Inflammatory cells of teleostean fish: a review focusing on mast cells/eosinophilic granule cells and rodlet cells

In contrast to the roles played by monocytes/macrophages, neutrophils and lymphocytes, the presence and functions of basophils, mast cells/eosinophilic granule cells, eosinophils and rodlet cells in teleosts are areas of controversy. The tissue distribution of mast cells/eosinophilic granule cells in species from a certain genus shows a characteristic pattern, and this pattern is usually also present at the family level. Functionally, the mast cells/eosinophilic granule cells of teleosts show close similarity to the mast cells of mammals. Acute tissue damage is causing mast cell/eosinophilic granule cell degranulation and release of mediators of inflammation, whereas an increase in the number of these cells is often found in chronically inflamed tissues. The mast cells/eosinophilic granule cells of teleosts show marked diversity in their staining properties, with both basophilic and acidophilic components in their granules. In some fish families, e.g. the labrids, the eosinophilic component is dominating, whereas in the pike the granules are strongly basophilic and show the metachromatic staining characteristics found in the granules of mast cells, but being more akin to the granules of the mucosal than to those of the connective tissue type of mast cells of mammals. With respect to rodlet cells, a cell type hitherto clearly demonstrated only in teleosts, a characteristic distribution pattern seems to be established in certain families. In other families rodlet cells are absent in some individuals and present in different tissues in others. However, there is a close relation between the presence of helminths or other noxious agents and the presence of rodlet cells. Massive aggregations of such cells can be seen in affected epithelia of gills or the intestinal tract, and in individuals of species from some fish families they also occur in association with mesothelial and endothelial tissues. The rodlet cell may represent a type of eosinophilic granulocyte that populates the tissues at its immature stage and mature in response to the appropriate stimuli, in a way similar to that of mast cell precursors. Present evidence points to a functional role for the rodlet cells of teleosts in host defence against parasites.     

The rodlet cells of teleostean fish: their potential role in host defence in relation to the role of mast cells/eosinophilic granule cells

The distribution and potential function of the rodlet cells of teleosts were studied by microscopic observations on tissue samples from the digestive tract and adjacent tissues, including the bulbus arteriosus. Fish representing 3-5 genera from each of the families Salmonidae, Cyprinidae, Gadidae and Labridae were included in the study. Great individual variations in the distribution of rodlet cells were found in all species of salmonids, gadids and labrids. The cells seemed to be absent in some individuals of a species and were associated with different epithelial tissues in others, but were not found in vascular endothelia. Their occurrence was common in all salmonids caught in their natural environment, whereas those in aquaculture, kept under controlled conditions with respect to water quality, showed extremely few rodlet cells. In species of the cyprinid family, the picture was different. Rodlet cells were consistently present under the endothelium of the bulbus arteriosus, and were very numerous at this location in individuals infected with blood flukes. In other epithelial tissues of cyprinids, rodlet cells were encountered in fairly high numbers, but in some tissues of individuals from all species they were occasionally absent. In all of the studied families rodlet cells seemed to be recruited when helminths affected epithelial tissues. Mast cells/eosinophilic granule cells were consistently very numerous in tissues of the intestine of cyprinids and labrids. In gadids, mast cells/eosinophilic granule cells seemed to be absent. Present evidence points to a role for the rodlet cells in defence functions, e.g. in combating helminths, and the suggestion earlier made for mast cells/eosinophilic granule cells, that evolution has created a "standing force" in particular tissues of teleosts consistently exposed to pathogens, whereas an efficient "mobilization force" has been an advantage in those living in more pathogen-free environments, may also be applied to rodlet cells, explaining the differences between teleostean families with respect to their distribution pattern.     

Piscidin 4, a novel member of the piscidin family of antimicrobial peptides

Piscidins are linear, amphipathic, antimicrobial peptides (AMPs) with broad, potent, activity spectrum. Piscidins and other members of the piscidin family appear to comprise the most common group of AMPs in teleost fish. All piscidins and related members of the piscidin family described to date are 18–26 amino acids long. We report here the isolation of a novel 5329.25 Da, 44-residue (FFRHLFRGAKAIFRGARQGXRAHKVVSRYRNRDVPETDNNQEEP) antimicrobial peptide from hybrid striped bass (Morone chrysops female x M. saxatilis male). We have named this peptide “piscidin 4” since it has considerable (to > 65%) N-terminal sequence homology to piscidins 1–3 and this distinctive, 10 to 11-residue, N-terminus is characteristic of piscidins. The native peptide has a modified amino acid at position 20 that, based upon mass spectrometry data, is probably a hydroxylated tryptophan. Synthetic piscidin 4 (with an unmodified tryptophan at position 20) has similar antibacterial activity to that of the native peptide. Piscidin 4 demonstrates potent, broad-spectrum, antibacterial activity against a number of fish and human pathogens, including multi-drug resistant bacteria. Its potent antimicrobial activity suggests that piscidin 4 plays a significant role in the innate defense system of hybrid striped bass.     

Immunocytochemical localization of piscidin in mast cells of infected seabass gill

Annual losses of ～5–10% of the juvenile stock of European seabass, Dicentrarchus labrax (L.) in the northern coast of the Adriatic Sea has been attributed to heavy infections of the gill monogenean Diplectanum aequans. Immunocytochemical, light and ultrastructural studies were carried out on seabass naturally parasitized with this monogenean. The site of the worm's attachment was marked by the common presence of haemorrhages and white mucoid exudate. In histological sections, infected gills showed hyperplasia, as well as proliferation of mucous cells and rodlet cells. Disruption and fusion of the secondary lamellae were common in all infected seabass, with several specimens also showing marked inflammation and erosion of the primary and secondary lamellar epithelium. Immunostaining of primary and secondary gill filaments with an antibody against the antimicrobial peptide piscidin 3 (anti-piscidin 3 antibody, anti-HAGR) revealed a subpopulation of mast cells that were positive. Mast cells were both within and outside the blood vessels of the primary and secondary lamellae, and often made intimate contact with vascular endothelial cells. Mast cells were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bound granules. Our data provide evidence showing the presence of piscidin 3 in the cytoplasmic granules of an important group of fish inflammatory cells, the mast cells resident in seabass gill tissue. There was no significant difference in the number of HAGR-positive mast cells between infected and uninfected fish (ANOVA, p > 0.05). However, mast cells in parasitized gills usually showed much stronger immunostaining intensity compared to those in unparasitized gills. These data are the first to document a response of piscidins or any other antimicrobial peptide of fish to parasite infection and suggest that mast cells may play a role in certain inflammatory responses without a detectable increase in their numbers.     

Mast cell responses to Ergasilus (Copepoda), a gill ectoparasite of sea bream

Immunocytochemical, light microscopy and ultrastructural studies were conducted on gill of sea bream, Sparus aurata L., naturally parasitized with the important parasitic copepod Ergasilus sp. to assess pathology and cellular responses. Thirty-seven S. aurata were examined from a fish farm; 26 (70%) were parasitized, with infection intensity ranging from 3 to 55 parasites per fish. Hosts were divided into two groups, lightly infected fish (<15 parasites per fish) and heavily infected fish (>15 parasites per fish). In histological sections, the copepod encircled gill lamellae with its second antennae, compressed the epithelium, provoked hyperplasia and hemorrhage, occluded arteries and often caused lamellar disruption. Fusion of the secondary lamellae due to epithelial hyperplasia was common in all infected fish; heavily infected fish showed more intense branchial inflammation. In both healthy and infected fish, mast cells (MCs) were free within the connective tissue inside and outside the blood vessels of the primary lamellae and made close contact with vascular endothelial cells, mucous cells and rodlet cells (RCs). MCs were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bound granules. Immunostaining of primary and secondary gill filaments with an antibody against the antimicrobial peptide (AMP) piscidin 3 (anti-piscidin 3 antibody, anti-HAGR) revealed a subpopulation of MCs that were positive. These MCs were more abundant in gills of heavily infected fish than in either lightly infected or uninfected fish (ANOVA, P<0.05). Our report documents the response of gill to ectoparasite infection and provides further evidence that mast cells and their AMPs may play a role in responding to branchial ectoparasite infections.     

Comparison of nuclear DNA content of rodlet cells and erythrocytes in some freshwater teleosts

DNA of rodlet cells and erythrocytes from three species of freshwater teleosts, Semotilus atromaculatus atromaculatus, Catostomus commersoni and Cyprinus carpio , was stained with the Feulgen reaction and examined by microdensitometry. Rodlet cells showed nuclear DNA content significantly different from erythrocytes of the same species, but the difference was less than a factor of C, assuming that erythrocytes reflect the normal 2C genome of somatic cells. In two species, S. atromaculatus and C. carpio , the rodlet cell nuclei contained less DNA than the erythrocytes; in C. commersoni they contained more. The identity of the rodlet cell is unknown; the results of these experiments lead to the rejection of the hypothesis that rodlet cells and erythrocytes of a species have the same DNA content, i.e. that the rodlet cell is a normal somatic component of fish tissue.     

Distribution of chloride cells in teleost larvae

Distribution and density of the chloride cells in the newly hatched larvae of teleosts vary depending on species and environmental salinity at hatching. In the euryhaline freshwater ayu (Plecoglossus altivelis), chloride cells are concentrated in the skin posterior to the pectoral fins and gradually decrease in number toward the head and tail. In the stenohaline sea water flounder (Kareius bicoloratus), most chloride cells are localized at the inner membrane of gill chambers and in the skin near the openings of gill chambers, but only a few cells appear in the skin of the yolk sac. In the stenohaline freshwater carp (Cyprinus carpio), only a few small chloride cells are scattered in the body skin. The density and abundance of chloride cells appears to be correlated with the different requirements for osmoregulation in teleost larvae.     

Host defense peptides in skin secretions of Odorrana tiannanensis: Proof for other survival strategy of the frog than merely anti-microbial

Genus Odorrana, among all amphibians studied, is generally reported to have the most abundant and diversified anti-microbial peptides even from a single individual frog. In our previous work, 46 cDNA sequences encoding precursors of 22 different anti-microbial peptides (AMPs) were characterized from the skin of frog, Odorrana tiannanensis. In this work, we reported the purification of three AMPs from skin secretions of O. tiannanensis. Their amino acid sequences matched well with the sequences deduced from cDNAs and they were designated as Odorranain-C7HSa, Brevinin-1-OT2 and Odorranain-G-OT, respectively. Furthermore, we selected to analyze the four most structurally diversified sequences among the 22 AMPs that are significantly different from all reported AMPs. By structural characterization, three of them were designated as pleurain-E-OT, odorranain-G-OT, odorranain-A-OT, belonging to AMP families already identified. The forth one with a unique 14-mer sequence of AILTTLANWARKFLa and C-terminal amidation represents the prototypes of a new class of amphibian AMP, and thereby named tiannanensin. Such broad diversity in sequences and structures are consistent with other species in Genus Odorrana. Multi-functions of the synthesized four special AMPs were screened, including anti-microbial, antioxidant, cytotoxic and hemolytic activities. The results suggest that these AMPs may employ sophisticated mechanisms of action in host defense in addition to anti-microbial, although their precise contribution to host defense still seems unclear.     

Atlantic Cod Piscidin and Its Diversification through Positive Selection

Piscidins constitute a family of cationic antimicrobial peptides that are thought to play an important role in the innate immune response of teleosts. On the one hand they show a remarkable diversity, which indicates that they are shaped by positive selection, but on the other hand they are ancient and have specific targets, suggesting that they are constrained by purifying selection. Until now piscidins had only been found in fish species from the superorder Acanthopterygii but we have recently identified a piscidin gene in Atlantic cod (Gadus morhua), thus showing that these antimicrobial peptides are not restricted to evolutionarily modern teleosts. Nucleotide diversity was much higher in the regions of the piscidin gene that code for the mature peptide and its pro domain than in the signal peptide. Maximum likelihood analyses with different evolution models revealed that the piscidin gene is under positive selection. Charge or hydrophobicity-changing amino acid substitutions observed in positively selected sites within the mature peptide influence its amphipathic structure and can have a marked effect on its function. This diversification might be associated with adaptation to new habitats or rapidly evolving pathogens.     

Structure-activity relationships of piscidin 4, a piscine antimicrobial peptide

Piscidin 4, an antimicrobial peptide recently isolated from mast cells of hybrid striped bass (Morone chrysops female × Morone saxatilis male), is unusual in that it is twice as long (44 amino acids) as the typical members of the piscidin family. We previously showed that native piscidin 4 had a modified amino acid at position 20, but synthetic piscidin 4 (having an unmodified Trp at position 20) had similar potent activity against a number of both human and fish bacterial pathogens. In this study, the structure and membrane topology of synthetic piscidin 4 were examined using liposomes as model bilayers. Circular dichroism analyses revealed that it had a disordered structure in aqueous solution and folded to form a relatively weak α-helical structure in both membrane-mimetic trifluoroethanol solutions and liposome suspensions. Fluorescence data (piscidin 4 embedded in liposomes) and leakage experiments indicated that piscidin 4 interacted strongly with the hydrophobic part of the liposome. Binding of piscidin 4 to liposomes induced significant blue shifts of the emission spectra of the single Trp residue (Trp20). Quenching of Trp20 by water-soluble quencher (either acrylamide or I-) indicated that the fluorescence of Trp20 decreased more in the presence of liposomes than in buffer solution, thus revealing that Trp20 is less accessible to the quenchers in the presence of liposomes. The relative leakage abilities of piscidin 4 (1 μM) with liposomes were in the following order: DPPC (100%)≥EYPC (94%)>DPPC/DPPG (65%)>EYPC/EYPG (0%). This high activity against DPPC and EYPC liposomes was contrary to our data suggesting that piscidin 4 has a much weaker tendency to form an α-helix than other piscidins, such as piscidin 1. However, the structural similarity of protozoan membranes to EYPC liposomes might explain our discovery of the potent activity of piscidin 4 against the important skin/gill parasite ich (Ichthyophthirius multifiliis), but its negligible hemolytic activity against vertebrate membranes (hybrid striped bass or human erythrocytes). It also suggests that other conformation(s) in addition to the α-helix of this peptide may be responsible for its selective activity. This differential toxicity also suggests that piscidin 4 plays a significant role in the innate defense system of hybrid striped bass and may be capable of functioning extracellularly.     

Distribution and development of rodlet cells in the gills and pseudobranch of the bass, Dicentrachus labrax (L)

Rodlet cells in various stages of development were found in large numbers in the bass gill and pseudobranch. In the gill, rodlet cells were found in the epithelium at the base of the secondary lamellae and on the filament between adjacent lamellae, whilst in the pseudobranch they were found over the whole area of the secondary lamellae as well as in the filament epithelium.
During early development, rodlet cells are characterised by their amorphous cell inclusions, prominent supranuclear Golgi complex and network of granular endoplasmic reticulum. Later, with formation of a fibrous border the arrangement of the cell organelles undergoes reorganisation; the endoplasmic reticulum becomes distended, numerous vesicles appear and the mitochondria aggregate in the apical region of the cell. One of the most striking features is the development of club-shaped sacs containing electron dense cores, which are orientated towards the open apex of the cell.
Various staining properties of rodlet cells for light and electron microscopy were compared with those of mucous cells found in the same tissues. Possible functions of the cell are discussed.     

IGF-I and branchial IGF receptor expression and localization during salinity acclimation in striped bass

The initial response of the IGF-I system and the expression and cellular localization of IGF type-I receptor (IGF-IR) were studied in the gill of a euryhaline teleost during salinity acclimation. Exposure of striped bass (Morone saxatilis) to hyperosmotic and hypoosmotic challenges induced small, transitory (<24 h) deflections in hydromineral balance. Transfer from freshwater (FW) to seawater (SW) induced an initial decrease in plasma IGF-I levels after 24 h in both fed and fasted fish. There was an overall decrease in liver IGF-I mRNA levels after SW transfer, suggesting that decreased plasma levels may be due to a decline in hepatic IGF-I synthesis. No changes were observed in gill IGF-I mRNA, but SW transfer induced an increase in gill IGF-IR mRNA after 24 h. Transfer from SW to FW induced an increase in plasma IGF-I levels in fasted fish. In fed fish, no significant changes were observed in either plasma IGF-I, liver, or gill IGF-I mRNA, or gill IGF-IR mRNA levels. In a separate experiment, FW-acclimated fish were injected with saline or IGF-I prior to a 24-h SW challenge. Rapid regain of osmotic balance following SW transfer was hindered by IGF-I. Immunohistochemistry revealed for the first time in teleosts that IGF-IR and Na(+)-K(+)-ATPase are localized in putative chloride cells at the base of the lamellae, identifying these cells in the gill as a target for IGF-I and IGF-II. Overall the data suggest a hyperosmoregulatory role of IGF-I in this species.     

Immunocytochemical studies on endothelin in mast cells and macrophages in the rat gastrointestinal tract

 To reveal the distribution of endothelin (ET)-containing stromal cells (mast cells and macrophages), we investigated the rat gastrointestinal tract immunohistochemically using antibodies to Big ET-1, Big ET-2, Big ET-3, and mature ETs. In all the regions of the gastrointestinal tract, immunoreactivity for all the antibodies used was found in stromal cells that were located mainly in the lamina propria (not in the submucosa). The number of these cells was largest in the small intestine and smallest in the colon. Moreover, Big ET-2, which was originally identified in the gastrointestinal tract, was also found in many stromal cells, but Big ET-3-containing cells, unexpectedly, were found in almost the same number as Big ET-2-containing cells, while Big ET-1-containing cells were few. These immunopositive stromal cells seemed to be mast cells and macrophages from their histological features. Double-immunohistochemical staining revealed that 92% of the mature ETs-positive cells were mast cells; the rest were macrophages. Furthermore, we confirmed that mature ETs coexisted with ET-A or ET-B receptors in identical cells. Hence, we presume that ETs are synthesized in and secreted from stromal cells in the rat gastrointestinal tract, that their main isotypes are not only ET-2 but also ET-3, and that ETs may act in an autocrine/paracrine fashion. Accepted: 4 November 1997     

两种有胃鱼消化道6种内分泌细胞的免疫组织化学比较研究

目的 应用6种胃肠激素抗血清对胡子鲶(Clarias fuscas)和瓦氏黄颡鱼(Pelteobagrus vachelli Richardson)消化道内分泌细胞进行了免疫组织化学比较.方法 应用链霉亲和素-生物素-过氧化物酶复合物技术(streptavidin biotin-peroxidase complex method,SABC法)免疫组织化学方法.结果 除胡子鲶后肠外,五羟色胺细胞在两种鱼的消化道各段均有分布,其中贲门部或幽门部密度最高,前中肠次之,食道最少;胡子鲶消化道中生长抑素细胞主要分布于胃贲门部和胃部,食管、胃幽门及肠道内未见分布,而瓦氏黄颡主要分布于食管和胃部,肠道内未见分布;胃泌素细胞在胡子鲶消化道内只在肠内检测到,瓦氏黄颡只在幽门胃和前肠两个部位检测到;胰多肽细胞只在瓦氏黄颡的幽门胃和前肠两个部位有分布;胰高血糖素和P-物质两种细胞在两种鱼的消化道各段均未见其分布.结论 两种有胃鱼消化道中六种内分泌细胞的分布既有一定的共性,体现了两者消化生理的共同点;同时又存在较大的种间差异,与各自食性及生活环境相适应.     

Piscidin,Fish Antimicrobial Peptide: Structure,Classification, Properties,Mechanism, Gene Regulation and Therapeutical Importance

Antimicrobial peptides (AMPs) are short molecules produced by almost all organisms. Fish AMPs contain innate immune components as their primary immune molecules. The fish AMPs include piscidins, hepcidins, defensins, cathelicidins and histone-derived peptides. Piscidin is potent and broad-spectrum; this peptide was conserved among Acanthopterygii superorder and is therapeutically important among other AMPs. It was present mainly in the tissues of gills, muscle, head-kidney, skin and intestine of teleost. Piscidin AMP family includes piscidin, moronecidin, pleurocidin, epinecidin, gaduscidin, misgurin, dicentracin, chrysophsin and myxinidin. This review reports the structural properties of various piscidin and their mode of action as it is important to know their mechanism how the peptide involved in antimicrobial activity. In addition, the gene expression of piscidin which influenced the immune responses, their pharmaceutical importance and biological applications were described. Overall, the review explains a broad spectrum of knowledge on piscidin, its classes and types, structure, cytotoxicity, membrane permeabilization, properties and therapeutical implications.

     

两栖类动物Cathelicidins家族 抗菌肽研究进展

两栖类动物皮肤裸露和湿润的特性易于微生物的生长,它们为了抵御病原微生物的侵袭,在长期自然进化过程中形成了以抗菌肽(AMPs)为主要防御机制的免疫系统。抗菌肽广泛分布于动物、植物、微生物中,是生物用于抵御细菌、真菌、病毒和原虫等病原体侵袭的重要武器之一,在进化上是一类非常古老而有效的天然防御物质。Cathelicidins是脊椎动物特有的重要抗菌肽家族之一,除具有高效广谱的抗菌活性,还具有如抗炎、抗氧化、伤口修复、抑制组织损伤和促进血管生成等多种重要活性,因此Cathelicidins家族抗菌肽已成为抗感染多肽类新药的研发热点。本文将从两栖类动物Cathelicidins家族抗菌肽的一般特点、来源分布、生物合成与结构、生物学活性、作用机制及应用前景等几个方面,综合阐述国内外的研究动态。     

Resistance to antimicrobial peptides in Gram-negative bacteria

Antimicrobial peptides (AMPs) are present in virtually all organisms and are an ancient and critical component of innate immunity. In mammals, AMPs are present in phagocytic cells, on body surfaces such as skin and mucosa, and in secretions and body fluids such as sweat, saliva, urine, and breast milk, consistent with their role as part of the first line of defense against a wide range of pathogenic microorganisms including bacteria, viruses, and fungi. AMPs are microbicidal and have also been shown to act as immunomodulators with chemoattractant and signaling activities. During the co-evolution of hosts and bacterial pathogens, bacteria have developed the ability to sense and initiate an adaptive response to AMPs to resist their bactericidal activity. Here, we review the various mechanisms used by Gram-negative bacteria to sense and resist AMP-mediated killing. These mechanisms play an important role in bacterial resistance to host-derived AMPs that are encountered during the course of infection. Bacterial resistance to AMPs should also be taken into consideration in the development and use of AMPs as anti-infective agents, for which there is currently a great deal of academic and commercial interest.