Peritoneal Exudate Leukocytes Research Articles

Histamine and mast cell activator compound 48/80 are safe but inefficient systemic adjuvants for gilthead seabream vaccination

Histamine has a key role in the regulation of inflammatory and innate immune responses in vertebrates. Gilthead seabream (Sparus aurata L.), a marine hermaphrodite teleost of great commercial value, was the first fish species shown to possess histamine-containing mast cells (MCs) at mucosal tissues. MCs are highly abundant in the peritoneal exudate of gilthead seabream and compound 48/80 (Co 48/80), often used to promote MC activation and histamine release, is able to promote histamine release from gilthead seabream MCs in vitro and in vivo. The aim of the present study was to analyze the effect of histamine and Co 48/80 on the immune responses of gilthead seabream. For this purpose, histamine and Co 48/80 were intraperitoneally injected alone or combined with 109 heat-killed Vibrio anguillarum cells and their effects on head kidney and peritoneal exudate were analyzed. The results indicated that although histamine and Co 48/80 were both able to alter the percentage of peritoneal exudate and head kidney immune cell types, only Co 48/80 increased reactive oxygen species production by peritoneal leukocytes. In addition, histamine, but not Co 48/80, was able to slightly impair the humoral adaptive immune response, i.e. production of specific IgM to V. anguillarum. Notably, both histamine and Co 48/80 reduced the expression of the gene encoding histamine receptor H2 in peritoneal exudate leukocytes. These results show for the first time in fish that although systemic administration of histamine and Co 48/80 is safe, neither compound can be regarded as an efficient adjuvant for gilthead seabream vaccination.

Enniatin B metabolites in liver and tissues of gilthead sea bream (Sparus aurata)

Histamine has a key role in the regulation of inflammatory and innate immune responses in vertebrates. Gilthead seabream (Sparus aurata L.), a marine hermaphrodite teleost of great commercial value, was the first fish species shown to possess histamine-containing mast cells (MCs) at mucosal tissues. MCs are highly abundant in the peritoneal exudate of gilthead seabream and compound 48/80 (Co 48/80), often used to promote MC activation and histamine release, is able to promote histamine release from gilthead seabream MCs in vitro and in vivo. The aim of the present study was to analyze the effect of histamine and Co 48/80 on the immune responses of gilthead seabream. For this purpose, histamine and Co 48/80 were intraperitoneally injected alone or combined with 109 heat-killed Vibrio anguillarum cells and their effects on head kidney and peritoneal exudate were analyzed. The results indicated that although histamine and Co 48/80 were both able to alter the percentage of peritoneal exudate and head kidney immune cell types, only Co 48/80 increased reactive oxygen species production by peritoneal leukocytes. In addition, histamine, but not Co 48/80, was able to slightly impair the humoral adaptive immune response, i.e. production of specific IgM to V. anguillarum. Notably, both histamine and Co 48/80 reduced the expression of the gene encoding histamine receptor H2 in peritoneal exudate leukocytes. These results show for the first time in fish that although systemic administration of histamine and Co 48/80 is safe, neither compound can be regarded as an efficient adjuvant for gilthead seabream vaccination.

Evaluation of Anti-Inflammatory Activity ofCitrus latifoliaTanaka Essential Oil and Limonene in Experimental Mouse Models

The genus Citrus (Rutaceae) includes several species of plants that produce some of the most cultivated fruits in the world, providing an appreciable content of essential oil. In folk medicine, they are used as a cholagogue, antipyretic, anti-inflammatory, sedative, and antitoxic effects. Lemon essential oil has been used since ancient times for its antiseptic, carminative, diuretic, and eupeptic effects. In this study, we investigated the anti-inflammatory activity of Citrus latifolia Tanaka essential oil (CLEO) and its main constituent LIM. In the cell viability assay, CLEO and LIM (3, 10, 30, and 90 μg/mL) had low cytotoxicity. In zymosan-induced peritonitis, LIM (500 mg/kg) decreased the infiltration of peritoneal exudate leukocytes and decreased the number of polymorphonuclear leukocytes. In vitro chemotaxis revealed that CLEO and LIM (1, 3, and 10 µg/mL) promoted a significant reduction of neutrophil migration toward fMLP and LTB4. LIM (500 mg/kg) also reduced TNF-α levels but did not alter IL-10 levels in the peritoneal exudate. In conclusion, this study showed that LIM isolated from CLEO had potential anti-inflammatory effects, likely by inhibiting proinflammatory mediators present in inflammatory exudate and leukocyte chemotaxis.

NK-like and oxidative burst activities are the main early cellular innate immune responses activated after virus inoculation in reservoir fish

Viral diseases are a major problem in fish farming and a deeper knowledge of the immunological mechanisms playing a part in the antiviral defence is still important. Moreover, fish farming practices (high densities, new areas of culture and egg/larvae/adult transport) are significantly increasing the spread of viruses and the number of susceptible or reservoir fish species. In this last point, no studies have focused on the immunological mechanisms playing a part in the antiviral responses in reservoir and non-susceptible fish species. Thus, we have evaluated the very early innate immune responses of gilthead seabream ( Sparus aurata) to the virus causing viral haemorrhagic septicaemia (VHSV) in salmonids since this virus has been found in seabream and neighbouring farmed marine fish species acting as a viral reservoir. The virus was detected in liver, head-kidney, spleen and peritoneal cavity suggesting that the virus reached these tissues but did not replicate as viral expression was almost absent by 72 h post-inoculation. Interestingly, VHSV provoked an influx of leucocytes to the peritoneal cavity and a redistribution of peritoneal exudate (PELs) and head-kidney (HKLs) leucocytes and their innate immune responses (non-specific cytotoxic (NCC or NK-like) activity, phagocytosis, reactive oxygen intermediate (ROI) production and myeloperoxidase (MPO) activity) were generally increased demonstrating that the immune system is activated and involved in the clearance of the virus. Strikingly, NK-like, ROI and MPO were the most enhanced by the presence of VHSV in both PELs and HKLs suggesting that these early innate immune events are crucial during early viral infection stages in non-susceptible or reservoir species. Differences in the immunological mechanisms between susceptible and reservoir species and with other particulate antigens are discussed.

Absence of endogenous interleukin-10 enhanced organ dysfunction and mortality associated to zymosan-induced multiple organ dysfunction syndrome

Experimental evidence suggests that interleukin (IL)-10 plays a pivotal role in generalized inflammation. Here we investigate the effects of IL-10 gene deletion on the acute phase of the multiple organ dysfunction syndrome (MODS) caused by zymosan in the mouse. MODS was induced by zymosan administration (500 mg/kg, suspended in saline solution, i.p.) in IL-10 wild-type and knockout mice; sham groups were treated with vehicle. Mice were sacrificed 18 h after zymosan or saline administration. In another set of experiments, animals were monitored for 12 days to assess systemic toxicity and survival rate. Mice lacking IL-10 displayed increased peritoneal exudate volume and leukocytes. Also, we observed a significant increase in myeloperoxidase activity and lipid peroxidation in ileum and lung tissues, as well as augmented levels of TNF-α, IL-1β and nitrogen-derived species in the plasma. With regard to organ injury, absence of IL-10 enhanced the renal, hepatocellular and pancreatic dysfunction caused by zymosan administration. All of these parameters significantly influenced the systemic toxicity and the overall survival at 12 days, which was significantly lower in IL-10 knockout mice. Therefore, this study demonstrates that the absence of endogenous IL-10 enhances the MODS induced by zymosan in mice.

C1 Inhibitor-Mediated Protection from Sepsis

C1 inhibitor (C1INH) protects mice from lethal Gram-negative bacterial LPS-induced endotoxin shock and blocks the binding of LPS to the murine macrophage cell line, RAW 264.7, via an interaction with lipid A. Using the cecal ligation and puncture (CLP) model for sepsis in mice, treatment with C1INH improved survival in comparison with untreated controls. The effect was not solely the result of inhibition of complement and contact system activation because reactive center-cleaved, inactive C1INH (iC1INH) also was effective. In vivo, C1INH and iC1INH both reduced the number of viable bacteria in the blood and peritoneal fluid and accelerated killing of bacteria by blood neutrophils and peritoneal macrophages. In vitro, C1INH bound to bacteria cultured from blood or peritoneal fluid of mice with CLP-induced sepsis, but had no direct effect on bacterial growth. However, both C1INH and iC1INH enhanced the bactericidal activity of blood neutrophils and peritoneal exudate leukocytes. C1INH-deficient mice (C1INH-/- mice) subjected to CLP had a higher mortality than did wild-type littermate mice. Survival of C1INH-/- mice was significantly increased with two doses of C1INH, one given immediately following CLP, and the second at 6 h post-CLP. C1INH may be important in protection from sepsis through enhancement of bacterial uptake by, and/or bactericidal capacity of, phagocytes. Treatment with C1INH may provide a useful additional therapeutic approach in some patients with peritonitis and/or sepsis.

Injection of xenogeneic cells into teleost fish elicits systemic and local cellular innate immune responses

The early innate immune response of the teleost gilthead seabream (Sparus aurata L.) against xenogeneic cells was studied. Fish received a single intraperitoneal injection of xenogeneic cells (tumour cell line), following which leucocyte mobilization, degranulation, peroxidase content, respiratory burst and phagocytic and cytotoxic activities were determined in both peritoneal exudate leucocytes (PELs) and head-kidney leucocytes (HKLs). The total number of PELs increased from 4 h post-injection until the end of the experiment (3 days). Interestingly, flow cytometric analysis of PEL and HKL suspensions revealed variations in the proportion of cell types. The percentage of HK acidophilic granulocytes significantly increased after 72 h, whereas PE acidophils increased after 4 h. Moreover, numbers of PE lymphocytes and monocyte-macrophages significantly increased during the experiment. The peroxidase content of the leucocytes was unaffected, although PEL degranulation was largely enhanced. This liberation of peroxidases correlated well with the enhancement of the oxidative respiratory burst activity in PELs, reflecting leucocyte activation. However, phagocytosis only increased in PELs 4 h after intraperitoneal injection, whereas the cytotoxic activity of HKLs increased 1 and 2 days post-injection but, in general, decreased in the PELs. Our data thus demonstrate that the appearance of xenogeneic cells involves leucocyte mobilization and innate immune-response activation at the site of invasion and in the head-kidney. Involvement of the various leucocyte types and potential modes of activation are discussed.

Early local and systemic innate immune responses in the teleost gilthead seabream after intraperitoneal injection of whole yeast cells

The early cellular innate immune responses of the teleost gilthead seabream ( Sparus aurata L.) against whole yeast cells were studied. Fish received a single intraperitoneal (i.p.) injection of Saccharomyces cerevisiae and leukocyte mobilization, degranulation, peroxidase content, respiratory burst, phagocytic and cytotoxic activities were assayed in both head-kidney leukocytes (HKLs) and peritoneal exudate leukocytes (PELs). The total number of PELs significantly increased from 4 h post-injection until the end of the experiment (3 days). Interestingly, flow cytometric analysis revealed variations in the proportion of cell-types in the PE. Thus, PE acidophilic granulocytes increased to a significant extent 4 h post-injection and were restored thereafter. Moreover, PE monocyte-macrophages started to increase from 24 h, the enhancement being statistically significant after 48 and 72 h. Degranulation was greater in PELs throughout the assay. The peroxidase content of the leukocytes was affected differently in HKLs and PELs. The respiratory burst activity was not affected in HKLs but significantly increased in PELs from 4 to 48 h post-injection with yeast cells. On the other hand, HKL phagocytosis had decreased 72 h post-injection with yeast cells while it increased after 4 and 24 h post-injection in the PELs. Conversely, the cytotoxic activity was significantly enhanced in HKLs from 24 to 72 h post-injection but slightly decreased in PELs. Finally, our data demonstrate that seabream injected with the yeast Saccharomyces cerevisiae show leukocyte mobilization and cellular innate immune response activation at the site of invasion and also in the head-kidney. The implications of the leukocyte-types and the immune responses observed, as well as analogies with other particulated antigens, will be discussed as possible models for investigating the effect of potential pathogens.

Acid exposure is an immune disruptor in adult Rana pipiens

Acidic environments are physiological stressors for amphibians. The objective of the present study was to document the effect of an acidic environment on innate immune system function under controlled experimental conditions in Rana pipiens. We developed an in vivo assay, by injecting a suspension of 1-microm fluorescent beads in fluid thioglycollate, to induce peritonitis. The number of peritoneal exudate leukocytes and their phagocytic activity did not increase with thioglycollate injection when frogs were exposed to pH 5.5 compared to when frogs were exposed to pH 7.0. An environment of pH 5.5 disrupted the inflammatory response of frogs compared to an environment of pH 7.0; at pH 5.5, more nonphagocytic leukocytes and fewer highly phagocytic leukocytes were found compared to those in frogs exposed to pH 7.0. Frogs stimulated by thioglycollate injection and exposed to pH 5.5 had a 50% increase in cells that did not exhibit phagocytosis and a 4- to 10-fold reduction in the number of highly phagocytic cells. This is evidence that acid exposure functions as an immune disruptor in adult R. pipiens under laboratory conditions.

Cloning, distribution and up-regulation of the teleost fish MHC class II alpha suggests a role for granulocytes as antigen-presenting cells

The major histocompatibility complex (MHC) class II alpha chain gene of the teleost fish gilthead seabream ( Sparus aurata), Spau-DAA, has been characterized. We cloned, sequenced and studied its polymorphism, before evaluating its expression in resting seabream leucocytes, tissues and tumor cells as well as in primed leucocytes. A complete allele was obtained by overlapping sequence fragments obtained by RT-PCR. The full-length Spau-DAA*101 comprises 1840 bp with a 5′-UTR region of 84 bp, an ORF of 729 bp and a 3′-UTR of 1027 bp. The putative protein of 242 residues shows homology with known MHC class II alpha genes, varying from 71 to 28% in other fish and humans, respectively. The protein sequence showed all the important features: leader peptide, α1, α2 and CP/TM/CYT regions, conserved cysteines and N-glycosylation site. The phylogenetic tree shows that it is included in the cluster containing the Percomorpha subclass and far from the human and shark genes. It is polymorphic, as seen when we sequenced the complete ORF of 11 alleles showing most of the amino acidic changes in the α1 domain, where the peptide-binding region (PBR) is found. Spau-DAA mRNA expression was mainly found in peritoneal exudate leucocytes, head-kidney, spleen, thymus and gill. Minor expression was detected in gut, brain, liver and PBLs. RT-PCR expression studies in isolated leucocyte subpopulations revealed, for the first time in the literature, that acidophilic granulocytes show high MHC class II gene expression. Apart from these granulocytes lymphocytes also express the Spau-DAA gene, although other cell types may also do the same. Finally, incubation of head-kidney leucocytes with yeast cells or pathogenic bacteria up-regulates Spau-DAA gene expression whilst incubation with ConA, ConA + LPS or PHA does not. The possible involvement of the seabream MHC class II alpha gene in the fish defence and antigen presentation are discussed.

The tumor necrosis factor alpha of the bony fish seabream exhibits the in vivo proinflammatory and proliferative activities of its mammalian counterparts, yet it functions in a species-specific manner.

Information on the bioactivities of non-mammalian cytokines is scant due to the lack of the recombinant molecules and specific antibodies. We produced the mature predicted peptide of tumor necrosis factor alpha (TNF alpha) from the bony fish gilthead seabream (Sparus aurata L.) (sbTNF alpha), and its biological role was determined in vitro and in vivo. We first demonstrated by analytical size-exclusion chromatography that sbTNF alpha is an oligomeric protein but the dimer appears to predominate over the trimeric form, in contrast to mammalian TNF alpha. Intraperitoneal injection of native sbTNF alpha resulted in (i) priming of the respiratory burst of the peritoneal exudate and head-kidney (HK) leukocytes, the latter being the bone marrow equivalent in fish; (ii) rapid recruitment of phagocytic granulocytes to the injection site, and (iii) induction of granulopoiesis in the HK. Interestingly, sbTNF alpha was able to induce a strong proliferation of HK cells in vitro, whereas human TNF alpha did not. Conversely, sbTNF alpha was not cytotoxic for murine L929 fibroblasts.

Acidophilic granulocytes of the marine fish gilthead seabream (Sparus aurata L.) produce interleukin-1beta following infection with Vibrio anguillarum.

The fish immune response to Gram-negative bacteria is poorly understood. In this study, we use a monoclonal antibody (mAb) specific to acidophilic granulocytes from the marine fish gilthead seabream (Sparus aurata L.), together with an antiserum specific to interleukin-1beta (IL-1beta) from this species, in order to investigate whether these cells are involved in the immune response against the pathogenic bacterium Vibrio anguillarum and, in particular, in the production of the pro-inflammatory cytokine IL-1beta. We found that gilthead seabream head-kidney, peritoneal exudate and peripheral blood leukocytes accumulated proIL-1beta intracellularly when challenged in vitro with V. anguillarum, whereas only peritoneal exudate and blood leukocytes were able to accumulate proIL-1beta following infection. Importantly, the blood leukocytes from infected animals that accumulated proIL-1beta were shown to be the acidophilic granulocytes. A rapid mobilization of such cells from the head-kidney to the site of inflammation following infection with V. anguillarum was also observed.

Apoptosis of sea bass ( Dicentrarchus labrax L.) neutrophils and macrophages induced by experimental infection with Photobacterium damselae subsp. piscicida

The infection of sea bass ( Dicentrarchus labrax L.) by intraperitoneal (i.p.) injection of the agent of fish pasteurellosis Photobacterium damselae subsp. piscicida resulted in the apoptosis of peritoneal neutrophils and macrophages. All the eight virulent and none of the two non-virulent strains tested exhibited apoptogenic activity. A secreted bacterial protein(s) is a likely candidate as the factor(s) responsible for this activity, since no apoptosis was induced by i.p. injected UV-killed virulent strains and the virulent culture supernatants exhibited a thermo-labile apoptogenic activity identical to that of live bacteria. The apoptotic process was characterized by the occurrence of DNA fragmentation detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining and DNA electrophoresis, and of typical ultrastructural alterations namely cell shrinkage, chromatin condensation, nuclear fragmentation and production of blebs with shedding of apoptotic bodies. In the apoptotic process induced by lethal doses of virulent bacteria or culture supernatants both peritoneal macrophages and neutrophils were extensively affected, the majority of these cells being apoptotic and reaching values around 10 7per peritoneal cavity for each cell type at 24 h post-injection. Moreover, the number of non-apoptotic macrophages was always below the initial number in the resting peritoneal cavity. Since macrophages are key cells in the elimination of both bacteria and apoptotic moribund cells and apoptotic bodies, the induction by Ph. damselae subsp. piscicida of simultaneous macrophage and neutrophil apoptosis results, on the one hand, in the destruction of the two phagocytic cell types involved in the restriction of multiplication of the bacteria and, on the other hand, in the uncontrolled progression of the apoptotic process towards secondary necrosis and eventual lysis of high numbers of moribund neutrophils and of neutrophilic apoptotic bodies, with the consequent extensive release of their highly cytotoxic components. Abundant apoptotic cells were also seen in sections of head-kidney from fish dying from experimental pasteurellosis. In contrast, no apoptosis was seen in vitro after the treatment with virulent culture supernatants of sea bass head-kidney macrophage cultures or after the treatment ex vivo of peritoneal exudate leukocytes with virulent bacteria or culture supernatants. The apoptotic process described here appears as a novel and very powerful microbial pathogenic strategy.

Characterisation of gilthead seabream acidophilic granulocytes by a monoclonal antibody unequivocally points to their involvement in fish phagocytic response.

The various cell types involved in fish phagocytic defence have not been properly established because of the morphological heterogeneity of leucocytes and the lack of appropriate cell-surface markers. In this study, we report the production and characterisation of a monoclonal antibody, G7, which specifically recognises gilthead seabream acidophilic granulocytes, as assayed by immunofluorescence and immunoelectron microscopy. The antibody reacted with 40%-50% of head-kidney and peritoneal exudate leucocytes and 10%-20% of spleen and peripheral blood leucocytes. More importantly, G7(+) acidophils constituted 85% of the head-kidney leucocytes showing phagocytic activity towards the fish pathogenic bacterium Vibrio anguillarum. The results are discussed in relation to the role played by this cell type in fish immune responses.

Nonspecific cell-mediated cytotoxicity in the seawater teleosts (Sparus aurata andDicentrarchus labrax): Ultrastructural study of target cell death mechanisms

In higher vertebrates, cytolytic lymphocytes lyse their targets by the mechanisms of cell death called necrosis and apoptosis. However, the mechanisms of target cell death mediated by fish cytotoxic cells have not been established. We report the ultrastructure of target cell death mediated by the seawater teleosts gilthead seabream (Sparus aurata) and sea bass (Dicentrarchus labrax). Head-kidney, blood, and peritoneal exudate leukocytes were incubated with HeLa or B16 melanoma tumor cells, as targets, and processed for transmission electron microscopic studies. After incubation of tumor cells with leukocytes, substantial ultrastructural changes typically associated with both necrosis and apoptosis were observed in the plasmalemma, nucleus, and cytoplasm of the tumor cells. These morphological changes included loss of microvilli, the formation of long cytoplasmic processes and blebs, condensation and margination of the chromatin, swelling of the organelles, and vacuolation and condensation of the cytoplasm. Our results indicate that leukocytes from gilthead seabream and sea bass are able to kill their targets by the mechanisms of necrosis and apoptosis, in a similar way to mammalian cytotoxic cells.

Effect of squalene on superoxide anion generation induced by a skin irritant, lauroylsarcosine

The effect of squalene on superoxide anion (O 2) generation was studied in rats in order to elucidate the mechanism whereby the compound decreases erythema induced by 1% lauroylsarcosine (LS) ointment. Topical application of superoxide dismutase (SOD) ointment (99000 U/g) was effective on the erythema as well as 10% squalene ointment, suggesting that O 2 is a major mediator responsible for skin irritation. LS (200–400 /xg/ml) caused overt production of O 2 from cultured keratinocytes and peritoneal exudate leukocytes; O 2 was significantly reduced by addition of squalene (100 /zg/ml). A similar response was observed with zymosan-induced O 2-generation. However, squalene had no effects on O 2 generation in a xanthine-xanthine oxidase system unlike the effect observed with SOD. These results suggest that a possible role of squalene for the alleviation of skin irritation is suppression of O 2 production depending on the different mechanism of action from SOD.

Phagocytic defence mechanism in sea bass (Dicentrarchus labrax L.): An ultrastructural study

The ultrastructure of the phagocytic process in fish has not been established in spite of the significant morphofunctional differences detected in the fish immune system with respect to the basic immunological pattern in vertebrates. We report the ultrastructure of the bacterial phagocytic defence mechanism in sea bass (Dicentrarchus labrax L.). Head-kidney, blood, and peritoneal exudate leukocytes were challenged with Aeromonas salmonicida and Escherichia coli and processed for transmission electron microscopic study. Macrophages challenged with bacteria showed changes in the cell outline, in the chromatin pattern, and in the ultrastructural features of the cytoplasm as a consequence of an activation process. The phagocytic process consists of the following: 1) Bacteria-macrophage contact. One or more spot contacts between the bacterial wall and the phagocyte membrane are observed. 2) Bacteria engulfment. Slight depressions, membrane invaginations, or cytoplasmic processes are formed at the phagocyte surface. Macrophage processes occasionally surround the bacteria, overlapping and roaming parallel, or a single, long pseudopod encircles a bacterium several times. 3) Endocytic vesicle formation. Macrophages show one or more bacteria inside membrane-bound cytoplasmic vesicles. 4) Phagolysosome formation. Some dense granules (lysosomes) fuse with the endocytic vesicle. 5) Intracellular killing/digestion. Bacteria inside the endocytic vesicles are observed both virtually intact or damaged at different digestion stages. Sea bass macrophages possess the mechanisms necessary to both engulf and kill bacteria. Cellular and subcellular events in the morphology of phagocytosis and lysosomal dissolution of bacteria fit the general pattern described for mammals.

Inhibitory activity of a series of coumarins on leukocyte eicosanoid generation.

Sixteen plant-derived or synthetic coumarins with different patterns of substitution were tested for their capacity to modify A23187-induced synthesis of leukotriene B4 and thromboxane B2 via the 5-lipoxygenase and cyclooxygenase pathways of arachidonate metabolism in rat peritoneal exudate leukocytes. Five of the 16 coumarins inhibited LTB4 production: all contain orthodihydroxy substitutions (approximate IC50 values 8-100 microM). The mechanism is likely to depend upon a combination of the coumarins' iron-chelating and iron ion-reducing abilities, properties which also confer beneficial activities of these compounds as scavengers of reactive oxygen species (Payá et al., Biochem. Pharmacol. 44, 205-214 (1992)). Inhibition of the cyclooxygenase pathway was only demonstrated by one compound, 5,7-dihydroxy-4-methylcoumarin, which did not inhibit 5-lipoxygenase, indicating that the cyclooxygenase inhibitory mechanism is different. Similar effects of the active coumarins were obtained using arachidonic acid as substrate for rat leukocyte eicosanoid generation, confirming that they act at the 5-lipoxygenase/cyclooxygenase level. The same profile of activity was also shown when the coumarins were tested against 5-lipoxygenase in human polymorphonuclear neutrophils. Taken together, these antioxidant and anti-eicosanoid properties of coumarins could be exploited for the design of potentially valuable non-toxic anti-inflammatory agents for treating diseases in which eicosanoid generation and the production of reactive oxygen species are involved.

Non-specific cytotoxic response against tumor target cells mediated by leucocytes from seawater teleosts, Sparus aurata and Dicentrarchus labrax: an ultrastructural study.

The in vitro cytotoxic reaction of leucocytes from the seawater teleosts gilthead seabream (Sparus aurata) and sea bass (Dicentrarchus labrax) against tumor target cells was studied by transmission and scanning electron microscopy. Head-kidney, blood or peritoneal exudate leucocytes were incubated with HeLa or B16 melanoma cells. After incubation, conjugates of leucocytes (effectors) and tumor cells (targets) were observed. Both the effector-target cell binding and target cell lysis steps of the cytotoxic process were characterized. Usually more than one effector was bound to the same target. The effectors exhibited ultrastructural features of either monocyte or lymphocyte cells. Monocyte-like effectors possessed an oval or kidney-shaped heterochromatinic nucleus and a few granules. They were flattened against the targets over a broad area or, occasionally, made several punctuated contacts. Lymphocyte-like effectors had a large, rounded or indented nucleus, numerous free ribosomes and occasional cytoplasmic granules. These effectors established spot contacts with the target. Contacts between monocyte-like and lymphocyte-like effectors were also recognizable. After incubation, some targets appeared virtually intact, while others had a smooth surface or showed cell processes and surface blebs. These ultrastructural changes in the targets are similar to those described as mediated by mammalian cytotoxic cells.

Highly lytic CD8+, alpha beta T-cell receptor cytotoxic T cells with major histocompatibility complex (MHC) class I antigen-directed cytotoxicity in beta 2-microglobulin, MHC class I-deficient mice.

Targeted disruption of the beta 2-microglobulin (beta 2m) gene results in major histocompatibility complex (MHC) class I deficiency and virtual disappearance of functional CD8+ cytotoxic T lymphocytes (CTLs) in beta 2m-deficient (beta 2m-/-) mice. We asked whether the beta 2m-/- mice are able to reject tumor cells injected i.p. and what is the cellular composition of peritoneal exudate leukocytes (PELs) from such mice. We found that beta 2m-/- mice do reject MHC class I-bearing tumor cells injected i.p. Surprisingly, analysis of PEL CTLs obtained from i.p. tumor-injected beta 2m -/- mice revealed the presence of a large proportion of functional, tumor-destroying CD8+, CD4-, alpha beta T-cell receptor-positive, CD3+, Thy-1+, MHC class I-negative CTLs with strong MHC class I-directed cytotoxic activity. These results call for careful studies of local accumulation of CD8+ CTLs in beta 2m -/- mouse models and suggest that the dramatic decrease in MHC class I expression caused by beta 2m gene disruption does not prevent CD8+/CD4- cell selection and expansion.