Introduction By using this model, it was shown that cytokines like

Brain Research 888 2001 227–234 www.elsevier.com locate bres Research report Effect of local sympathectomy on 24-h changes in mitogenic responses and lymphocyte subset populations in rat submaxillary lymph nodes during the preclinical phase of Freund’s adjuvant arthritis a a a b ´ Ana I. Esquifino , Patricia O. Castrillon , Fernando Chacon , Rodolfo Cutrera , b , Daniel P. Cardinali a ´ ´ Departamento de Bioquımica y Biologıa Molecular III , Facultad de Medicina, Universidad Complutense, Madrid, Spain b ´ Departamento de Fisiologıa , Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina Accepted 3 October 2000 Abstract Wistar male rats received a bilateral superior cervical ganglionectomy or sham-operation and 10 days later were injected with Freund’s complete adjuvant or its vehicle. Two days later, rats were killed at six different time intervals throughout a 24-h cycle. The mitogenic effect of lipopolysaccharide LPS and concanavalin A Con A and the relative size of lymphocyte subset populations were measured in submaxillary lymph nodes. Cells from sympathectomized lymph nodes showed a lower response to Con A. Freund’s adjuvant injection decreased amplitude of daily rhythm in Con A response, an effect prevented by denervation. Generally, ganglionectomy increased Con A response at the early phase of arthritis. Acrophases for Con A and LPS effect occurred at early afternoon and did not change after ganglionectomy. Administration of Freund’s adjuvant caused a 10-h advance in acrophase of LPS mitogenic activity, an effect prevented by ganglionectomy. Significant 24-h rhythms were observed in relative size of lymph node B and T cells. Denervation augmented amplitude of rhythm in B cells in adjuvant’s vehicle-injected rats. As far as T lymphocyte subsets, acrophases occurred at the afternoon 1 1 1 1 1 1 CD4 and CD4 –CD8 cell types or at night CD8 cell types. Immunization augmented amplitude of 24-h rhythms in CD4 –CD8 1 cells regardless of innervation whereas denervation counteracted the suppression of daily rhythm in CD8 cells seen in arthritis. The results indicate that some of the changes seen in 24-h organization of immune responses in lymph nodes at an early phase of arthritis are modified by severing the local sympathetic nerves.  2001 Elsevier Science B.V. All rights reserved. Theme : Endocrine and autonomic regulation Topic : Neural–immune interactions Keywords : Superior cervical ganglion; Submaxillary lymph node; Lymphocyte subset; Circadian rhythm; Freund’s adjuvant arthritis

1. Introduction By using this model, it was shown that cytokines like

interleukins 1, 2 or 6, granulocyte-macrophage colony- Injection of Freund’s complete adjuvant to rats leads to stimulating factor or interferon-a are responsible for many an inflammatory disease of the joints that shows four of the symptoms observed as a response to inflammation stages in its time-course: preclinical first week, acute e.g. anorexia, depressed activity, also described as ‘sick- weeks 2–4, postacute weeks 5–8 and recovery weeks ness behavior’ [3,21]. 9–11 [4]. This inflammatory disease has been widely One feature of mycobacterial adjuvant arthritis less often employed as an experimental model to examine the addressed is the existence of changes in circadian relationships between the brain and the immune system. rhythmicity of biologic responses during the immune reaction. The rationale for circadian organization is the predictive value of the mechanisms enabling an organism Corresponding author. Tel.: 154-11-4508-3698; fax: 154-11-4963- to keep equilibrium in response to anticipated variations of 6287. E-mail address : cardinalmail.retina.ar D.P. Cardinali. the environment [26]. One essential circadian rhythm in 0006-8993 01 – see front matter  2001 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 3 0 6 0 - 2 228 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