Carmen Barriga and Ana Beatriz Rodriguez
Department of Animal Physiology, Faculty of Science, University of Extremadura 

https://doi.org/10.36866/pn.47.20

Phagocytosis is an important element of the non-specific immune response and represents a fundamental mechanism of defence against infection. The leukocytes responsible for carrying out the process are attracted to microorganisms by the chemotactic substances that the latter release. They engulf their target (phagocytosis) and then destroy it by the action of enzymes which form oxygen-derived free radicals by means of a series of oxidation-reduction reactions which lead to what is known as the “respiratory burst”. In this process, various chemically aggressive species are formed, such as superoxide anions, hydrogen peroxide, or hydroxyl radicals. Their function is to injure and eventually destroy the microorganism.

The presence of free radicals in these phagocytes is beneficial for the organism, since it is thanks to their formation within those cells that pathogenic microorganisms are destroyed – an effective adaptation and solid defence adopted by the organism in its natural habitat. Evidently, a lowering of free radical production in phagocytes, or their neutralisation by antioxidants before these cells can do their work of destroying antigens, would hardly represent an immunological advantage. What would really be an advantage would be if once the radicals had fulfilled their goal they were then sequestered and/or eliminated from the phagocytes, as this would have the effect of guaranteeing the integrity of these important cells.

In recent years, many reports have shown that melatonin is a broad-spectrum antioxidant due to its ability to scavenge free radicals and to stimulate antioxidant enzymes (*1) (Tan et al. 2000). We have observed that, in ring dove (Streptopelia risoria) heterophils, pharmacological concentrations of melatonin (23 x 106 pg/ml) reduce super-oxide anion levels by modulating the activity of the enzyme superoxide dismutase, and at the same time induce a marked decline in lipid peroxidation (*2) (Rodríguez et al. 1999).

In light of these results, we asked ourselves the following question: Could melatonin both favour the phagocytic activity of heterophils, and subsequently neutralise the free radicals formed in the phagocytes to avoid causing them cellular damage? With this in mind, we performed an in vitro study, evaluating the effect of different incubation times with the hormone melatonin on phagocytosis, and the formation of superoxide anion levels in heterophils after the ingestion of microorganisms. We used the diurnal (50 pg/ml) and nocturnal (300 pg/ml) physiological concentrations of melatonin that had previously been detected in our avian model, ring dove (*3) (Rodríguez et al. 1999). We also used pharmacological concentrations of 23 x 106 pg/ml melatonin, a value that had previously been determined as highly effective in the modulation of the phagocytic process and the free radical levels following phagocytosis (*2) (Rodríguez et al. 1999). The phagocytes, in this case heterophils, were isolated from ring dove blood by brachial vein puncture followed by density gradient centrifuging. We used Candida albicans as the antigen to be ingested in evalu-ating the capacity for ingestion, destruction, and free radical formation. We determined the phagocytic index (no. of candidae phagocytosed in 100 heterophils), the candidicide power (no. of candidae killed of the total phagocytosed), and the superoxide anion production following the inges-tion by the heterophils, using the nitroblue tetrazolium reduction tech-nique. The determinations were made after 30 and 60 min of incubation.

Figure 1 gives a summary for the different tests carried out in this study of the melatonin concentrations that were found to be significant with respect to the control (free of hormone), and to the diurnal melatonin concentration (50 pg/ml). We observed that, in the presence of nocturnal and pharmacological concentrations of melatonin, the antigen-ingesting capacity of the heterophils was stimulated due to their greater efficacy in engulfing and phagocytosing antigens. Both melatonin concentrations also stimulated the candida-killing capacity (candidicide power) of the heterophils at both the shorter (30 min) and longer (60 min) incubation times.

With respect to the superoxide anion levels formed after ingestion in the heterophils incubated with melatonin, there was a clear decline in these levels following 30 min incubation of the heterophils with all three concentrations (diurnal, nocturnal, or pharmacological). The lowest superoxide anion levels were obtained after 60 min incubation and at the maximum (nocturnal) physiological and pharmacological concentrations, with the latter concentration giving the lowest levels of the free radical. Therefore, as indicated by the results of the 60 min incubation trials, the effect of melatonin on phagocyte oxidative metabolism seems to be concentration and time dependent. The finding that at greater hormone concentrations and longer incubation times melatonin has a greater effect on the suppression of superoxide anion levels could be explained on the basis of the possibility that, at short incubation times, the hormone probably acts alone when the other antioxidant mechanisms of the phagocytes are only partially activated, while at longer times the results may point to a sum of the effects of the melatonin and of other enzyme systems and antioxidant cellular components (catalase, superoxide dismutase, vitamins C and D, glutathione, etc.) which protect the cells against the activated forms of oxygen deriving from the respiratory burst.

It is therefore of interest that extraction of the pineal gland in the ring dove leads to a rise in superoxide anion levels (*4) (Rodríguez & Lea, 1994), and these levels are observed to fall following in vitro incubation with melatonin at pharmacological concentrations using inert particles as antigen to be ingested (*5) (Rodríguez et al. 1997). The present findings also complement the negative correlation that we have recently observed between serum melatonin concentrations and superoxide anion levels (*3) (Rodríguez et al. 1999). Also, as was noted above, incubation of ring dove heterophils with the pharma-cological concentration of the hormone (23 x 106 pg/ml) led to a modulation of different biochemical parameters, all related to the respiratory burst, such as the superoxide dismutase activity and lipid peroxidation.

This is thus corroboration that melatonin in phagocytes behaves as an antioxidant in vivo and as a good scavenger in vitro of both free radicals and non-free radicals alike6 (Reiter et al. 2000). In view of the present results, we can conclude that melatonin favours heterophil phagocytic activity at the same time as neutralising superoxide anion levels when the hormone acts at the greatest physiological concentration (which is attained during the hours of darkness) or at pharmacological concentrations, with its effectiveness increasing with greater incubation time.

*REFERENCES

1: Tan, DX, Chen, LD, Poeggeler, B, Manchester, LC & Reiter, RJ (1993). Melatonin: a potent endogenous hydroxyl radical scavenger. Endocrine Journal. 1, 57-60.

2: Rodriguez, AB, Nogales, G, Marchena, JM & Barriga, C (1999). Suppresion of both basal and antigen-induced lipid peroxidation in ring dove heterophils by melatonin. Biochemical Pharmacology. 58, 1301-1306.

3: Rodriguez, AB, Marchena, JM, Nogales, G, Duran, J & Barriga, C (1999). Correlation between circadian rhythm of melatonin and phagocytosis and superoxide anion levels in ring dove heterophils. Journal Pineal Research. 26, 35-49.

4: Rodriguez, AB & Lea, RW (1994). Effect of pinealectomy upon the nonspecific immune response of the ring dove (Streptopelia risoria). Journal of Pineal Research 16, 159-166.

5: Rodriguez, AB, Ortega, E, Lea, RW & Barriga, C (1997). Melatonin and the phagocytic processor heterophils from ring dove (Streptopelia risoria). Molecular and Cellular Biochemistry 168, 185-190.

6: Reiter, RJ, Tan, DX, Manchester, LC, Karbownik, M & Calvo, JR (2000). Pharmacology and physiology of melatonin in the reduction of oxidative stress in vivo. Biol. Signals Recept 9, 160-171.