M. Kepka

Adrenergic regulation of the innate immune response in common carp (Cyprinus carpio L.).

Catecholamines exert their physiological actions through α and β adrenergic receptors (ARs). As ARs are not exclusively expressed on neuroendocrine cells, but also on leukocytes, they may facilitate neuroendocrine modulation of immune responses. We sequenced the β(2a)-AR in common carp, and studied its expression profile and involvement in the regulation of teleost innate immune responses. β(2a)-AR messenger RNA was found to be constitutively expressed in brain areas, especially in the preoptic nucleus (NPO, homologous to the mammalian hypothalamus), and in immune organs. During the active phase of an in vivo inflammatory response, induced by i.p. zymosan treatment, β(2a)-AR gene expression was up-regulated in the peritoneal leukocytes. Additionally, adrenaline in vitro reduced the synthesis of oxygen radical species and nitric oxide, while it enhanced arginase activity in fish phagocytes. Furthermore, in vitro adrenaline administration inhibited expression of pro-inflammatory cytokines, chemokines and their receptors. It is therefore hypothesized that adrenaline will down-regulate phagocyte skewing toward classical/innate polarization.

Mechanisms involved in apoptosis of carp leukocytes upon in vitro and in vivo immunostimulation

During inflammation leukocyte activity must be carefully regulated, as high concentrations and/or prolonged action of pro-inflammatory mediators e.g. reactive oxygen species (ROS) can be detrimental not only for pathogens but also for host tissues. Programmed cell death - apoptosis is a most effective regulatory mechanism for down regulation of leukocyte activity, but little is known about this process in fish. We aimed to reveal the mechanisms of initiation and regulation of apoptosis in carp neutrophilic granulocytes and macrophages. During zymosan-induced peritonitis in carp, activated inflammatory neutrophilic granulocytes and monocytes/macrophages died by apoptosis. This correlated with a strong production of ROS, but pretreatment of the fish with NADPH oxidase inhibitor only slightly decreased late apoptosis. Interestingly in vitro incubation with zymosan or phorbol ester, but not lipopolisaccharide and poli I:C induced apoptosis of head kidney neutrophilic granulocytes. This coincided with loss of mitochondrial membrane potential. Moreover, in zymosan-stimulated neutrophilic granulocytes NADPH oxidase inhibitor not only reduced the production of ROS but also apoptosis. A similar effect was not observed in cells stimulated with phorbol ester, where DPI reduced ROS production, but not apoptosis. In PMA-stimulated neutrophilic granulocytes both the respiratory burst and apoptosis were reduced by protein kinase inhibitor. Furthermore, a short neutrophil stimulation either with PMA or with zymosan did induce caspase-independent apoptosis. These results show that in carp, apoptosis is an important regulatory process during in vitro and in vivo immunostimulation. In neutrophils, protein kinase, but not NADPH oxidase, is involved in PMA-induced apoptosis while apoptosis induced by zymosan is ROS-dependent.

Neuroendocrine modulation of the inflammatory response in common carp: Adrenaline regulates leukocyte profile and activity

Inflammatory responses have to be carefully controlled, as high concentrations and/or prolonged action of inflammation-related molecules (e.g. reactive oxygen species, nitric oxide and pro-inflammatory cytokines) can be detrimental to host tissue and organs. One of the potential regulators of the inflammatory process are stress mediators including adrenaline. In vivo effects of adrenaline were studied during zymosan-induced (Z) peritoneal inflammation in the common carp Cyprinus carpio L. Adrenaline injected together with zymosan (ZA) did not change the number of inflammatory leukocytes in the peritoneal cavity, however at 24h post-injection it significantly reduced the percentage of monocytes/macrophages. Moreover, compared to cells retrieved from fish treated with PBS or zymosan only, adrenaline increased the percentage of apoptotic leukocytes in the focus of inflammation. Furthermore, adrenaline significantly reduced the expression of chemokine CXCL8_L1 (a functional homolog of mammalian IL-8) and its receptors (CXCR1 and CXCR2), indicating changes in leukocyte recruitment after stress. We conclude that adrenaline may contribute to a coordinated reaction by influencing the inflammatory response via direct regulation of leukocyte migration and/or apoptosis.

Activity of the hypothalamus–pituitary–interrenal axis (HPI axis) and immune response in carp lines with different susceptibility to disease

The stress response transmitted by the HPA axis is one of the best examples of neuroendocrine–immune interactions that are critical for survival. Analogous to the situation in mammals, the stress response in fish is characterized by the activation of the hypothalamo–pituitary–interrenal axis (HPI). Effects of cortisol on the fish immune system comply with findings in mammals and suggest that the differences in sensitivity to stress will influence the immune response and as a consequence of survival. Therefore, we studied the stress response and its immunity-related effects in four different carp lines (R3, R3xR8, K and R2) that display a differential pathogen susceptibility. Previous studies indicate that R3xR8 and R3 carp are susceptible to bacterial and parasite infection, while R2 and K are relatively resistant to infection. Interestingly, the most striking effect of stress on leukocyte composition and activity was observed in the pathogen-resistant K carp, even though no robust changes in gene expression of stress-involved factors were observed. In contrast, R3 carp showed no spectacular stress-induced changes in their immunological parameters with concurrent significant activation of the HPI axis. Upon stress, the R3 carp showed up-regulation of crf,pomc and gr2 gene expression in the hypothalamus. Furthermore in R3 carp, at all levels of the HPI axis, stress induced the highest up-regulation of il-1β gene expression. Although we are aware of the complexity of the interactions between stress and pathogen susceptibility and of the risk of interpretation based on correlations, it is noteworthy that the fish more susceptible to infection also exhibited the highest response to stress.

A role for melatonin in maintaining the pro- and anti-inflammatory balance by influencing leukocyte migration and apoptosis in carp.

Melatonin is responsible for the synchronization of many physiological processes, including the immune response. Here we focus on the expression of melatonin MT1 receptors in/on leukocytes, and on the effects of melatonin administration on the inflammatory processes of carp. For the first time, we showed that fish leukocytes express MT1 receptors, implicating direct responsiveness to melatonin stimulation. Moreover, both in vitro and in vivo, melatonin modulated the immune response. The most potent effects of melatonin concerned the regulation of leukocyte migration. Melatonin reduced chemotaxis of leukocytes towards CXC chemokines in vitro. In vivo, during zymosan induced peritonitis, i.p. administration of melatonin reduced the number of neutrophils. This correlated with a melatonin-induced decrease of gene expression of the CXCa chemokine. Moreover, melatonin induced a decrease of the respiratory burst in inflammatory leukocytes. Although these data do suggest a potent anti-inflammatory function for this hormone, melatonin-induced inhibition of leukocyte apoptosis clearly indicates towards a dual function. These results show that also in carp, melatonin performs a pleiotropic and extra-pineal function that is important in maintaining the delicate pro- and anti-inflammatory balance during infection. They furthermore demonstrate that neuroendocrine-immune interaction via melatonin is evolutionary conserved.

An iminosugar-based heparanase inhibitor heparastatin (SF4) suppresses infiltration of neutrophils and monocytes into inflamed dorsal air pouches.

Local infiltration of inflammatory cells is regulated by a number of biological steps during which the cells likely penetrate through subendothelial basement membranes that contain heparan sulfate proteoglycans. In the present study, we examined whether administration of heparastatin (SF4), an iminosugar-based inhibitor of heparanase, could suppress local inflammation and degradation of heparan sulfate proteoglycans in basement membranes. In a carrageenan- or formyl peptide-induced dorsal air pouch inflammation model, the number of infiltrated neutrophils and monocytes was significantly lower in mice after topical administration of heparastatin (SF4). The concentration of chemokines MIP-2 and KC in pouch exudates of drug-treated mice was similar to control. In a zymosan-induced peritonitis model, the number of infiltrated cells was not altered in drug-treated mice. To further test how heparastatin (SF4) influences transmigration of inflammatory neutrophils, its suppressive effect on migration and matrix degradation was examined in vitro. In the presence of heparastatin (SF4), the number of neutrophils that infiltrated across a Matrigel-coated polycarbonate membrane was significantly lower, while the number of neutrophils passing through an uncoated membrane was not altered. Lysate of bone marrow-derived neutrophils released sulfate-radiolabeled macromolecules from basement membrane-like extracellular matrix, which was suppressed by heparastatin (SF4). Heparan sulfate degradation activity was almost completely abolished after incubation of lysate with protein G-conjugated anti-heparanase monoclonal antibody, strongly suggesting that the activity was due to heparanase-mediated degradation. Taken together, in a dorsal air pouch inflammation model heparastatin (SF4) potentially suppresses extravasation of inflammatory cells by impairing the degradation of basement membrane heparan sulfate.