228 A
adaptive responses is that of the immune reaction. Circa- daily. Rats had access to food and water ad libitum.
dian changes in the circulation of T, B, or natural killer Surgery was performed under tribromoethanol anesthesia
NK lymphocyte subsets in peripheral blood and in the 250 mg kg, i.p.. Adequate measures were taken to
density of epitope molecules at their surface, which may be minimize pain or discomfort, in accordance with the
related to cell reactivity to antigen exposure, have been principles and procedures outlined in European Com-
reported for references see [7]. Changes in lymphocyte munities Council Directives 86 609 EEC.
subset populations
depend on
time-of-day-associated Rats subjected to a SCGx or its sham-operation 10 days
changes in cell proliferation in immunocompetent organs earlier were s.c. injected at the base of the tail with
and or on diurnal modifications in lymphocyte release and Freund’s complete adjuvant 0.5 mg heat-killed Mycobac-
traffic among lymphoid organs. terium butyricum rat or its vehicle 0.5 ml paraffin oil
During recent years we have examined the regulation of containing 15 mannide monooleate at 1100 h. On the
circadian rhythmicity of lymphoid cell proliferation taking second day after Freund’s adjuvant or adjuvant’s vehicle
as a model the rat submaxillary lymph nodes. The bilateral injection, groups of 5–7 rats from each experimental group
anatomical location of submaxillary lymph nodes and their were killed by decapitation at six different time intervals
easily manipulable autonomic innervation allowed us to throughout a 24-h cycle and their submaxillary lymph
carry out the analysis of some of the humoral and neural nodes were removed aseptically, weighed and placed in
mechanisms regulating the lymphoid organs. Both in Petri disks containing balanced salt solution, the cells
immunized and in non-immunized rats a significant diurnal variation of cell proliferation in submaxillary lymph nodes
was reported, displaying maximal activity at early after- noon [5]. Such a maximum coincided with peak mitotic
responses to lipopolysaccharide LPS and concanavalin A Con A in incubated lymph node cells [17]. A purely
neural regulatory pathway including as a motor leg the sympathetic and parasympathetic fibers innervating the
lymph nodes was identified. In addition, a hormonal pathway involving the circadian secretion of melatonin
was also instrumental in producing rhythmicity [6,8].
Studies to be reported herein were addressed to assess further the role of the sympathetic nervous system in the
regulation of circadian immune function. We examined the effect of surgical sympathetic denervation of submaxillary
lymph nodes,
achieved by
superior cervical
gang- lionectomy SCGx, on the 24-h changes in the in vitro
mitogenic effect of LPS and Con A and in the relative size of lymphocyte subset populations in submaxillary lymph
nodes at an early phase of mycobacterial adjuvant arthritis i.e. on the second day after injection of Freund’s adjuvant
or its vehicle.
2. Materials and methods
2.1. Chemicals
3
Thymidine [methyl- H] specific activity 20 Ci mmol was purchased from NEN Research Products Boston,
MA, USA. Freund’s complete adjuvant was purchased from Difco Detroit, MI, USA. All other drugs and
Fig. 1. Twenty-four hour changes in mitogenic activity of Con A and
reagents employed were obtained from Sigma St. Louis,
LPS in submaxillary lymph nodes of rats on the second day after the
MO, USA.
injection of Freund’s complete adjuvant or adjuvant’s vehicle. Rats were subjected to SCGx or its sham-operation 10 days earlier. Groups of 4–7
rats were killed by decapitation at six different time intervals throughout a
2.2. Animals and experimental design
24-h cycle. Shown are the means6SEM. Asterisks designate groups in which time-of-day changes were significant, as detected in a one-way
Experiments were carried out in adult male Wistar rats
ANOVA P,0.01; n.s.: not significant time-related changes in a one-way
180–220 g, kept under light between 08:00 and 20:00 h
ANOVA. Further analysis employing
COSINOR
is described in Table 1.
A .I. Esquifino et al. Brain Research 888 2001 227 –234
229
being gently teased apart. After removing the clumps by studies, we used the following monoclonal antibodies:
centrifugation, the cells were suspended in sterile sup- Anti-rat LCA OX-33 for B lymphocytes Serotec, Ox-
plemented medium RPMI 1640, containing 10 heat- ford, UK, anti-rat TCR a b R7.3 for T lymphocytes
inactivated, fetal bovine serum, 20 mM
L
-glutamine, 0.02 Serotec, Anti-rat CD4 OX-35 which recognize a rat T
mM 2-mercaptoethanol and gentamicin 50 mg ml, and helper cell differentiation antigen Pharmingen, San Diego,
were counted. CA, USA, and anti-rat CD8a OX-8 which recognize the
reactive antigen expressed on rat T cytotoxic suppressor 2.3. Mitogen assays
cells Pharmingen.
Lymphocytes from
submaxillary lymph nodes isolated as indicated above, were washed in
Mitogen assays were performed as described in detail cold PBS with 0.02 sodium azide and then incubated
5
elsewhere [17]. Submaxillary lymph node cells were 3310 cells tube with appropriate primary antibodies for
obtained as described above and used at a final number of 30 min at 48C. Following two washes, the cells were
5
cells well 0.1 ml of 5310 . Control and experimental incubated with 1 ml of PBS–BSA 1, for 5 min at 48C,
cultures were run in triplicate. Mitogens were added to the washed three times, resuspended in 1 paraformaldehyde
cultures at final supramaximal concentrations of 5 mg ml. in PBS. Fluorescence intensity was analyzed by fluores-
plus
The cultures were incubated in a humidified 378C in- cence activated cell sorting FACStar
; Beckton Dickin- cubator in an atmosphere of 5 CO . After 48 h incuba-
son, Mountain View, CA, USA. Dead cells were excluded
2 3
tion, H-thymidine 0.2 mCi was added to each well in a by gating with propidium iodide.
volume of 0.02 ml. Cells were harvested 5 h later using a automated sample harvester, and the filters were counted in
2.5. Statistical analysis a liquid scintillation spectrometer. The proliferation index
was estimated as the ratio between stimulation in the Statistical analysis of results was performed by employ-
presence of mitogens controls. Results were expressed as ing a one-way analysis of variance ANOVA, followed by
proliferation index number of cells. Student–Newman–Keuls or Dunnett’s t-tests, and a
COSINOR
analysis. The latter was used to analyze general 2.4. Lymphocyte subsets
rhythmic parameters, i.e. acrophase the maximum of the sinus function fit by the experimental data, mesor the
The relative size distributions of lymphoid cells in statistical estimate of the mean and amplitude half the
submaxillary lymph nodes of rats were determined by difference between calculated maximal and minimal val-
FACS analysis, as previously described [11,12]. For these ues. P values lower than 0.05 were considered significant.
Table 1
COSINOR
analysis of the effect of SCGx on 24-h changes of Con A and LPS mitogenic activity in rat submaxillary lymph nodes, on the second day after
a
injecting Freund’s adjuvant or its vehicle Mesor
Amplitude Acrophase
Percent h, min
of rhythm Sham-operation,
Adjuvant’s vehicle Con A
8.261.2 7.360.9
14:11602:04 40.765.6
LPS 5.060.7
2.260.5 13:27603:00
31.863.4 SCGx,
Adjuvant’s vehicle Con A
4.860.6 1.760.3
15:29601:20 61.967.5
LPS 6.060.9
2.560.6 12:26602:00
46.966.5 Sham-operation,
Freund’s adjuvant
[
Con A 6.761.1
4.460.5 12:24602:11
71.768.7 LPS
5.560.7 2.660.5
03:21601:45 44.866.5
SCGx, Freund’s adjuvant
Con A 15.262.5
5.061.0 07:25600:32
94.068.7 LPS
N.S. N.S.
N.S. N.S.
a 5
Shown are the means6SEM n54–7 group. Mesor and amplitude values are expressed as proliferation index number of cells310 . Asterisks designate
[
significant differences in a one-way ANOVA followed by a Student–Newman–Keuls test, , P,0.01, , P,0.05 as compared to the remaining groups, , P,0.05 as compared to sham-operated, adjuvant’s vehicle-injected rats. N.S., not significant daily changes in a one-way ANOVA; 2: not significant
changes in
COSINOR
.
230 A
3. Results phase for Con A effect occurred at early afternoon and did
