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In Vitro Effect of the Reproductive Hormones on the Oxidative Burst Activity of Polymorphonuclear Leucocytes from Cows: A Flow Cytometric Study

2009; Wiley; Linguagem: Inglês

10.1111/j.1439-0531.2009.01519.x

1439-0531

A. Chaveiro, Fernando Moreira da Silva,

Milk Quality and Mastitis in Dairy Cows

In this study, the effect of reproductive hormones and substances with hormonal activity on the oxidative burst activity of blood polymorphonuclear leucocytes (PMN) high yielding dairy cows was evaluated. Different concentrations of: progesterone, oestradiol 17β, FSH, LH, GnRH, cortisol and PGF2α were incubated in vitro for 4 h with PMN of seven high milk yielding cows, during the period of anoestrous postpartum. Controls were run in parallel in which each hormone was replaced by its solvent. After incubation with hormones the competence of PMN to generate H2O2 was monitored by flow cytometry. A down-regulation on the oxidative burst activity of PMA-stimulated PMN was observed when cells were incubated with progesterone. Significant (p ≤ 0.001) differences between control and progesterone incubated cells were observed from 6.56 μg/ml. The same predisposition was observed when PMNs were incubated with cortisol. Besides for all concentrations employed, a decrease in the burst activity was observed, only beyond 0.19 mg/ml, statistical differences between the results obtained by the control and the cortisol incubated cells were obtained. Concerning oestradiol 17β, an increase on H2O2-production was observed when PMN were incubated with 15 pg/ml and 45 pg/ml of this steroid (p ≤ 0.05), followed by a depression of the cell’s activity when unphysiological concentrations were employed. Significant (p ≤ 0.05) differences between the obtained with the control and oestradiol 17β incubated cells were observed only in the highest concentration of oestradiol. No statistical differences were observed in the metabolic burst activity of PMN incubated with FSH, GnRH and LH when compared with the results obtained by the control. Phagocytosis and subsequent intracellular killing are the most important activities of polymorphonuclear leucocytes (PMN) against invading pathogens. Upon activation in the bloodstream, the PMN become more adhesive, allowing receptor-mediated margination and adhesion to the vasculature (Lawrence and Springer 1991; Zimmerman et al. 1992; von Andrian and Arfors 1993). Then, adherent PMN undergo shape change and crawl on the surface of the endothelial cells, followed by transendothelial migration into the extracellular compartment (Smith et al. 1989; Butcher 1993; Springer 1994; Smolen and Boxer 1995). Consequently, PMN migrate along a chemotactic gradient towards the offending stimulus to finally kill the invading agent by phagocytosis or release of granule contents and reactive oxygen metabolites (Klebanoff and Clark 1978; Zigmond 1978; Babior 1984). Considerable amount of literature indicates hormonal influences, directly or indirectly, on different immune mechanisms. Kelley (1980, 1988) reported that stress induced hormonal changes, alter the resistance of domestic animals to infectious diseases by affecting the cells of the immune system, which mostly are controlled by stimuli that are not immunogenic (Ader and Cohen 1985; Moreira da Silva et al. 1994). A significant increase in granulocyte numbers was found during pregnancy and in the luteal phase when compared with the follicular phase of the normal ovarian cycle (Northern et al. 1994; Apseloff et al. 2000; Faas et al. 2000; Bouman et al. 2001; Veenstra van Nieuwenhoven et al. 2002), suggesting a role for progesterone and oestrogen in increasing granulocyte numbers. During the periparturient period, cows are subjected to major changes in sex steroid hormone levels that may induce suppression of immune function. In the last week before parturition, plasma concentrations of 17β-oestradiol may rise steeply, peaking in the last 3 days before delivery but then falling rapidly to regain basal values directly after calving. During the last month of gestation, the high plasma concentration of progesterone declines, but a dramatic fall usually occurs only in the last 2 days before delivery. Studies develop by Moreira da Silva et al. (1998) showed a reduced metabolic neutrophil respiratory burst activity during the week before parturition, reaching the minimum 3 days after calving. As well as observed during pregnancy, in the oestrous cycle, hormonal fluctuations are observed including low progesterone and high 17β-oestradiol (E2) plasma concentrations in the follicular phase, and high progesterone and low E2, in the lutheal phase. The decrease in the burst activity is not yet fully understood, and how these changes in sex steroid hormonal concentrations relate to changes in immune response affect immune cell function remains obscure and the results published to now are not consistent. During the past decade, several researchers observed depressed bovine PMN functions near parturition at the moment that 17β-oestradiol peaks (Moreira da Silva et al. 1998; Hoeben et al. 2000), even at the neutrophil gene expression level (Madsen et al. 2004). Changes in 17β-oestradiol disturb the accurate function of the immune system (Watson and Gametchu 2001; Edwards 2005). Lamote et al. (2006) demonstrated the presence of oestrogen receptors in bovine blood PMN at the protein and mRNA level, and suggested that 17β-oestradiol influences PMN function after binding with its receptor, which initiates a specific oestrogen response (Farach-Carson and Davis 2003; Edwards 2005). Concerning neutrophil’s oxidative burst, it has been shown to be increased (Molloy et al. 2003), decreased (Békési et al. 2000) or not affected (Cassidy 2003) when incubated with these hormones. Recent studies developed by Chaveiro and Moreira da Silva (2008) showed a significant increase on the PMN’s burst activity in the oestrous period, decreasing during the lutheal phase of the oestrous cycle. In summary, it is not clear from the current literature data if changes in ovarian, hypothalamus and pituitary plasma levels contribute to the bactericidal activity of bovine. Therefore, the present investigation was designed to study whether alteration in the oxidative burst activity of PMN from high yielding dairy cows could be detected after incubation of PMN with physiological and high concentrations of FSH, LH, GnRH, progesterone and oestradiol 17β, cortisol and prostaglandin F2 alpha. Cows were in the anoestrous postpartum period, to avoid interference from endogenous physiological hormones found in cycled animals. Polymorphonuclear leucocytes’s oxidative burst was evaluated using a flow cytometric (FCM) technique by measuring the oxidation of 2′,7′ dichlorofluorescin (DCFH) to high green fluorescent 2′,7′dichlorofluorescein (DCF) by H2O2-production, after activation of cells with phorbol 12-myristate 13-acetate (PMA). All chemicals used in this study were obtained from Sigma Chemical Company (St. Louis, MO, USA) unless otherwise stated. Seven clinically healthy high yielding dairy cows of Holstein Friesian breed, in their first to third lactation (1.7 ± 0.4), among 11–25 days after calving (15.4 ± 1.8) were used in this experiment as blood donors. All cows were calved normally and showed no clinical signs of periparturient diseases. They were fed with corn and grass silage ad libitum, and concentrate according to their milk production. Water intake was provided for ad libitum. Milkings were at 7:00 hours and 19:00 hours. To confirm their post-partum anoestrus, plasma progesterone levels were evaluated by a solid-phase radioimmunoassay (Diagnostic Products Corp., Los Angeles, CA, USA) as previously described (Robinson et al. 1989) and validated for bovine plasma. The detection limit is 0.095 nm. As a complement, to confirm the absence of the ovary activity, cows were observed for oestrus. Blood samples (100 ml) were aseptically collected from the external jugular vein in tubes containing 10% of sodium citrate (38 g/l) as an anticoagulant, and it was processed within 30 min after collection. After being centrifuged for 20 min at 1000 × g, the plasma was recuperated and frozen at −30°C for further analysis, and the remaining suspension diluted with isotonic saline solution to the original volume, it was then centrifuged for a second time at 1000 × g for 20 min. Supernatants were removed and 200 ml of H2O was added for 1 min to the rest. Isotonicity was restored by addition of 100 ml hypertonic saline solution (27 g/l NaCl). After centrifugation for 15 min at 1000 × g the pellet was washed twice in 10 ml of phosphate buffer saline of Dulbecco’s (PBS) (Gibco BRL, Paisley, UK) and centrifuged each time for 15 min (1000 × g). The final cell pellet was suspended in 1 ml of PBS; cells were counted with an electronic cell counter (Coulter Counter ZF, Coulter Electronics Ltd, 3RH Luton, UK) and diluted with PBS at a concentration of 107 cells/ml. To minimize cell clumping, tubes and reagents were kept in ice throughout the procedure. Just before incubation with the hormones, cells were suspended with PBS supplemented with 2% of bovine serum albumin (BSA) (Merk FR, Germany) at a concentration of 106 cells/ml. A suspension of 5 ml of PMN was incubated in the dark for 4 h in a gentle horizontal shaking water bath at 37°C with physiological and high concentrations hormones, increasing by a factor 3: Progesterone (4-pregnene-3,20-dione) (1 ng/ml to 177 μg/ml), 0.05% ethanol, Oestradiol 17β (1,3,5[10] estratriene-3,17 beta-diol) (15 pg/ml, to 2.65 μg/ml) dissolved in 0.05% sterile dimethyl sulphoxide (DMSO), FSH (from 15 ng/ml to 2.65 mg/ml), dissolved in PBS-BSA, LH (Sigma Chemicals) (from 5 ng/ml to 885 μg/ml), dissolved in PBS-BSA, GnRH (ICN Biomedical Inc., Costa Mesa, CA, USA) (From 15 pg/ml to 2.65 μg/ml), dissolved in PBS-BSA, Cortisol (from 10 ng/ml to 0.59 mg/ml) prepared in 0.05% DMSO, Prostaglandin F2α (Pitman-Moore, Inc., Mundelein, IL, USA) (from 7.6 pg/ml to 0.45 μg/ml) prepared in PBS-BSA, Controls were run in parallel in which each hormone was replaced by the solvent. After incubation with hormones or the solvents, PMN’s oxidative burst was evaluated with a flow cytometer FACScan™ (FCM) (Becton Dickinson, San Jose, CA, USA), equipped with a 15 mW argon Laser, by measuring the oxidation of DCFH to the high green fluorescent DCF by H2O2 production, essentially according to a previously published method by Chaveiro and Moreira da Silva (2008). Briefly, DCFH oxidation was evaluated by incubating, in the dark, the suspension of 5 ml of PMN with 5 μm (final concentration) of DCFH-DA (Eastman Kodak Company, Rochester, NY. USA) prepared in DMSO in the horizontal shaking water bath at 37°C for 15 min. Afterwards, PMA in DMSO was added (final concentration = 0.1 μm) and the incubation was continued for a further 15 min before FCM measurements. Cell population was separated on the basis of the forward-angle light scatter (FALS) and the side angle light scatter, and a bitmap was drawn around the PMN. For each measurement, the green DCF fluorescence intensity of the gated PMN population was analysed on a constant number of 3000 PMN. Viability of leucocytes was measured in a Bürker chamber by exclusion of 0.2% trypan blue in PBS. Viability tests were made immediately after isolation and after H2O2 measurements, for each concentration of hormones and for controls. All statistical procedures were computed on a microcomputer package (Statistix® NH Analytical Software; Roseville, MN, USA) according to Snedecor and Cochran (1968). A mean and a standard error of the mean (SEM) were determined for each parameter considered. A paired t-test was computed between the results obtained for each tested hormone as well as the results obtained in the respective controls. For the results of progesterone incubated leucocytes, a linear regression was computed in which the different concentrations of this steroid were considered as the independent variable, and the difference between the results obtained in the control and progesterone treated cells were considered as the dependent variable. Different cows were used as a covariant. Measurements of progesterone (average of 4.95 ± 0.97 ng/ml) confirmed that all the employed cows were in the anoestrous postpartum. The results, shown in Fig. 1, indicated that for all employed concentrations of progesterone, a down regulation of the H2O2-production was observed, decreasing to very low levels in the highest concentrations employed, when compared with the control. The higher was the concentration of this steroid in the incubation medium, the lower was the oxidative burst activity of PMN. Statistical differences (p ≤ 0.001) between the results of the control and progesterone incubated cells were observed from 6.56 μg/ml. Incubation of leucocytes with oestradiol 17β produced two different reactions on the oxidative burst activity of PMN. With physiological concentrations of this steroid (15 and 45 pg/ml), an increase (p ≤ 0.05) on the oxidative burst activity of PMN were observed, when compared with the control measurements. For the unphysiological concentrations employed, a down regulation of the activity of cells was observed (Fig. 2). As observed with Progesterone treated cells, the higher was the concentration of oestradiol in the incubation medium, the lower was the activity of the cells. For the control measurements, there was no significant difference in the PMN’s H2O2-production for all concentrations of DMSO employed. Significant (p ≤ 0.05) differences between the results obtained with the control and oestradiol 17β incubated cells were observed only in the highest concentration of oestradiol 17β employed. Effect of progesterone on the oxidative burst activity of polymorphonuclear leucocytes (PMN) isolated from cows in the anoestrous postpartum. Cells were incubated with progesterone (b) or DMSO (a) for 4 h. Afterwards, the PMN’s oxidative burst activity was monitored by flow cytometry (see Materials and Methods for details). Each point represents the mean ± SEM of seven cows, (*p ≤ 0.001) Effect of oestradiol 17β on the oxidative burst activity of polymorphonuclear leucocytes (b) isolated from cows in the anoestrous postpartum. Controls were run in parallel, in which the steroid was replaced by the solvent (a). Each point represents the mean ± SEM of seven cows, (*p ≤ 0.05) For the cortisol and prostaglandin F2α, it was observed that prostaglandin F2α did not interfere with the metabolic burst activity of the PMN. After H2O2 measurements, no statistical differences were found between the results of the control PMN and the PMN incubated with this hormone. Nevertheless, for the cortisol, a decrease on PMN’s H2O2 production was observed when this steroid was present in the incubation medium (Fig. 3). Besides, for all concentrations employed, a decrease in the burst activity was observed, only beyond 0.19 mg/ml, statistical differences between the results obtained by the control and the cortisol incubated cells were obtained. Effect of cortisol on the oxidative burst activity as measured by flow cytometry. Cells were incubated with cortisol (b) or DMSO (a) for 4 h. Afterwards, the polymorphonuclear leucocytes’s oxidative burst was monitored by flow cytometry (see Materials and Methods for details). Each point represents the mean ± SEM of seven cows, (*p ≤ 0.05) As far as hypothalamus/hypophysis hormones were concerned, no statistical differences were observed between the metabolic burst activity of the cells incubated with these hormones and the respective controls. Hormones did not appear to be harmful for the cells, even in the highest concentrations employed, as no statistical differences were observed between cell’s viability of controls and hormones incubated cells for both diluters employed. Studies on the relation between the immune system and reproduction/reproductive factors are important for several reasons, being the most important that, as known, immune responses regulate various reproductive processes, so that deviations from normal immune responses may interfere with fertility. Concerning oestradiol 17β, this steroid hormone was shown to have a dual effect on the PMN oxidative burst activity. In physiological concentrations, an increase of the microbicidal activity of PMN was observed, whereas in unphysiological concentrations, cells presented a down regulation for H2O2-production after the phagocytosis stimulus. The mechanism of these actions is not clear. It has been suggested that during phagocytosis stimulus, in association with changes in cell granules an oxidase is activated leading to increased production of NADP+ and stimulation of the pentose pathway for glucose oxidation (Zatti et al. 1965). When added in high concentrations, in vitro, steroids inhibit various oxidative enzyme systems, perhaps by allosteric effects (Williams-Ashman and Liao 1964). Research developed by Bodel et al. (1972) show that steroids also suppressed the glucose oxidation after phagocytosis. Myeloperoxidase (MPO), as well as H2O2, forms a potent antimicrobial system effective against variety of micro-organisms, appearing to contribute significantly to the antimicrobial activity of PMN. Klebanoff (1979) observed that high concentrations of steroids had the capacity to modify the activity of MPO-mediated microbicidal system of neutrophils, decreasing the microbicidal activity of PMN. Bodel et al. (1972), working with high concentrations of oestradiol 17β, related that concentrations of this steroid 104 times greater than the physiological concentration inhibit the oxidative metabolism of human neutrophil in vitro. Nevertheless, Jacobs et al. (1973) related that oestradiol and oestrone in physiological concentrations increased the bactericidal activity of guinea pig PMN. Oestradiol may have thus two separate effects which are unrelated. However, as the relation between oxidative activity-induced changes in PMN and the metabolic consequences of H2O2-production is not clear, the relation between the two actions of oestradiol effect in our system is also not known. As observed with unphysiological concentrations of oestradiol, progesterone did have a significant decrease on the oxidative burst activity of the PMN. In this study, the effect of progesterone on the activity of cells was further emphasised by the comparable effects observed with oestradiol 17β. Lamote et al. (2006) demonstrated the presence of oestrogen receptors in bovine blood PMN at the protein and mRNA level and suggested that, by binding of 17β-oestradiol to its receptor causes an immunomodulating effect on different PMN functions. The previous studies have shown that high concentrations of progesterone, in vitro, inhibit human activity of lymphocyte blastogenesis (Shiff et al. 1975; Clemens et al. 1979). It has been postulated that the ability of progesterone in high concentrations to inhibit lymphocyte reactivity is very important for maintenance of pregnancy (Clemens et al. 1979). In most species, the trophoblast cells of the placenta produce progesterone, resulting in a high local concentration of progesterone at the maternal-foetal interface. It can thus be admitted that the ability of progesterone to depress the immune system activity can also be a mechanism of maintenance of pregnancy. Another point to consider is that oestradiol and progesterone are present concurrently in cow blood in varying concentrations: the ratio of the two hormones may be an important determinant of the effect on the immune system. In a previous research in which the metabolic burst activity of bovine PMN was evaluated during the oestrous cycle, a significantly increase of the metabolic oxidative burst was observed in the oestrous period (Chaveiro and Moreira da Silva 2008). A fall was then observed, in which a steady state was observed during the lutheinic phase of the oestrous cycle, reaching the minimum value when progesterone levels were maximum. Although prostaglandin F2 α and cortisol also augment during the oestrous period, the results of the present experiment demonstrate that the increase of the metabolic burst activity of PMN can not be associated to the increase of these hormones. In studies developed by Fox and Heald (1981), it was observed that physiological concentrations of cortisol enhance phagocytosis. However, as in this study, the killing capacity in physiological cortisol treated PMN was not different from control, it can be concluded that although phagocytosis may be enhanced with physiological concentrations of cortisol, the ultimate capability of the PMN to reduce bacterial numbers is not affected. This is in agreement with Smith (1977) and Ignarro (1977), who demonstrated that physiological concentrations of glucocorticoids had no effect on PMN Iysossomal enzyme release in vitro. As viability lost was not observed, the decrease of activity observed on the cortisol incubated cells can not be attributed to an increase of dead cells. From our study, it can be concluded that pharmacological doses of glucocorticoids will have an adverse effect on the bactericidal function of the bovine PMN, while prostaglandin F2α does not influence this immune function. In conclusion, it can be postulated that progesterone decreases the metabolic burst activity in bovine PMN booth in physiological and unphysiological concentrations, while with oestradiol 17β; a decrease is only observed with unphysiological concentrations. As far as hypothalamus/hypophysis hormones are concerned, no interference of the burst activity of PMN is observed when incubated either with physiological or with unphysiological concentrations of these hormones. As leucocytes are important mediators of inflammation, endogenous hormones may similarly modify the inflammatory response in diseases. The first author is supported by DRCT (Direcção para a Ciência e Tecnologia, Portugal). The authors express their appreciation to the technician Sofia Pires for her excellent assistance. F Moreira da Silva developed the study design and as project leader supervised and critically revised the manuscript drafting. A Chaveiro was responsible for the data acquisition and analysis and also drafted the manuscript for publication.