid cells to phytochemaaglutinin (PHA). A rapid photoelectric procedure for reading cell migrations enabled the study of MI over a wide range (10 log) of antigen concentrations in vitro. Hi/PHA mice required immunization with a 10 times higher dose of ovalbumin (OVA) in Freund’s complete adjuvant (FCA) than Lo/PHA mice for a comparable response in DTH (footpad swelling) and MI oftheir induced peritoneal exudate cells (PEC). Lo/PHA spleen showed marked bizonal MI on Day 5 after immunization with low doses (0.1 and 0.5 pg) of OVA in FCA, one peak being obtained in presence of in vitro concentrations of IO-’ or lo-’ &ml OVA and another peak at 1 or IO &ml, whereas Hi/PHA spleen showed stimulation of migration. In contrast, MI in Lo/PHA spleen failed to persist beyond Day 19, whereas it appeared progressively in Hi/PHA spleen, being maximal by Day 27. Low-zone inhibition in Hi/PHA spleen and PEC was lacking or poor even after immunization with higher doses of OVA in FCA. The implications of these findings are discussed.
INTRODUCTION Genetic selection for the intensity of the response of mice lymphoid cells to phytohemagglutinin (PHA) has led to the development of two lines of mice characterized by either a high (Hi/PHA) or a low (Lo/PHA) response (1). The marked difference, 20-fold, in their in vitro response to this T-cell mitogen was shown to be accompanied by differences as marked in the development of T-cell-mediated immune reactions both in vitro and in vivo. Thus the mixed-lymphocyte reaction (MLR) (2) and graftversus-host reactivity (GVHR) (3) were shown to be much stronger in the Hi/PHA line. It was therefore of interest to investigate whether another parameter of T-cell reactivity, the development of delayed-type hypersensitivity (DTH) and its in vitro correlate, cell migration inhibition (MI) (4, 5), had also been affected during the course of selection, and, if so, in the same sense as the response to alloantigens and mitogen. ’ French Government Scholarship holder. This work was supported by CRL Inserm 821042. 249 0008-8749183 $3.00 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
250
GAUTHIER-RAHMAN
ET
AL.
The development in this laboratory of a rapid photoelectric procedure for reading cell migrations from capillary tubes (6) and, later, from agarose drops (7) enabled us to study the effect of a wide range (10 log) of antigen concentrations on the migration of cells of induced peritoneal exudates (PEC) or spleen (SC) from mice immunized with different doses of ovalbumin (OVA) in Freund’s complete adjuvant (FCA) and to follow the kinetics of this phenomenon. As is well known, DTH to simple protein antigens may not be easy to assessin mice by the footpad assay, due to the occurrence of anaphylactic reactions after local challenge with antigen (8- 10). However, a recent method involving the use of heat-aggregated antigen for elicitation of DTH (10) enabled us to establish a notable difference in the response of the Hi/PHA and Lo/ PHA lines, which correlated with that observed in vitro in the development of migration inhibition of their PEC and SC. The main finding was that the Hi/PHA line needs to be immunized with a dose of antigen 10 times higher in order to develop a reaction similar to that of the Lo/PHA line. MATERIALS
AND
METHODS
Mice These experiments were performed in mice of the Hi/PHA and Lo/PHA lines belonging to F22 to F24 generations from our own breeding colonies and in outbred Swiss albino mice purchased from Iffa Credo (69210J’Arbresle, France). It is recalled that the Hi/PHA and Lo/PHA lines were developed from a starting parental population of out-bred Swiss albino mice provided by different breeders. Antigen Salt-free Grade V crystallized and lyophilized Sigma Chemical Company.
ovalbumin
(OVA) was obtained from
Immunization For migration inhibition experiments and serum antibody determination, ovalbumin in a dose of 0.1, 0.5, 5, 50, or 500 pg/mouse, comprised in 0.1 ml of sterile saline, was emulsified with an equal volume of Freund’s complete adjuvant (Difco) and injected id at six sites, three on each side of the shaved dot-sum. For DTH experiments, the antigen dose per mouse was comprised in 25 ~1 of saline emulsified with an equal volume of FCA, and the emulsion mixture was injected subcutaneously on either side of the base of the tail. Delayed-Type
Hypersensitivity
The footpad assay was performed according to the method of Titus and Chiller (10) with minor modifications. Briefly, aggregated ovalbumin (AOVA) was prepared by heating a 2% solution of OVA in saline at 70°C for 1 hr, followed by slow cooling. The precipitate was washed twice and then made to the original volume in saline. DTH was elicited in groups of 5 to 8 mice on Day 8 after immunization as above with different doses of OVA. The suspension of AOVA (30 ~1) was injected subcutaneously in the right hind footpad with a 27-gauge needle, and the same volume of saline was injected into the left hind footpad. Footpad thickness was measured at 4, 24,48, 72, and 96 hr after challenge under ether anaesthesia using an “Oditest” skin-
DELAYED
HYPERSENSITIVITY
AND
MIGRATION
INHIBITION
251
thickness gauge (Kroplin, GMBH, 6490 Schluechtern, 1, W. Germany). The extent of swelling was calculated by subtracting the thickness of the saline-injected footpad from that of the antigen-injected footpad. Cell Migration
Inhibition
Experiments using the capillary-tube technique with peritoneal exudate cells and photoelectric readings were begun as described elsewhere (6). When the photoelectric procedure for reading migration from agarose microdroplets was developed, migration of spleen cells was studied using this latter method (7). (I) Peritoneal exudate cells. It was found by prior experiment that large numbers of PEC could be obtained from one mouse after ip injection of Freund’s incomplete adjuvant (FIA) (Difco). Groups of mice immunized with different doses of OVA in FCA were sacrificed by cervical section on Days 5, 12, 19, 60, or later, after immunization and 4 days after ip injection of 0.5 ml FIA. The peritoneal cavity was washed out with a total of 20 ml of cold 199 medium supplemented with antibiotics and 1% heat-inactivated fetal bovine serum (FBS). The cell suspension was washed twice and the number of viable cells ascertained by trypan blue exclusion. At the same time, the number of phagocytic cells in the exudate was determined by counting the cells containing oil droplets. In general, 70-75% of the cells contained oil droplets and were considered to be macrophages. About 30 to 60 X 1O6 cells could be obtained from one mouse by this method. Much fewer PEC were obtained from Lo/PHA mice as compared to Swiss or Hi/PHA mice. Inhibition of migration of PEC from individual mice was studied in many cases in the two latter strains, but pools of two or more mice had to be used in Lo/PHA mice. A cell concentration of 18-20 X 106/ ml was found suitable for photoelectric reading using the capillary-tube technique. About 4 ml of suspension was required for the 80 capillaries needed for each experiment. The cell suspension was drawn up into capillary tubes of O.l-mm internal diameter. One end of the capillary tube was sealed by flaming. After centrifugation the capillaries were cut just below the cell-liquid interface. The end containing the cell pellet was fixed individually in the well of a sterilin migration plate with a dab of sterile silicone grease, and the well was filled immediately with 0.45 ml of medium with or without antigen. Six to eight capillaries were mounted per concentration of antigen, with 8 to 16 controls (medium without antigen). Migrations were read at 18 hr generally and also at 48 hr in some cases. (2) Spleen cells. The spleen was excised after the peritoneal exudate had been removed and minced in a drop-by-drop stream of cold 199 medium with 1% FBS. The cell suspension (20 ml vol) was passed through a stainless-steel sieve (60 pm), the cells were washed, and cell viability was ascertained by trypan blue exclusion. In a few experiments, red cells were lysed with 0.83% cold NH&l. Cell suspensions and pellets were maintained in an ice bath. Inhibition of migration of SC suspensions from individual spleens was studied with the agarose microdroplet technique. In many cases migration inhibitions of PEC and SC from the same animal were studied in parallel. Antigen concentrations OVA concentrations of 10e5, 10m4, 10m3, lo-‘, IO-‘, 1, 10, 100, and 250 &ml were prepared in 199 medium containing 20% heat-inactivated FBS by the adjunction
252
GAUTHIER-RAHMAN
ET
AL.
of an appropriate concentration of OVA in sterile saline in l/ 100 of the final volume. Concentrations of 1, 2.5, 5, and 10 mg/ml were obtained by adding weighed dry OVA to medium. Antigen concentrations were prepared prior to sacrifice of the animals and maintained in an ice bath. The Agarose Microdroplet bition
Technique for Photoelectric
Readings of Migration
Inhi-
This technique was a modification of the usual technique ( 11, 12) and is described in detail elsewhere (7). Briefly, spleen cell suspensions of 4 X lO’/ml were prepared in 0.2% seaplaque agarose (final) in 199 medium supplemented with antibiotics and 15% FBS. The suspension was drawn up into a 50-~1 Hamilton syringe fitted with an automatic dispenser. A sterilin migration plate was previously prepared by greasing the rims of its wells and cooling or an LKB plate was held at strict horizontal on an ice slurry. Four l-p1 microdroplets were dispensed per well of the sterilin plate. After a uniform gelation time of 6 min the wells were filled with 0.45 ml of medium without antigen (controls) or with one of the 11 different concentrations of OVA prepared and covered with round 22-mm coverslips. The plates were then placed together with the LKB table at strict horizontal at 37°C. Eight microdroplets per concentration and 16 controls were usually studied. In addition, 8-12 microdroplets were allowed to dry before the wells were filled with control medium (no migration). These served as background controls. Migrations were read in general at 18 hr. Photoelectric Readings of Cell Migrations The migrating cells are viewed under dark-field illumination. According to the technique used, the glass stub of the cut capillary or the agarose microdroplet is hidden by a rectangular or a circular black mask placed in the ocular. The light reflected by the migrating cells is directed to the photosensitive surface of the Polaroid camera of an apparatus for automatic microphotography (Nachet, Model N 8901, Levallois-Perret, France) and the migration may, if desired, be photographed. The light diffracted is measured as electrical potential by the electronic exposure meter of the apparatus, which is connected to a voltmeter of sufficient sensitivity. The voltage, in volts, corresponds logarithmically to the amount of light diffracted by the migration in lumens. Inhibition is indicated by a fall in voltage. Thus, % inhibition
=
LC - Lo”* x loo L”
where L’ is the mean luminosity of migrating cells in control medium (no antigen) and Lo”* the mean luminosity of migration at a given antigen concentration. The luminosity, in lumens, is equal to the antilog of the observed voltage (in volts) minus the background luminosity. Migration inhibition of 20% or more was considered significant. Serum Antibody Determination Blood samples were obtained by retro-orbital puncture from groups of 5-6 mice on Days 7, 11, 19, 27, and 60 after immunization with 0.5, 5, 50, or 500 pg of OVA in FCA as described above. Anti-ovalbumin titers of individual set-awere determined
DELAYED
HYPERSENSITIVITY
by passive hemagglutination bent assay) (13). Cyclophosphamide
AND MIGRATION
and also by micro-ELISA
253
INHIBITION
(enzyme-linked
immunosor-
(Cy) Pretreatment
Mice were given an ip injection of 100 mg/kg Cy 72 hr prior to immunization with 0.5, 5, or 50 pugOVA emulsified in 0.1 ml FCA. Migration inhibition of their PEC was studied on Day 5. RESULTS Delayed-Type
Hypersensitivity
The results of the footpad assay in mice of the two strains performed on Day 8 after immunization with 0.5, 5, or 50 pg in FCA are presented in Fig. 1. It can be seen that Lo/PHA mice show stronger reactions than Hi/PHA after immunization with lower doses of OVA, 0.5 and 5 pg. After immunization with 50 pg OVA, Hi/PHA mice show the stronger reactions, those in Lo/PHA being depressed. Optimal immunization for DTH was obtained in Lo/PHA after 5 pg OVA in FCA. Furthermore, footpad swelling was found to be more persistent in Lo/PHA mice. Maximal DTH was observed at 24 hr, but some swelling was still present at 48 and 72 hr. These late reactions were always higher in Lo/PHA mice whatever the immunizing dose of antigen (Fig. 1). However, there was great variation in the DTH reactions of individual mice and the differences observed between the two lines were at the limit of significance.
Immunizing
c! ,o
50.
; E 0
40.
.-,” =u
30
Dose:
LO ..I.. H,
pg
#
t
t
0.5
5
50
_
4
24
40
Hours
72
96
after
4
24
48
72
96
4
24
48
72
challenge
FIG. 1. Delayed-type hypersensitivity response of Hi/PHA and Lo/PHA mice. Eight days after immunization with 0.5, 5, or 50 pg OVA in FCA, mice were injected with 30 ~1 of heat-aggregated OVA (AOVA) in the right footpad and the same volume of physiological saline in the left footpad. The difference between the swelling of the right and left footpad is shown j, SEM at various times after the challenge. Each curve was obtained with the mean readings for 5-8 mice.
254
GAUTHIER-RAHMAN
Migration Inhibition Antigen
ET
AL.
in Induced PEC and the Efect of the Immunizing
Dose of
On Day 5 after immunization of Swiss, Hi/PHA, or Lo/PHA mice with 0.1, 0.5, 5,50, or 500 Kg of OVA in FCA, MI was present in PEC. In accordance with classical data, it was generally observed in the presence of a high concentration of OVA in vitro, 1 to 5 or even 10 mg/ml. However, in some circumstances MI was observed in a zone of a much lower antigen concentration. The results obtained on Day 5 in the presence of high concentrations of OVA in vitro are summarized in Fig. 2. A marked effect of the immunizing dose of OVA can be seen, particularly in Hi/PHA mice. MI on Day 5 is quasi-maximal (30-40%) in
50
40
: .w .n ..c
30
C
20
ix!
10
0
-10
-20
-30
lmmunizir dose
50
500
(P9
FIG. 2. Effect of the immunizing dose of antigen on the development of migration inhibition (MI) in induced peritoneal exudate cells of Hi/PI&A, Lo/PHA, and Swiss mice. MI was studied by the capillarytube technique with photoelectric readings (as described under Methods) on Day 5 after immunization with different doses of OVA in FCA and in the presence of in vitro concentrations of OVA ranging from lo-’ &ml to 10 mg/ml. The means of the greatest MI, generally observed in the high zone of antigen concentration (250 fig to 10 m&ml), are shown with SEM. The figures near the tops of the columns indicate the number of experiments.
DELAYED
HYPERSENSITIVITY
AND
MIGRATION
INHIBITION
255
Swiss or Lo/PHA mice after immunization with as little as 0.5 pg OVA, whereas in Hi/PHA mice this is obtained only after immunization with 50 pg. After the largest immunizing dose, 500 pg, MI is diminished in both Hi/PHA and Lo/PHA mice, this effect being most marked in Lo/PHA mice, where MI is replaced by stimulation and least in Swiss mice, where mean MI is still 32.5%. Similarly, after the lowest immunizing dose, 0.1 pg, only Swiss mice show significant MI (27%). MI of PEC on Day 5 was often seen as a sharp peak obtained in the presence of a narrow range of high in vitro concentrations of antigen, l-5 mg/ml, particularly after immunization with nonoptional doses of OVA. After optimal immunization, in Lo/PHA MI was spread over a much wider range of antigen concentrations, with the appearance of a second peak at very low antigen concentrations, 1O-‘-1O-3 pg/ml. In contrast, in Hi/PHA mice MI on Day 5 remained a single peak in the high zone of antigen concentration even after optimal immunization (Fig. 3). The response of PEC of Swiss mice was similar to that of Lo/PHA (data not shown). Kinetics of Development
of MI in Induced PEC
The results of sequential study on Days 5, 12, 19, 30, and 60 after immunization with 0.5 or 500 pg OVA in FCA are summarized in Table 1. MI increased somewhat from Day 5 to Day 12, whatever the immunizing dose. This effect was only marked in Hi/PHA mice, where it was present after both high and low immunizing doses, and in Lo/PHA mice only after the higher dose. After immunization with a low dose, 0.5 pg OVA, mean MI in Hi/PHA mice was less than in Lo/PHA at all times studied (Table 1). On the contrary, after immunization with a high dose, 500 pg, mean MI was higher in Hi/PHA on Days 5, 12, and 60. Lo/PHA mice, showing no MI on Day 5 after immunization with 500 pg, developed a mean MI (2 1.35%) only half that of Hi/PHA (42.25%) by Day 12, which increased
40 I
Log,,
Cont.
Ova.
p9
I ml
FIG. 3. Lack of early low-zone inhibition in induced peritoneal exudate cells of Hi/PHA mice. The curves show the mean MI observed at different antigen concentrations in 3 experiments for each line of mice performed on Day 5 after immunization with 5 pg OVA in FCA.
256
GAUTHIER-RAHMAN
ET AL.
TABLE 1 Kinetics of Migration Inhibition in Induced Peritoneal Exudate Cells of Three Lines of Mice after Immunization with a Low or a High Dose of Antigen in FCA Immunizing dose of OVA (rg) 0.5
Days after immunization
Percentage inhibition observed in PEC Expt. No.
5
I 2 3
12
1 2 3
19
1 2 3
30 60 500
5
12
1 2
19
I 2 3
60
1 2 3
120
1
Lo/PHA
Hi/PHA 18.6 (I mg) 46.0 (10 mg) 19.0 (10 mg) Mean 27.83 f 9.08 27.6 (10 mg) 35.5 (5 mg) 43.0 (2.5 mg) Mean 35.33 f 4.47 7.7 (5 mg) 32.2 (1 mg) 54.5 (2.5 mg) Mean 31.46 + 13.51 28.4 (10 mg) 25.5 (5 mg) 33.8 (5 mg) 9.9 (2.5 mg) 21.5 (10 mg) Mean 2 I .70 f 6.90 42.3(1 ccg) 42.2 (1 d Mean 42.25 k 0.05 33.3 (10 rg)
32.2(1 4 20.9 (I mg) 4.1 (10 a9 Mean 19.06 f 8.16 ND
66.0 (10 mg) 38.4 (2.5 mg) 38.6 (IO mg) Mean 44.83 + 5.84 43.6 (1 mg) 47.0 (10 mg) 51.6 (1 mg) Mean 47.40 + 2.31 53.2 (2.5 mg) 12.9 (2.5 mg)
Swiss 44.5 (10 mg) 46.2 (10 mg) 33.8 (5 mg) Mean 41.5 f 3.88 30.6 (2.5 mg)
ND”
Mean 33.00 + 20.20 ND 41.6 (10 mg) 44.6 (5 mg) 44.5 (10 mg) -35.2 -40.0
(2.5 mg) (5 mg)
Mean -37.60 + 2.4 20.9 (1 4 21.8 (1 /.a) Mean 21.35 + 0.45 34.5 (10 pg) 42.8 (1 mg) Mean 38.65 f 4. I5 6.35 (100 pg) 4.65 (1 mg) Mean 5.50 f 0.85 ND
30.9 (2.5 mg) 35.5 (2.5 mg) Mean 33.2 + 2.30 44.2 (0. I fig) 39.5 (5 mg) 26.1 (5 mg) 48.5 (100 pg) Mean 38.03 + 6.50 54.0 (100 pg) 33.8 (I mg) 16.2 (100 /.& Mean 34.50 f 10.92 44.5 (2.5 mg)
No@. The maximal MI in each experiment is noted with, in parentheses, the antigen concentration in vitro where it was obtained. Means are indicated with standard error. Negative figures indicate stimulation percentage. ’ ND, not done.
to 38.65% on Day 19. By Day 60, however, Lo/PHA no longer showed significant MI as compared to Hi/PHA, where MI was still present in two out of three experiments (Table 1). Swiss mice appeared to retain practically unchanged MI from Day 5 to Day 60 after immunization with 0.5 or 500 pg, the titers after the latter dose being somewhat lower (Table 1). Bizonal Inhibition
in PEC
MI in PEC of Lo/PHA and Swiss mice was frequently bizonal with a high zone at 100 pg to 10 mg/ml OVA in vitro and a low zone around lo-‘- lop3 &ml (Fig.
DELAYED
HYPERSENSITIVITY
AND MIGRATION
INHIBITION
257
3). After immunization with 0.5 pg OVA, a small peak in the low zone was seen in Lo/PHA mice in two of three experiments on Day 5, one of three on Day 12, and one of two on Day 19. Hi/PHA mice did not show the low-zone peak on Day 5, but small low-zone peaks appeared on Days 12 and 19 in half the experiments if culture was prolonged to 48 hr. Low-zone inhibition was more marked in PEC of all three lines of mice at times later than Day 5 and after immunization of Hi/PHA with higher doses. In Lo/PHA mice, the low-zone peak could become as high as the other one (Fig. 4). Bizonal inhibition of PEC was observed in Swiss mice as late as 10 months after immunization with 50 pg OVA in FCA (data not shown). After immunization with 500 pg OVA, MI in Hi/PHA was shifted to the left on Days 12 and 19, the major peaks being at 1 or 10 pg/ml and 10m4 to 10e2 pg/ml with little or no inhibition in the usual high zone (around 1 mg/ml) (Table 1, Fig. 5). In Lo/PHA mice, under these circumstances, inhibition in the high zone still occurred (Fig. 4) together with an additional peak at 1 or 10 pg/ml, or was observed over a wide range of Ag concentrations. Efect of Cyclophosphamide Treatment of Lo/PHA or Hi/PHA mice with 200 mg/kg Cy ip 72 hr prior to immunization with 0.5, 5, or 50 pg OVA in FCA was without notable effect on MI of induced PEC on Day 5. Migration
Inhibition
in Spleen and the Efect of the Immunizing
Dose of Antigen
Except for some early experiments where pools of 2-3 spleens were employed, individual spleens were studied using the agarose microdroplet technique with photoelectric readings. OVA concentrations from 10m5 pg/ml to 5 mg/ml were used. 50.
X,
:p
a? ;--
2”
. . ..-I
/
10.
0
l-‘:
:
r
:
* -3
-10
L....
-2
-1
0
1
.
2
: :
:.
5 f
‘j 3 : b
4 ‘8 i
-20.
-30. L-lo
Cone.
Owe.
pglml
FIG. 4. Marked bizonal inhibition in induced peritoneal exudate cells and spleen of a Lo/PHA mouse on Day 19 after immunization with 500 pg OVA in FCA.
258
GAUTHIER-RAHMAN
ET AL. -
PE s.” sp,..n
50 I
40. 30.
20.
10 0 10.
. -4
-3
-2
-,
0
1
2
\' .
.v
3
4
-20
-30.
%
Cow.
Ova.
pa/ml
v\
.
FIG. 5. Bizonal inhibition in induced peritoneal exudate cells and spleen of a Hi/PHA mouse on Day 12 after immunization with SOOjtg OVA in FCA.
On Day 5 after immunization with low antigen doses, 0.1 or 0.5 pg OVA, in FCA MI of 35-40s was observed in Lo/PHA spleen, whereas this was absent in Hi/PHA (Figs. 6 and 7, Table 2). However, MI on Day 5 appeared in Hi/PHA spleen after immunization with 5 or 50 pg OVA (Fig. 7). MI in Lo/PHA spleen was quasi-maximal
LO%0
Cont.
Ova.
pglml
FIG. 6. Comparison of migration inhibition in the spleen of Hi/PHA and Lo/PHA mice on Day 5 after immunization with a low dose of OVA, 0.5 rg in FCA. Ml was studied in individual spleens by the agarose microdroplet technique with photoelectric readings (see Methods). The means of MI observed in four spleens of each line of mice at the different in vitro antigen concentrations of OVA are shown with the SEM. Marked bizonal inhibition is observed in Lo/PHA and only stimulation in Hi/PHA spleen.
DELAYED
HYPERSENSITIVITY
AND MIGRATION
A
I3
50 :
..P r -c o\
259
INHIBITION
I
I
HI
40. 30. 20
10. 0, -10 -2oL lmmunislng
Dose:
0.1
0.5
5
50
500
0.1
0.5
5
50
500
(U9) FIG. 7. Effect of the immunizing dose of antigen on the development of migration inhibition in spleens of Hi/PHA and Lo/PHA mice. Mice were sacrificed on Day 5 aher immunization with different doses of OVA in FCA. MI was studied in each experiment in the presence of in vitro concentrations of 10e5 to 2500 &ml. The means of the greatest MI observed in the low zone of antigen concentration, 10-3-10-2 &ml (A), and those of the high zone, l- 10 &ml (B), are shown. The figures near the tops of the columns represent the number of experiments.
after immunization with the lowest dose, 0.1 pg, whereas in Hi/PHA 5 pg appeared to be optimal. MI was markedly reduced in Lo/PHA spleen after immunization with 50 pg and less so in Hi/PHA. After 500 pg, MI was markedly reduced in both lines (Fig. 7). Bizonal Inhibition
in Spleen
Migration inhibition on Day 5 after immunization with low doses was found to be markedly bizonal in Lo/PHA spleens with one peak in the presence of OVA concentrations of 10e2 and 10e3 pg/ml and another peak generally at 1- 10 &ml, in some cases at 100 Clg/ml (Table 2, Figs. 6 and 7). In contrast, MI in Hi/PHA spleen was observed in the high zone of Ag concentration with but little inhibition in the low zone even after optimal immunization (Fig. 7). Kinetics of Migration
Inhibition
in Spleen
Sequential study was performed on Days 5, 12, 19, 27, and 40 or 60 after immunization with 0.5 or 500 fig OVA in FCA. The evolution of MI in spleen after immunization with 0.5 pg is shown in Fig. 8 and Table 2. Bizonal inhibition was maximal (about 40%) on Day 5 in Lo/PHA spleen. Both peaks decreased thereafter progressively on Days 12 and 19 and were insignificant by Day 27. On the contrary,
1 2 3 4 Mean SEM
1 2 3 4 Mean SEM
1 2 3 Mean SEM
12
19
27
(lo-’ (10-r ( lO-2 (10-Z
(10-r ( lO-3 (IO-’ (lo-3
(10-r (lo-3 (lo-3 ( lO-3
pg) rg) rg) pg)
pg) pg) pg) jJg)
pg) pg, 10-r rg) rg) pg)
34.4 ( 1om3 rg) 10.8 ( lO-3 pg) -20.8 (IO-’ pg) 8.13 kl3.05
0 16.6 27.5 37.5 20.40 k8.02
3.5 6.0 -6.5 -13.3 -2.57 k4.29
-41.2 7.1 -20.0 -24.0 -19.77 +10.17
A
Hi/PHA
(1 (1 (1 (1
Pia rg) rd d
-11.0 (10-r 26.7 (IO-’ 7.85 k18.85
Mean SEM
14.83 k21.15
1 2
Mean SEM rg) pg)
52.7 (10m2 pg) 12.2 (lo-2 pg) -20.4 ( 10m2 pg)
/*g) pg) pg) pg)
1 2 3
(lo-2 (lo-3 ( 1om2 ( lO-2
(250 (100 (100 (10
pg) pg) pg) pg)
5.70 kl9.50
1 2 3 Mean SEM
A
Swiss
rg)
B
The figures
rg) rid 15.7 (100 rg) 17.66 k3.62
24.7 (1 12.6 (1
52.5 (100 rg)
-0.20 k31.90
are
-32.1 (1 j& pool 36 31.7 (10 rg) pool 36
in parentheses.
33.2 (IO-’ pg) 9.1 (lo-3 pg) 12.7 (10m3 pg) 18.33 k7.50
48.5 (lO-3
-1.20 k27.8
-29.0 (IO-* fig) 26.6 ( 10m3 pg)
in FCA
in vitro where it was found
25.2 (1 pg) -13.8 (1 fig)
12.90 k5.02
14.8 (10 rg) 20.5 (100 rg) 3.4 (100 Pd
1
Mean SEM
1 2
Expt.
0.5 pg OVA
IO.2 (1 PI9 37.2 (100 rg) 6.4 (100 pg) 10.8 (100 pgg) 16.15 k7.08
37.7 70.2 34.1 18.0 40.00 210.93
B
of
with
in spleens
(IO-‘, lO-2 rg) ( 10-3, lO-2 pg) (lo-), 1om2 /Jg) (lo-‘, 10-r rg)
A
Lo/PHA
observed
Immunization
10.2 7.1 20.0 45.6 20.72 k8.73
24.0 54.6 41.0 27.8 36.85 f6.94
inhibition
Low-Dose
1 2 3 4 Mean SEM
1 2 3 4 Mean SEM
Expt.
Percentage
in Spleen after
(1 pg, 10 pg) (1 4 (10 pg) (1 pg, 10 pg)
(1 fig) (1 fig, 10 a) (10 /a) (10 jtg)
B
Inhibition
57.0 (10 rg) 1.65 (1 rg) 37.0 (1 @cg, 10 pg) 31.88 kl6.18
29.1 24.40 k5.10
27.5
31.7 9.3
-15.85 42.1 17.4 21.1 16.18 fll.98
-68.5 -23.32 + 16.62
-6
-26.9 7.1
of Migration
2
Nofe. The inhibition observed in the low zone (A) or high zone (B) is noted with the antigen concentration for individual spleens except where pools are indicated. Negative figures indicate percentage stimulation.
I 2 3 4 Mean SEM
Expt.
5
Days after immunization
Kinetics
TABLE
DELAYED
50 .-: 5 .I c
40.
30. * 20.
10.
O-
-10 -
-20.
-30 -
HYPERSENSITIVITY
AND MIGRATION
261
INHIBITION
4 4 I 4 1 5
12
19 Days
27 after
5 lmmunizat
12
19
27
ion
FIG.8. Kinetics of migration inhibition in Hi/PHA and Lo/PHA spleen after immunization with a low antigen dose, 0.5 pg OVA in FCA. Mice were sacrificed on Days 5, 12, 19, and 27 after immunization and MI was studied in individual spleens as under Fig. 7. The means of the greatest MI observed in the low zone, 10-3-10-2 pg/ml (A), and in the high zone, I-10 pdrnl (B), of in vitro Ag concentrations are shown with the SEM. The figures near the tops of the columns represent the number of spleens. Lo/PHA spleens show maximal bizonal inhibition on Day 5, which declines rapidly thereafter. In contrast, Hi/PHA spleens, after initial enhancement of migration, develop increasing MI particularly in the high zone, which is maximal by Day 27.
after initial stimulation on Day 5 (approx -20%) MI appeared in Hi/PHA spleen from Day 12 onward and increased progressively until Day 27 (3 1.88%). It was observed only in the high zone at 1 or 10 pg/ml except on Day 19, when a small peak (20.4%) was also present in the low zone (Fig. 8). MI was no longer observed in Hi/PHA spleen by Day 40. Swiss spleen was found to be like Lo/PHA spleen in that it responded to low immunizing doses of OVA, 0.1 and 0.5 pg, with marked bizonal inhibition on Day 5, but only in one of two experiments, and was like Hi/PHA spleen in that mean
262
GAUTHIER-RAHMAN
ET
AL.
MI appeared to increase with time, to 33 and 50% on Day 12 after immunization with 0.1 and 0.5 Kg, respectively (Table 2). After an immunizing dose of 500 pg OVA (Table 3), MI in both Lo/PHA and Swiss spleen was insignificant on Days 5 and 12 with presence of stimulation on Day 12, whereas in Hi/PHA spleen, bizonal MI in a low degree was present on Days 5 and 12. By Day 19, bizonal inhibition was observed in Lo/PHA and Swiss spleen, whereas only a low-zone peak was retained in Hi/PHA spleen. Practically unchanged bizonal MI of 33% was still present in Swiss spleen on Day 60, by which time Lo/ PHA and Hi/PHA spleen no longer showed significant MI (Table 3). Serum Antibody On Day 5, after ovalbumin antibody immunization with titers were found Hi/PHA mice.
immunization with 0.5, 50, or 500 pg OVA in FCA, no antiwas detected by passive hemagglutination or by ELISA. After 0.5 or 500 pg OVA in FCA, Day 12, 19, and 27 serum antibody to be very similar by both techniques in Lo/PHA and
DISCUSSION Earlier studies performed in the two lines of mice genetically selected for their low or high response to phytohemagglutinin, a T-cell mitogen, showed a marked difference to exist between them in reactions involving the T-cell response to cell-associated Ag, namely mixed-lymphocyte culture (2) and graft versus host (3). The Hi/PHA line was shown to have the much greater T-cell reactivity. The present study utilizing a soluble protein Ag, OVA in FCA, brings to light differences in the behavior of the two lines in reactions of DTH and MI. These differences are related to the optimal immunizing dose of Ag, to the dose response in vitro in MI of cells sensitized in vivo, and to the kinetics of MI in spleen and PEC of immunized mice. The use of a rapid photoelectric procedure for reading cell migration (6, 7) permitted the study of MI over a wide range (10 log) of Ag concentrations in vitro and led to the observation of the phenomenon of bizonal inhibition. The optimal immunizing dose of Ag was found to be 10 times higher in the case of Hi/PHA mice, both for the in vivo footpad assay of DTH (Fig. 1) and for in vitro MI of spleen and PEC (Figs. 2 and 7). Early (Day 5) marked low-zone inhibition in presence of in vitro concentrations of 10e3 to 10m2 &ml OVA, with a second peak at 1 to 100 lg/ml, was a characteristic feature of Lo/PHA spleen after immunization with low Ag doses of 0.1 or 0.5 pg, but was found not to persist, decreasing rapidly by Days 12 and 19. On the contrary in Hi/PHA spleens, after initial stimulation, MI progressively appeared and became maximal by Day 27, the major or single peak always being in the high zone, 1 to 10 pg/ml (Fig. 8, Table 2). Similarly, early lowzone inhibition was lacking in PEC of optimally immunized Hi/PHA mice (Fig. 3). Bizonal inhibition was also observed in Swiss PEC and spleen. It suggests the existence of two cell populations responding to very different in vitro concentrations of Ag, 2 to 4 log apart. The lack of marked bizonal inhibition in Hi/PHA spleen and PEC at early times after immunization would suggest that cells responding to very low doses of Ag with liberation of MIF are either lacking or too few in number or prevented from expressing themselves in this line of mice. Even at times later than Day 5 and after immunization with larger Ag doses, low-zone inhibition, although present in Hi/PHA spleen and PEC, was not as high as in Lo/PHA (Figs. 4-7, Table
DELAYED
HYPERSENSITIVITY
AND
MIGRATION
INHIBITION
264
GAUTHIER-RAHMAN
ET
AL.
3). That antibody was responsible for the phenomena observed is unlikely, since no circulating antibody was found on Day 5 after immunization with 0.5 or 500 )~g OVA in FCA, and the antibody titers by passive hemagglutination at later times were very similar in the two lines. The lack of persistance of bizonal inhibition in Lo/PHA spleen could be due to failure of multiplication of the cells concerned, their migration out of the spleen, or the development of suppressive phenomena. It has been shown in the case of DTH that two cell populations are involved, one of which is replicating as shown by its sensitivity to vinblastine (14). On the other hand, the progressive increase of MI in Hi/PHA spleen after low-dose immunization could be due to slow multiplication of a rare cell population and/or the progressive removal of a suppressive effect. The response in MI of Swiss mice was superior to that of both lines. Swiss PEC responded on Day 5 after immunization with very low (0.1 pg) or very high (500 pg) doses of Ag, whereas the dose-response range of the two lines was much more restricted, Hi/PHA failing to respond after low and Lo/PHA after high immunizing doses. Persistance of MI in PEC and spleen was also much longer in Swiss mice (Tables 1 and 3). It would appear that the adequate presence or expression of both cell populations, responding respectively to high or low Ag doses in vitro, was necessary for the better development of MI seen in Swiss mice. Parallel study of MI in individual spleens and PEC showed that the two did not appear or disappear simultaneously. Thus, the distribution or expression of cells responding in MI was not homogenous in the different lymphoid cell populations of the same mouse, particularly at early or later times (Day 60) after immunization (compare Tables 1 and 3). MI in the spleen appeared to be a more transient phenomenon than in PEC. The time after immunization when MI appeared in the spleen and its duration were related both to genetic character and the immunizing dose of Ag (Tables 1 and 3). Hanington ( 15) found that MI in the spleen after cancerization appeared around Day 11 and became maximal by 3 weeks, long after the rejection of ip-injected allogenic tumor cells. Our data would suggest that this does not necessarily preclude the role of cells responding in MI in the rejection of the tumor allograft. The significance of bizonal migration inhibition observed herein in both spleen and PEC remains to be established. Two types of MIF have been isolated in vitro ( 16, 17) with different molecular weights and isoelectric points. Could the two types of MIF be produced by two different cell populations responding in vitro to very different Ag concentrations? Cells producing MIF after antigenic or mitogenic stimulation have generally been found to be of Lyt l+ phenotype (18-20). However, in some cases, such as immunization with active influenza virus or DNFB, involvement in DTH of T cells of different phenotype has been observed (2 1, 22). The possibility that the poor low-zone inhibition in Hi/PHA mice is due to the relative lack of cells of a particular Lyt phenotype is presently being explored. Moreover, the greater Tcell reactivity of the Hi/PHA line was demonstrated in response to cell-associated Ags in MLC and GVH. MIF production during MLC was found to be by both Lyt l+ and Lyt 2+ cells (23). It is possible that the Hi/PHA line would be a better producer of MIF in MLC than the Lo/PHA, and this study is under way. Bizonal inhibition has not hitherto been described. However, MI at low in vitro concentrations of OVA was observed by us in PEC of guinea pigs after treatment with Corynebacterium parvum (24). MI of leukocytes from some human subjects was observed at extremely low concentrations of PPD (5 X 10e7 &ml) by McCoy et al.
DELAYED
HYPERSENSITIVITY
AND MIGRATION
INHIBITION
265
(25) and also by us (unpublished data). Our experiments would suggest that this type of response could be related to genetic character. Finally, migration stimulation, sometimes of marked degree, seen in Hi/PHA spleen, particularly after immunization with small doses of Ag (Table 2, Figs. 6-8), would suggest the presence of a migration stimulation factor. Such a factor has been shown to be produced by certain human T-cell subsets (26, 27) and also in mice (27, 28). Suppressor T cells of the Lyt 2+,2 phenotype were found to be involved (28). Although no suppressor cells sensitive to cyclophosphamide were found in our experiments, suppressor T cells producing migration stimulation factor may be present with higher frequency in Hi/PHA mice. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 1I. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
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