Research Article

Histamine is stored in mast cells of most evolutionarily advanced fish and regulates the fish inflammatory response

Iván Mulero, M. Pilar Sepulcre, José Meseguer, Alfonsa García-Ayala, and Victoriano Mulero
PNAS December 4, 2007 104 (49) 19434-19439; https://doi.org/10.1073/pnas.0704535104

  1. Edited by Martin Flajnik, University of Maryland, College Park, MD, and accepted by the Editorial Board October 23, 2007 (received for review May 15, 2007)

Abstract

Mast cells are important as initiators and effectors of innate immunity and regulate the adaptive immune responses. They have been described in all classes of vertebrates and seem to be morphologically and functionally similar. However, early studies had shown that fish and amphibian mast cells were devoid of histamine. In this study, we take a fresh look at the evolution of histamine and find that the mast cells of fish belonging to the Perciformes order, the largest and most evolutionarily advanced order of teleosts, are armed with histamine. More importantly, histamine is biologically active in these fish where it is able to regulate the inflammatory response by acting on professional phagocytes. In addition, the actions of histamine in these immune cells seem to be mediated through the engagement of H1 and H2 receptors, which, together with the H3 receptor, are well conserved in bony fish. We propose that the storage of histamine in vertebrate mast cells and its use as an inflammatory messenger was established in primitive reptiles (Lepidosauria) ≈276 million years ago. This same feature seems to have developed independently in Perciform fish much more recently in the Lower Eocene, between 55 and 45 million years ago, a short period during which the great majority of Percomorph families appeared.

The presence of mast cells (MCs) has been reported in all classes of vertebrates, including fish (1–4), amphibians (5), reptiles (6), birds (7), and mammals (8, 9). Several reports have described numerous granular cells in the intestinal mucosa, dermis, and gills of many teleost fish families, such as salmonids (10, 11), cyprinids (12, 13), and erythrinids (2, 14). In contrast, other studies have failed to find granular cells in the same tissues of other fish species (4) or have observed only very few granular cells (15, 16). Anyway, all these studies have shown that fish MCs constitute a heterogeneous cell population, which is exemplified by their heterogeneous morphology, granular content, sensitivity to fixatives, and response to drugs (17, 18). As regards this heterogeneity, one of the most controversial aspects is related to the staining properties of the cytoplasmic granules of MCs, which are frequently described as either basophilic or eosinophilic. Because of this, different authors have referred to them in different species as: mast cells, basophilic granular cells, or acidophilic/eosinophilic granule cells (EGCs) (17, 19).

Despite this heterogeneity, the main functional properties of MCs in teleosts are fairly similar to those of MCs in mammals. Thus, the granules of fish MCs are also known to contain components common to their mammalian counterparts, e.g., alkaline and acid phosphatases, arylsulphatase and 5′-nucleotidase (20, 21), lysozyme (22), and peptide antibiotics known as piscidins (23). As regards the two biogenic amines present in the granules of mammalian MCs, namely serotonin and histamine, all of the studies performed to date in fish have shown that fish MCs contain the former but lack the latter (12, 24, 25). In fact, it has been hypothesized that the storage of histamine in MCs and the powerful actions of histamine on smooth muscle have evolved together at least twice: in the immediate ancestors of lungfish and in the immediate ancestors of primitive reptiles (24). This hypothesis is upheld by (i) the observation that histamine is stored in the MCs of all studied descendants of primitive reptiles but absent from fish and amphibians (12, 26) and (ii) many physiological experiments have demonstrated that fish, excluding lungfish, are unable to respond to the intravascular injection of histamine (24). However, the presence of histamine in the gastric mucosa, as a regulatory molecule of acid gastric secretion, is a general feature in all vertebrates (27). In this study, we look again at the evolution of histamine and find that MCs of fish belonging to the Perciformes order, the largest and most evolutionarily advanced order of teleosts, are endowed with histamine. More importantly, histamine is biologically active in these fish and able to regulate the inflammatory response by acting on professional phagocytic granulocytes.

Results

Gilthead Seabream MCs Are Found in the Gills and Intestine.

We observed numerous cells in the connective tissue of the gills and intestine with the morphological and staining features of teleost MCs, i.e., the presence of numerous cytoplasmic granules that were strongly stained with eosin (Fig. 1 A and B). Interestingly, two different eosinophilic cell types were observed in these organs by using the G7 monoclonal antibody (mAb), which is specific to gilthead seabream acidophilic granulocytes (AGs) (28): MCs (G7− cells) and AGs (G7+ cells) (Fig. 1 B). MCs were numerous in the gill arch, in the connective tissue surrounding the central cartilage of the gill filaments (Fig. 1 B), and in the end of the gill filaments. In the intestine, MCs were numerous in the submucosa layer (Fig. 1 A). Neither MCs nor AGs of gilthead seabream showed the metachromatic staining characteristic of mammalian MCs after being stained with toluidine blue at low pH (Fig. 1 C).

Fig. 1.
Fig. 1.

Gilthead seabream MCs contain histamine and lack serotonin. (A–C) Sections of intestine (A and C) and gills (B) were stained with H&E (A), immunostained with the G7 mAb and slightly counterstained with eosin (B), or stained with toluidine blue at low pH (C). (D–F) Section of the intestine immunostained with the G7 mAb (D), with the G7 mAb and the anti-histamine antibody (E), or with the anti-histamine antibody alone (F). (G–L) Consecutive sections of healthy (G–I) or hyperplasic (J–L) gills were stained with H&E (G and J), immunostained with the G7 and the anti-histamine antibodies (H and K), or immunostained with the anti-histamine antibody (I and L). (M–O) Sections of the gills were immunostained with the anti-serotonin antibody (M and O) or with the G7 mAb (N) and then counterstained with eosin. M and N are consecutive sections. (P–R) Transmission electron micrographs of granular cells present in intestinal submucosa (P and Q) and immunoelectron microscopy using the anti-histamine antibody (R). (A) Cells showing a strong staining with eosin (black arrowheads) are numerous in the submucosa layer (SB) of the intestine. EP, intestinal epithelium. (B) AGs (black arrowheads, G7+) and MCs (red arrowheads, G7−) are seen in the gill filament. Note that both cell populations are eosinophilic but that only AGs are immunostained with the G7 mAb. (C) The cytoplasmic granules of MCs and AGs found in the intestine do not show metachromasia when stained with toluidine blue at low pH after being fixed in 10% formalin and 5% acetic acid in methanol. Note the strong metachromatic reaction of the goblet cells (red arrowheads) of the intestinal epithelium (EP). M, smooth muscle layer. SB, submucosa. (D–F) MCs present in the intestinal submucosa contain histamine (black arrowheads, G7−), whereas AGs (red arrowheads, G7+) do not. V, blood vessel. (G–I) Eosinophilic MCs (black arrowheads, G7−) observed in the connective tissue surrounding the cartilage (C) of the gill filament contain histamine. An AG (G7+) lacking histamine is depicted with a red arrowhead. Note that H and I correspond to the same section. (J–L) Eosinophilic MCs (black arrowheads, G7−) containing histamine (black arrowheads) were numerous in gills showing hyperplasia in the secondary lamellae (L). Several AGs (G7+) lacking histamine are depicted with red arrowheads. Note that K and L correspond to the same section. GF, gill filament. (M–O) Eosinophilic MCs (black arrowheads, G7−) lack serotonin. AGs (red arrowheads, G7+) and serotonergic neuroepithelial cells of the gills (white arrowheads) are observed. (P–R) Two granular cell types are observed in intestinal submucosa: a granular cell type showing the ultrastructural features of AGs and whose granules are not immunostained with the anti-histamine antibody (P) and a larger granular cell type with a more heterogeneous granular population (Q), which is ImmunoGold-labeled with the anti-histamine antibody (R). CL, collagen.

Gilthead Seabream MCs Contain Histamine but Lack Serotonin.

It is widely accepted that teleost fish and amphibians MCs lack histamine and, therefore, it is believed that the storage of histamine in these cells evolved in the immediate ancestors of primitive reptiles (24). Surprisingly, however, we found that gilthead seabream MCs (i.e., eosinophilic and G7− cells) were strongly immunostained with an antibody against histamine (Fig. 1 D–L). The specificity of the reaction was confirmed by preabsorption of anti-histamine with histamine [supporting information (SI) Fig. 6]. In contrast, gilthead seabream MCs were not immunostained with an antibody against serotonin (Fig. 1 M–O), which, however, stained serotonergic neuroepithelial cells in the gills (Fig. 1 M and O) and the fibers that innervate the smooth muscle of the intestine (data not shown). Importantly, AGs were negative for both histamine (Fig. 1 E, H, and K) and serotonin (Fig. 1 M and N), as shown by a double immunohistochemistry (IHC) using either the anti-histamine or anti-serotonin antibodies together with the G7 mAb. We also found that the number of MCs containing histamine, as well as the number of AGs, increased considerably in some specimens that naturally showed slight hyperplasia of the secondary lamellae of the gills (Fig. 1 J–L). Finally, two granular cell populations were found in the intestinal submucosa by transmission electron microscopy (Fig. 1 P and Q). One of them showed the ultrastructural features described for seabream AGs (28), whereas the other is larger (10 vs. 5 μm) and showed a more heterogeneous granule population. Notably, only the granules of the latter are immunostained with the anti-histamine antibody (Fig. 1 R).

Histamine Kills Gilthead Seabream When Injected i.p. and Induces Contraction of the Intestinal Smooth Muscle ex Vivo.

The above results prompted us to investigate whether histamine was able to exert any pharmacological effect on the gilthead seabream, because earlier studies showed that histamine had no great impact on the cardiovascular system, including blood pressure, heart rate, vascular smooth muscle, and permeability of capillaries or in the contraction of extravascular smooth muscle (24). We observed that gilthead seabream specimens rapidly died upon i.p. injection of 500 mg/kilogram of body weight of histamine, although lower concentrations had no effect (SI Table 1). Interestingly, compound 48/80, which is a potent histamine-releasing agent primarily from MCs (29), was also lethal at concentrations known to promote histamine release (SI Table 1). In addition, this effect was also induced by the H2 receptor agonist dimaprit, but not with the H1 receptor agonist pyridilethylamine or the H3 receptor agonist immethridine (SI Table 2). However, we were unable to block this systemic effect of histamine and dimaprit by using nonlethal concentrations of different histamine receptor antagonists (SI Table 3). Therefore, we used the well established intestinal smooth muscle contraction assay to further study the pharmacological effects of histamine in the gilthead seabream. Again, it was observed that histamine, dimaprit, and compound 48/80 caused a strong contraction of the intestinal smooth muscle and constriction of branchial blood vessels, whereas pyridilethylamine failed to do so (Fig. 2 A–I and SI Table 4). In addition, all these effects of histamine were reversibly blocked by the H2 receptor antagonist ranitidine (SI Table 4). Strikingly, ex vivo incubation of intestine and gill fragments with compound 48/80 resulted in the degranulation of MCs from both tissues (Fig. 2 J and K), whereas i.p. injection of this agent caused the degranulation of gill MCs (Fig. 2 L and M).

Fig. 2.
Fig. 2.

Histamine regulates smooth muscle contraction in gilthead seabream. (A–D) An intestine fragment was treated with 5 mg/ml histamine, and images were taken every 4 s. (E and F) Sections of intestinal fragments treated for 1 min with PBS (E) or histamine (5 mg/ml) (F) were stained with H&E. Smooth muscle cells of intestine are relaxed (white arrows) and show round-oval nuclei (red arrows) after being incubated with PBS, whereas they are contracted (white arrows) and show very thin nuclei (red arrows) after being incubated with histamine. (G–I) A gill fragment was treated with 7.5 mg/ml compound 48/80, and images were taken every 20 s. Note that blood moves away from the branchial arteries after the addition of compound 48/80. (J and K) Sections of a gill fragment treated with compound 48/80 was immunostained with the anti-histamine serum and then counterstained with eosin. Note that eosin-positive cells show a strong degranulation (arrowheads), and a few of them are weakly immunostained for histamine. (L and M) Sections of the gill arch from PBS- (L) or compound 48/80-injected (M) fish were immunostained with the anti-histamine antibody. Note that cells showing immunoreactivity with the anti-histamine antibody are not observed in the gill of compound 48/80-injected fish, but several cells with the morphological features of MCs are present (black arrowheads). C, cartilage; GA, gill arch; GF, gill filament; SB, submucosa; M, smooth muscle layer; V, blood vessel.

Histamine Regulates the Respiratory Burst of Gilthead Seabream Leukocytes.

The presence of histamine in the granules of gilthead seabream MCs suggests that this molecule might play a role in the regulation of the fish inflammatory response, as occurs in higher vertebrates. Therefore, we stimulated total head kidney leukocytes and purified AGs with VaDNA in the presence of histamine or the specific H1 and H2 receptor agonists. The results show that histamine inhibited the Vibrio anguillarum (VaDNA)-primed respiratory burst of total head kidney leukocytes (Fig. 3 A) and purified AGs (Fig. 3 B) in a dose-dependent manner. Interestingly, the H1 agonist pyridilethylamine exerted the same effect and with a potency similar to histamine, whereas the H2 agonist dimaprit at 10−4 M had the opposite effect and further increased the respiratory burst of gilthead seabream phagocytes (Fig. 3 A and B). Cell viability was not significantly affected by the treatments and was ≈90%, except with 10−3 M (SI Fig. 7) and 10−2 M (data not shown) dimaprit, which reduced cell viability to 70 and 20% in total head kidney leukocytes, and 22 and 3% in AGs, respectively (data not shown and SI Fig. 7). Notably, only ≈20% of live AGs were responsible for the chemiluminescence response of these cells after being treated with 10−3 M dimaprit, suggesting that the highest concentrations of this compound led to a strong activation of AGs that, in turn, induced their death. In addition, only AGs, but not lymphocytes, died in the presence of 10−3 M dimaprit, ruling out a toxic effect of this concentration of the compound on seabream leukocytes (SI Fig. 7).

Fig. 3.
Fig. 3.

Histamine regulates the respiratory burst of gilthead seabream phagocytes. Total head kidney leukocyte (A) or purified AG (B) suspensions were stimulated for 16 h with 50 μg/ml VaDNA in the absence or presence of 10−5 to 10−2 M histamine, pyridilethylamine (H1 receptor agonist), or dimaprit (H2 receptor agonist). The respiratory burst activity was then measured as the luminol-dependent chemiluminescence triggered by PMA (1 μg/ml). Data are presented as mean ± SE. of triplicate cultures and are representative of three independent experiments. Different letters denote statistically significant differences between the groups according to a Waller–Duncan test. The groups marked with “a” did not show statistically significant differences from cells stimulated with VaDNA alone, which is indicated with a horizontal line. RLU, relative light units.

Histamine Is Present Only in MCs of Perciformes Fish.

We next studied whether MCs of the other main phylogenetic groups of teleosts and of lungfish also contain histamine. First, we studied the European seabass, which also belongs to the Perciformes order but to a different family (Moronidae), and found that MCs are quite similar to those of the gilthead seabream. Thus, seabass MCs were numerous in the gills and intestine, and their cytoplasmic granules were strongly stained with eosin (Fig. 4 A), contained histamine (Fig. 4 B), and were devoid of serotonin (data not shown). In sharp contrast, the granules of MC from the rainbow trout, which belongs to the order Salmoniformes, were not stained red with eosin but were stained purple with Giemsa (Fig. 4 C), as described (30). These cells were observed in the connective tissue surrounding the gill filaments (data not shown) and in the intestine (Fig. 4 C), where they formed a layer of one or two rows of cells between the stratum compactum and the circular muscle layer. More interestingly, rainbow trout MCs were devoid of histamine (Fig. 4 D) and serotonin (data not shown). Similarly, we did not find any histamine-positive cells in the gills and intestine from any of the other species analyzed, including zebrafish (Cypriniformes), European eel (Anguilliformes), and turbot and Senegalese sole (both Pleuronectiformes) (data not shown). In addition, both H&E and Giemsa staining failed to identify “typical” MCs, i.e., those showing eosinophilic granules, in the gills and intestine of all these species. Notably, no serotonin-immunoreactive cells were found in the intestinal submucosa of all these species, although a few serotonin-immunoreactive enteroendocrine cells were located in the intestinal mucosa of trout and eel (data not shown). Finally, numerous eosin-positive/metachromatic-negative cells were observed in the intestinal submucosa and gills of lungfish (Fig. 4 E and F), but they were devoid of histamine (Fig. 4 G) and serotonin (data not shown).

Fig. 4.
Fig. 4.

Histamine is present only in MCs of Perciformes. Consecutive sections of seabass gills were stained with H&E (A) or immunostained with the anti-histamine antibody (B). Sections of rainbow trout intestine were stained with Giemsa (C) or immmunostained with the anti-histamine antibody (D). Sections of lungfish intestine (E and G) and gills (F) were stained with H&E (E), toluidine blue (F), or anti-histamine antibody (G). Sections of frog intestine (H and I) immunostained with the anti-serotonin (H) or anti-histamine (I) antibodies and then counterstained with toluidine blue (TB). (A and B) Numerous eosinophilic MCs containing histamine (black arrowheads) are observed at the end of the gill filaments. C, cartilage; V, blood vessel. (C and D) Numerous MCs (black arrowheads) showing purple granules are seen between the smooth muscle layer (M) and the stratum compactum (ES) in the intestine. No histamine-positive cells are found in the intestine. EP, intestinal epithelium; SB, submucosa layer. (E–G) Numerous eosin-positive and metachromatic-negative MCs (red arrowheads) showing birefringent granules are observed in the intestinal submucosa and gills. No histamine-positive cells are found in the intestine. V, blood vessel; asterisk, pigment cell. (H and I) Several MCs located in the intestinal submucosa (SB) showing metachromasia after being stained with toluidine blue (blue arrowheads). Metachromatic MCs are not immunostained with anti-serotonin or anti-histamine sera. Note the presence of two serotonin-immunoreactive enteroendocrine cells (white arrowheads) in the intestinal epithelium (EP).

We also extend our study to two of the main groups of amphibians: frogs and newts. MCs of the intestinal submucosa of the frog showed metachromasia after being stained with toluidine blue at low pH but are devoid of histamine and serotonin (Fig. 4 H and I). Although no histamine-immunoreactive cells were observed in the intestine of the frog, some enteroendocrine cells located in the intestinal epithelium were immunostained with the anti-serotonin antibody (Fig. 4 H). Similar results were obtained in the newt; that is, MCs showed metachromasia after being stained with toluidine blue but they do not contain histamine or serotonin (data not shown). Strikingly, the i.p. injection of up to 500 mg/kg of histamine and 15 mg/kg of compound 48/80 are not lethal for the frogs, and both molecules failed to induce the contraction of the intestinal smooth muscle of this species (data not shown).

Discussion

MCs are present in adult mammals in virtually all vascularized tissues (31). They have been described in all classes of vertebrates and seem to be morphologically and functionally similar to their mammalian counterparts. Perhaps one of the most controversial aspects of the biology of MCs of early vertebrates concerns the presence of histamine in their granules. Using a spectrofluorometric method (32), earlier studies had shown that fish and amphibian MCs were devoid of histamine (12, 26), whereas its presence in the gastric mucosa, as a regulatory molecule of acid gastric secretion, was a general feature of all vertebrates (27). More recently, however, the presence of very low amounts of histamine in the granules of frog MCs has been revealed by IHC (33). More importantly, these observations were further supported by numerous physiological and pharmacological studies showing that most fish and amphibian species were unable to significantly respond to the intravascular injection of histamine (24). To the best of our knowledge, however, no Perciformes species were included in any of these pharmacological studies and most morphological studies involving histamine (24). In the present study, we use IHC to describe the presence of histamine in the MCs of teleost fish belonging to the order Perciformes, the largest and most evolutionarily advanced order of teleosts, as well as the absence of this molecule in lungfish and amphibian MCs. We also show that teleost, lungfish and amphibian MCs are devoid of serotonin, another important inflammatory mediator released by mammalian MCs upon activation (7, 8). In addition, these morphological observations are accompanied by several functional assays that demonstrate that histamine is able to induce specimen death, contraction of the intestinal smooth muscle, and constriction of branchial blood vessels in gilthead seabream. Strikingly, compound 48/80, which is a powerful histamine-releasing agent from mammalian MCs (29), induced the degranulation of seabream MCs and exerted the same physiological effects as histamine, suggesting that the release of endogenous histamine from seabream MCs mediates the observed pharmacological effects of this agent. In sharp contrast, however, histamine and compound 48/80 were not lethal for frog, which further supports our observation on the absence of histamine in amphibian MCs. Further, all of the observed action of histamine in seabream MCs may be mediated by the engagement of H2 receptor, because only the H2 agonist dimaprit, but not H1 or H3 agonists, was also able to induce specimen death and smooth muscle contraction. Although future studies aimed at the characterization of the fish histamine receptors are required, well conserved (60% amino acid similarity between fish and mammals) homologues for H1, H2, and H3 receptors have been identified in the zebrafish, shown to be expressed in the brain and a few other tissues, and found to regulate swimming behavior of young larvae (34). In addition, it has been reported that several antagonists specific for human histamine receptors can effectively inhibit fruit fly histamine receptors in vivo and ex vivo (35). Collectively, these data support our pharmacological experiments and, therefore, strongly suggest a role for histamine in fish.

We also show that histamine is important in the regulation of fish professional phagocyte activity. Histamine and the H1 receptor agonist pyridilethylamine were both found to strongly inhibit the phagocyte respiratory burst activity primed by bacterial DNA, whereas an H2 agonist had the opposite effect; that is, it further increased this activity. Hence, histamine may regulate fish phagocyte functions in a complex manner through the engagement of different receptors, as has been shown in murine macrophages, where histamine acting on H1 and H2 receptor restricts the growth of Mycobacterium bovis bacillus Calmette–Guérin via the production of interleukin-18 (36). In contrast, in an Escherichia coli infectious peritonitis model, histamine acting on H1 and H2 receptors impaired neutrophil recruitment, which delayed the elimination of bacteria (37). Whatever the outcome, our results suggest that fish phagocyte functions may be regulated by the release of histamine from MCs upon their activation. One interesting question that remains to be clarified is the mechanism by which fish MCs are activated, because IgE is not present in this group of animals, and the “classical” activation mechanism of mammalian MCs involves engagement of the high-affinity IgE receptor (FcεRI) by IgE bound to specific antigens (38). Nevertheless, apart from activation via FcεRI, MCs are also activated to release some of the same mediators after the aggregation of surface FcγRIII by IgG/antigen complexes and after exposure to a range of small peptides, including chemokines, the anaphylatoxins C3a and C5a, and fragments of fibrinogen and fibronectin (31, 38). More recently, heterotypic aggregation with activated T cells (39) and the direct recognition of soluble mediators derived from infectious agents, such us lipopolysaccharide, have also been found to induce MC activation and mediator release (40). All these “alternative” mechanisms may operate in the activation of fish MCs, because the presence of receptors for IgM (41), anaphylatoxins (42) and soluble mediators derived from infectious agents (43) are all functional in these animals.

Based on our data, we propose an alternative model for the origin and evolutionary history of histamine in MCs (Fig. 5). It is possible that although the storage of histamine in MCs and its use as an inflammatory messenger was established in primitive reptiles (Lepidosauria) ≈276 mya (44), this same feature developed independently in Perciform fish in the Lower Eocene, between 55 and 45 mya, a short period during which the great majority of Percomorph families appeared (47). It is striking that piscidins had a similar evolutionary history, because only the MCs of Perciformes are endowed with these antimicrobial peptides (48). However, a related family of antimicrobial peptides, called pleurocidins, are synthesized by MCs of the Atlantic halibut (49), a flat fish belonging to the order Pleuronectiformes and the family Pleuronectidae. This sharply contrasts with our results in two fish species representative of the order Pleuronectiformes, turbot (Scophthalmidae) and Senegalese sole (Soleidae), whose MCs are not stained with eosin and lack histamine. Collectively, these results suggest great heterogeneity in the mediators produced by the MCs of different species and point to the complexity of the evolutionary history of these immune cells.

Fig. 5.
Fig. 5.

Evolutionary model for the storage of histamine in vertebrate MCs and its utilization as an inflammatory messenger. (A) Phylogeny showing the dates of the last common ancestors between the represented classes and human beings. Dates are given as molecular clock estimates (44), and commonly used model species are enclosed in parentheses. The emergence of various physiological and immunological features is shown at the top of the figure. Adapted from DeVries et al. (45). (B) Cladogram showing the phylogenetic relationships between the most important orders of ray-finned fishes (Actinopterygii). Adapted from Trapani (46). The orders analyzed in this study are shown in different colors.

Materials and Methods

Animals.

Gilthead seabream (Sparus aurata L., Perciformes, Sparidae) specimens (10–150 g) were kept in 260 liters of running-seawater aquaria (flow rate 1,500 liter/h) at 23°C under a 12-h light/dark cycle and fed with a commercial pellet diet (Skretting) at a feeding rate of 15 g of dry diet per kilogram biomass of fish per day. Zebrafish (Danio rerio H., Cypriniformes, Cyprinidae) of the TL genetic background were kindly provided by the Zebrafish International Resource Center and maintained as described in the Zebrafish handbook (50). Other fish and amphibian species used in this study are described in SI Materials and Methods .

Microscopy and IHC.

Intestine and gill sections were subjected to an indirect IHC method (51) using the G7 mAb, which is specific to gilthead seabream AGs (28), or the polyclonal antibodies against histamine (Sigma) and serotonin (INC). Sample processing, section staining and IHC protocols are described in detail in SI Materials and Methods .

Pharmacological Assays.

This section is described in SI Materials and Methods .

Respiratory Burst Assay.

Total gilthead seabream head kidney leukocytes were obtained as described (28). AGs, the professional phagocytic granulocytes of this species, were isolated by magnetic-activating cell sorting (MACS) using the G7 mAb (28). Total head kidney leukocyte or purified AG suspensions were stimulated for 16 h with 50 μg/ml phenol-extracted genomic DNA from the bacterium Vibrio anguillarum ATCC19264 (VaDNA) in the absence or presence of 10−5 to 10−2 M histamine, pyridilethylamine, or dimaprit. The respiratory burst activity of phagocytes was then measured as the luminol-dependent chemiluminescence triggered by phorbol myristate acetate (PMA) (43). Cell viability was checked in parallel samples by flow cytometry analysis of cells stained with 40 μg/ml propidium iodide.

Statistical Analysis.

Data were analyzed by ANOVA, and a Waller–Duncan multiple range test was used to determine differences between groups.

Acknowledgments

We thank Drs. B. Novoa (Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Cientificas, Spain), A. Estepa (Miguel Hernandez University, Spain) R. Barrera (Valenciana de Acuicultura, Spain) M. L. Ribeiro (Universidade Federal do Pará, Brazil) and R. DaMatta (Universidade Estadual do Norte Fluminense, Brazil) for providing some of the specimens or tissue samples used in this study. This work was supported by Spanish Ministry of Education and Science Grant BIO2005-05078 (to V.M.) and a Fundación Séneca-Murcia Fellowship (to I.M.).

Footnotes

  • *To whom correspondence should be addressed. E-mail: vmulero{at}um.es

  • Author contributions: I.M. and V.M. designed research; I.M. and M.P.S. performed research; I.M., M.P.S., J.M., A.G.-A., and V.M. analyzed data; and I.M. and V.M. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission. M.F. is a guest editor invited by the Editorial Board.

  • This article contains supporting information online at www.pnas.org/cgi/content/full/0704535104/DC1.

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Histamine is stored in mast cells of most evolutionarily advanced fish and regulates the fish inflammatory response
Iván Mulero, M. Pilar Sepulcre, José Meseguer, Alfonsa García-Ayala, Victoriano Mulero
Proceedings of the National Academy of Sciences Dec 2007, 104 (49) 19434-19439; DOI: 10.1073/pnas.0704535104
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