1
Title: Influence Of The Specific Immune Response On Some Consistent
Murine Behaviors
Author: Jose Vidal
Affiliation: University of Barcelona School of Psychology, 08035
Barcelona, Spain.
Correspondence to: Jose Vidal; Departamento de Personalidad, Facultad de
Psicologia, Universidad de Barcelona; Passeig de la Vall d'Hebron,
171; 08035 Barcelona, Spain.
Phone: 34-93-4021072, ext. 3124
Fax: 34-93-4021362
e-mail: jvidal@psi.ub.es
Running head: immune response and behavior
2
Abstract
The goal was to discover if the generation and maintenance of the
specific immune reponse resulted in alterations of reliable behaviors (i.e.,
behaviors correlated over time). CD1 male mice were measured in a small
open field (behaviors recorded: ambulation, rearing, and interaction with a
conspecific) and, several days later, were immunized with antigens (either
splenocytes from C57BL/6 mice or a mixture of sheep erythrocytes and goat
serum); the same behaviors were recorded again some hours, or some days,
after immunization. Immunizations and behavioral measurements were
repeated a few more times. Blood levels of antibodies to the antigens were
measured 6 days after immunization.
The recorded behaviors turned out to be consistent (according to Kendall
coefficient of concordance). The mice mounted an antibody response to the
antigens, yet no behavioral changes were apparent during the response. On
the contrary, a single injection of E. coli lipopolysaccharide decreased
ambulation and rearing. It is proposed that, in healthy mice kept in normal
conditions, the specific immune response may be unrelated to reliable
behaviors.
Key words: immune response, behavior, ambulation, rearing, interaction
with conspecific, DIG-ELISA.
3
Psychoneuroimmunology studies, among other things, the interactions of
behavior and the immune response. One issue is whether the generation and
maintenance of the immune response, be it innate (natural) or acquired
(specific), cause alterations of behavior. It is now established that generation
of innate immunity to microorganisms causes neuroendocrine and
behavioral changes (sickness behavior; for reviews, see Berczi &
Szentivanyi, 1996; Dantzer, Bluthé, Kent, & Goodall, 1993). When it comes
to acquired immunity, there are some reports describing the neuroendocrine
alterations that take place at the peak of the immune response (Besedovsky
& Del Rey, 1991; Carlson, Felten, Livnat, & Felten, 1987; Catania, Airaghi,
Manfredi, & Zanussi, 1990; Saphier, 1989; Stenzel-Poore et al., 1993;
Zalcman, Shanks, & Anisman, 1991) and scarce reports on the behavioral
alterations that occur at the time of the immune response (Gates et al., 1992;
Zacharko et al., 1997).
A related issue is whether the elicitation of the acquired immune response
(be it the antibody response or cellular immunity) alters consistent behavior
(i.e., a component of a trait) or fluctuating behavior (i.e., a state). For
reasons to be discussed later, Zacharko et al. (1997), reported probably the
effect of the antibody response on a state, and Gates et al. (1992) reported
probably the effect of several factors on a putative trait (fear). I prefer to
investigate the effect of the antibody response on traits rather than on states,
because consistent behavior is more characteristic and, therefore, more
predictable. In this context, a previous publication of ours (Vidal & Rama,
1994) reported, in random-bred CD1 mice of either sex, the lack of
correlation of the antibody response to one antigen (keyhole limpet
4
hemocyanin) with consistent behaviors in the open field (ambulation and
rearing). The results from that study could not disclose if the generation of
the antibody response altered behavior because: i) immunization took place
between 1 and 2 weeks after behavioral tests, ii) the study was correlational,
and therefore unsuitable to establish causal relations. Besides, it is not clear
whether states or traits were assessed then, since each behavior and the level
of antibodies were recorded once. Consequently, the present report aims at
finding out whether a persistent immune response can alter consistent
behavior, which presumably reflects a trait.
The antigen chosen to activate the immune system should be i)
innocuous, noninfective, and nonneoplastic, to avoid the elicitation of innate
immunity that would confound the effects of acquired immunity on behavior
(Besedovsky & Del Rey, 1991), and ii) immunogenic enough to elicit a
strong immune response for, according to Besedovsky (Besedovsky & Del
Rey, 1991) and other authors (Stenzel-Poore, et al., 1993), only moderate, or
large, doses of antigen (which presumably elicit large immune responses)
are able to cause neuroendocrine changes. Consequently, in the present
experiments, mice were repeatedly immunized with either a mixture of
antigens (sheep erythrocytes plus goat serum), which presumably stimulate a
large immune response, or with allogeneic leukocytes; which elicit strong
cell-mediated immunity and humoral (antibody) immunity (Colombe, 1994).
The behaviors recorded in this report were taken from the ethogram
developed by Grant and Mackintosh (1963) and from the murine behaviors
in the open field (Gomá & Tobeña, 1978; Ivinskis, 1968). From all those
behaviors, ambulation and rearing were chosen because they were reliable
5
(Gomá & Tobeña, 1978; Ivinskis, 1968).
Method
Subjects. CD1 mice, of both sexes, were purchased from Charles River
(CRIFFA, Barcelona, Spain); those mice were mated in our laboratory and
their offspring were the subjects for the experiments reported here. The
mice, housed 3 or 4 per cage, lived under a 12-hour light-dark cycle: lights
on from 8:00 hr to 20:00 hr. The mice received at libitum food and water.
The temperature of the room was 22  1 oC. At the beginning of the
experiments, the mice were 2-3 months old.
Behavioral test: Activity and interaction with a conspecific. This test took
place in a (24 x 24 x 14 cm) square open-field whose floor was marked off
by diagonal lines in 4 triangles; the open-field was placed in a room
illuminated by 2 fluorescent lamps. The interaction-with-conspecific test
was the test used by Bluthé, Dantzer, & Kelley (1992) with these
modifications: i) mice, not rats, were used, ii) behavioral observations took
place in the open field, not in the home cage, iii) the conspecific was an
adult C57BL/6 male mouse previously sedated with haloperidol (0.7 mg/kg,
intraperitoneally, ip.); the conspecific was sedated to reduce its interaction
with the test mice. During 4 minutes and 30 seconds, these behaviors in the
open field were recorded from each test mouse: interaction time (time, in
seconds, spent exploring the conspecific), ambulation (number of lines
crossed by the mouse), and rearing (number of times the mouse stood
completely erect on its hindlegs). The open field was wiped clean, with
soapy water, before each mouse was placed in it. The test was carried out
between 15:00 and 18:00 hours.
6
Immunization and antibody measurement. In one experiment, the mice
were injected ip. with sheep erythrocytes (SRBC, from Cappel) and
subcutaneously (sc.) with goat serum (Sigma) from a single goat; goat serum
was given sc., and not ip., to reduce the risk of anaphylactic-like shock.
Throughout the experiment, the mice were immunized five times, with
increasing doses of antigens.
In another experiment, the mice were injected ip. with spleen cells from
C57BL/6 male mice (allogeneic leukocytes); before injection, the cells were
depleted of erythrocytes by treatment with Tris-ammonium chloride buffer.
Throughout the experiment, the mice received four injections of allogeneic
leukocytes, the dose being 2x107 cells/mouse/injection. At the end of this
experiment, the mice were injected (ip.) once with lipopolysaccharide from
E. coli 055:B5 (LPS, from Sigma; this compound elicits sickness behavior),
75 g/mouse (about 1.4 mg/kg).
Blood levels of antibody to each antigen were measured by diffusion-ingel enzyme-linked immunosorbent assay (DIG-ELISA). The procedure was
initially as described (Vidal, 1996), although two modifications were
introduced for the measurement of the antibody level to allogeneic cells: i)
monolayers of allogeneic cells were created on poly-L-lysine-coated
hydrophilic petri dishes (nunclon delta dishes; Nunc, Denmark), ii) areas of
antibody content were revealed by the silver intensification method of
Merchenthaler, Stankovics, and Gallyas (1989); this procedure worked
better than the one previously used (Przepiorka & Myerson, 1986). The level
of antibody to a given antigen in a test serum is expressed as fraction of the
level in a standard serum (standard sera for IgM and IgG were different).
7
The mice were bled, between 11:00 and 13:00 hours, from the
retroorbital plexus while they were anesthetized with ethyl ether.
Design. About half of the mice in every cage were randomly assigned to
either the experimental or the control groups (the mice in a cage came from
the same litter). Two experiments were performed: In experiment 1, three
measurements of the behaviors (baseline) were taken before immunizations;
thereafter, experimental mice were immunized 5 times, at 7-day intervals,
with various doses of SRBC and goat serum (control mice were injected
with the vehicle), and behaviors were recorded at different times after some
immunizations; the number of mice in the control and experimental groups
was respectively 10 and 12. In experiment 2, two measurements of the
behaviors (baseline) were taken before immunizations; thereafter,
experimental mice were immunized 4 times, at 14-day intervals, with 2x107
allogeneic leukocytes per mouse (control mice were injected with the
vehicle), and the behaviors were recorded at different times after each
immunization. The number of mice in the control and experimental groups
was 11.
The time points for behavioral recordings were taken from the literature:
neuroendocrine changes after immunization take place at the peak of the
antibody response: 4 days post-immunization (Carlson, Felten, Livnat, &
Felten, 1987; Zalcman, Shanks, & Anisman, 1991), 5 days postimmunization (Besedovsky & Del Rey, 1991; Stenzel-Poore et al., 1993), 8
days post-immunization (Saphier, 1989), or 4 hours post-immunization
(Catania, Airaghi, Manfredi, & Zanussi, 1990); consequently, behaviors
were recorded, in this paper, usually 5 days post immunization, although
8
occasionally at 3 hours, 2 days, or 4 days post-immunization. Fourteen days
after the last injection of allogeneic leukocytes, the mice were injected with
LPS, and behaviors were recorded between 2 and 4 hours later.
Statistics. To assess consistency of a given behavior, the various (6 or 7)
measurements of that behavior in the control mice, throughout the
experiment, were correlated; the overall magnitude and significance of the
correlations was estimated by Kendall's coefficient of concordance.
The design was a pretest-posttest design (i.e., a between-within design
with a baseline). In this case, differential changes of the experimental group,
relative to the control group, from baseline (pretest) to treatment (posttest)
are to be assessed either by analysis of covariance or by "treatment x trial"
interaction (Edwards, 1985); the interaction test was used here.
The statistical packages used were CSS Statistica for DOS (release 3.1)
and Statistica for Windows, v. 5.1 (Statsoft, Tulsa, OK).
Results
Consistency of behaviors. Table 1 shows that each of the 3 behaviors
(interaction with a conspecific, ambulation, and rearing) was consistent (i.e.,
the several measurements of any one behavior, in control mice, were
moderately and significantly correlated).
Effect of immunization with sheep erythrocytes and goat serum on
behavior. Figure 1 shows the means ( SEM) of both experimental and
control groups along the experiment. By inspection of the graph, both
groups behaved in parallel throughout the experiment (i.e., immunization
did not alter the spontaneous behavioral changes). This result was confirmed
by calculating the "treatment x trial" interaction for each behavior: none of
9
the interactions was significant (not shown).
The antigens did activate the immune system of mice, because the
experimental mice mounted antibody responses to SRBC and goat albumin
(Table 2).
Effect of immunization with allogeneic leukocytes on behavior. Figure 2
shows the means ( SEM) of experimental and control groups along the
experiment. By inspection of the graph, the experimental group seems to
score lower than the control group on day 49. Yet, those differences were
apparent in the baseline, before immunization, and thus the "treatment x
trial" interactions, from days 12 to 80, did not reach significance (for
interaction time: F(6, 114) = 0.29, p= 0.94; for ambulation: F(6, 114) = 0.23,
p= 0.96; for rear: F(6, 114) = 0.21, p= 0.97). Therefore, it cannot be said that
immunization caused significant behavioral alterations.
The allogeneic cells activated the immune system of recipient mice,
because the latter mounted an antibody response to the foreign cells (Table
2). In this experiment, foreign splenocytes were not treated with mitomycin
C, but treatment of cells with that drug (to prevent donor cell proliferation)
yielded similar results (not shown).
Once the experiment was completed, the experimental mice were treated,
on day 91, with LPS: this was done to show that the behavior of those mice
could indeed be altered. Figure 2 shows that LPS caused a decrease in
ambulation and rearing, and therefore, control and experimental groups no
longer followed parallel courses ("treatment x trial" interaction, days 12 92: for interaction time: F(7, 133) = 0.41, p= 0.89; for ambulation: F(7, 133)
= 2.95, p= 0.007; for rear: F(7, 133) = 2.58, p= 0.016).
10
Discussion
The behaviors recorded here were moderately reliable (in Table 1, the
average correlation coefficient for the varios measurements of ambulation
was on the order of 0.40 and so was the average correlation coefficient for
the various measurements of rearing; the reliability of the interaction time
with a conspecific had not been assessed previously, but this behavior also
turned out to be consistent, although less than ambulation and rearing
[average correlation coefficient about 0.25 ). These results agree with
previous reports on the reliability of ambulation and rearing in the open field
(Gomá & Tobeña, 1978; Ivinskis, 1968.).
The results reported here are in line with our previous results (Vidal &
Rama, 1994); namely, in adult, healthy mice, the antibody response is
uncorrelated with some consistent behaviors in the open field. Then (Vidal
& Rama, 1994), the correlational method was used to arrive at such a
conclusion; now, an experimental approach (activation of the immune
system) was chosen to explore the occurrence of causal relations between
consistent behaviors and the antibody response. Because the results reported
here are negative (generation of specific antibodies did not seem to alter
consistent behavior; Figures. 1 and 2), they do not prove the lack of relation
of behavior and the antibody response; yet, when one considers that the
correlational approach and the experimental one yield parallel results, one
may tentatively propose the lack of association of the specific immune
response and consistent behavior.
Nevertheless, a comment is in order. The failure of the antibody response
to alter behavior could be due to the stability of the recorded behaviors. This
11
was tested by giving the mice a pulse of LPS and, in fact, LPS decreased
ambulation and rearing (Fig. 2 and Results), which agrees with previous
reports (Dunn, Chapman, & Antoon, 1992; Kozak, Conn, & Kluger, 1994;
Yirmiya, Rosen, Donchin, & Ovadia, 1994). It is surprising, although, the
lack of effect of LPS on interaction with a conspecific (Fig.2 and Results).
This result is at variance with a previous report describing the reduction in
interaction with a conspecific brought about by LPS (Bluthé et al., 1992).
The explanation may reside in the time at which LPS was given: whereas
Bluthé et al. used naive rats, I used habituated mice that scored close to zero
at the time of LPS injection (on day 79, the scores [mean and SEM of
control and experimental groups were 5.1 1.4 and 1.9  0.8; on day 91, 2-4
hours after LPS, the scores of the same groups were 2.5  1.1 and 0.3  0.2).
It could well be that the scores were so close to zero that no further decrease
was possible. It is worth mentioning that LPS administration to less
experienced mice also decreased their interaction with another mouse (my
own unpublished results).
The present experiments aimed at finding out whether the "pure"
elicitation of acquired immunity (by antigens devoid of additional effects
other than eliciting the specific immune response; Besedovsky & Del Rey,
1991) resulted in alteration of known consistent behaviors. This approach
differs in two respects from two published reports on the effect of
immunization on behavior. One of the articles reported the capacity of the
secondary immune response to decrease fear (Gates et al., 1992). Gates et al.
challenged sheeps previously immunized with the nematode Haemonchus
contortus with two suboptimal doses of the same parasite and measured the
12
distance the sheeps stayed away from a human: boosted animals stood closer
to the human than control animals, which was interpreted as fear reduction.
The authors used as antigen a worm, a complex organism able to elicit
several effects (e.g., natural immunity) besides triggering the acquired
immune response, and those effects may explain the reduction in fear. Other
paper reported the anhedonic effect of the specific immune response in mice
(Zacharko et al., 1997). In this case, the antigen (sheep erythrocytes) was
appropriate, but the recorded behavior (stimulation by electrodes in the
nucleus accumbens) may be more akin to mood than to characteristic
behavior; i.e., it may reflect a state rather than a trait. This raises the
possibility, not studied in the present paper, that the specific immune
response may alter states but not traits.
In conclusion, the present results suggest that, in adult, healthy mice, the
specific immune response may not be related to consistent behavior, and this
agrees with the previously reported orthogonality of behavior and the
antibody response (Vidal & Rama, 1994).
Acknowledgements
This work was supported by a grant (DGICYT No. PB95-0458) from the
Spanish Ministry of Education.
13
References
Berczi, I., & Szentivanyi, A. (1996). The pituitary gland,
psychoneuroimmunology, and infection. In H. Friedman, T.W. Klein,
& A.L. Friedman (Eds.) Psychoneuroimmunology, stress, and
infection (pp. 79-109). Boca Raton, Florida: CRC Press.
Besedovsky, H.O., & Del Rey; A. (1991). Physiological implications of the
immune-neuro-endocrine network. In R. Ader, D.L. Felten, & N.
Cohen (Eds.) Psychoneuroimmunology 2nd edition (pp. 589-608). San
Diego, California: Academic Press.
Bluthé, R.-M., Dantzer, R., & Kelley, K.W. (1992). Effects of interleukin-1
receptor antagonist on the behavioral effects of lipopolysaccharide in
the rat. Brain Research , 573 318-320.
Carlson, S., Felten, D.L., Livnat, S., & Felten, S.Y. (1987). Alterations of
monoamines in specific central autonomic nuclei following
immunization in mice. Brain, Behavior, and Immunity , 1, 52-63.
Catania, A., Airaghi, L., Manfredi, M.G., & Zanussi, C. (1990). Hormonal
response during antigenic challenge in normal subjects. International
Journal of Neuroscience , 51, 295-296.
Colombe, B.W. (1994). Histocompatibility testing. In D.P. Stites, A.I.
Terr,& T.G. Parslow (Eds.) Basic and clinical immunology, 8th
edition (pp. 237-255). London: Prentice Hall International.
Dantzer, R., Bluthé, R-M., Kent, S., & Goodall, G. (1993). Behavioral
effects of cytokines: An insight into mechanisms of sickness behavior.
In E.B. de Souza (Ed.) Neurobiology of cytokines (pp. 131-150).
Orlando, Florida: Academic Press.
14
Dunn, A.J., Chapman, Y., & Antoon, M. (1992).Endotoxin-induced
behavioral changes of mice in the multicompartment chamber are
distinct fom those of interleukin-1. Neuroscience Research
Communications, 10, 63-69.
Edwards, A. L. (1985). Multiple regression and the analysis of variance and
covariance (2nd edition). New York: Freeman.
Gates, G.R., Fell, L.R., Lynch, J.J., Adams, D.B., Barnett, J.P., Hinch, G.N.,
Munro, R.K., & Davies, H.I. (1992). The link between immune
responses and behaviour in sheep. In A.J. Husband (Ed.) Behaviour
and immunity (pp. 23-41). Boca Raton, Florida: CRC Press.
Gomá, M., & Tobeña, A. (1978). Reliability of various measures obtained in
the open-field test. Psychological Reports, 43, 1123-1128.
Grant, E.C. & Mackintosh, J.H. (1963). A comparison of the social postures
of some common laboratory rodents. Behaviour, 21, 246-259.
Ivinskis, A. (1968). The reliabilty of behavioural measures obtained in the
open-field. Australian Journal of Psychology, 20, 173-177.
Kozak, W., Conn, C.A., & Kluger, M.J. (1994). Lipopolysaccharide induces
fever and depresses locomotor activity in unrestrained mice. American
Journal of Physiology, 266, R125-R135.
Merchenthaler, I., Stankovics, J., & Gallyas, F. (1989). A highly sensitive
one-step method for silver intensification of the nickeldiaminobenzidine endproduct of peroxidase reaction. Journal of
Histochemistry and Cytochemistry, 37, 1563-1565.
Przepiorka, D. & Myerson, D. (1986). A single-step silver enhancement
method permitting rapid diagnosis of cytomegalovirus infection in
15
formalin-fixed, paraffin-embedded tissue sections by in situ
hybridization and immunoperoxidase detection. Journal of
Histochemistry and Cytochemistry, 34, 1731-1734.
Saphier, D. (1989). Neurophysiological and endocrine consequences of
immune activity. Psychoneuroendocrinology, 14, 63-87.
Stenzel-Poore, M., Vale, W.W., & Rivier, C. (1993). Relationship between
antigen-induced immune stimulation and activation of the
hypothalamic-pituitary-adrenal axis in the rat. Endocrinology, 132,
1313-1318.
Vidal, J. (1996). Differences of nu/+ and nu/nu mice in some behaviors
reflecting temperament traits. Physiology and Behavior, 59, 341-348.
Vidal, J. & Rama, R. (1994). Association of the antibody response to
hemocyanin with behavior in mice bred for high or low antibody
responsiveness. Behavioral Neuroscience, 108, 1172-1178.
Yirmiya, R., Rosen, H., Donchin, O., & Ovadia, H. (1994). Behavioral
effects of lipopolysaccharide in rats: involvement of endogenous
opioids. Brain Research, 648, 80-86.
Zacharko, R.M., Zalcman, S., MacNeil, G., Andrews, M., Mendella, P.D., &
Anisman, H. (1997). Differential effects of immunologic challenge on
self-stimulation from the nucleus accumbens and the substantia nigra.
Pharmacology, Biochemistry, and Behavior, 58, 881-886.
Zalcman, S., Shanks, N., & Anisman, H. (1991). Time-dependent variations
of central norepinephrine and dopamine following antigen
administration. Brain Research, 557, 69-76.
16
Table 1
Reliability of the behaviors (interaction with conspecific, ambulation, and
rearing)
experiment 1
experiment 2
behavior W
2
P
r
W
2
p
r
tinter
0.40
21.4
0.011
0.28
0.34
23.9
0.008
0.23
amb
0.48
26.1
0.002
0.38
0.54
37.7
0.00004
0.46
rear
0.50
27.1
0.001
0.40
0.56
39.3
0.00002
0.49
In experiment 1 (mice immunized with SRBC + goat serum), the 10
control mice were measured in 6 occasions; in experiment 2 (mice
immunized with allogeneic leukocytes), the 11 control mice were measured
in 7 occasions.
W: Kendall coefficient of concordance; 2: statistic for W; p: significance
of 2 ; r: average (Spearman) correlation coefficient.
tinter: interaction time with conspecific; amb: ambulation; rear: rearing.
17
Table 2
Antibody response of mice immunized with antigens
experimental group
control group
anti-SRBC antibodies
IgM1
0.70 (0.13); N=12
0.21 (0.02 ); N=10
IgG2
0.13 (0.04); N=10
0.00; N=10
IgG3
0.18 (0.05); N=10
0.01 (<0.001): N=10
anti-goat albumin antibodies
IgM1
0.09 (0.03); N=12
0.01 (<0.010); N=10
IgG2
4.02 (0.69); N=10
0.03 (<0.001); N=10
IgG3
2.20 (0.32); N=10
0.03 (<0.001); N=10
Anti-allogeneic cells antibodies
IgGBL
0.001 (0.0007); N=11
0.001 (0.0007); N=11
IgG1
0.001 (0.0005); N=11
0.0005 (0.00007);N=11
IgG2
0.36 (0.08); N=10
0.006 (0.002); N=11
IgG3
1.15 (0.14); N=10
0.01 (0.002); N=11
Anti-SRBC (and goat albumin) antibodies: IgM1: IgM 6 days after 1st immunization,
IgG2: IgG 6 days after 3rd immunization, IgG3: IgG 6 days after 5th immunization. Doses of
antigens are indicated in the legend to Figure 1.
Anti-allogeneic cells antibodies: IgGBL: baseline IgG, IgG1: IgG 6 days after 1st immunization, IgG2: IgG 6 days after 2nd immunization, IgG3: IgG 6 days after 3rd immunization.
Only traces of IgM were detected 6 days after the 1st immunization. Dose of antigen: 2x107
splenocytes/mouse.
Figures in the columns show the mean, the standard error of the mean (in parentheses), and
the number of mice.
18
Figure 1. Effect of immunization with SRBC and goat serum on
behavior.
o: mean of immunized mice; *: mean of control mice. Error bars are
standard error of means. Each arrow indicates one immunization.
Doses of antigens: 1st immunization: 2.5x107 SRBC + 0.01 ml goat
serum; 2nd immunization: 5.0x107 SRBC + 0.02 ml goat serum; 3rd
immunization: 1.0x 108 SRBC + 0.04 ml goat serum; 4th immunization:
2x108 SRBC ; 5th immunization: 4x108 SRBC + 0.05 ml alum-adsorbed
goat serum (35 mg gel / ml).
Number of mice are indicated in the columns of Table 2.
Figure 2 . Effect of immunization with allogeneic cells on behavior.
o: mean of immunized mice; *: mean of control mice. Error bars are
standard error of means. Each arrow indicates one immunization (the last
one, on day 91, corresponds to LPS injection). On days 30 and 91, behaviors
were recorded from 2 to 4 hr after immunization.
Antigen: viable splenocytes from C57BL/6 mice, 2x107 cells per
injection. LPS: 75 g/mouse.
Number of mice are indicated in the columns of Table 2.
19
Figure 2
interaction time (sec)
Influence
Effect ofofimmunization
alloinmunization
on interaction
on interaction
time time
40
35
24
30
18
25
20
12
15
10
6
5
0
0
-5
0
10
10
20
20
3030
4040
5050
60 60
70 70
80 80
90 90
days
100 100
LPS
ammbulation
ambulation
Influence
Effect of
ofimmunization
alloimmunization
on ambulation
on ambulation
60
70
55
60
50
50
45
40
35
30
30
20
25
10
20
150
0
10
10
20
20
3030
4040
5050
60 60
70 70
80 80
90 90
days
100 100
LPS
rearing
rear
Influence
Effect of
ofimmunization
alloimmunization
on rearing
on rearing
70
55
50
60
45
50
40
40
35
30
30
25
20
20
10
15
100
0
10
10
20
20
3030
4040
5050
60 60
70 70
80 80
90 90
days
100 100
LPS
------------------------------------------------------------------Figure 1
interaction time (sec)
Effect of immunization on interaction time
40
35
30
25
20
15
10
5
0
-5
0
10
20
30
40
50
60
70
80
90
100
80
90
100
80
90
100
days
ambulation
Effect of immunization on ambulation
60
55
50
45
40
35
30
25
20
15
0
10
20
30
40
50
60
70
days
rearing
Effect of immunization on rearing
55
50
45
40
35
30
25
20
15
10
0
10
20
30
40
50
days
60
70