Stress-induced adaptation of neutrophilic granulocyte activity in K and R3 carp lines.

Both in mammals and fish, stress induces remarkable changes in the immune response. We focused on stress-induced changes in the activity of neutrophilic granulocytes in the R3 and K lines of common carp, which showed differential stress responses. Our study clearly demonstrates that a prolonged restraint stress differentially affects the activity of K and R3 carp neutrophils. In the K line, stress decreased the respiratory burst, while in the R3 line it reduced the release of extracellular DNA. Surprisingly, the stress-induced changes in ROS production and NET formation did not correlate with changes in gene expression of the inflammatory mediators and GR receptors. In neutrophilic granulocytes from K carp, gene expression of the stress-sensitive cortisol GR1 receptor was significantly higher than in neutrophils from R3 fish, which will make these cells more sensitive to high levels of cortisol. Moreover, upon stress, neutrophilic granulocytes of K carp up-regulated gene expression of the anti-inflammatory cytokine IL-10 while this was not observed in neutrophilic granulocytes of R3 carp. Therefore, we can hypothesize that, in contrast to R3 neutrophils, the more cortisol sensitive neutrophils from K carp respond to stress with up-regulation of IL-10 and consequently reduction of ROS production. Most probably the ROS-independent NET formation in K carp is not regulated by this anti-inflammatory cytokine. These data may indicate a predominantly ROS-independent formation of NETs by carp neutrophilic granulocytes. Moreover, they underline the important role of IL-10 in stress-induced immunoregulation.

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-1ß 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.

Production of inflammatory mediators and extracellular traps by carp macrophages and neutrophils in response to lipopolysaccharide and/or interferon-γ2.

Neutrophilic granulocytes and macrophages are crucial for the innate immune response against infections. They migrate into the focus of inflammation, where they efficiently bind, engulf and kill bacteria by proteolytic enzymes, antimicrobial peptides, reactive oxygen (ROS) and nitrogen (RNS) species. Moreover, activated neutrophils and macrophages can form extracellular traps (ETs). Fish neutrophils and macrophages are morphologically, histochemically, and functionally similar to their mammalian counterparts, but their significance for regulation of inflammatory responses and pathogen killing needs further elucidation. We compared the activity of head kidney monocytes/macrophages and neutrophilic granulocytes of common carp and established that upon lipopolysaccharide stimulation, not only neutrophils, but also carp monocytes/macrophages release extracellular DNA and are capable to form macrophage extracellular traps (METs). To clarify whether many specific LPS functions reported for piscine phagocytes might be due to impurities in the commonly used LPS preparations we studied expression of inflammatory mediators, release of DNA, ROS and RNS in cells stimulated with LPS or its highly purified form (pLPS). Also IFN-γ2 stimulation and its synergism with LPS/pLPS in stimulating expression of pro-inflammatory mediators was studied. Results substantiate that a classical stimulation of TLR4 by LPS may indeed be absent in carp as most of the classically reported LPS effects are abolished or diminished when pLPS is used. Interestingly, we also observed a potent IL-10 expression in neutrophilic granulocytes upon LPS stimulation, which, apart from their pro-inflammatory function, clearly indicates a role in restrictive control of the inflammatory reaction.

Characterization and expression analysis of an interferon-γ2 induced chemokine receptor CXCR3 in common carp (Cyprinus carpio L.).

Chemokine and chemokine receptor signalling pairs play a crucial role in regulation of cell migration, morphogenesis, and cell activation. Expressed in mammals on activated T and NK cells, chemokine receptor CXCR3 binds interferon-γ inducible chemokines CXCL9-11 and CCL21. Here we sequenced the carp CXCR3 chemokine receptor and showed its relationship to CXCR3a receptors found in other teleosts. We found high expression of the CXCR3 gene in most of the organs and tissues of the immune system and in immune-related tissues such as gills and gut, corroborating a predominantly immune-related function. The very high expression in gill and gut moreover indicates a role for CXCR3 in cell recruitment during infection. High in vivo expression of CXCR3 at later stages of inflammation, as well as its in vitro sensitivity to IFN-γ2 stimulation indicate that in carp, CXCR3 is involved in macrophage-mediated responses. Moreover, as expression of the CXCR3 and CXCb genes coincides in the focus of inflammation and as both the CXCb chemokines and the CXCR3 receptor are significantly up-regulated upon IFN-γ stimulation it is hypothesized that CXCb chemokines may be putative ligands for CXCR3.

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.

Carp neutrophilic granulocytes form extracellular traps via ROS-dependent and independent pathways.

Neutrophil extracellular traps (NETs) have recently been described as an important innate defense mechanism that leads to immobilization and killing of invading pathogens. NETs have been identified in several species, but the mechanisms involved in NET formation and their role in infection have not been well determined yet. Here we show that upon in vitro stimulation with different immunostimulants of bacterial, fungal or viral origin, carp neutrophilic granulocytes rapidly release NET structures. We analyzed the composition of these structures and the kinetics of their formation by confocal microscopy, by quantifying the levels of extracellular DNA and the release of enzymes originating from neutrophilic granules: myeloperoxidase, neutrophil elastase and matrix metalloproteinase 9 (MMP-9). Profiles of NET release by carp neutrophils as well as their enzyme composition are stimulus- and time-dependent. This study moreover provides evidence for a stimulus-dependent selective requirement of reactive oxygen species in the process of NET formation. Collectively the results support an evolutionary conserved and strictly regulated mechanism of NET formation in teleost fish.

Neuroendocrine-immune interaction: regulation of inflammation via G-protein coupled receptors.

Neuroendocrine- and immune systems interact in a bi-directional fashion to communicate the status of pathogen recognition to the brain and the immune response is influenced by physiological changes. The network of ligands and their receptors involved includes cytokines and chemokines, corticosteroids, classical pituitary hormones, catecholamines and neuropeptides (e.g. opioids), as well as neural pathways. We studied the role of opioid, adrenergic and melatonin G-protein coupled receptors (GPCR) on carp (Cyprinus carpio) leucocytes. Ligand interaction by morphine and adrenaline both in vitro and in vivo resulted in considerable decrease of chemotaxis and expression of CXC chemokines and chemokine CXC receptors. These effects may have substantial influence on the process of inflammation, the efficacy of which is crucial for an effective immune response. Both opioid receptors and chemokine receptors are G-protein coupled receptors (GPCRs), and were classically assumed to function as monomers. This paradigm is now challenged by the emerging concept of homo- and hetero dimerization which may represent the native form of many receptors. G-protein coupling, downstream signaling and regulatory processes such as receptor internalization are largely influenced by the dimeric nature. The true functional importance of GPCR interactions remains enigmatic, but it certainly has implications with respect to the specificity of currently used medications. This review focuses on the important function of chemokine GPCRs during inflammation and the potential neuroendocrine modulation of this process through "neuroendocrine" GPCRs.

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.

Neuroendocrine-immune interaction in fish: differential regulation of phagocyte activity by neuroendocrine factors.

Coping with physical, chemical and biological disturbances depends on an extensive repertoire of physiological, endocrinological and immunological responses. Fish provide intriguing models to study bi-directional interaction between the neuroendocrine and the immune systems. Macrophages and granulocytes are the main actors in the first and rapid innate immune response. They are resident in different organs and are moreover rapidly recruited and activated upon infection. They act in response to recognition of pathogen-associated molecular patterns (PAMPs) via a repertoire of surface and intracellular receptors by inducing a plethora of defense reactions aiming to eradicate the pathogen. Subsequent production of inflammatory mediators stimulates other leukocytes required to develop an adaptive and specific antibody response. The type of phagocyte reaction will therefore depend on their differentiation state, specific receptor repertoire and their specific location. Apart from these pathogen induced responses, immune reactivity may be modulated by neuroendocrine factors. Over the last years we extensively studied changes in carp stress axis activity and the effect of its end-products on the immune system in an acute stress paradigm. We focus on specific neuroendocrine receptors on leukocytes and their effect on crucial phagocyte activities. We performed identification and functional analyses of different glucocorticoid, opioid and adrenergic receptors on carp phagocytes. Results show that their ligands of neuroendocrine origin may have substantial impact on specific phagocyte functions in a differential way. Inflammatory and microbicidal responses fight pathogens but may be detrimental to the host tissue. Neuroendocrine modulation may regulate inflammation to reach an optimum defense while preventing excessive host cell damage.

Functional analysis of carp interferon-gamma: evolutionary conservation of classical phagocyte activation.

In teleost fish two IFN-gamma gene sequences were found for which two phylogenetic clusters can be distinguished. Our previous analysis of expression of these in carp led us to hypothesize that a classical IFN-gamma function is associated with the IFN-gamma2 cluster. We investigated the evolutionary conservation of the IFN-gamma function, inducing classical activation of phagocytes, thus skewing towards a Th1-like profile of immune activation. Recombinant proteins for the carp IFN-gamma sequences of both clusters were made and we studied their effects on expression of proinflammatory mediators. Carp IFN-gamma2, in contrast to carp IFN-gamma1, was powerful in inducing a proinflammatory reaction in phagocytes: a classical synergistic response with lipopolysaccharide was observed for the induction of iNOS expression and NO release, for expression of CXCL9-11-like chemokines and the expression of proinflammatory cytokines IL-1beta, TNFalpha and the IL-12 subunits p35 and p40. In contrast, like in mammals, the CXCL8-like cytokines are LPS but not IFN-gamma sensitive. These results corroborate an evolutionary conserved nature of IFN-gamma function in lower vertebrates including classical activation of phagocytes.

Morphine-induced changes in the activity of proopiomelanocortin and prodynorphin systems in zymosan-induced peritonitis in mice.

We have shown that supplementation of proinflammatory agent with a high dose of morphine not only abolishes inflammation-related pain symptoms but also inhibits influx of leukocytes to the inflamed peritoneal cavity. Present investigations focused on effects of morphine on proopiomelanocortin and prodynorphin systems during zymosan-induced peritonitis. Males of SWISS mice were ip injected with zymosan (Z, 40 mg/kg) or zymosan with morphine (ZM, 20 mg/kg). At time 0 (controls) and 4 and 24h after stimulation, peritoneal leukocytes (PTLs) were counted, PTL levels of opioid peptides (beta-endorphin and dynorphin) measured by radioimmunoassays, while mRNAs coding their respective precursors (POMC and PDYN) and receptors (MOR and KOR) determined by QRT-PCR. Influx of inflammatory PTLs, mainly PMNs, was significantly delayed by morphine co-injection. Total levels of beta-endorphin and dynorphin corresponded with PTL numbers, while levels per cell were similar in all groups except of beta-endorphin, decreased in ZM at 4h. Levels of both peptides in peritoneal fluid were increased in Z and ZM groups at 4h, while at 24h only in case of beta-endorphin in Z group. POMC was increased only in ZM group at 4h of peritonitis, while PDYN in both Z and ZM groups at the same time. MOR mRNA was increased 24h after injection in Z and ZM groups, while KOR mRNA was similar in all groups except of decrease in Z at 24h. In conclusion, endogenous opioids and their receptors are involved in zymosan-induced peritonitis and affected in various ways by morphine co-injection.

Strain differences in some immune parameters can be obscured by circadian variations and laboratory routines: studies of male C57BL/6J, Balb/c and CB6 F1 mice.

This is a report on the potential influence of circadian changes and laboratory routines on some immune parameters (thymic and splenic weights, the numbers of bone marrow, peripheral blood, and peritoneal leukocytes) in: (1) males of C57BL/6J, Balb/c, and CB6 F1 mice kept under identical laboratory conditions; (2) males of CB6 mice kept under the same laboratory conditions, except for opposite light/dark regimes, either light/dark (LD) or dark/light (DL). All the animals were purchased from the same supplier and adapted for 4-5 weeks to strictly controlled housing conditions. Some parameters were similar at certain time points but statistically significantly different at others due to strain-specific daily variations. In order to make the interstrain comparisons more reliable, the data collected around the day/night cycle were pooled for calculations of mean values. Several immune parameters of CB6 mice kept under DL conditions were significantly different than those in mice under the conventional LD conditions. In conclusion, the extrapolation of results (especially in the field of neuroimmunology) to other strains (or species) should be done with great caution; and all interstrain (interspecific) comparisons, especially those from various laboratories, should always be related to specific time points and laboratory conditions.

Expression of proenkephalin (PENK) mRNA in inflammatory leukocytes during experimental peritonitis in Swiss mice.

Zymosan- or thioglycollate-induced experimental peritoneal inflammation in mice may serve as a convenient model for investigations of involvement of opioid peptides derived from exudatory leukocytes in the inflammatory processes. During peritonitis, the influx of neutrophils and monocytes/macrophages correlated with a sequential appearance of proinflammatory cytokines (IL-1beta and TNFalpha). After both kinds of stimulation, the expression of PENK mRNA was much higher in exudatory peritoneal leukocytes than its basal level in steady state.

Inhibitory effects of morphine on some inflammation-related parameters in the goldfish Carassius auratus L.

Acute peritonitis induced in the goldfish by intraperitoneal injection of a sterile Thioglycollate solution shows a typical pattern with intraperitoneal exudation of serum proteins followed by influx of leucocytes (mainly heterophils/macrophages) correlated with elevated levels of chemotactic factors in peritoneal fluid and blood plasma. Supplementation of Thioglycollate with morphine (20 mg kg(-1) b.w.) does not affect the leakage of serum proteins into peritoneum. In contrast, it reduces the number of exudate peritoneal leucocytes (among them heterophils/macrophages) to the control level and decreases the level of peritoneal fluid/plasma chemoattractants, both effects being reversed by naltrexone pretreatment. Morphine itself acts as a chemokinetic factor for fish leucocytes as it increases their random movements. Therefore inhibitory effects of morphine on accumulation of exudate cells might be explained by inhibition of the production/release of chemotactic factors and/or reduced sensitivity of leucocytes to chemotactic signals. The effects of morphine on the goldfish peritonitis are in concordance with those described recently in Atlantic salmon and CB6 mice.

Morphine modulation of peritoneal inflammation in Atlantic salmon and CB6 mice.

Peritoneal inflammation is a convenient model for comparisons of modulatory effects of morphine in phylogenetically distant vertebrates. Both in salmon and mice morphine injected intraperitoneally together with an irritant (thioglycollate) significantly inhibits inflammation as estimated by the number of peritoneal leukocytes. The low number of exudate cells in morphine-treated animals seems to be compensated by their high activity, as evidenced by the enhanced phorbol myristate acetate-induced respiratory burst. The morphine-inhibited influx of leukocytes into the irritated peritoneal cavity correlates with the morphine-lowered level of plasma chemotactic factors both in fish and mice. It implies that morphine impairs the level of plasma chemotactic factor either directly (affecting their release from the resident peritoneal cells) or indirectly (decreasing the number of inflammatory leukocytes by inhibition of their migration from hemopoietic sites). The inhibitory effects of morphine on both the cell number and chemoattractant level are completely reversed by the naltrexone pretreatment, which implicates the involvement of opioid receptors.

Depletion of head kidney neutrophils and cells with basophilic granules during peritoneal inflammation in the goldfish, Carassius auratus.

The head kidney morphology of the goldfish with the experimental peritoneal inflammation (on day 2 after i.p. injection with sterile 3% Thioglycollate) is compared with that in the control fish (i.e. on day 2 after i.p. injection with a strile physiological saline PBS) with special emphasis on identification of granulocytes on semithin and consecutive ultrathin sections. The most striking feature of head kidneys of goldfish in the course of peritoneal inflammation is a severe depletion of mature neutrophils and cells with basophilic granules (basophils/mast cells). These observations suggest the involvement of the head kidney, the main hematopoietic organ of teleosts, in the inflammatory process.

Morphine reverses the inhibitory effects of repeated saline injections on peritoneal inflammation in mice.

Morphine sc injections reversed the inhibitory effects of daily sc saline injections on the thioglycollate-elicited peritoneal inflammation in Swiss mice.

Morphine modulation of thioglycollate-elicited peritoneal inflammation in the goldfish, Carassius auratus.

Intraperitoneal (i.p.) injection of adult goldfish with 3% thioglycollate (TG) induces an acute peritoneal inflammation connected with a massive influx of inflammatory leukocytes mainly of the head kidney origin. The number of peritoneal exudate cells retrieved on day 2 of the inflammatory response is significantly lower in fish injected with TG and morphine (20 mg/kg b.w.) than in animals injected with TG only. The morphine effect was totally antagonized in fish injected 20 min earlier with naltrexone (1 mg/kg), a well-known blocker of opioid receptors. Light microscopy of the head kidney Epon sections revealed that basophilic granulocytes are common in the control PBS-injected fish and even more frequent in fish injected with morphine only. In sharp contrast basophils are very rare in the head kidneys from animals with the TG-induced peritoneal inflammation, while they are more numerous in fish injected with TG and morphine. Supposedly, the basophilic granulocytes might be involved in the inhibitory effects of morphine on the acute inflammation in goldfish. An involvement of the head kidney in the morphine modulation of the inflammatory response is strongly supported by the detection of opioid receptors in the head kidney cells suspension.