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Endogenous glucocorticoids modulate several physiological responses, such as glucose metabolism, cardiovascular activity and immune reactions (Wilckens and De Rijk, 1997; Marik and Zaloga, 2002). Indeed, regulation of leukocyte recruitment (Flower et al., 1986; Moraes et al., 1987; Abe et al., 1995; Rovai et al., 1998; Leech et al., 2000), increase in vascular permeability, secretion of cytokines, expression of adhesion molecules, phagocytic and microbicidal activities (Filep et al., 1997; Fassbender et al., 1999; Nakagawa et al., 1999; Torsteinsdottir et al., 1999; Weber et al., 2001, 2004), which occur in the course of an inflammatory or stress injury, are modulated by an increase in the concentration of endogenous glucocorticoids in the plasma. In addition to the effects exerted by endogenous hormones, synthetic hormones are important therapeutic tools for the treatment of inflammatory diseases and as immunosuppressor agents (Rhen and Cidlowski, 2005; Song et al., 2005).
A broad range of anti-inflammatory effects elicited by high concentrations of endogenous glucocorticoids and therapeutic doses of synthetic hormones are mediated through an intracellular receptor, the glucocorticoid cytoplasmic receptor (GR), a member of the steroid hormone receptor superfamily located in the cytoplasm. On binding to the hormone, the GR is released from heat-shock protein 90 and other regulatory proteins, allowing it to gain access to the nucleus. Domains within the N-terminal region of the receptor interact with either negative or positive glucocorticoid response elements (GREs) in promoter regions of genes, with subsequent initiation or repression of transcription (Adcock, 2000; Lu and Cidlowski, 2004). Notwithstanding this classical mechanism of action, it has been suggested that the activated GR has an action directly on nuclear factor-κB (NF-κB) or on activating protein-1, inhibiting their binding to DNA and thus impeding transcription (van der Burg and van der Saag, 1996; Wissink et al., 1998; Reichardt et al., 2001; Lu and Cidlowski, 2004). In addition, a non-genomic mechanism has been proposed as it has been observed that administration of the GR antagonist RU 38486 or of protein synthesis blockers does not alter the reduced phagocytic and microbicidal activities of neutrophils or macrophages evoked by high doses of synthetic steroids (Liu et al., 2005; Long et al., 2005).
Our previous data have shown that endogenous glucocorticoids exert a physiological control on neutrophil mobilization, that is in the absence of an inflammatory response they help to keep neutrophils in circulation, as adrenalectomy or blocking of the GR resulted in increased rolling and adherence of leukocytes to postcapillary venules (Farsky et al., 1995). In addition, endogenous glucocorticoids accelerate neutrophil maturation in the bone marrow and its transference into the peripheral compartment, with consequent neutrophilia (Cavalcanti et al., 2006). The effects of endogenous glucocorticoids are most probably modulated by L-selectin, as adrenalectomy induced a decrease in the expression of L-selectin in mature neutrophils from bone marrow and an increased expression of L-selectin in circulating neutrophils. The expression of L-selectin in these cells is controlled by post-translational mechanisms, as the concentrations of L-selectin mRNA were the same in cells obtained from control and adrenalectomized (ADX) animals (Cavalcanti et al., 2006).
Physiologically, L-selectin has an important role in the mobilization of leukocytes from bone marrow (Takeshita et al., 2004) and in their movement into tissue, being responsible for the rapid onset of the adherence of neutrophils to the endothelium (Petri and Bixel, 2006). Furthermore, our data (Cavalcanti et al., 2006) indicate that endogenous glucocorticoids, by modulating L-selectin expression, maintain mature neutrophils in different compartments during their life span, avoiding the inappropriate mobilization of leukocytes.
The results from the present study reveal the physiological role of endogenous glucocorticoids in the movement of neutrophils in the absence of any injury or stress conditions. The interaction of endogenous glucocorticoids with GR was evaluated in bone marrow, circulating and endothelial cells after treatment with RU 38486. By screening the passage of neutrophils in each phase, we showed that via GR, endogenous glucocorticoids act on neutrophils by modulating their initial contact with the endothelium, whereas they act on the endothelium by controlling the events responsible for the subsequent firm adherence of neutrophils to the vessel wall. In addition, the differential control by the hormones on the expression of adhesion molecules in each microenvironment may involve distinct intracellular mechanisms, as the translocation of NF-κB into the nucleus was only altered in endothelial cells.
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The results presented here show that endogenous glucocorticoids have an important role in the physiological control of neutrophil mobilization displaying different actions on neutrophils and endothelial cells, and that the control of the expression of adhesion molecules in each cell microenvironment may involve distinct intracellular mechanisms.
Total counts and relative differential proportions of blood leukocytes provide an important representation of the state of activation of the immune system, and of the pattern of distribution of leukocytes in the body. Cell–cell and cell–stromal contact, mediated by adhesion molecules on cell surfaces, are responsible for the maintenance of leukocytes in the bone marrow and peripheral compartments (van Eeden et al., 1997; Bauer et al., 2001). L-selectin is involved in these processes, as its expression is evident in all phases of granulocyte maturation and peaks in mature neutrophils (Lund-Johansen and Terstappen, 1993; Takeshita et al., 2004). In addition, it mediates the rolling behaviour of neutrophils in peripheral blood (Petri and Bixel, 2006).
Our previous results obtained in ADX animals (Cavalcanti et al., 2006) have now been corroborated by the results obtained in RU 38486-treated animals and indicate that secreted glucocorticoids regulate, via GR, different phases of neutrophil mobilization. It is likely that high concentrations of endogenous glucocorticoids control the number of leukocytes in circulation in stress conditions as leukocytosis is one of the characteristics of hormone therapy (Harris et al., 1995; Liles et al., 1997; Nakagawa et al., 1998; Weber et al., 2001, 2004). The mechanism involved in these conditions seems to be related to a change in the longevity of the cell in the circulation induced by altering apoptosis (Chang et al., 2004; Madsen-Bouterse et al., 2006) or by impairing the synthesis and expression of L-selectin (Nakagawa et al., 1999; Weber et al., 2001, 2004). Our data suggest that endogenous glucocorticoids act as a selective modulator of the delivery of granulocyte cells from the bone marrow, as ADX (Cavalcanti et al., 2006) and RU 38486-treated rats presented an increased rate of neutrophil maturation in the bone marrow with consequent neutrophilia, but with no effects on the lymphocyte/mononuclear cell lineages. Also, our data do not support the possibility that endogenous glucocorticoids affect the death of circulating neutrophils. Nevertheless, modifications in the expression of L-selectin may be an important control target of endogenous glucocorticoids, as hormone deficiency (Cavalcanti et al., 2006) or blockage of GR caused a decrease and increase in L-selectin expression in bone marrow and peripheral neutrophils, respectively, that was not dependent on pre-translational mechanisms (Cavalcanti et al., 2006). Next, we hypothesized that these hormones regulate L-selectin shedding. The effect of exogenous glucocorticoids on L-selectin expression by post-translational mechanisms has already been implicated by observations that annexin-1, a protein indicated as a mediator of the anti-inflammatory effects of glucocorticoids, hinders the migration of neutrophils to inflammatory sites (Perretti and Ahluwalia, 2000) by inducing L-selectin enzymatic cleavage from the surface of neutrophils (Strausbaugh and Rosen, 2001; De Coupade et al., 2003). However, recent data have shown that the action of annexin-1 on L-selectin shedding is dependent on concentrations of the protein and its bioactive peptide Ac2-26, and on the state of neutrophil activation (Hayhoe et al., 2006). Conversely, higher doses of annexin-1 caused an upregulation of L-selectin expression in human neutrophils (Hayhoe et al., 2006). From these findings and our results, it is probable that a differential molecular control by physiological and therapeutic doses of hormones occurs during healthy and stress-related conditions.
Furthermore, our data show that the expressions of P- and E-selectin in endothelial cells were not altered by a reduction in endogenous glucocorticoids levels. As selectins are responsible for the rolling behaviour of leukocytes (Sperandio, 2006), we suggest that endogenous glucocorticoids physiologically regulate the rolling behaviour by acting mainly on neutrophils.
Conversely, the role that endogenous glucocorticoids play in the firm adherence of leukocytes to the endothelium, the subsequent step to rolling during leukocyte transference into tissue, may reflect their actions on endothelium. This hypothesis is supported by our previous data demonstrating that neutrophils from ADX animals had a smaller than normal capacity to adhere to endothelial cells in vitro and that the expression of β2 integrin was normal in both basal and FMLP-stimulated conditions. Also, in the present study, β2 integrin expression was not altered in cells obtained from RU 38486-treated rats compared to controls (data not shown). Furthermore, the ability of endothelial cells from ADX animals to adhere to neutrophils in vitro was enhanced and the synthesis and membrane expression of ICAM-1, VCAM-1 or PECAM-1 in the endothelium of both ADX or RU 38486-treated animals were increased. Moreover, these data indicate the differential actions of endogenous glucocorticoids on neutrophils and endothelial cells during the firm adhesion process, and point to the long-lasting effects of endogenous glucocorticoids, as in vitro adherence and adhesion molecule mRNA concentrations were measured in primary cultured cells. Inhibition of the expression of endothelial adhesion molecules has been demonstrated previously after the administration of these hormones, at doses used in hormonal therapy (Tailor et al., 1999; Pitzalis et al., 2002). This is the first time that endogenous glucocorticoids have been shown to be involved in these physiological control mechanisms and the synthesis and expression of immunoglobulins, and indicates that the action of endogenous glucocorticoids on endothelial cells functions as an important physical barrier that contributes to the cessation of inappropriate mobilization of neutrophils from the circulation.
Repression of many pro-inflammatory genes via the inhibition of transcription factors, including NF-κB and activating protein-1, has been shown to be an important mechanism of the anti-inflammatory actions of endogenous and exogenous glucocorticoids (Pitzalis et al., 2002; Hermoso and Cidlowski, 2003). As the synthesis of adhesion molecules investigated here is mediated by NF-κB, the translocation of this factor into the nucleus was assessed. While NF-κB translocation was markedly enhanced in endothelial cells obtained from ADX and RU 38486-treated rats, no alteration was detected in neutrophils from either group of animals. Hence, in addition to corroborating the mechanism of action of exogenous glucocorticoids in the expression of adhesion molecules in endothelial cells (Pitzalis et al., 2002), our data show that these hormones are involved in the control of neutrophil mobilization, and that the expression of L-selectin on neutrophils is not dependent on NF-κB transcription.
Results from recent studies have led to the awareness that endogenous biochemical routes must be promptly activated to defend the host and then inactivated when resolution is achieved (Serhan et al., 2007). Here, by studying neutrophil mobilization, we highlight the physiological role secreted glucocorticoids play in a host's defence mechanism and disclose the effects of each component of this important system, which needs to respond promptly to any aggression but must not be activated inappropriately. Also, it is now clear that the fine adjustment induced by endogenous glucocorticoids on the mobilization of neutrophils may differ from that evoked by hormone therapy.