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  • 1. Rainbow trout (Oncorhynchus mykiss) monocytes in culture with cortisol: Expression of CD18, PU.1/Spi-1, TNF-α, IL-1β and Glucocorticoid Receptor (GR) Javier Infante Lourido September 2007 Tutors: Cellular biology, Physiology and Immunology Department Faculty of Biosciences Lluís Tort Autonomous University of Barcelona Simon Mackenzie
  • 2. INDEX I. ABSTRACT................................................................................................................................2 1. INTRODUCTION.......................................................................................................................3 2. OBJECTIVES..........................................................................................................................10 3. MATERIAL AND METHODS..................................................................................................11 4. RESULTS AND DISSCUSION................................................................................................15 4.1 Cellular culture under different conditions with cortisol....................................15 4.2 CD18.........................................................................................................................17 4.3 Glucocorticoid receptor (GR)................................................................................19 α β 4.4 Cytokines (TNF-α and IL-1β)..................................................................................22 4.5 PU.1 (Spi-1)..............................................................................................................26 5. CONCLUSIONS......................................................................................................................28 6. REFERENCES........................................................................................................................30 ACKNOWLEDGMENTS............................................................................................................ 36 1
  • 3. I. ABSTRACT In this study primary cultures of rainbow trout monocytes/macrophages were treated with the corticosteroid hormone, cortisol, simulating physiological concentrations during the acute stress response. Cortisol at high concentrations (300ng/ml) affects the capacity of monocytes to adhere to the culture plate surface therefore decreasing the number of macrophages. Moreover, expression studies with one day culture monocytes show that cortisol in high concentrations downregulates the expression of the inflammatory cytokines, TNF-α and IL-1β and the transcription factor PU.1, which may indicate the activation and reprogramming of homeostatic energy saving processes in the cultured cells. This may mimic a glucocorticiod- induced acute stress response in vitro. However, the cellular response to conditions mimicking chronic stress, 30ng/ml cortisol, is not observed indicating that such processes are only activated at high cortisol concentrations. These results suggest that the induction of differential regulatory mechanisms by cortisol in in vitro cultured monocytes/macrophages are concentration and context-dependent (differentiation status of cells). This study provides the basis of an in vitro cell culture model to investigate immune-endocrine interactions at the level of the transcriptome. 2
  • 4. 1. INTRODUCTION The characteristics of immune system of fishes are the results of the characteristics of the environment were they live and the evolution of the immune system. The aquatic medium allows easy transport and growth of microorganisms and this results in a high concentration of 24 prokaryotic cells in oceans, estimated in 10 prokaryotic cells in oceans (Press and Evensen, 10 1999), and are very important in marine biomass, and viruses may account for 10 in aquatic habitats (Wilhelm and Suttle, 1999). Fish immune system Fish are the most diverse group in vertebrates, and are considered a crossroad between the innate and adaptive immunity (Tort et al., 2003) inside a gradual transition from the innate immune system of invertebrates to the recombinatorial immune system of higher vertebrates (Khalturin et al., 2004). Fig 1: Evolution of inmune system (Tort et al., 2004). Adaptive immunity requires an induction by the antigen, the differentiation of self from non-self, the specificity towards the antigen and an immunological memory. A clonally diverse anticipatory repertoire in which lymphocyte bears a unique antigen receptor is the central feature of the adaptive immune response. All jawed vertebrates, assemble their antigen- 3
  • 5. receptor genes through recombinatorial rearrangement of different immunoglobulin or T cell receptor gene segments (Cooper and Alder, 2006) and recently it has been discovered that agnathans also have an adaptive immune system that is based on recombinatorial assembly of a different type of modular genetic units to generate a highly diverse repertoire of lymphocytes, each with a unique anticipatory receptor (Alder et al., 2005; Cooper and Alder, 2006; Pancer et al., 2004). These facts suggest that lymphocyte progenitors evolved in the most basal vertebrates or in a protochordate ancestor (Cooper and Alder, 2006). Innate responses are induced by an antigen and differentiates the own from the strange, witch allows to the innate immune system to detect the presence of an infectious microbe, but also to determine the type of the infecting pathogen (Medzhitov and Janeway, 2000). The binding between TLRs (Toll-like receptors) and PAMPs (pathogen associated molecular patterns) is the basis of this differentiation and triggers acute inflammation through production of reactive oxygen and glucocorticoids and expression of cytokines and their receptors and chemokines and their receptors (Stavitsky, 2007). The majority of pluricellular organisms are able to maintain their integrity thanks to an innate immune system based on cell phagocytosis and secretion of soluble antimicrobial molecules (Tort et al., 2003). This innate immunity system is divided into physical and chemical barriers, cellular and humoral components (Magnadottir, 2006). Physical barriers, like scales, mucous surfaces of skin and the epidermis, are the first line of defence against pathogens (Bernstein et al., 1996; Harding et al., 1990). Mucus possesses different substances including antibacterial peptides, lysozyme, lectins and proteases (Ellis, 2001) and is very important as a first barrier in trout (Haynes and McKinney, 1991; Rast and Litman, 1994) and in other teleost species(McKinney et al., 1986; Pettey and McKinney, 1988; Walsh and Luer, 1998). Humoral parameters include growth inhibitors, lytic enzymes, agglutinins, opsonins, interferon, lectins, cytokines, chemokines, bacterial peptides and complement system, which is a multicomponent system that involves 20-30 plasma proteins and several receptors (Magnadottir, 2006; Magnadottir et al., 2005). Cellular components of innate immune system involve many cells, including phagocytic cells, like granulocytes, mainly neutrophils, monocytes/macrophages, and including B lymphocytes, which recently has been discovered that are capable of efficient phagocytosis (Li et al., 2007). The macrophages: origin and functions. Macrophages perform pinocytosis and phagocytosis, and are cytotoxic against foreign cells (McKinney et al., 1986; Walsh and Luer, 1998). They differentiate from monocytes precursors after the expression of fms and L-plastin genes (Crowhurst et al., 2002). Another important gene involved in the differentiation of myeloid cells is PU.1 (SPI-1), whose concentration inside immunological stem cells determine the destination of this cells, that in 4
  • 6. conditions of high concentration of the transcription factor of PU.1 turn into macrophages and in low concentrations differentiate to B lymphocytes (DeKoter and Singh, 2000). In short, macrophages are fundamental immune cells and are fundamental in defence against pathogens, making diverse functions, like the phagocytosis, pathogen killing through respiratory burst or antigen presentation (Secombes et al., 2001). As regards to the origin of these immune cells, the thymus, kidney and spleen are the major lymphoid organs in teleosts (Zapata et al., 2006). Rainbow trout, like the other teleost species, do not have bone marrow or lymph nodes (Rombout et al., 2005; Zapata et al., 1995; Zwollo et al., 2005). The head kidney (pronefros, anterior kidney) is the primary site for haematopoiesis in teleosts, instead of bone marrow (Bromage et al., 2004; Fange, 1986; Rombout et al., 2005; Zapata, 1979; Zwollo et al., 2005). The trunk kidney (opisthonephros, posterior kidney) mainly consists in renal tissue (Zapata et al., 1995). Kidney is located in trout ventrally across the backbone extending from the base of the cranium (head kidney) to the caudal region (trunk kidney) (Wake, 1997). In this organ takes place the multiplication and differentiation of hematopoietic stem cells (Zapata et al., 1995). The head kidney has no renal function, does not have nephrons and it is the place where hemopoiesis occurs. The hemopoietic tissue is interdigitated with adrenal- Fig 2: Morphology and location of kidney. 5
  • 7. like tissue, while trunk kidney posses renal and immune tissues (Grassi et al., 1997; Zapata, 1990). Furthermore, head kidney in teleosts has a system of macrophages that concentrate and separate phagocytosed material, the reticuloendothelial system (Roberts, 1989). Spleen is implicated in haematopoiesis, but its role is mainly limited to erythropoiesis and thrombopoiesis (Rowley et al., 1999). In short, trout kidney is a complex organ that houses four different functions, hemopoietic, reticuloendothelial, endocrine and excretory functions (Zapata, 1990). Monocytopoiesis in teleosts usually occurs in the head kidney and/or spleen monocytes. Cells are released to the blood stream and after arrival at the place of inflammation, increase their phagocytic potential and enhance their antimicrobial activity (Reite and Evensen, 2006; Rowley et al., 1999). The mobilization of leucocytes from haematopoietic tissues to the place where the antigenic particles are present is a very important episode in the inflammation process and is essential for the development of a full immune response (Cuesta et al., 2006). Inflammatory response. Inflammation is a local response to protect the organism from pathogenic invasions, an essential component of pathogen clearance, resolution of sepsis and repair of injury and many diseases, and it is controlled by at least two different general mechanisms (Tracey, 2002) During the more rapid, local, neural regulation, pro-inflammatory cytokines (IL-1β, IL-18, TNF-α) are released. But inflammatory responses can be more dangerous than the original inciting stimulus, so the activation is inhibited or limited by stimulation of the acetylcholine-cholinergic pathway through the vagus nerve (Tracey, 2002). Anti-inflammatory cytokines, such as IL-10 and transforming growth factor-β (TGF-β) specifically inhibit the release of TNF and other proinflammatory mediators. Glucocorticoids, adrenaline, α-melanocyte-stimulating hormone (α- MSH) and other classical stress hormones inhibit cytokine synthesis and intracellular transduction. The conjunction of the signals that arrive to the nervous system through sensory systems is integrated in the brain and triggers appropriated responses, controlling inflammatory mechanisms. Adhesion molecules and migration process. A very important success in immune is the migration of immune cells towards the inflamed tissue, process in which adhesion molecules play a very important role. Inside this group of molecules three fundamental classes are present: Selectins, Integrins and immunoglobulin superfamily, and other like cadherins, hyaluronate receptors or the receptor protein tyrosine phosphatases (Freemont and Hoyland, 1996). 6
  • 8. Selectins are proteins that bind their intracellular domain to carbohydrates. There are three major groups of selectins: L-selectin, E-selectin and P selectin, which are expressed mainly in leucocytes, endothelial cells and platelets and endothelium respectively (Bevilacqua and Nelson, 1993; Lasky, 1992). Integrins are heterodimeric transmembrane glycoproteins consisting of non-covalently associated α and β subunits and the distinctive in leucocytes is the expression of integrins containing a β2 (CD18) chain paired with one of α chains (Harris et al., 2000). Immunoglobulin superfamily is a large and diverse family of molecules that have one or more immunoglobulin- like domains (Springer, 1990a) and include among others ICAMs (intercellular adhesion molecules) VCAMs (vascular cell adhesion molecules), LFA-2, LFA-3 or MacCAM-1. Adhesion molecules determine the leukocyte egress from blood to the extravascular matrix, process that can be divided into four sequential steps which include adhesion and signalling events (Pitzalis et al., 2002). In the first step (the transient leukocyte adhesion) leukocytes leave the central stream and start rolling along the vessel walls. The cell adhesion molecules (CAMs) that are involved in this step are mainly the selectins. L-selectin is expressed mainly in leukocytes, whereas E and P-selectin are expressed by endothelial cells and platelets, respectively. Usually, rolling cells detach from the vessel walls under the influence of shear stress, but during inflammatory reactions many more leukocytes arrest onto the endothelium (Atherton and Born, 1972). The arrest of leukocytes onto epithelial cells is principally mediated by integrins, particularly β1 (VLA- 4) and β2 (LFA-1 and MAC-1) and their respective ligands in epithelial cells, VCAM-1 and ICAM- 1, ICAM-2 and ICAM-3 (Hogg, 1993; Springer, 1990b). The second step in this process is the cell activation. Integrins are normally expressed in a low-avidity and non functional state to prevent random adhesion in the circulation (Hogg and Landis, 1993; Springer, 1990a) but can be activated by inflammatory mediators, particularly proinflamatory chemokines (Baggiolini et al., 1997; Schall and Bacon, 1994). Activated integrins can subsequently mediate the third step of leukocyte strong adhesion to endothelial cells, forming bonds with specific endothelial receptors. Finally these interactions make possible the fourth step, diapedesis, the transendothelial migration to the inflamed tissues (Pitzalis et al., 2002), where macrophages perform their functions against the pathogenic particles. 7
  • 9. Immune responses to stress Stress is a very important concept in physiology but is difficult to define and in fish has been studied not only for theoretical interest, but also for the economic importance of intensive fish culture. It could be defined as a real or symbolic (perception of a potential threat) state produced by an environmental or other factor that extends the adaptive responses beyond its normal physiological range (Brett, 1958). From a physiologic point of view, a specific activation of stress system is useful to the animal, because it allows restoring homeopathic balance of inner medium altered by a stressor. The animal diverts energy from normal metabolism to adapt physiological systems to the stressor. However, if the stress situation continues, the adaptive value could be lost and originate problems to the individual. In these circumstances (maladaptation), stress could originate problems in growth, reproduction, osmorregulation and immunity. Stress produces a series of alterations, which are classified in three groups (Wedemeyer and McLeay, 1981): Primary alterations, like the release of ACTH from adenohypophysis or stress hormones (catecholamines and corticosteroids) from the interrenal. Secondary alterations (physiological), like blood chemistry and hematological changes (hyperglycemia, hyperlacticemia, hypochloremia, leucopenia, reduced blood clotting time), tissue changes (depletion of liver glycogen, and inter-renal vitamin C), metabolic changes (negative nitrogen balance and oxygen debt) or in the diuresis (blood electrolyte loss). Tertiary effects affect to whole animal, like the impaired growth, parr-smolt transformation, spawning success, migratory behaviour or increased disease incidence (Ottaviani and Franceschi, 1996). Generally stress induces immunosuppresion; however different factors, like the nature of the stressor and organism, type, intensity and duration of stress generate different responses. Even so, it is clear that stressors of long or medium duration generate immunosuppresion and maladaptation, especially if the stressor is chronic or repeated (Tort et al., 2004). The investigation of the evolutionary bases of stress argues that the basic mechanisms and molecules involved in stress, like CRH, ACTH, glucocorticoids, biogenic amines and cytokines are fundamentally similar and well preserved throughout evolution (Ottaviani and Franceschi, 1996). One of the most important changes occurring in stress conditions is the activation of hormonal axis, increasing plasma levels of cortisol (Herbert and Cohen, 1993). 8
  • 10. The role of cortisol on immune response The perception of a stressor activates the Central Nervous System (CNS), specifically in the hypothalamus, where are the two mayor axis, the sympatico-chromafin axis, that innervates chromafin cells in head kidney, involved in catecholamines synthesis, and the HPI (Hypothalamus-Pituitary-Interrenal) axis, that through the synthesis of two hormones, CRH (corticotrophin-releasing hormone) and TSH (Thyroid-releasing hormone) stimulates the synthesis and liberation of cortisol in interrenal cells of head kidney. Cortisol is the major stress steroid and plays multiple roles in organisms, like the catabolic action of this glucocorticoid, that allow to the fish increases energetic availability to maintain homeostasis (Schreck, 1981). Table 1: Effects of different stressors on immune indicators. Values are calculated as the reduction in percentage of each indicator compared to respective controls, which were set at 100%. PI = Phagocytic index. (Tort, 2004) In short, cortisol causes multiple effects in the immune system (Table 1), but in seems that cortisol could even act as an activator of the early immune response and thus it could help to maintain key defence mechanisms, whereas other actions of cortisol at long term normally would end in immunosuppression (Tort et al., 2004). In this work we study the effects of cortisol on several indicators of the immune system, particularly in adhesion and cellular differentiation of monocytic cells. To this purpose were studied different molecules: the adhesion molecule CD-18, the transcription factor PU.1, cortisol receptor (CR) and cytokines TNF-α and IL 1-β using the housekeeping gene 18S as reference. 9
  • 11. 2. OBJECTIVES The objective of this work is to determine the effect of cortisol in the expression of key molecules, as the cytokines, adhesion proteins or transcription factors. This general objective is split in the following experimental tasks: 1. Development of a primary cell culture to observe the behaviour of the macrophages under diverse treatments with cortisol. 2. To determine the expression of the different genes of specific cytokines, the TNF-α and Interleukin 1-β (IL 1-β) under conditions of high and low cortisol levels. 3. To determine the expression of the adhesion protein CD18 in conditions of high and low cortisol concentration. 4. To determine the expression of the transcription factor PU.1 (spi1) under conditions of high and low cortisol levels. 10
  • 12. 3. MATERIALS AND METHODS 2.1 Animals Rainbow trout (Oncorhynchus mykiss) were obtained from the “Truchas Jose Andrés” (Olot, Girona) and held in 150 litres tanks with recirculating freshwater at room temperature. The tanks were provided with a biofiltration system, with oxygen, salinity, pH, temperature and nitrogen compounds. The fish were subjected to 12 h light:dark cycle. The fish were fed with commercial pellets provided by “Proaqua” at a daily ration of 0.5 % of the body weight. The fish were at least 15 days under the above conditions prior experiments. 2.2 Cell culture The fish were killed by overanesthetization in 2-phenoxy-ethanol and the head kidneys were dissected and placed in sterile 200 µm cell strainer (BD Biosciences). The tissue was squeezed through the cell strainer using sterile forceps into Dulbecco`s Modified Eagle Medium (PAA laboratories) containing high glucose, 10% heat inactivated Foetal Bovine Serum (PAA laboratories) and primocin (500ng, InvivoGen). The samples were centrifuged for 1 minute at 1500 rpm (Hettich, D-78532 Tuttlingen), and the pellet was taken and resuspended in DMEM medium with FBS and primocin. This TM mixture of cells and medium were plated in 60mm of diameter plates (NUNC ) and put in an incubator (Sanyo MC0175), maintained at 15 ºC and 5% of CO2. 2.2.1 One day culture After the cells were plated with medium with FBS and Primocin we added to each plate cortisol, ethanol and/or RU-486 (cortisol antagonist) or none according to the condition in each plate. Conditions: 1. Control (nothing) 2. Ethanol (60µl in each plate). 3. Cortisol (30 ng/ml) 4. Cortisol (300 ng/ml) 5. Cortisol (300 ng/ml) + RU-486 (100ng/ml) 6. RU-486 (100ng/ml) 11
  • 13. After stimulation the plates were stored during 24 hours and then start the RNA extraction. 2.2.2 Five days culture No adhering cells were removed at 24 hours and new medium was added and the cells were incubated for an additional 4 days. After 48 hours no adhering cells were removed and the medium was replaced. At day 5 we took pictures of each culture condition to demonstrate the cortisol effects in culture. 2.3 RNA extraction Fig 3: Methodology diagram. After 24 hours of culture at 15 ºC and 5% of CO2 the medium was extracted from the plate and centrifugated, the RNA was isolated from the cells using 1ml of Tri Reagent (SIGMA) per 60 mm dish, which was added to the pellet, after to the culture plate (Fig 3) and after to a clean tube. The homogenate was stored at room temperature for 5 minutes and after addition of 0,2 ml of chloroform (SIGMA) it was mixed vigorously and samples were stored for 2-15 minutes at room temperature. The samples were then centrifuged at 12000 G for 15 min at 4 ºC. After, 12
  • 14. the aqueous phase was transferred to a clean tube, 0.5 ml of 2-propanol (SIGMA) was added and mixed. The tubes were stored for 5-10 minutes at room temperature and centrifuged at 12000 G for 8 min (4-25 ºC). The pellet was washed with 1 ml of 75% ethanol and centrifuged at 7500 G for 5 min (4-25 ºC). The RNA pellet was air dried for 5-10 minutes and after, 10 µl of DPC water was added. These samples were incubated at 55-60 ºC for 10 minutes to dissolve the RNA pellet. To measure the concentration of RNA and determine RNA quality a RNA gel was done. 2.4 Reverse transcription PCR Total RNA from cells was reverse transcribed with SuperScript III reverse transcriptase and an anchored oligo-dT primer (Biotools). Reverse transcription was performed following the next protocol: Denaturation at 95 ºC for 7 min followed by 30 cycles of: denaturation at 95 ºC for 1 min; annealing at 50 ºC for 1 min; extension at 72 ºC for 1 min 30s. The final step was an extension at 72 ºC for 5 min. The cDNA was stored al -20 ºC. 2.5 PCR reaction The conditions for PCR were as follows: denaturation for 5 min at 94 ºC followed by 30 cycles of denaturation at 94 ºC for 45 s, annealing for 45 s and extension at 72 ºC for 30 s, with a final extension at 72 ºC for 10 min. The cDNA was then used to obtain partial cDNAs of different genes using Taq DNA polymerase (Biotools). The genes used in this experiment were 18S, CD18, Cortisol Receptor and SPI1. CD18 primers, synthesized by Bonsai technologies group (Alcobendas, Madrid), were designed based on rainbow trout CD18 partial sequence data generated (Goetz et al., 2004a) (accession number CF753098). CD18 sense 5´- CAGGATTGGCTGGAGGAATA-3´and antisense 5-´ GCCTATTGTAGGCAGCTTGG-3´ primers amplified a PCR product of length 347 bp. Annealing temperature in PCR was 58 ºC. PCR Primers for PU.1 (SPI1), synthesized by Bonsai Technologies Group (Alcobendas, Madrid), were designed based on rainbow trout SPI1 (PU.1) complete sequence (AY606036) (Ribas et al, 2007). PCR temperature was 55 ºC. 13
  • 15. Cortisol receptor primers were designed by Acerete (Acerete et al., 2007), forward 5′- AGTGCTCCTGGCTGTTYCTNATG-3′ and reverse 5′-TTTCGGTAATTGGTTGCTGATGAT-3′. Annealing temperature was 55ºC. TNF-α and IL-1β primers were designed based on rainbow trout sequences (AJ277604) and (AJ223954) respectively. TNF-α Forward: 5’ GGATCCATGGAGGGGTATGCGATG 3’ and TNF-α Reverse: 5’ AAGCTTTCATAGTGCAAACACACC 3’. IL-1β Forward: 5’ GGAACCGAGTTCAAGGACAA 3’ and IL-1β Reverse: 5’ GGCTACAGGTCTGGCTTCAG 3’. PCR products were visualized on 1% agarose gels containing 0,5 µg/mL ethidium bromide. 14
  • 16. 4. RESULTS AND DISCUSSION. 4.1 Cellular culture under different conditions with cortisol A macrophage culture was performed to observe changes induced by cortisol at high and medium concentrations in macrophages. Fig 4: Pictures of 5 days culture plates in different conditions: Control, ethanol, cortisol 30 ng/ml, cortisol 300 ng/ml, cortisol 300 ng/ml plus RU-486 (100 ng/ml) and RU-486 (100 ng/ml). The results obtained (FIG 4) show that in case of cortisol at 300 ng/ml there are less number of cells than in the other plates, while after 30 ng/ml of cortisol this decrease of cell number was not observed. At the same time, RU486, a cortisol antagonist blocks the effects of cortisol in culture. The presence of a lesser number of cells after 300 ng/ml cortisol treatment could be caused by induction of apoptosis or due to a decrease of cellular adhesion molecules expression. 15
  • 17. There are diverse studies that have shown that glucocorticoids enhance cellular apoptosis (Deane et al., 2006; Walsh et al., 2002; Weyts et al., 1998a); however, other studies showed that glucocorticoids, including cortisol, have a protective role by decreasing cellular apoptosis (Deane et al., 2006; Liles et al., 1995; MacKenzie et al., 2006; Weyts et al., 1998b), which agrees with the results performed in our laboratory, where apoptosis was not observed (Mackenzie et al., personal observations). In this way, the hypothesis after observing these results is that under conditions of high concentrations of cortisol, similar to acute stress plasma cortisol concentrations, a downregulation of cellular adhesion molecules could be induced leading to a lesser number of cells. As a physiological response this could end up in an inhibition of leucocyte migration under conditions of acute stress with de purpose of saving energy to be directed to respond to the stressor and the stress situation. However, under moderate concentrations of cortisol (30 ng/ml) no noticeable changes were observed, which from a physiological point of view, could mean that under conditions of chronic stress the cellular adhesion is not modified. 16
  • 18. 4.2 CD18 Integrins are cell surface receptors that mediate cell- cell and cell-matrix interactions and bidirectional signal transduction between extracellular matrix and cytoplasm (Huhtala et al., 2005). Integrins bind to different ligands, like extracellular matrix proteins, such as collagens, laminins and fibronectin, cell surface immunoglobulin superfamily counter receptors such intercellular adhesion molecules (ICAMs) and blood proteins such as fibrinogen (Hynes, 2002). Integrins are heterodimeric glycoproteins composed by one α-chain and one β-chain and are classified in families in function of their β chain. There are at least nine different β chains (β1, β2, β3, β4, β5, β6, β7, β8, βu) which bind to Fig 5: Integrins structure. (Harris different α chains, so there are a great number of possible et al., 2000). combinations, of which 20 have been identified (Hynes, 1992; Postigo and Sanchez-Madrid, 1993). β2 chain is CD18 and this subunit binds to different α chain to form different integrins, in this sense β2 chain (CD18) binds to αL (CD11a), αM (CD11b) and αX (CD11c) and form LFA-1 Mac-1 and gp 150.95 integrins (Haynes and McKinney, 1991; Postigo and Sanchez-Madrid, 1993; Springer, 1990a). Integrins are very important in cellular adhesion and migration to inflammated tissues, particularly for leukocyte to endothelial cells (Pitzalis et al., 2002), a very important process for the transport of immune cells to inflammated tissues. Fig 6: Results of PCR reaction of expression of CD18 in different conditions: Control, ethanol, cortisol 30 ng/ml, cortisol 300 ng/ml, cortisol 300 ng/ml plus RU-486 (100 ng/ml) and RU-486 (100 ng/ml). Results obtained (Fig 6) show that there are no influence of cortisol concentration in expression of CD18 by monocytes of one day culture, at least employing PCR reaction, which it is a not a quantitative technique. 17
  • 19. These results do not support the initial theory that cortisol downregulates the expression of cell adhesion molecules (CAMs) that was explained in anterior point, although this regulation could be done on other adhesion molecules different of CD18. Nevertheless the fact that cortisol does not induce noticeable variations in the expression of this molecule does not mean that cortisol don’t have effects in cellular adhesion process, since in addition to genetic mechanisms, cortisol triggers a set of non-genetic mechanisms, which could have an important role. For example it is known that cellular adhesion molecules, including integrins, are expressed in a low-binding avidity state to inhibit random binding to endothelium, a state which quickly can change to high binding avidity (Hogg et al., 1993; Hogg and Landis, 1993). Moreover, redistribution of cell adhesion molecules in cell surface can modify the cellular adhesion process, and reorganizations are quickly possible (Hogg et al., 1993; Hogg and Landis, 1993). In this way, there are experiments that show the fast non-genetic mechanisms that modulate molecular adhesion processes, for example altering binding avidity or surface distribution, (Pitzalis et al., 2002; Smith et al., 1988) and these mechanisms could play an important role in the cellular adhesion process and could be inhibited by glucocorticoids. 18
  • 20. 4.3 Glucocorticoid Receptor (GR) Cortisol, binds to GR and the GC-GR complex moves to the nucleus where it has the capacity to bind to specific DNA sequences called “glucocorticoid response elements”, inducing the inhibition or stimulation of the relevant genes (Jewell et al., 1995; Pratt, 1993). To allow binding of the ligand, the heat shock proteins Hsp90 and Hsp70 are essential as chaperones to open the hydrophobic cleft of receptor (Morishima et al., 2000). So, the principal role of the GR is as a transacting transcription factor. This key role of GR in the cortisol action leads us to study this gene expression to help us to better understand the mechanisms of action of cortisol in fish monocytes. The GR structure and function is well established in mammals and conserved among the vertebrates analyzed (Stolte et al., 2006). The GR belongs to the nuclear receptor superfamily. This structure was predicted for the GR (Giguere et al., 1986) and consists in several domains: N-terminal region, DNA-binding region, Hinge region and ligand-binding region. Fig 7: Glucocorticoid receptor cDNA schematic representation showing primers situation (Fw: Forward; Rv: Reverse). A/B, N-terminal region; C, DNA-binding region; D, Hinge region; E, ligand-binding region. The N-terminal region (Fig 7: A/B) is the most variable and it is involved in transactivation of downstream genes. The DNA-binding region (Fig 7: C) is the central domain and binds to glucocorticoid responsive elements (GRE) in promoter regions to stimulate the expression of glucocorticoid responsive genes. This region is strictly conserved in vertebrates (Stolte et al., 2006). The hinge region (Fig 7: D) is involved in conformational changes during receptor-ligand binding and it is quite variable in sequence and length. The ligand-binding region (fig 7: E), situated at the C-terminus, is involved in joining to the ligand, including cortisol. This region is relatively well conserved, what explains why multiple receptors can bind same ligand and trigger a similar in vitro transactivation (Stolte et al., 2006). Although the structure and function of GR is very well conserved in vertebrates there are some differences. Unlike mammals, fish do not synthesize aldosterone, and it was hypothesized that cortisol would act like a unique glucocorticoid to control mineralocorticoid (MR) and glucocorticoid functions in fish, taking over also osmorregulatory functions (Dean et al., 2003; Ducouret et al., 1995; Greenwood et al., 2003; Mommsen et al., 1999). As a result of this MR and GR may act as cortisol receptors in fish species. 19
  • 21. Moreover later investigations have modified the initial supposition of a unique cortisol receptor, appearing duplicated genes (GR1 and GR2) and also splicing isoforms (Prunet et al., 2006). In rainbow trout concentrations of cortisol required to induce activation of downstream genes are significantly different between the duplicate GR proteins, so GR2 requires lower concentrations of cortisol than GR1a (with nine-amino-acids insert) to induce transactivation (Bury et al., 2003). These differences could be involved in differential functions of different forms in fish physiology. Fig 4: Results of PCR reaction of expression of GR in different conditions: Control, ethanol, cortisol 30 ng/ml, cortisol 300 ng/ml, cortisol 300 ng/ml plus RU-486 (100 ng/ml) and RU-486 (100 ng/ml). The results obtained in this experiment were inconclusive because the huge differences between the individuals analyzed, which prevent us to draw conclusions about the cortisol action mechanisms. In spite of the evidence on the mammalian GR regulation, there are few studies in fish. In general, it has been shown that the GR expression and abundance is downregulated by the presence of high concentrations of cortisol (Bellingham et al., 1992; Mommsen et al., 1999; Pottinger et al., 2000; Rosewicz et al., 1988; Weyts et al., 1998c; Yudt and Cidlowski, 2002). Moreover downregulation of GR expression, GR activity and half-life are modulated by GR phosporylation (Webster et al., 1997), which is itself up-regulated by GRs (Orti et al., 1992). This mechanism, with other influences, can modulate the stress response of organisms, particularly when the stress is chronic (Sapolsky et al., 2000). As regard to fishes in general there is an inverse correlation between GR abundance and cortisol concentrations in fish. This fact was shown in different tissues and fish species, like in peripheral blood leukocytes in carp (Cyprinus carpio L.) (Weyts et al., 1998c) and in liver (Pottinger, 1990) and brain (Lee et al., 1992) of rainbow trout (Oncorhynchus mykiss). However experiments performed in vitro (Sathiyaa and Vijayan, 2003) and in vivo (Vijayan et al., 2003) with rainbow trout hepatocytes showed that, besides the downregulation of GR there was an upregulation of GR mRNA, unlike the mammals. And other studies showed 20
  • 22. that the proteasome was involved in trout hepatocytes (Boone and Vijayan, 2002), which coincide with mammal’s results (DeFranco, 2002; Wallace and Cidlowski, 2001). In conclusion, abundance of GRs is highly variable between species, individuals and from cell to cell (Bellingham et al., 1992). Moreover the differences between the results found by the different authors in fish and the inconclusive dates obtained in this experiment stresses the interest in elucidating the question of regulation of expression and abundance of GR in trout monocytes/macrophages and posttranscriptional control mechanisms. 21
  • 23. α β 4.4 Cytokines (TNF-α and IL-1β) An inflammatory process triggers a cytokine cascade where Tumor necrosis factor α (TNF-α) is released, followed by interleukin-1β (IL-1β) and then IL-6. This cascade triggers the releasing of other cytokines, and globally functions like potent chemoattractans to induce migration of neutrophils and macrophages to the site of infection (Secombes et al., 2001). Cytokines are large (8–60 kDa) and soluble polypeptide mediators that regulate growth, differentiation and function of different cellular types. The expression of these molecules and their receptors is not restricted to cells of the immune system, and are expressed in other tissues, like brain and endocrine glands (Turnbull and Rivier, 1999). Moreover cytokines are involved in different physiological functions outside the immunoregulation, like induce fever, sleep, anorexia, malaise and alterations in neuroendocrine secretions. So, cytokines are key regulators of coordinated local and systemic responses to tissue trauma, infection and disease. Cytokines play an important role in interactions within and between nervous, endocrine and immune systems. Cytokines are the molecules that act like messengers that stimulate the hypothalamic-pituitary-adrenal (HPA) axis when the organism is under the action of a stressor agent or an infection (Haddad et al., 2002; Harris and Bird, 2000; Liberman et al., 2007; Tracey, 2007). IL-1 and TNF-α act as auto/paracrine or endocrine factors, regulating pituitary development, cell proliferation, hormone secretion and feedback control of HPA axis (Hall et al., 1985; Holland et al., 2002; Safieh-Garabedian et al., 2002a; Safieh-Garabedian et al., 2002b; Woiciechowsky et al., 1999). Two events that confirm this question are the fact that intra- peritoneal injection of recombinant trout IL-1β increase cortisol concentrations in plasma (Holland et al., 2002) while cortisol is able to inhibit TNF-α preparation (Saeij et al., 2002). These two cytokines, TNF-α and IL-1β, are two of the most important and the most well- characterized of fish cytokines (Plouffe et al., 2005), and was taken to study their expression under cortisol treatments. 4.4.1 Tumor necrosis factor α (TNF-α) TNF-α is described as a “β- jellyroll” family of cytokines, in which eight antiparallel β- strands form a sandwich 3D structure (Idriss and Naismith, 2000). TNF-α gene contains four exons and three introns. 22
  • 24. TNF are involved in diverse functions, like tumor cytotoxicity, broad-ranging immunological activities, induction of many other cytokines, immunostimulant, or mediators of the inflammatory response (Turnbull and Rivier, 1999). TNF-α was identified at first time in Japanese flounder (Paralychthys olivaceus) (Hirono et al., 2000), followed by mRNA identification in rainbow trout (Laing et al., 2001), brook trout (Salvelinus fontinalis), carp (Saeij et al., 2003b) and sea bream (Garcia-Castillo et al., 2002). The TNF molecule consists in rainbow trout of two forms, TNF-α y TNF-β, with two and three isoforms respectively (Zou et al., 2002); nevertheless other authors (Goetz et al., 2004b) affirm that TNF-β does not exist in fishes. Within TNF-α two genes presents in rainbow trout, TNF1 and TNF2, TNF2 is highly expressed than TNF1. TNF-α in rainbow trout is expressed constitutively in head kidney and gill and inducible in isolated head kidney cells (Laing et al., 2001). TNF-α is present in two different forms, a soluble 17 kDa form and a transmembrane 26 kDa form. Soluble 17 kDa form is synthesized by photolytic cleavage of the extracellular portion of the 26 kDa form. The majority of actions mediated by TNF-α correspond to soluble and mature 17 kDa form, which is found as homotrimers. However, the transmembrane form could be too active and it may be responsible for some of local actions of TNF-α (Goetz et al., 2004b). Fig 8: Results of PCR reaction of expression of TNF-α in different conditions: Control, ethanol, cortisol 30 ng/ml, cortisol 300 ng/ml, cortisol 300 ng/ml plus RU-486 (100 ng/ml) and RU-486 (100 ng/ml). The results obtained (Fig 8) show a down regulation of TNF-α expression in conditions of cortisol 300 ng/ml, however these results do not shown noticeable differences at the cortisol concentration of 30 ng/ml. These results are in concordance with data obtained by other authors, showing that cortisol down-regulates the expression of TNF-α in different species and cells, like in human macrophages (Joyce et al., 2001; Steer et al., 2000; Steer et al., 1998), rainbow trout macrophages (MacKenzie et al., 2006), rainbow trout head kidney leukocytes (Holland et al., 2003) and carp head kidney macrophages (Saeij et al., 2003a). 23
  • 25. 4.4.2 Interleukin 1-β (IL 1-β) Interleukin have numerous and diverse immunoregulatory actions, some of them have proinflamatory actions (IL-1α, IL-1β, IL-8, IL-9), while other interleukins have anti-inflammatory effects (IL-1ra, IL-4, IL-10, IL-13) and IL also induce systemic aspects of acute phase response, like the fever (Turnbull and Rivier, 1999). IL-1β is a proinflamatory cytokine and is involved in induction of other immunomodulatory cytokines and other transcripts of immune response, such as COX2 and MHC class II (Major histocompatibility complex class II) (Plouffe et al., 2005). Rainbow trout IL-1β was the first to be cloned in fish teleosts (Zou, 1999), and a second IL-1β gene has been discovered in rainbow trout, and these two genes were called IL-1β1 and IL-1β2, and both genes are expressed in head kidney leukocytes after LPS stimulation (Secombes et al., 2001). Unlike mammals, trout IL-1β haven’t got a clear caspase-1 cut site (Zou et al., 2002), but it seems that trout IL-1B must be cleaved by interleukin converting enzyme (ICE) before to be activated and secreted (Secombes et al., 2001). Of the two IL-1β types receptors to IL-1: IL-1R type I and IL-1R type II, only the binding of IL-1α or IL-1β to type 1 receptor work as antagonists by capturing excess of IL-1. IL-1β plays an important role in interaction between immune, nervous and endocrine systems, affecting directly to hypothalamic-pituitary-adrenal (HPA) axis function, stimulating cortisol secretion, like it was demonstrated in rainbow trout with an intra-peritoneal injection of recombinant trout IL-1β (Haddad et al., 2002; Holland et al., 2002). Fig 9: Results of PCR reaction of expression of IL 1-β in different conditions: Control, ethanol, cortisol 30 ng/ml, cortisol 300 ng/ml, cortisol 300 ng/ml plus RU-486 (100 ng/ml) and RU-486 (100 ng/ml). The results obtained (Fig 9) show a down regulation of IL-1β expression in conditions of cortisol 300 ng/ml, which simulates an acute stress condition, but do not show noticeable differences at cortisol 30 ng/ml, corresponding to chronic conditions. 24
  • 26. These results are in concordance with the dates obtained by others authors, in which cortisol acts as down-regulating expression of IL-1β in different conditions, like in human leucocytes, rainbow trout culture leucocytes (MacKenzie et al., 2006; Zou et al., 2002) and carp head kidney monocytes (Saeij et al., 2003a). 25
  • 27. 4.5 PU.1 (SPI-1) PU.1 was identified as the putative oncogene Spi-1. The PU.1 locus is a high-frequency integration site for spleen focus-forming virus (SFFV) in Friend murine acute erythroleukemias and was called SFFV provirus integration site-1 (Spi-1) (Moreau-Gachelin et al., 1989; Moreau- Gachelin et al., 1988). PU.1 is a member of the transcription factor family Ets that plays an essential role in the development and differentiation of both myeloid (granulocytes and monocytes/macrophages) and lymphoid cells (Fisher and Scott, 1998; Hsu et al., 2004) and it is involved in expression regulation of different genes, like heavy and light chains of immunoglobulins, cytokines (IL-1β, MIP-1α, TNF-α, IL-1ra), complement receptors, growth factors, different components related with phagocytosis and cellular development, enzymes, MHC class II and other genes (Henderson and Calame, 1998; Lloberas et al., 1999). During haematopoiesis PU.1 is up-regulated in myeloid development and down- regulated in erythrocyte differentiation (Voso et al., 1994). In this way PU.1 deficient mouse exhibits a group of functional deficiencies in macrophages, granulocytes and progenitors of B and T lymphocytes (Scott et al., 1994). Different studies have shown that PU.1 blocks erythroid differentiation by directly antagonizing GATA-1 activity. This reciprocal negative regulation determines myeloid versus erythroid fate (Rekhtman et al., 2003; Rekhtman et al., 1999; Rhodes et al., 2005; Zhang et al., 2000). Moreover, recent studies have demonstrated that the concentration of PU.1 determinates haematopoietic progenitor cells fate to B lymphocytes or macrophages. Low concentrations of PU.1 induces the B lymphocyte differentiation, while high concentrations of PU.1 triggers macrophage differentiation and development and blocks B cells proliferation (DeKoter and Singh, 2000). Fig 10: Results of PCR reaction of expression of PU.1 in rainbow trout monocytes (SPI-1 in different conditions: Control, ethanol, cortisol 30 ng/ml, cortisol 300 ng/ml, cortisol 300 ng/ml plus RU-486 (100 ng/ml) and RU-486 (100 ng/ml). The results obtained (Fig 10) show a down-regulation in expression of PU.1 (Spi-1) gene in 300 ng/ml cortisol concentration, but do not reveal noticeable changes in 30 ng/ml of cortisol condition. 26
  • 28. These results suggest that cortisol repress the monocyte development and differentiation, at least in conditions of high levels of cortisol, which simulate conditions of a acute stress, but it seems that in conditions of moderate concentrations of cortisol, which are similar to the plasma concentrations in chronic stress, this downregulation does not occur. These results may help us to better understand the immune fish responses to stress. In this sense, the downregulation of PU.1 gene in rainbow trout monocytes under high concentrations of cortisol, and not the downregulation at moderate concentrations could be explained as the fish response to an acute stress blocking myeloid differentiation and development, and thus saving energy to cope with the acute stress situation. Moreover, a possible interpretation for no downregulation of PU.1 at moderate concentrations of cortisol could be that, in a chronic stress situation, blocking of myeloid and lymphoid cells differentiation could not be beneficial in the long term, because it could compromise immune system and their defence against pathogens. 27
  • 29. 5. CONCLUSIONS. The effect of cortisol addition to trout macrophages culture shows that high concentration of cortisol, similar than under conditions of acute stress, induces a reduced number of cells compared with the conditions in which the cortisol is absent or it is counteracted with its antagonist, RU-486. However, under conditions of cortisol at moderate concentrations, similar than under chronic stress conditions, no noticeable variations are recorded respect to control plates. These results could be explained by two theories, the increment of cellular apoptosis or the inhibition of cell adhesion to the matrix. Experiments performed in our laboratory show that increase of apoptosis do not takes place, so the reduction of adhesion molecules could be the cause of this effect. From a physiological point of view the downregulation of cellular adhesion molecules in conditions of acute stress has a functional sense, since the organism reduce the cellular adhesion molecules expression decreasing cellular trafficking, with the consequent savings in energy, which can be redirected to cope with the stress situation. However, the results obtained to the adhesion molecule tested (CD18) reveal that cortisol does not significantly affect the expression of this molecule, although this regulation could exist in other adhesion molecules different than CD18. Moreover, the lack of down-regulation of expression of CD18 does not mean that cortisol doen not have effects in the cellular adhesion phenomenon. Cortisol could regulate the expression of other genes, or through non-genetic mechanisms, like the changing of avidity binding of cellular adhesion molecules or the alteration of their situation in cellular surface, which could play an important role in cellular adhesion process. As regards to glucocorticoid receptor (GR) the results obtained in this work were inconclusive because of the enormous variation between de individuals. Nevertheless, this question could be very interesting to study because it could provide a better understanding of cortisol mechanisms of action. Cortisol showed a clear effect on the expression levels of the cytokines TNF-α and IL- 1β, showing similar results than previously observed. This effect is opposite to the observed in response to LPS in rainbow trout macrophages. As regards to the PU.1 (Spi-1) gene, the results found show that cortisol down-regulates the expression of this gene in conditions of high concentration of cortisol, but not under 28
  • 30. moderate concentrations. These situations are very similar to the concentrations of cortisol in plasma under acute and chronic stress conditions. In this way, a possible functional and physiologic interpretation of these results is that in conditions of acute stress PU.1 is downregulated, and so, differentiation and development of monocytes to mature macrophages are blocked or attenuated. This fact could have the purpose of save the energy of myeloid differentiation to use that energy to an overall systemic response in front of an acute stressor. Nevertheless, under conditions of chronic stress, the inhibition of myeloid differentiation could be not adequate at long term, because it could compromise the defence of the organism against pathogens, and for this reason the expression of this transcription factor (PU.1) could not be downregulated in these conditions. In short, the results obtained in this work support the conclusions obtained by other authors (MacKenzie et al., 2006), who, using functional microarrays have arrived to the conclusions that in general cortisol have generally contrary effects to LPS. These responses included the cortisol cell protection of the suppression of genes involved in basic cellular functions and maintenance of activities, including protection against apoptosis. In conclusion, cortisol inhibits the expression of the inflammatory cytokines TNF-α and IL-1β and the transcription factor PU.1, with a possible purpose of saving energy in order to cope with the response to acute stress conditions; however, the same response to conditions of chronic stress is not observed, as this downregulation could be negative in the long term. Our results also indicate the induction of differential mechanisms involved in the regulatory roles of cortisol which are depending on the concentration levels. 29
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