Circadian variations have been observed in several immune system functions, allowing the immune cells to be active and ready at specific times of the day for greater effectiveness. The daily and seasonal rhythms of immune functions are regulated and co-ordinated by the neuroendocrine homeostatic system. In the present work we evaluated the circadian rhythm of phagocytosis and superoxide anion levels in macrophages in control and stress situations. Male Wistar rats (Rattus norvegicus), 3 months old, were maintained under a 12 h light and 12 h dark photoperiod. The stressor was forced swimming to exhaustion, with the rats subjected to free swimming for 2 h and 15 final minutes swimming on a rotating rod test that is a modified version of the automated Porsolt test (Thornton et al. 1990). The animals were subjected to this stress every 2 h over one circadian period. After killing the animals by decapitation, the macrophages were obtained from the peritoneal cavity. The functional capacity of macrophages was evaluated by the phagocytosis index (PI, number of latex beads ingested by 100 macrophages) and the superoxide anion levels by a quantitative nitroblue tetrazolium test (Rodriguez et al. 2001). The rats were handled according to the guidelines of the European Community Council Directive 86/6091 EEC. All data are expressed as mean values ± S.D. Data were compared by Student’s unpaired t test. Values of P < 0.05 were considered significant.

In conclusion, these data suggest that stress leads to alterations in the non-specific immune response.

The authors thank Elena Circujano Vadillo for technical assistance. The research was supported in part by Grant 01/09 from the Junta de Extremadura-Consejeria Bienestar Social.

The results (Fig. 1) showed that in the control rats, the phagocytic activity is higher during darkness with maximum values at 24.00 h (639 ± 25 PI, n = 6), and declines during daylight to minimum values at 14.00 h (153 ± 34 PI). For the stressed animals, there were also day/night variations in the phagocytic activity, but now the dark-period values were higher than those of the basals, with the maximum values being obtained at 02.00 h (698 ± 30 PI). For the superoxide anion levels (Fig. 2) produced in the macrophages during oxidative metabolism, the cells from the control animals presented a rise during the daylight period, with maximum levels reached at 10.00 h. The stress led to a loss of the circadian rhythm in superoxide anion levels, being raised in the stressed animals in a general fashion over the length of the circadian cycle relative to those found in the control situations.

All procedures accord with current national guidelines.