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Cetaceans have adapted to aquatic life by evolving various anatomic and physiologic traits, but biological defense mechanisms specific to aquatic mammals that protect against pathogenic microorganisms in the aquatic environment have not been elucidated. In this study, we investigated the migration of polymorphonuclear leukocytes in bottlenose dolphins in response to various chemotactic factors and compared the migration response with that of terrestrial animals such as cows and humans to characterize biological defense mechanisms unique to cetaceans. Bottlenose dolphin neutrophils showed strong chemotactic activity toward zymosan-activated serum and recombinant human interleukin-8 but no chemotaxis toward N-formyl-methionyl-leucyl-phenylalanine or leukotriene B 4 at any concentration examined. Bovine and human neutrophils showed the strongest chemotactic activity at 37°C, whereas chemotactic activity declined significantly at 15°C and 4°C. By contrast, bottlenose dolphin neutrophils exhibited relatively strong chemotactic activity even at 15°C. These results suggest that the mechanism by which bottlenose dolphin neutrophils detect chemoattractants differs from that of terrestrial mammals and that the immune system of cetaceans may be cold-adaptive, allowing the animals to exert robust host defense responses in aquatic habitats, which tend to be colder than the terrestrial environment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Objective: To evaluate effects of extracellular lactate on viability, shape change, lactate metabolism, and reactive oxygen species (ROS) production in equine polymorphonuclear leukocytes (PMNs)., Sample: PMNs isolated from equine venous blood samples., Procedures: PMNs were incubated with 0 to 300mM lactate for 30 minutes before each experiment. Viability was assessed via trypan blue exclusion. Shape change was assessed via flow cytometry and light microscopy. Relative quantification of monocarboxylic acid transporter and lactate dehydrogenase lactate dehydrogenase (LDH) isotype mRNAs was performed with a real-time PCR assay. Effects of lactate at a pH of 7.4 to 6.0 on ROS production in response to phorbol 12-myristate 13-acetate, opsonized zymosan, or N-formyl-methionyl-leucyl-phenylalanine was assessed by luminol-dependent chemiluminescence., Results: Lactate had no effect on viability of PMNs but did alter their size and density. Monocarboxylic acid transporter 1 and lactate dehydrogenase B mRNA values were not altered. Monocarboxylic acid transporter 4 and lactate dehydrogenase A mRNA values were significantly decreased. Lactate incubation of cells significantly decreased PMN-derived luminol-dependent chemiluminescence and induced different sensitivities to stimulants (phorbol 12-myristate 13-acetate, opsonized zymosan, and N-formyl-methionyl-leucyl-phenylalanine). The response ratio to N-formyl-methionyl-leucyl-phenylalanine revealed that PMNs were primed by incubation with up to 50mM lactate, significantly increasing the production of ROS. Incubation with lactate and acidic pH caused a synergistic effect on ROS production., Conclusions and Clinical Relevance: Extracellular lactate potentially has a direct effect on the capacity to produce ROS by equine PMNs, which may be associated with alterations in innate immune functions within a short period after high-intensity exercise.

This study describes a chemotaxis assay of ferret polymorphonuclear cells (PMNs). The optimal conditions for this chemotaxis assay were investigated for three chemoattractants: zymosan activated serum (ZAS), recombinant human interleukin-8 (rhIL-8) and N-formyl-Met-Leu- Phe (fMLF). In this study, ferret polymorphonuclear cells (PMNs) reacted to ZAS and rhIL-8, but not fMLF. The optimal concentration of ZAS and rhIL-8 were 5% and 100 ng/ml, respectively. The optimal incubation time of each reagent was 60 min. Due to the lack of response shown from fMLF, the existence of formyl peptide receptors (FPR) on ferret PMNs was investigated by evaluating FPR binding using flow cytometry. The receptor was not detected, implying that ferret neutrophils may lack FPR. This study confirms the fundamental experimental conditions for ferret PMNs chemotaxis and elucidates new findings concerning FPR in ferret neutrophils., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The authors produced a monoclonal antibody (mAb) against dolphin neutrophils by fusing mouse myeloma cells with lymph node cells from a Wistar rat immunized with bottlenose dolphin (Tursiops truncatus) polymorphonuclear leukocytes (PMNs). This mAb (DN1) was reactive against 77.1 +/- 8.6% of dolphin peripheral blood PMN by flow cytometric analysis; furthermore, there was no cross-reactivity with human or bovine leukocytes. The DN1-positive cells isolated with a sorting cytometer were almost all (99.7%) neutrophils. By using DN1 in conjunction with magnetic-activated cell sorting (MACS), the authors isolated neutrophils and eosinophils from density gradient-fractionated PMN with 100% and 95.6 +/- 4.8% purities, respectively. These results suggest that this mAb specific for bottlenose dolphin neutrophils is useful as a potential reagent to study bottlenose dolphin neutrophils and eosinophils.

Ferret polymorphonuclear cells (PMNs) and peripheral blood mononuclear cells (PBMCs) were separated from whole blood by density gradient centrifugation. Using a 50% Percoll solution (density=1.066), PMNs and PBMCs were successfully isolated after centrifugation; the purities of the PMNs and PBMCs were 94.2% and 95.6%, respectively. To evaluate the function of isolated ferret PMNs, we measured the superoxide generation with a MCLA-dependent chemiluminescence assay. The isolated ferret PMNs responded to phorbol 12-myristate 13-acetate (PMA) with kinetics similar to that of human PMNs. The ferret PMNs did not respond to N-formyl-Met-Leu-Phe (fMLF), unlike human PMNs, which rapidly responded. Thus, authors established a method for the rapid separation of highly purified populations of functional PMNs from the whole blood of ferrets.

To study different activation states in polymorphonuclear leukocytes (PMN) in mice, we compared the function of murine PMN obtained from the bone marrow (BMPMN) with those of PMN obtained by intraperitoneal induction with thioglycolate (TGPMN) or uric acid (UAPMN). When stimulated with chemotactic peptides, e.g., formyl-methionyl-leucyl-phenylalanine (fMLF), WKYMVM, or WKYMVm, the TGPMN and UAPMN showed greatly enhanced generation of reactive oxygen species (ROS) compared with BMPMN, which suggests that exudation to the peritoneum per se induces a primed state in the cells. The WKYMVm peptide was the most potent stimulant of ROS generation, and it desensitized for subsequent stimulation with fMLF or WKYMVM. This desensitization was broken by the addition of cytochalasin B. The TGPMN and UAPMN appeared to be fully primed, since no increase in response was induced by pretreatment with tumor necrosis factor alpha (TNF-alpha). In contrast, the BMPMN response was increased 2.5- to 3-fold. The differences in oxidative responses were supported by degranulation studies. Preincubation with TNF-alpha promoted CR3 expression on BMPMN, and this level of expression was also enhanced by WKYMVm. In contrast, CR3 expression on untreated TGPMN and UAPMN was already similar to that on TNF-alpha-primed BMPMN and could be only slightly enhanced by TNF-alpha treatment. Taken together, these results indicate that BMPMN are in a resting state and have the capacity to become primed, while peritoneal exudate PMN are already fully primed upon isolation. These results have major implications for murine neutrophil research and show the importance of defining which PMN subsets to use when investigating murine models.

Neutrophils of ayu (Plecoglossus altivelis) were previously shown to have unusually high respiratory burst activity (RBA). To understand this unique character of ayu neutrophils, the RBAs of resting and inflammatory neutrophils of ayu and carp (Cyprinus carpio) were compared. Inflammation was induced in the peritoneal cavity by injecting killed-bacteria. The RBA of peritoneal-exudate (inflammatory) neutrophils was measured after stimulation with phorbol myristate acetate (PMA). Resting neutrophils were obtained from kidney and blood of non-injected fish. In carp, the RBA of inflammatory neutrophils was much higher than that of resting neutrophils. On the other hand, in ayu no significant difference was observed. The RBA of neutrophils was already high in the kidney stock. The process of inflammation did not further enhance RBA. In addition to PMA, other stimulants (zymosan, opsonized-zymosan, and zymosan-treated serum) were used to measure RBA. Even with these stimulants, the RBA of inflammatory neutrophils was always higher than that of kidney neutrophils in carp. On the other hand in ayu, the RBA of kidney neutrophils was already high in the kidney stock, and no significant difference was observed between peritoneal and kidney neutrophils in ayu. These results indicate ayu neutrophils have spontaneously activated characteristics with the respect to the ROS generation in the kidney hematopoietic-stock.

Immunocytes, which include antigen-presenting cells, B cells, natural killer cells and neutrophils, can be stimulated directly or indirectly with bacterial DNA and synthetic oligodeoxynucleotides (ODNs) with different structures and sequences. In the present study, we investigated the effect of synthetic ODNs on the respiratory burst of dolphin neutrophils using a chemiluminescence assay. Phosphorothioate (PS)-ODNs dose-dependently induced the respiratory burst, while phosphodiester (PO)-ODNs did not, regardless of CpG-content. The PS-ODN-induced activity was completely abolished by the flavoprotein inhibitor diphenyleneiodonium, which indicates that the NADPH-oxidase is activated by PS-ODNs. These results reveal that PS-ODNs induce dolphin neutrophil NADPH-oxidase activation in a CpG motif-independent but phosphorothioate-dependent manner.

Bovine colostrum and milk contain many immunomodulatory components. The low-molecular-weight fraction (< 10 kDa) was separated from colostrum and milk by gel filtration chromatography, and its effect on the oxidative burst of bovine polymorphonuclear leukocytes (PMNL) was investigated in vitro. The oxidative burst activity induced by Staphylococcus aureus was considerably enhanced when PMNLs were incubated with this low-molecular-weight fraction. However, phorbol 12-myristate 13-acetate did not trigger a burst after priming with this fraction. The oxidative burst activity enhanced by this fraction was reduced after heating. These results confirmed that a low-molecular-weight substance(s) of less than 10 kDa, present in bovine milk and colostrum, enhances the oxidative burst activity of PMNL.

Neutrophils of vertebrates undergo respiratory burst activity (RBA) as a defense mechanism against bacterial infections. We report here that ayu (Plecoglossus altivelis) have unusually high RBAs even when they are in a healthy condition. Kidney and blood leukocytes were obtained from ayu, rainbow trout (Oncorhynchus mykiss), carp (Cyprinus carpio), eel (Anguilla japonica), and pond smelt (Hypomesus nipponensis). Neutrophil RBA was measured by flow cytometry using dihydrorhodamine after stimulation with phorbol myristate acetate. The amount of RBA of neutrophils from both blood and kidney was significantly higher in ayu than in the other species (e.g. the fluorescence intensity of ayu blood neutrophils was about 3-7 times higher than that from trout and carp, and that of ayu kidney neutrophils was 2-19 times higher than that of rainbow trout, carp, eel, and pond smelt). This unique character of ayu neutrophils was invariable even at different ages, locations, and sex-maturation stages.

Bovine milk contains various immunoreactive components, and the activation of polymorphonuclear leukocytes (PMNLs) function in breast-fed infants has been reported. In this study, the effect of milk on the oxidative burst of bovine PMNLs was investigated in vitro. When PMNLs were incubated with 0.1% colostrum or normal milk, the oxidative burst induced by serum-opsonized Staphylococcus aureus was enhanced, and the enhancement declined dose-dependently. The enhancement of the oxidative burst by milk was not due to opsonins but the priming activities. Also, the phorbol 12-myristate 13-acetate (PMA)-induced oxidative burst increased after incubation with 0.1% colostrum, but the colostral enhancement of the oxidative burst was unaffected by the incubation time. These results suggest that bovine milk contains oxidative burst promoting factor(s).

We studied the effects of recombinant dolphin tumor necrosis factor alpha (rdoTNFalpha) on the respiratory burst activity of dolphin neutrophils. rdoTNFalpha enhanced the luminol-dependent chemiluminescence response of dolphin neutrophils induced by concanavalin-A, opsonized zymosan, and heated plasma, but not that induced by phorbol myristate acetate. The TNF-associated priming activity was concentration- and preincubation time-dependent, and heat-instable. These data suggest that, as in human neutrophils, TNFalpha enhances the respiratory burst in dolphin neutrophils that follows short-term incubation with various receptor-mediated agonists.

To investigate the early host defense function in aquatic animals, the respiratory burst activity of bottlenose dolphin neutrophils against soluble and particulate stimulants was measured by luminol-dependent chemiluminescence assays and compared with those of bovine and human. Dolphin neutrophils generated the respiratory burst in response to phorbol 12-myristate 13-acetate (PMA), concanavalinA (ConA), heated-plasma (HP), and homologous-plasma opsonized zymosan except N-formyl-Met-Leu-Phe (fMLP). However, the respiratory burst of dolphin neutrophils stimulated by lipopolysaccharide and Staphylococcus aureus was inferior to those of bovine and human. Furthermore, DP-OZ also induced the respiratory burst of bovine and human neutrophils. In conclusion, dolphin neutrophils responded to several soluble and particulate stimulants as well as human neutrophils, but were refractory or slightly responded to bacterial agents.

Bovine colostrum contains a variety of essential nutrients, antibodies, cytokines, hormones, and growth factors that are important for nutrient supply, host defense, growth and for general neonatal adaptation. We have investigated the effect of bovine colostrum on the phagocytic activity for latex particles by normal peripheral blood polymorphonuclear leukocytes using flow cytometric analysis. The phagocytosis promoting effect was observed in colostrum. The promoting activity showed dose-dependent increase up to 25% at a concentration of colostrum. This activity was maximum in the colostrum obtained at parturition and gradually decreased with the time course of lactation as well as IgG level. Colostrum possessed the higher promoting activity than milk and normal serum. These results indicate that bovine colostrum strongly activates polymorphonuclear leukocyte phagocytosis, suggesting the concernment with development of nonspecific immune system in newborns., (Copyright 2001 S. Karger AG, Basel)

Oxygen radical generation by stimulation with phorbol myristate acetate (PMA) was evaluated in bottlenose dolphin neutrophils. A Cypridina luciferin analog-dependent chemiluminescent assay demonstrated that dolphin neutrophils generate superoxide by the addition of PMA, and that its superoxide-forming activity is completely suppressed by diphenylene iodonium, a specific inhibitor of NADPH oxidase. These results indicate that dolphin neutrophils possess NADPH oxidase activity. Furthermore, the NADPH oxidase activity (hydrogen peroxide production) in dolphin neutrophils, as well as in human neutrophils, was greater at 37 degrees C than at a lower temperature. RT-PCR with specific primers revealed that dolphin neutrophils expressed the mRNAs of the major NADPH oxidase components, which included membrane-associated flavocytochrome b (gp91(phox) and p22(phox)) and cytosolic factors (p40(phox), p47(phox), and p67(phox)), implying the existence of these protein homologues in dolphin neutrophils.

Using an antibody against the synthetic peptide corresponding to the COOH-terminal region of human cytochrome b558 large subunit, a broad band was specifically detected in neutrophil lysates from 6 marine fish and 2 freshwater fish by western blotting. Immunofluorescence assay showed that the antibody recognized the epitopes in eel and tilapia neutrophils permeabilized with detergent. These results suggest that the cytochrome b large subunit universally exists in fish neutrophils and that the epitopes are exposed to the cytoplasmic side of fish neutrophils as well as human neutrophils. Furthermore, a synthetic peptide corresponding to the COOH-terminus of the large subunit apparently blocked superoxide production in a specific and dose-dependent fashion in eel and tilapia neutrophils, indicating that the region equivalent to the COOH-terminus of cytochrome b large subunit is responsible for superoxide generation in fish neutrophils.

When western blotting analysis of Japanese eel neutrophil lysate was performed using antibody against the synthetic peptide corresponding to the carboxyl terminal region of human cytochrome b558 large subunit, a broad band was specifically detected at approximately 90 kDa. The antibody recognized the epitope present in eel neutrophils only after the cells were permeabilized with detergent. These results indicate that the large subunit of cytochrome b exists in fish neutrophils and the epitope is exposed to the cytoplasmic side of membrane, and suggest that cytochrome b is conserved across species.

Two oxygen radicals, superoxide and hydrogen peroxide, were measured by ferricytochrome C reduction and scopoletin fluorescent assays to investigate oxygen metabolism during the respiratory burst in Japanese eel neutrophils. Maximal superoxide production was obtained using 0.1 microgram/mL of PMA. Oxygen consumption was almost equivalent to superoxide production, indicating that the consumed oxygen was almost fully converted to superoxide in eel neutrophils. Hydrogen peroxide production was approximately half that of oxygen consumption or superoxide production in neutrophils elicited by killed bacteria or casein. The amounts of superoxide and hydrogen peroxide production, by neutrophils elicited by irritants, were about 11.5 and 5.0 nM/10(7) cells/ min, respectively, which were significantly higher than those of unelicited neutrophils (2.4 and 0.3 nM/10(7) cells/min, respectively).