Elsevier

Molecular Immunology

Volume 55, Issues 3–4, October 2013, Pages 231-236
Molecular Immunology

Carp head kidney leukocytes display different patterns of oxygen radical production after stimulation with PAMPs and DAMPs

https://doi.org/10.1016/j.molimm.2013.01.016Get rights and content

Abstract

Wound healing and tissue regeneration are essential mechanisms to ensure the survival and health of any organism. Despite this, only a few studies have been devoted to study tissue regeneration during wound healing in fish. Reactive oxygen species (ROS), in particular hydrogen peroxide, play an important dual role both for promoting tissue repair, but also for eradication of pathogens. This study aims at dissecting the contribution of PAMPs (using β-glucan) and DAMPs in the respiratory burst response of carp head kidney-derived leukocytes, and address their contribution to wound healing processes. Consistent with a pathogen eradication strategy, ROS responses to PAMP stimulation (β-glucan) was fast, vigorous and highly dominated by production of superoxide anion. In contrast, stimulation with DAMPs led to a slow, subtle but long-lasting production of oxygen radicals dominated by hydrogen peroxide. Using an in vitro model of scratch-wounded CCB fibroblast cell cultures and a novel PhotoID proliferation assay, stimulation with low and continuous levels of hydrogen peroxide (5 μM) led to a slight increase in the percentage of wound recovery and thus promoted wound closure. In contrast, high doses of hydrogen peroxide (300 μM) impaired fibroblast scratch-wound recovery and caused cell death. These results elucidate the capacity of hydrogen peroxide to influence the fate of tissue regeneration through the establishment of environments suitable for promoting either tissue regeneration or oxidative stress and thereby potential tissue damage. Direct in vitro stimulation with β-glucans did not impact fibroblast scratch-wound recovery, which further suggests that interaction with tissue-resident leukocytes or other components of the fish immune system are required to induce fibroblast proliferation and thus for the accelerated wound healing promoted by β-glucan stimulation.

Highlights

► Carp head kidney leukocytes can recognize DAMPS and PAMPs and produce different ROS patterns in response to them. ► Low and continuous doses of hydrogen peroxide seemed to improve wound closure. ► High doses of hydrogen peroxide impaired fibroblast scratch-wound recovery and caused cell death. ► Direct β-glucan stimulation of scratch-wounded fibroblasts cultures did not enhance wound recovery.

Introduction

A normal healing response starts when the tissue is injured. Work on mammalian models has shown that damage initiates a cascade of events with a coordinated interaction of different cell types which are involved in several overlapping phases including coagulation, early/late inflammation, cell proliferation and tissue remodelling (Diegelmann and Evans, 2004, Schaffer and Barbul, 1998, Mescher and Neff, 2005, Werner et al., 2007, Matsubayashi et al., 2011). Conversely, in fish little is known about the interactions between the immune system and the later phases of the wound healing process, albeit the inflammatory phases have been studied (Gonzalez et al., 2007, Forlenza et al., 2011).

Detection of tissue damage in the host is accomplished by the immune system, which uses a series of receptors displayed by the immune cells to identify the nature of the injury and act towards its repair. Such receptors are able to discriminate between the scenery related with an open wound, (implicating the intrusion of pathogens in the injury), and those consistent with pathogen-free trauma (evoking sterile inflammation) (Schreml et al., 2010, Rock et al., 2010). The aforementioned receptors are able to distinguish the different injury sceneries based on molecular signals. For example, pathogens display series of specific molecular motifs called pathogen-associated molecular patterns (PAMPs), which are not found in the host. On the other hand, identification of pathogen-free trauma is accomplished by the recognition of damage-associated molecular pattern (DAMPs), molecules from the host which in healthy cells are contained in the cells and hidden from the immune cells (Lotze et al., 2007). Both situations activate signalling pathways leading to the destruction and elimination of the pathogens and infected or damaged cells (Bianchi, 2007).

The involvement of compounds such as reactive oxygen species (ROS), nitric oxide (NO), cytokines, chemokines and growth factors during pathogen eradication and tissue regeneration have been studied (Bianchi, 2007, Werner and Grose, 2003). Immune cells like neutrophils, macrophages and lymphocytes are known to produce these molecules after they encounter pathogens or sense DAMPs (Rock et al., 2010, Lotze et al., 2007, Werner and Grose, 2003, Witte and Barbul, 2002, Kanta, 2011, Sen, 2009). However, there is growing evidence that non-immune cells such as fibroblasts, smooth muscle cells, endothelial cells and keratinocytes are able to reproduce this signal molecules, although to a lesser degree (Werner et al., 2007, Witte and Barbul, 2002, Finkel, 1999, Schaffer et al., 1997). An extraordinary coordination for the quantities and timing in the production of these molecules is needed, and exerts an important impact in the outcome of wound healing (Diegelmann and Evans, 2004, Wallach-Dayan et al., 2007). For example, while ROS in low concentrations (1–25 μM) function as a second messengers and promote cell proliferation, at higher concentrations (25–50 μM) have microbicidal effects, and in excessive amounts (>50 μM) can impair cell growth and cause apoptosis (Kanta, 2011).

Fibroblasts are recognized as a link between the immune system and the tissue regeneration machinery, since they are involved in the production of collagen and other extracellular matrix components (ECM), and are major regulators of inflammation during wound healing (Diegelmann and Evans, 2004, Ingerslev et al., 2010, Trengove et al., 1999). Cytokine profiles produced by leukocyte subsets can influence macrophages and fibroblast to reproduce the same cytokine profile, which can lead to tissue repair with normal architecture or favours fibroblast activation tower higher collagen production rates and fibrogenesis (Mescher and Neff, 2005). Thus, the cellular responses of leukocytes and fibroblasts, and their interactions during wound healing are of great importance for the understanding of tissue regeneration processes.

β-Glucans are glucose polysaccharides present in the cell wall of yeast, mushrooms, bacteria, algae and plants (Dalmo and Bøgwald, 2008). In mammalian systems, the immune stimulatory activity of β-glucans, especially the enhancement of tissue regeneration processes has been reported (Brown and Gordon, 2003, Reynolds et al., 1980, Son et al., 2005, Chen, 2007). Such immune stimulatory effects have also been described in several fish species (Dalmo and Bøgwald, 2008) however information on the effect of β-glucan in tissue regeneration processes in fish is scarce. We have previously reported that β-glucan enriched bath promotes wound closure in common carp (Przybylska et al., in press), however the mechanisms through which β-glucan accelerates the wound healing process remains to be understood.

Since leukocytes, reactive oxygen species and fibroblasts have been shown to play a key role during tissue regeneration, this paper studies the oxygen radical response of carp head kidney-derived macrophages to DAMPs and β-glucans. Furthermore, the direct effect of β-glucans and hydrogen peroxide on carp fibroblast scratch-wound recovery is discussed.

Section snippets

Fish

European Common carp (Cyprinus carpio carpio) were obtained from the central fish facility ‘De Haar-vissen’ (Wageningen, The Netherlands). R3 × R8 carp are the offspring of a cross between fish of Hungarian origin (R8 strain) and the Polish origin (R3) (Irnazarow, 1995). The fish used ranged between 50 and 100 g and were kept at 23 °C (±1 °C) with 12:12 h light:dark photoperiod.

Carp head kidney leukocyte isolation and culture

Head kidney leucocyte (HKL) isolation and culture were performed based on the protocol described by Joerink et al. (2006).

Carp head-kidney leukocytes (HKL) display different oxygen radical production kinetics after β-glucan and DAMPs stimulation

The ROS production of carp HKL after stimulation with Zymosan, DAMPs supernatant or DAMPs debris was measured using RT-luminol assay. Stimulation with Zymosan generated a fast and vigorous oxygen radical response with a clear peak of 4000 RLU magnitude, 30 min post-stimulation (See Fig. 1). The peak was followed by a uniform reduction of magnitude until the end of the measurement; significant differences are described in Table 1. Stimulation of HKL with DAMPs supernatant showed lower magnitudes

Discussion

Wound closure and tissue remodelling is very important to keep the health integrity of any organism (Singer and Clark, 1999, Martin, 1997, Noga, 2000). However, the mechanisms by which the organisms can alert and guide the immune system to react appropriately in different scenarios such as during infection or wound healing are not completely understood. In this study the role of oxygen radical species was analysed due to their involvement in infection clearance and tissue repair processes (

Acknowledgments

The authors would like to thank Thomas Lindenstrøm for his review of the paper content and text. This work has received funding from the Seventh Framework Program FP7/2007-2013 under grant agreement no. 214505.10

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