Ultrastructure of granulocytes of teleost fish (Salmoniformes, Cypriniformes, Perciformes)

Analysis of data on ultrastructure of granulocytes of freshwater and marine bony fish of orders Salmoniformes, Cypriniformes, and Perciformes showed that in all studied species there were revealed two types of granulocytes-neutrophils and eosinophils. The exception was the bluefish Pomatomus saltatrix L. whose pronephros hemopoietic tissue was found to contain one type of the granulocyte line-neutrophils. The identification parameters of granular leukocytes are specific granules filling the cytoplasm. The main form of specific granules in neutrophils of bony fish of various phylogenetic groups is an elongated granule with different distribution of fibrils or a granule that has fibrils-formed crystalloid. Eosinophil granules are mainly of large, electron-dense, homogenous form.     

Phagocytosis by B-cells and neutrophils in Atlantic salmon (Salmo salar L.) and Atlantic cod (Gadus morhua L.)

Phagocytosis by fish cells has mostly been studied using adherent leucocytes, excluding suspended cells such as the majority of B-cells and neutrophils, but a recent study describes professional phagocytosis of latex beads and bacteria by B-cells from rainbow trout. In the present study, phagocytosis by B-cells and neutrophils from salmon and cod was studied. Leucocytes were isolated from peripheral blood (PBL) and head kidney (HKL). By flow cytometry analyses, proportions of MAb labelled cell populations with internalized fluorescent beads, as well as the number of beads within each cell, could be determined. Phagocytic capacity and ability were demonstrated in B-cells and neutrophils from salmon and cod. In salmon, B-cells had higher phagocytic ability than neutrophils in HKL, but not in PBL. For cod the phagocytic ability of B-cells were lower than for neutrophils in both HKL and PBL, but the phagocytic capacity of cod B-cells were higher than for neutrophils in both HKL and PBL. For salmon B-cells the phagocytic capacity was lower than or similar to neutrophils in HKL and PBL. The total phagocytic ability of leucocytes was different in the species studied. The highest phagocytic ability was observed in cod, showing similar values for PBL and HKL. Salmon PBL displayed about twice the phagocytic ability of cod PBL. There seemed to be some major differences between the two fish species concerning phagocytosis. In salmon, a rather large proportion of phagocytic leucocytes were phagocytic B-cells, indicating that B-cells may have an important function in particle clearance in this species. In cod, phagocytic leucocytes in HKL and PBL were mostly neutrophils, and only a small proportion of B-cells were phagocytic, supporting the more prominent role of innate immune functions in cod neutrophils.     

Morphological and phagocytic characteristics of peritoneal exudate cells in tilapia, Oreochromis niloticus (Trewavas), and carp, Cyprinus carpio L.

The morphology and phagocytic activity of peritoneal exudate cells (PEC) obtained by an intraperitoneal injection of liquid paraffin into tilapia, Oreochromis niloticus , and carp, Cyprinus carpio , were studied with light and electron microscopy. PEC consisted of monocyte-macrophage series cells (M-Mø), neutrophils, eosinophils (granular cells) and others. Cells exhibiting the same morphology as mammalian macrophages but different from monocytes of the same species were identified with light and electron microscopy and designated as peritoneal macrophages. Light and electron microscopy revealed that M-Mø, neutrophils and eosinophils (granular cells) phagocytozed foreign materials added in vivo and in vitro. Eosinophils appeared later in the peritoneal exudate and less actively phagocytic as compared with M-Mø and neutrophils. Small and large phagosomes were formed in M-Mø, neutrophils and eosinophils (granular cells). Large phagosomes were common in neutrophils. Fusion of cytoplasmic granules with the phagosome membrane was observed. The in vitro experiment on phagocytosis revealed that the phagocytic rates in M-Mø and neutrophils were positively correlated with the doses of foreign materials. The results indicated that these two cell types have the highest capacity of phagocytosis.     

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.     

Nonpathogenic, environmental fungi induce activation and degranulation of human eosinophils

Eosinophils and their products are probably important in the pathophysiology of allergic diseases, such as bronchial asthma, and in host immunity to certain organisms. An association between environmental fungal exposure and asthma has been long recognized clinically. Although products of microorganisms (e.g., lipopolysaccharides) directly activate certain inflammatory cells (e.g., macrophages), the mechanism(s) that triggers eosinophil degranulation is unknown. In this study we investigated whether human eosinophils have an innate immune response to certain fungal organisms. We incubated human eosinophils with extracts from seven environmental airborne fungi (Alternaria alternata, Aspergillus versicolor, Bipolaris sorokiniana, Candida albicans, Cladosporium herbarum, Curvularia spicifera, and Penicillium notatum). Alternaria and Penicillium induced calcium-dependent exocytosis (e.g., eosinophil-derived neurotoxin release) in eosinophils from normal individuals. Alternaria also strongly induced other activation events in eosinophils, including increases in intracellular calcium concentration, cell surface expression of CD63 and CD11b, and production of IL-8. Other fungi did not induce eosinophil degranulation, and Alternaria did not induce neutrophil activation, suggesting specificity for fungal species and cell type. The Alternaria-induced eosinophil degranulation was pertussis toxin sensitive and desensitized by preincubating cells with G protein-coupled receptor agonists, platelet-activating factor, or FMLP. The eosinophil-stimulating activity in Alternaria extract was highly heat labile and had an M(r) of approximately 60 kDa. Thus, eosinophils, but not neutrophils, possess G protein-dependent cellular activation machinery that directly responds to an Alternaria protein product(s). This innate response by eosinophils to certain environmental fungi may be important in host defense and in the exacerbation of inflammation in asthma and allergic diseases.     

Staphylococcus aureus Panton-Valentine Leukocidin Is a Very Potent Cytotoxic Factor for Human Neutrophils

The role of the pore-forming Staphylococcus aureus toxin Panton-Valentine leukocidin (PVL) in severe necrotizing diseases is debated due to conflicting data from epidemiological studies of community-associated methicillin-resistant S. aureus (CA-MRSA) infections and various murine disease-models. In this study, we used neutrophils isolated from different species to evaluate the cytotoxic effect of PVL in comparison to other staphylococcal cytolytic components. Furthermore, to study the impact of PVL we expressed it heterologously in a non-virulent staphylococcal species and examined pvl-positive and pvl-negative clinical isolates as well as the strain USA300 and its pvl-negative mutant. We demonstrate that PVL induces rapid activation and cell death in human and rabbit neutrophils, but not in murine or simian cells. By contrast, the phenol-soluble modulins (PSMs), a newly identified group of cytolytic staphylococcal components, lack species-specificity. In general, after phagocytosis of bacteria different pvl-positive and pvl-negative staphylococcal strains, expressing a variety of other virulence factors (such as surface proteins), induced cell death in neutrophils, which is most likely associated with the physiological clearing function of these cells. However, the release of PVL by staphylococcal strains caused rapid and premature cell death, which is different from the physiological (and programmed) cell death of neutrophils following phagocytosis and degradation of virulent bacteria. Taken together, our results question the value of infection-models in mice and non-human primates to elucidate the impact of PVL. Our data clearly demonstrate that PVL acts differentially on neutrophils of various species and suggests that PVL has an important cytotoxic role in human neutrophils, which has major implications for the pathogenesis of CA-MRSA infections.     

The activity of several components of the innate immune system in diploid and triploid turbot

The use of triploid fish may be of interest in research, e.g. study of how this condition affects the size and activity of cells. In addition, triploid fish are sterile and production of triploids in fish species that are marketed after reaching sexual maturity may be of economic interest. In the present study, the effects of triploidy on the activity of several components of the innate immune system of turbot (Psetta maxima L) were determined. Triploid turbot had bigger cells (erythrocytes and neutrophils) but the number of blood erythrocytes, leucocytes and thrombocytes was lower than in diploid fish. The differential cell count was similar in both types of fish. The respiratory burst and the phagocytic activities were higher in neutrophils of triploid turbot. However, because the number of neutrophils was higher in diploids, the total respiratory burst activity and the phagocytosis per microliter of blood was similar in both types of fish. No differences were found in serum complement, lysozyme or bactericidal activities. The results indicate that the activities of the humoral components of the innate immune system tested are similar in diploid and triploid fish and that the lower leucocyte number found in triploids is compensated for by higher cell activity.     

Eye colour and aggression in juvenile guppies, Poecilia reticulata peters (Pisces: Poeciliidae)

A series of four related experiments on guppy, Poecilia reticulata, juveniles indicates that eye colour is related to motivational state. In juveniles of this species two distinct eye colour states are easily discernible, with a transient intermediate state infrequently seen. These states are dark, light, and intermediate. In aggressive interactions involving both both dark- and light-eyed fish, dark-eyed fish won 98.6% of the encounters (N=222 encounters). Eye colours are significantly correlated with bout lengths of certain behaviours (aggressive encounters, swimming, substrate biting); total time spent in certain behaviours (swimming, aggressive encounters); and type and intensity of aggressive encounters. Eye colour appears to be related to motivational state and may serve a signal function.     

Immunostimulants,adjuvants, and vaccine carriers in fish: Applications to aquaculture

Use of immunostimulants, adjuvants, and vaccine carriers in fish culture offers a wide range of attractive methods for inducing and building up protection against diseases. Immunostimulants and adjuvants can be administered before, with, or after vaccines to amplify the specific immune response generating elevations of circulating antibody titers and numbers of plaque-forming cells. Special applications of immunostimulants include assisting shower or other regimens to increase topical uptake of vaccines. In addition, immunostimulants may be used alone, inducing elevated activities in the nonspecific defense mechanisms such as increased oxidative activity of neutrophils, augmented engulfment activity of phagocytic cells, or potentiating cytotoxic cells. In cases where disease outbreaks are cyclical and can be predicted, losses may be reduced by elevating the nonspecific defense mechanisms, and the immunostimulants may be used in anticipation of events to prevent losses from diseases. Complete Freund's adjuvant was one of the first immunostimulants used in animals to elevate the specific immune response, and it has also been successfully used in conjunction with injection of fish bacterins. Other adjuvants, immunostimulants, and biological response modifiers that have been used in fisheries research include levamisole, salt baths, and bacterial lipopolysaccharides. Vaccines have been adsorbed to inert particles, such as bentonite on latex beads, to carry the immunogens to maximize in vivo uptake for bath immunization and to facilitate in vitro phagocytosis. Each substance presents special problems in timing and method of administration (injection, immersion, oral—by feed—or flush treatments), dosage adjustments for size and fish species, storage stability, and cost. An additional consideration is that the nonspecific defense mechanisms and immune responses in fish are highly variable among individuals and statistical validation requires appropriate sample numbers and carefully controlled experiments.This article reviews the literature and present concepts of use of immunostimulants, adjuvants, and vaccine carriers in fish. Cautions for use are noted, as some of these potent substances can suppress or alter biological pathways if used inappropriately. Recent research, defining pathways of the action of immunostimulants, adjuvants, and vaccine carriers, helps explain how these substances activate the protective mechanisms in fish. In addition, immunostimulants used alone hold tremendous potential for use in fish farms, hatcheries, and aquaculture facilities to reduce losses from infectious diseases. Research on the immunostimulant, levamisole, and the light oil adjuvants for use in food fish is in progress. Applications for use of these immunostimulants are proposed.     

Schistosoma mansoni: Complement and antibody damage,mediated by human eosinophils and neutrophils,in killing schistosomula in vitro

The time and course of damage to Schistosoma mansoni schistosomula mediated by human eosinophils and neutrophils and by antibody (A) and/or complement (C) was studied. The rate of schistosomula death was significantly higher in the complement containing systems (i.e., “A + C” or “C alone”) when compared to A alone. In general, at all the time points studied, the percentage of killing in the three systems was A + C > C alone > A alone irrespective of whether the effector cells were neutrophils or eosinophils. Preferential killing of schistosomula by eosinophils, as compared to neutrophils, was observed with C alone and A + C, but only when eosinophils and neutrophils from the same donor were compared. In contrast, eosinophils and neutrophils appeared to be equally effective in killing antibody-coated schistosomula.A comparison was made of the susceptibility to killing of schistosomula prepared mechanically or by skin penetration. There was no appreciable difference in terms of susceptibility to killing by the various combinations of eosinophils, neutrophils, antibody, and complement between these two forms of schistosomula.The preferential killing of complement-coated, as compared to antibody-coated schistosomula by eosinophils appears to be relatively rapid, an observation which may be of significance both in natural and acquired immunity to migrating larval helminths.     

Phagocytosis in Dictyostelium: Nibbling, Eating and Cannibalism

ABSTRACT. Phagocytosis is a highly conserved biological process that serves numerous functions in a wide variety of organisms. Over the past few decades Dictyostelium has proven to be an excellent organism for investigations in cell biology and this is certainly no less the case for a study of phagocytosis. This review examines three distinct phagocytic activities which have been characterized in Dictyostelium. The first, "vegetative phagocytosis," represents the classical eukaryotic microbial uptake of food particles (bacteria). The second, a predatory form of phagocytosis, arises when one species such as Dictyostelium caveatum attacks another species of slime mold, engulfing small pieces of the target prey. This has been termed "cell nibbling." The third phagocytic process is "sexual cannibalistic phagocytosis." In this situation a zygote giant cell, having arisen from the fusion of gametic amoebae, attracts unfused nonzygotic amoebae of the same species and engulfs them as a food source. While cell nibbling has not been actively studied, vegetative and sexual cannibalistic phagocytosis have received varying amounts of attention leading to the idea that some of the elements (e.g., glycoprotein receptors and a G_αs subunit) involved in certain of these phagocytic events may be the same. On the other hand, some unique events (e.g., filopodial induction in prey by D. caveatum ) are also worthy of further investigation. Among other things, the presence of self-nonself recognition, the existence of opsonin-like substances and the presence of signal transduction elements (e.g., an A2-like receptor that negatively modulates sexual phagocytosis) once considered to be extant only in higher organisms suggest that much can be learned about phagocytosis in general by further studies in the classic, eukaryotic microbe Dictyostelium discoideum and related species.     

Observations on the granulocyte peroxidase of teleosts: a phylogenetic perspective

Observations on granulocyte peroxidase of 123 teleosts are reported and correlated with previous studies to put occurrence of fish granulocyte peroxidase in a phylogenetic perspective. Eosinophil peroxidase occurs in archaic groups such as lungfish and sturgeons, but is usually weak and infrequently observed in chondrichthyans, and in teleosts occurs in some elopomorphs, is consistently observed in stomiiforms, is rarely observed and usually weak in acanthopterygians and absent from salmoniforms, scopelomorphs and paracanthopterygians. Peroxidase is absent from lungfish and sturgeon neutrophils, is present in neutrophils of some elopomorphs and clupeimorphs. both of which are basal teleost groups, but in salmoniforms and higher teleosts is consistently observed in neutrophils. although often weak in pleuronectiforms.
Loss of eosinophil peroxidase and development of neutrophil peroxidase in teleosts may be due to inefficient phagocytosis and loss of peroxidase with degranulation in eosinophils, being superseded by concentration of peroxidase in phagosomes in neutrophils.     

New insights regarding the regulation of chemotaxis by nucleotides, adenosine, and their receptors

The directional movement of cells can be regulated by ATP, certain other nucleotides (e.g., ADP, UTP), and adenosine. Such regulation occurs for cells that are "professional phagocytes" (e.g., neutrophils, macrophages, certain lymphocytes, and microglia) and that undergo directional migration and subsequent phagocytosis. Numerous other cell types (e.g., fibroblasts, endothelial cells, neurons, and keratinocytes) also change motility and migration in response to ATP, other nucleotides, and adenosine. In this article, we review how nucleotides and adenosine modulate chemotaxis and motility and highlight the importance of nucleotide- and adenosine-regulated cell migration in several cell types: neutrophils, microglia, endothelial cells, and cancer cells. We also discuss difficulties in conducting experiments and drawing conclusions regarding the ability of nucleotides and adenosine to modulate the migration of professional and non-professional phagocytes.     

THE MOBILIZATION AND EXTRACELLULAR RELEASE OF GRANULAR ENZYMES FROM HUMAN LEUKOCYTES DURING PHAGOCYTOSIS

The importance of granular (lysosomal) enzymes from neutrophils in producing the tissue damage of acute inflammation has been suggested by much indirect and some direct evidence. This study has investigated the kinetics of release and subsequent fate of granular enzymes from phagocytizing human leukocytes The following observations are made: (a) During phagocytosis, the granular enzyme lysozyme is released from leukocytes into the extracellular medium. (b) Release of lysozyme increases as phagocytic challenge increases, but attains a maximum. (c) Release of lysozyme accompanies phagocytosis and is not a delayed event. (d) The lack of release of a nongranular enzyme, lactic dehydrogenase, indicates that cell damage is not a necessary condition of enzyme release. (e) Like lysozyme, β-glucuronidase is released from phagocytizing leukocytes. Acid α-naphthyl phosphatase and cathepsin also appear to be released, but are not found in appreciable amounts in the extracellular medium, in part because of their lability in solution. These results support the concept that extracellular release of granular enzymes may be a useful secretory function of inflammatory leukocytes which becomes damaging to the host in certain circumstances.     

Different aspects of tubulin polymerization in spermatids of cyprinodontidae (fish,teleost)

Our study concerns 10 genera and 26 species of cyprinodontid fish. In the cytoplasm of spermatids tubulin polymerizes in various forms according to species. We have demonstrated the presence of classic microtubules with a diameter of 24 nm and also of tubules of smaller diameter (15 run) and greater diameter (30 to 50 nm). Microtubules are very numerous in the cytoplasm of certain species. They may be arranged without any order or form bundles which may contain several hundred parallel elements. They never form a manchette. In two species (Aplocheilichthys normani and Epiplatys fasciolatus) only spermatids that degenerate show this peculiarity. The microtubules present two kinds of decorations. The first type are small elements composed of MAP which enable two or more microtubules to link up. The second type are curved tubulin elements that give the microtubule that bear them the appearance of an incomplete doublet. Doublets and triplets may also be formed. Cyprinodontidae spermatocytes and spermatids probably synthesize a very large quantity of tubulin which polymerizes in certain species.     

The life cycle of Euclinostomum heterostomum (Rudolphi, 1809) (Trematoda: Clinostomatidae)

In small forest streams of West Nigeria the snail species Bulinus globosus was found to be the first intermediate host of a clinostomatid species which proved in successful infection experiments with the cichlid fish Tilapia zillii as second intermediate host to be Euclinostomum heterostomum (Rudolphi, 1809). Final hosts are herons. The morphology of the redia and the morphology and behaviour of the cercaria are described for the first time. The metacercaria which settles dorsal to the swim-bladder or in the kidney of the host fish requires more than two months (at tropical temperatures) for full development. Histological sections of the metacercaria revealed that its pharynx is provided with only few muscle fibres but surrounded by many myoblast-like cells, and the fact that the interior surface of this organ has a dense brush-like lining consisting of long microtriches. The pathology of infected fish is briefly described, and remarks about the geographical distribution of the parasite are given.     

濒危鱼类稀有白甲鱼外周血细胞特征

为了研究濒危鱼类稀有白甲鱼（Onychostoma rara）外周血细胞的特征,以采自长江中游沅江水系清水江共计21尾稀有白甲鱼的血液为材料,采用常规方法对稀有白甲鱼外周血细胞的组成、形态、大小和数量进行了观测。结果显示,稀有白甲鱼红细胞数量为（1.75±0.44）×106 个/ L,白细胞数量为（4.91±1.95）×105 个/ L。在血涂片上共计观察到了5种白细胞,包括淋巴细胞、血栓细胞、单核细胞、嗜中性粒细胞和嗜酸性粒细胞,没有发现嗜碱性粒细胞。其5种白细胞数量比例差异较大,其数量比例关系为：淋巴细胞>血栓细胞>嗜中性粒细胞>单核细胞>嗜酸性粒细胞。这5种白细胞的大小也有所不同,其大小关系为：单核细胞>嗜中性粒细胞>嗜酸性粒细胞>淋巴细胞>血栓细胞。与已报道的鱼类相比,稀有白甲鱼白细胞的数量明显较高,红细胞数量较多、体积相对较小,可能与其适应流水生活相关。     

A-, B- and C-type prymnesins are clade specific compounds and chemotaxonomic markers in Prymnesium parvum

Harmful blooms formed by planktonic microalgae (HABs) in both freshwater and coastal waters regularly lead to severe mortalities of fish and invertebrates causing substantial economic losses of marine products worldwide. The mixotrophic haptophyte Prymnesium parvum is one of the most important microalgae associated with fish kills. Here 26 strains of P. parvum with a wide geographical distribution were screened for the production of prymnesins, the suspected causative allelochemical toxins. All investigated strains produced prymnesins, indicating that the toxins play an important role for the organism. The prymnesins can be classified into three types based on the length of the carbon backbone of the compound and each algal strain produced only one of these types. Biogeographical mapping of the prymnesin distribution indicated a global distribution of each type. In addition, phylogenetic analyses based on internal transcribed spacer (ITS) sequences revealed monophyletic origin of all prymnesin types and clades could therefore be defined based on the toxic compound. It might be that evolution of new species within the P. parvum species complex is driven by changes in toxin type or that they are a result of it. Such a correlation between chemotype and phylotype has never been documented before for a harmful microalga. Chemotaxonomy and ITS-type classification may thus be used to further delimit the P. parvum species complex.     

β-Glucan Induces Reactive Oxygen Species Production in Human Neutrophils to Improve the Killing of Candida albicans and Candida glabrata Isolates from Vulvovaginal Candidiasis

Vulvovaginal candidiasis (VVC) is among the most prevalent vaginal diseases. Candida albicans is still the most prevalent species associated with this pathology, however, the prevalence of other Candida species, such as C. glabrata, is increasing. The pathogenesis of these infections has been intensely studied, nevertheless, no consensus has been reached on the pathogenicity of VVC. In addition, inappropriate treatment or the presence of resistant strains can lead to RVVC (vulvovaginal candidiasis recurrent). Immunomodulation therapy studies have become increasingly promising, including with the β-glucans. Thus, in the present study, we evaluated microbicidal activity, phagocytosis, intracellular oxidant species production, oxygen consumption, myeloperoxidase (MPO) activity, and the release of tumor necrosis factor α (TNF-α), interleukin-8 (IL-8), IL-1β, and IL-1Ra in neutrophils previously treated or not with β-glucan. In all of the assays, human neutrophils were challenged with C. albicans and C. glabrata isolated from vulvovaginal candidiasis. β-glucan significantly increased oxidant species production, suggesting that β-glucan may be an efficient immunomodulator that triggers an increase in the microbicidal response of neutrophils for both of the species isolated from vulvovaginal candidiasis. The effects of β-glucan appeared to be mainly related to the activation of reactive oxygen species and modulation of cytokine release.     

Actin polymerization in human eosinophils,unlike human neutrophils,depends on intracellular calcium mobilization

Eosinophils represent major effector cells in the allergic inflammation. In contrast to neutrophils, the mechanism of eosinophil activation during the inflammatory response is poorly understood. In this study, the relation between calcium fluxes, chemotaxis, and actin polymerization in eosinophils from healthy non-atopic donors was investigated. Pre-incubation of eosinophils with the intracellular calcium chelator BAPTA dose-dependently prevented an increase in the intracellular calcium concentration ([Ca²⁺]_i), whereas the depletion of extracellular calcium in the test medium had no effect. The chemotactic response of eosinophils, which was measured by the modified boyden chamber technique upon stimulation with RANTES, C5a and PAF, was dose-dependently inhibited by the chelation of intracellular calcium as well as inactivation of the cells in Ca²⁺-depleted medium. To evaluate whether other cell functions which are involved in the migratory response of eosinophils might be dependent on intracellular and extracellular calcium, actin polymerization was investigated. Flow-cytometric measurement of F-actin with NBD-phallacidin revealed that actin polymerization in human eosinophils in response to RANTES, C5a, and PAF was dose-dependently inhibited by the intracellular calcium chelator BAPTA. Since it is well known that actin polymerization in neutrophils is not affected by chelation of intracellular calcium, actin polymerization in these cells was investigated under the same conditions as for eosinophils. In contrast to eosinophils, BAPTA did not inhibit actin polymerization in neutrophils. In summary, these data demonstrate that intracellular calcium fluxes represent a prerequisite for eosinophil chemotaxis and actin polymerization in human eosinophils. Furthermore, regulation of actin polymerization in eosinophils differed from that of neutrophils on the level of intracellular calcium fluxes. © 1996 Wiley-Liss, Inc.