E França, A Junior, S de Oliveira, A Honório-França
blood phagocytes, chronoimmunomodulation, e. coli and bactericidal activity, melatonin, superoxide
E França, A Junior, S de Oliveira, A Honório-França. Chronoimmunomodulation of melatonin on bactericidal activity of human blood phagocytes. The Internet Journal of Microbiology. 2008 Volume 6 Number 2.
In this work we studied the chronoimmunomodulation effect of melatonin on blood phagocytes. This cells stimulated with melatonin and incubated with EPEC presented enhanced superoxide. Mononuclear (MN) and polymorphonuclear (PMN) phagocytes stimulated with melatonin in the presence of bacteria showed enhanced superoxide release.EPEC killing by MN phagocytes stimulated with melatonin was time dependent. The highest bactericidal index were observed over the period of 60 and 120 minutes of incubation. PMN phagocytes stimulated with melatonin, independently of time, showed increased in the bactericidal activity. Interactions between melatonin and phagocytes support the hypothesis of pineal chronomodulation microbicidal processes.
Several works have suggested that hormones and neuropeptides act as potent immunomodulators participating in various aspects of immune system function, both in health and disease (Dardenne & Savino,1994; Blalock 1994; Lissoni et.al, 1997; Srinivasan et al., 2005).
The pineal gland, with its indole metabolism and particularly melatonin synthesis, is characterized by a series of oxidative processes involving photochemical reactions and redox reactions with the participation of free radicals or peroxides inhibitors or generators. Melatonin is an endogenous neurohormone and its actions are related to its capability to scavenge free radicals, increase the antioxidant activity of enzymes (Keplac et al, 2005; . Sudnikovich et al, 2007) mainly due to its scavenging capacity against reactive oxygen species (Keplac et al, 2005). These observations have led to the hypothesis of pineal involvement in the body oxidative processes (Ianãs, et al., 1991). Some works have reported a functional connection between the pineal gland and the immune system (Liebmann et al., 1997; Pandi-Perumal et al, 2008).
The pineal hormone melatonin due to its lipophilic nature, has access to every cell and every part of a cell in the body, suggesting that it could exert effects on blood immune cells. Probably the effects of melatonin on the immune cells, may be by indirect influence on the synthesis and release the other hormones or cytokine or by direct action on phagocytosis and phagocytic bichemical process (Liebmann et al., 1997; Rodriguez et al., 1999).
The bactericidal activity is probably due to the activation of superoxide anion-dependent killing mechanism operating on the cell surface (Asad, 1994). There is considerable evidence that phagocytes rapidly increase oxygen consumption during phagocytosis or upon stimulation of their with a variety of agents (Badwey, et al, 1983).
The products of oxygen reduction and excitation have been implicated in the destruction of bacteria, yeast, viruses and mycoplasmas by phagocytes, constituting key components in the microbicidal mechanisms of phagocytes (Babior, 1984; Badwey, et al, 1983; Honorio-França, et al, 1997).
Some studies have shown that melatonin has an antioxidant effect which includes the scavenging of highly reactive oxygen free radicals and there is evidence that melatonin may protect the effects of free radical-induced neuronal damage (Sandyk, 1990; Reiter,1993; Tan, 1994; Rodriguez et al., 1999; Keplac et al, 2005; . Sudnikovich et al, 2007)., whereas others demonstrated a prooxidant effect (Ianãs, et al., 1991) and probably acts amplifying cellular activation reactions (Fjaerli et al., 1999).
In the present study we investigated the chronoimmunomodulation effect of melatonin on superoxide release and the bactericidal activity by blood phagocytes.
Materials And Methods
Blood Cell Separation: Blood samples were collected from 51 volunteer donors 18 to 35 years of age whose
Bactericidal Index = (1 - NT/NO) x 100
NT = number of colony forming units at times 30, 60 and 120 minutes after phagocytosis
NO = number of colony forming units at time 0
Melatonin Effect On Blood Phagocytes Superoxide realease
MN or PMN blood phagocytes are able to spontaneous superoxide release. When we analyzed superoxide release by phagocytes stimulated with PMA, we observed that PMA enhanced superoxide release over the spontaneous release in both kind of cells (Table 1).
Melatonin stimulated enhance in superoxide release from either PMN or MN phagocytes. The effect was higher in PMN phagocytes, where melatonin effect was equivalent to PMA (Table 1).
PMN phagocytes stimulated with melatonin showed superoxide release at levels equivalent to those of PMA-stimulated phagocytes (Table 1).
*p< 0.001 comparing superoxide release the groups of phagocytes treated with the group of phagocytes without bacteria.
Melatonin Is Able to Stimulate Superoxide Anion Release by Blood Phagocytes in the Presence of EPEC.
MN and PMN phagocytes in the presence of bacteria increase in superoxide anion release compared with spontaneous release (Figure 1).
MN blood phagocytes stimulated with melatonin in the presence of bacteria showed enhance release superoxide anion when compared with spontaneous release and levels equivalent to those of bacteria stimulated cells (Figure 1). PMN phagocytes stimulated with melatonin in the presence of bacteria showed increase superoxide release when compared with spontaneous release and with the cells only stimulated with bacteria (Figure 1)
Blood phagocytes showed bactericidal activity even in the absence of melatonin, although this activity was lower than observed when the cells were stimulated with melatonin (Table 2).
The bactericidal activity of MN phagocytes stimulated with melatonin was time dependent. The highest bactericidal index was observed over the period of 60 and 120 minutes of incubation. PMN phagocytes stimulated with melatonin independently of the time, showed increased in the bactericidal activity (Table 2).
*p<0.001 comparing the melatonin-treated group with the untreated group for the same phagocyte.
†p<0.001 comparing the values of each incubation time with the preceding time in the same group.
Our results demonstrate that blood phagocytes stimulated with melatonin are able to kill EPEC, supporting the idea that this hormone is able to stimulate microbicidal activity by blood phagocytes.
The modulatory role of hormones in the regulation of immunity and the pathogenesis of infection has been the focus of many studies (Stanisz et al, 1994). The evidence that melatonin is part of the intrinsic regulation of the immune cells (Liebmann et al., 1997). Studies indicate that neurohormones exert immunomodulatotory effects (Pierpaoli & Maestroni, 1987; Besedovsky & Rey, 1996) and have showed that melatonin hormone probably acts in the body oxidative processes (Ianãs, et al., 1991). Melatonin is an endogenous neurohormone produced by the pineal gland in mammals, and its beneficial action has been linked to its ability to scavenge different free radicals and increase the antioxidant activity of enzymes (Keplac et al, 2005; . Sudnikovich et al, 2007; Pandi-Perumal et al, 2008). Many studies have postulated that melatonin has a stimulatory action on the immune cells (Cutolo et al, 1999; Skwarlo-Sonta , 2003; Pawlak et al, 2005).
In the present study, melatonin showed an chronoimmunomodulatory effect on blood phagocytes
In addition, the effects of melatonin on the immune cells, may be a direct action of on phagocytosis and the phagocytic bichemical process or an indirect acts on the synthesis and release the others hormones or cytokine (Liebmann et al., 1997, Rodriguez et al., 1999; Pandi-Perumal et al, 2008).
At this time , we do not know why melatonin induces enhanced superoxide release by PMN cells compared to MN cells. In response to infections, PMN and MN phagocytes (Babior,1984; Forman et al, 1986; Honorio-França et al, 1997) engage in the respiratory burst as a host cell-mediated immune reaction (Babior,1984).
During oxidative stress, cells generate high contents of superoxide radicals (Rodriguez et al, 2004). Free radical generation has been reported as an important mechanism for body protection in infectious processes, mainly intestinal infections (Honorio-França et al,1997; Honorio-França et al, 2001; França-Botelho et al, 2006).This microbicidal activity may be considered one of first mechanisms in host defense against bacteria and other microorganisms.
In the present study we demonstrated that increased superoxide release by blood phagocytes stimulated with melatonin has an effect on bactericidal activity.
Blood phagocytes showed bactericidal activity when stimulated with melatonin. The highest bactericidal index was observed for phagocytes that were incubated simultaneously with EPEC and melatonin. Ours results suggest that melatonin hormone acts more quickly in PMN phagocytes. The bactericidal activity these phagocytes was similar in every incubation time. By the other hand, the bactericidal activity of MN phagocytes stimulated with melatonin was dependent of time. The effects of melatonin hormone to MN phagocytes only were observed after 60 minutes the incubation. The results suggest that probably activation of microbicidal mechanism, via melatonin, to blood phagocytes may be different. These results support the idea that superoxide is important in microbicidal mechanisms. It is still not known if melatonin activity is only linked to superoxide release. Interaction between melatonin and other free radicals may possibly be involved in microbicidal mechanisms.
This work support the idea that interactions between melatonin and blood phagocytes may induce superoxide release and increased bactericidal activity, resulting in an additional mechanism of protection against infections. In conclusion, we may state that these results support the hypothesis of pineal involvement through its main hormone melatonin as a possible chronoimmunomodulator of microbicidal processes.
We wish acknowledge the Conselho Nacional Pesquisa (CNPq) and the Post Graduated Program of São Paulo State University (UNESP), São Paulo, Brazil.