Adhesion and ingestion activities of fish phagocytes induced by bacterium Aeromonas salmonicida can be distinguished and directly measured from highly diluted whole blood of fish

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Abstract

The phagocytes of fish play an important role in innate host defense against bacterial infection, and participate in various immunoregulatory processes. Here, we investigated the effects of various opsonins in the ingestion and adhesion processes by examining respiratory burst (RB) activity in blood and head kidney (HK) fish phagocytes. RB activity was induced in rainbow trout phagocytes with the bacterium Aeromonas salmonicida (strain MT004) in the presence of various opsonins [purified antibodies (Ab), immune serum (IS), normal serum (NS) and heat-inactivated immune serum (HI-IS)], and measured in terms of luminol-amplified chemiluminescence (CL) emission at 20 °C for 210 min. The RB activity of blood phagocytes was measured directly from highly diluted whole blood and compared to that observed in isolated head kidney (HK) phagocytes measured under similar conditions. In addition, the extracellular RB activity of adhesion (extracellular degranulation) and the intracellular RB activity of ingestion were distinguished through their inhibition by gelatin and cytochalasin D. Our results showed that the first CL peak appeared within 50 min, and decreased or vanished when gelatin was added to the reaction or when the active complement was destroyed by heating. The second CL peak appeared after 50 min, depending on the utilized opsonin, and vanished when cytochalasin D was added to the reaction. Our results indicate that adhesion and ingestion compete for consumption of reactive oxygen intermediates. Specific IgM without an active complement was a relatively inefficient opsonin, whereas specific IgM with an active complement increased the magnitude of ingestion-mediated RB activity and accelerated the ingestion of target bacteria. Taken together, these results indicate that adhesion and ingestion responses competed for limited phagocyte resources and that the bacterial uptake by blood phagocytes can be measured directly from highly diluted blood.

Introduction

Phagocytosis, a fundamental defense mechanism in bony fish, is mediated by phagocytic cells such as neutrophils, monocytes and macrophages. The role of these cells in host defense is to limit the dissemination of infectious organisms and bring about their final destruction [1]. The antibacterial defense mechanisms of phagocytic cells rely on production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) during a respiratory burst (RB) [1], [2]. The production of these compounds has been correlated with killing of bacteria in fish [3], [4], [5] and humans [6], [7].

Aeromonas salmonicida, the causative agent of furunculosis, is one of the most widely studied fish pathogens. It is classified into typical and atypical strains, with typical strains being the most virulent [8], [9]. The virulent strains are covered with the so-called A-layer, which is composed of a surface protein [10], [11], [12] that gives the bacterium its hydrophobic nature [13] and other special characteristics that are believed to be associated with virulence [13], [14]. Some strains of the pathogen have been reported to be toxic to phagocytic cells of fish [14], [15], [16]. Additionally, the A-layer and the LPS structure of A. salmonicida affect the interaction of the bacterium with trout macrophages [14], [17], [18] and complement-mediated killing [19].

In the serum of teleost fish, two types of immunoglobulin (belonging to the IgM and IgD subpopulations) have been characterized. Fish IgM is an avid agglutinator that can activate the classical complement pathway. The fish IgD was recently characterized in serum [20], and is not yet well-defined in terms of its characteristics or function. Among activation of classical pathway of complement another important function of immunoglobulin M along complement is to enhance bacterial phagocytosis by neutrophils and macrophages in fish [4], [21], [22], [23], [24]. Moreover, other serum compounds, such as mannose binding lectin [5] and C-reactive protein [25], have been shown to enhance phagocytosis of bacteria in fish. Shen et al. [26] demonstrated that channel catfish NK-like cells expressed a putative FcμR that participated in antibody dependent cell-mediated cytotoxicity reactions. However, to our knowledge the existence of opsonin receptors (including Fc- or complement receptors) has not yet been definitely confirmed in the phagocytes of salmonid species. Froystad et al. [27] studied isolated head kidney (HK) macrophages of rainbow trout and proposed that neither Fc- nor complement receptors were important for phagocytosis of beads coated with IgM or complement. Froystad postulated that phagocytosis by these cells mainly involved scavenger receptors, which are integral membrane proteins that mediate binding and internalization of a wide range of negatively charged macromolecules [28]. In contrast, O'Dowd et al. [29] showed that peripheral blood leukocytes of Atlantic salmon bound immune complexes (IC) in an antibody-dependent fashion via what the authors proposed to be Fc receptors.

The most commonly used technique for measuring RB activity in fish is the nitroblue tetrazolium (NBT) reduction technique, which requires isolated phagocytes. However, the isolation process has been shown in human studies to activate the cells [30], [31], [32]. Another important disadvantage of the NBT method is that it is quite laborious to measure the kinetics of the RB reaction with this method, and in practice only a few time points are generally used. Unfortunately, the kinetics of RB activity are affected by temperature [33], and the variations among individual fish may be considerable, making the NBT method less than optimal. These limitations are overcome by another widely used technique, which utilizes luminescent emission of the reaction between luminol and ROS to measure RB kinetics. In fish, the RB activity of phagocytes has been monitored using this technique in isolated phagocytes [24], [34], [35], [36], [37], [38], [39] and diluted blood [33]. Diluted blood has a major advantage over isolated phagocytes, in that the isolation process is eliminated and the phagocytes are in an ex vivo state. However, the induction of the RB activity of blood phagocytes has been reported [33] to require a powerful activator such as zymosan to achieve a measurable chemilumienscence emission. Here, we describe a luminol-enhanced chemiluminescence method for separately evaluating adhesion and ingestion respiratory burst activities of fish blood and HK phagocytes. Using bacteria cells as cell activator instead of zymosan two separate processes are measurable separately and thus the effects of various opsonins and receptors involved in phagocytosis are evaluated.

Section snippets

Bacteria and culture conditions

The bacterial fish pathogen Aeromonas salmonicida (strain MT004) was a generous gift from Dr. Ian Bricknell (FRS Marine Laboratory, Aberdeen, Scotland). The strain has been demonstrated to be susceptible to phagocytosis in the presence of various opsonins [40] and was reported to be avirulent [4], [21], [41], [42], [43], [44], [45]. The bacteria were grown at 20 °C in tryptone soy broth (TSB, Merck, Germany) with agitation to late log phase, harvested by centrifugation at 2500×g for 10 min,

Results

The titer of the anti-A. salmonicida MT004 antibodies in pooled serum from vaccinated rainbow trout was 1:738, the titer of anti-A. salmonicida MT004 in purified antibodies (Ab) from the same sera was 1:735, and the protein concentration in the Ab fraction was 4.8 μg/μl.

Discussion

The respiratory burst (RB) activity of rainbow trout phagocytes, measured through luminol-amplified chemiluminescence (CL), showed a biphasic response that was dependent on the number of effector (E) cells. When the number of HK cells exceeded 5×104 cells/well, the two peaks merged into one large peak, and it became impossible to accurately estimate the magnitude of the ingestion process (data not shown). The same phenomenon occurred when the number of target bacteria (T) exceeded 5×107

Acknowledgements

This study was supported by Employment and Economic Development Centre of Finland.

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