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Tilapia mast cell lysates enhance neutrophil adhesion to cultured vascular endothelial cells
Fish & Shellfish Immunology (2002) 13, 243–250 doi:10.1006/fsim.2001.0398 Available online at http://www.idealibrary.com on
Tilapia mast cell lysates enhance neutrophil adhesion to cultured vascular endothelial cells TOMOMASA MATSUYAMA1* AND TAKAJI IIDA2 1
Fish Pathology Division, National Research Institute of Aquaculture, Nansei, Mie 516-0193, Japan 2 Faculty of Agriculture, Miyazaki University, Gakuen Kibanadai-Nishi 1-1, Miyazaki 889-2192, Japan (Received 17 September 2001, accepted 16 November 2001, published electronically 20 February 2002) The possible role of fish mast cells in regulating neutrophil adhesion to vascular endothelial cells was studied using primary cultures of tilapia vascular endothelial cells. The endothelial cell monolayer, which was cultured in 96 well plates, was stimulated for appropriate periods with tilapia mast cell (tMC)-lysates or with Leibovitz-15 (L-15) medium, as a control, and peripheral neutrophils were added into each well after removal of the lysates. After 30 min incubation, cells in the wells were fixed with formalin and non-adherent neutrophils were removed. The cells were stained with Giemsa and neutrophil adhesion was observed microscopically. Although some neutrophils attached to the endothelial cells without stimulation, neutrophil adhesion was enhanced after the incubation of the endothelial cells with tMC-lysates. Neutrophil adhesion was maximal 6 h after the lysate stimulation, with a six-fold increase compared to the control. Neutrophil adhesion also increased when the endothelial cells were stimulated with neutrophil lysates, lipopolysaccharide and zymosan-treated tilapia sera. These results indicate that fish vascular endothelial cells express some neutrophil adhesion molecule(s) after stimulation with various substances. 2002 Published by Elsevier Science Ltd.
Key words:
neutrophil adhesion, vascular endothelial cell, fish mast cell, eosinophilic granule cell, tilapia.
I. Introduction The interaction of circulating neutrophils with vascular endothelial cells is an initial event in acute inflammation. The recruitment of neutrophils from the blood-stream to infected sites not only depends on chemoattractants generated at these sites, but also requires interactions with vascular endothelial cells. During inflammation, neutrophils are tethered to and roll on endothelial cell surfaces. The cells then adhere, and finally migrate out of blood vessels to reach inflammatory sites [1]. Stimulation of channel catfish (Ictalurus punctatus) neutrophils by phorbol dibutyrate intensified their *Corresponding author. E-mail: [email protected]#rc.go.jp 243 1050–4648/02/$-see front matter
2002 Published by Elsevier Science Ltd.
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adhesion to the extracellular matrix [2]. The full-length cDNA beta 2 (CD18) molecule, which is responsible for the tight adhesion of leucocytes, was identified from channel catfish neutrophils [3]. However, adhesion molecules expressed by vascular endothelial cells have not been studied in fish. It is well documented in mammalian species that some of the adhesion molecules, e.g. selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), expressed on endothelial cell surfaces contribute to the adhesion and transmigration of neutrophils [1]. Mammalian mast cells induce expression of adhesion molecules on endothelial cell surfaces by releasing granular components [4]. Mast cell-like cells of fish (fMCs) have been termed eosinophilic granular cells (EGCs) since their granules are eosinophilic unless special fixatives and staining techniques are used [5]. The arrangement of fMCs closely associated with blood vessels suggest an interaction between fMCs and endothelial cells. In fact, we previously reported the vascular permeability promoting e#ect of the tilapia mast cell (tMC) products in the inflamed tilapia [6]. In the present study, we have examined e#ects of tMC-lysates and some other substances on adhesion of tilapia neutrophils to cultured vascular endothelial cells in vitro. II. Materials and Methods FISH
Tilapia (Oreochromis niloticus) weighing 37 to 92 g (average weight S.E., 69·213·8 g) were maintained at 30 C in 180 l tanks in a recirculating water system and were fed daily dry pelleted diet (Chubu Shiryo, Aichi, Japan). ISOLATION AND CULTURE OF TILAPIA VASCULAR ENDOTHELIAL CELLS
Tilapia vascular endothelial cells were isolated from the rete mirabile of swim bladders and cultured in a 96-well flat bottom tissue culture plate (NUNC, New York, U.S.A.) [7]. Endothelial cells were cultured for 10 days and confluent monolayers were used for later analyses. NEUTROPHIL ADHESION ASSAY
Adhesion of neutrophils was examined as follows. Neutrophils were isolated from peripheral blood using a discontinuous density gradient [8]. To induce neutrophilia, fish were injected with formalin-killed Escherichia coli IAM 1239 (1 mg fish 1) into the swim bladder 12 h before the taking of blood. Isolated neutrophils were finally suspended in Leibovitz-15 (L-15) medium containing 10% foetal bovine serum (FBS) to prevent aggregation of the cells (1106 cells ml 1). Cultured endothelial cells were washed twice with L-15 medium and stimulants (or medium alone in control wells) were poured into each well (100 l well 1). After the cells were incubated at 30 C for di#erent time intervals, the stimulants were removed and the endothelial cells were gently washed twice with L-15 medium. The peripheral neutrophils were then added to each well (1105 cells well 1). The cultured endothelial cells of the tilapia readily peeled o# the culture plate by agitation and vortexing of the wash fluid
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altered the distribution of the adherent neutrophils causing them to concentrate in a peripheral ring [9]. To overcome these problems, non-adherent neutrophils were removed after fixing the cells. Briefly, after 30 min incubation at 30 C without agitation, 100 l of 20% formalin in phosphate-bu#ered saline (PBS) was carefully added to each well. The cells were fixed for 30 min. The wells were washed twice with PBS by vortex for 30 s on a plate mixer (Kawakizaki, Tokyo, Japan) to remove non-adherent cells. Neutrophil adhesion was observed microscopically after the staining of cells with Giemsa. Some endothelial cells still peeled o# during the procedure, so only the neutrophils adhering to the endothelial cells were counted. The adhesion activity was expressed as an index of adhesion using the following formula: Index of adhesion=(number of neutrophils adhering to endothelial cells)/ (number of observed endothelial cells)100. Five fish were used for each test. The kinetics of neutrophil adhesion induced by tMC-lysates was measured after cultured endothelial cells had been stimulated with tMC lysates (1105 cells ml 1) or L-15 medium for 30 min, 1 and 2, 4, 6, 8, 12, 16 and 24 h. The tMCs were isolated and pooled from 25 fish, and the tMC lysates were made according to Matsuyama and Iida [6]. Neutrophil adhesion induced by various agents was measured after incubating the endothelial cells for 6 h with the following substances: tMC lysates (1105 cells ml 1), neutrophil lysates (1105 cells ml 1), substance P (100 g/ml 1; Sigma, MI, U.S.A.), lipopolysaccharide from E. coli LPS) (100 g ml 1, Sigma), 10% zymosan-treated tilapia sera (supernatant of normal tilapia sera incubated with zymosan, 1 mg ml 1 for 1 h at 25 C), 10% heat-inactivated tilapia sera, and L-15 medium. Neutrophil lysates were made according to Matsuyama et al. [10]. All reagents were suspended in L-15 medium supplemented with penicillinstreptomycin-fungizone (Bio Whittaker, New York, U.S.A.). The analysis was performed at 30 C in air, and the medium or the stimulants were warmed to 30 C before use. STATISTICS
The results were expressed as the mean ( S.E.). Statistical analysis was performed using Student’s t-test; P<0·05 was considered significant. III. Results CELL COMPOSITION
The isolated and pooled tMCs were 94% pure with 6% neutrophils. The enriched neutrophils isolated from peripheral blood contained neutrophils (61·43·2%), monocytes (6·25·1%), lymphocytes and thrombocytes (32·49·2%) in 15 fish analysed. MORPHOLOGY OF ADHERENT NEUTROPHILS
Neutrophils adhering to vascular endothelial cells became polarized, having a hand mirror shape [Fig. 1(a)]. On the other hand, the neutrophils
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Fig. 1. Neutrophils adhered to vascular endothelial cells (a) or uncovered area in the culture plate (b). These two photographs are of the same well. Arrowheads indicate neutrophils.
attaching to the plastic where endothelial cells had peeled away were oval or rounded [Fig. 1(b)]. Neutrophils adhered tightly to vascular endothelial cells and were not detached by washing. KINETICS OF NEUTROPHIL ADHESION INDUCED BY TMC LYSATES
Some neutrophils attached to endothelial cells without stimulation (Index of adhesion at time 0 was 13·35·4) or after incubation with L-15 medium (minimum was 7·62·1 at 4 h after incubation and maximum was 13·93·9 at 30 min after incubation) (Fig. 2). Incubating the vascular endothelial cells with tMC lysates enhanced the neutrophil adhesion with a peak of adhesion after 6 h incubation: a six-fold increase compared to the stimulation with L-15 medium. Stimulation with the lysates for more than 6 h resulted in a decrease in neutrophil adhesion. After stimulation for 18 and 24 h, there was no significant di#erence between the tMC lysate-stimulated group and the control. NEUTROPHIL ADHESION INDUCED BY VARIOUS AGENTS
Neutrophil adhesion was significantly enhanced by the stimulation of endothelial cells, not only with tMC lysates, but also with neutrophil lysates, LPS and zymosan-treated sera. No significant di#erence was detected with other substances tested (Table 1).
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80 70
*
Index of adhesion
60
*
50 *
*
40 30 20 10 0
0
1
2
4
6 8 12 18 Time after stimulation (h)
24
Fig. 2. Kinetics of neutrophil adhesion after stimulation with tMC lysates (1105 cells ml 1) () or L-15 medium ( ). n=5, *Significantly di#erent from L-15 mediumstimulated control. Table 1. E#ect of stimulants on neutrophil adhesion Stimulants
Index of adhesion
tMC lysates Neutrophil lysates Substance P LPS 10% zymosan-treated sera 10% heat-inactivated sera L-15 medium No stimulation
48·511·7* 33·68·4* 29·612·2 55·414·6* 49·513·2* 8·01·3 9·71·5 6·61·5
Neutrophil adhesion was measured after incubating the endothelial cells for 6 h with various substances. *Significantly di#erent from L-15 medium control. n=5.
IV. Discussion In this study, a quantitative monolayer adherence assay was used to investigate the adhesion of tilapia neutrophils to cultured vascular endothelial cell monolayers. This is the first report investigating the interaction of fish vascular endothelial cells and neutrophils. When the endothelial cells were stimulated with tMC lysates, the intensity of neutrophil adhesion increased gradually to be six times the control value after 6 h stimulation. In vivo, tMCs are apposed to blood vessels, and rapidly release granular material, which enhances neutrophil migration and the permeability of blood vessels, on injection of non-self materials [6, 11, 12]. Because of the close association of tMCs with vascular endothelial cells, during inflammation, the tMC components probably act on the endothelial cells more e#ectively in vivo than
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in vitro. So, a stronger and prolonged response would be expected in vivo as reported in salmonid experiments [13–15]. The tMC component(s) which acts on vascular endothelial cells, was heat stable as neutrophil adhesion was not diminished by heating the tMC lysates at 100 C for 10 min (data not shown). We reported previously that vascular permeability enhancing components of tMC, which activate cultured vascular endothelial cells to increase the uptake of Ca2+ , were also heat stable [6]. May be the product of tMC enhancing the neutrophil adhesion is the same substance as that promoting the vascular permeability. Neutrophils were spread on the endothelial cells and adhered very tightly. The adhesion molecule integrin (CD18) has been identified on channel catfish neutrophils [3] and the interaction of such adhesion molecule(s) may be involved in the neutrophil adhesion to cultured endothelial cells. The expression of integrin by the neutrophils must be up-regulated before the binding to immunoglobulin superfamily molecules on endothelial cell surfaces. In mammalian species, tumour necrosis factor alpha (TNF-) [16], IL-8 [16] and formyl peptides [18] are known to increase the expression of integrin by neutrophils. In vitro stimulation with phorbol esters also increases the integrin (CD18) expression by channel catfish head kidney neutrophils [3]. The peripheral neutrophils used in this study are expected to be up-regulated in their expression of integrin, following priming by injection of killed E. coli [19–21]. Neutrophil adhesion was also enhanced when the vascular endothelial cells were stimulated with neutrophil lysates, LPS and zymosan-treated sera. In mammals vascular endothelial cells express adhesion molecules after stimulation with various substances. The mast cell granular component TNF- increases the expression of ICAM-1 and VCAM-1 on endothelial cells [4], and histamine enhances the expression of P-selectin [22]. Neutrophil elastase, a granule serine proteinase, enhances ICAM-1 expression [23]. Substance P [24] and LPS [25] are known to induce the expression of ICAM-1 on vascular endothelial cells. Neutrophil adherence was increased after incubating cultured vascular endothelial cells with complement fragments [9]. As observed in mammals, vascular endothelial cells in fish probably regulate the progress of inflammation by reacting to several stimuli. The neutrophils that adhered to the plastic plate at gaps in the monolayer were oval or rounded. On the other hand, those attached to endothelial cells resembled a hand mirror in shape. This indicates that the neutrophils adherent to the endothelial cells were moving on the surface of the monolayer, while the cells adherent to the plastic surface were not. It is probable that stimulated endothelial cells release chemokines (i.e. IL-8) which bind to endothelial cells as reported in mammals [26]. Peripheral neutrophils may adhere to endothelial cells and transmigrate to sites of inflammation by following chemokines presented on the endothelial cell surface. We express our sincere thanks to Dr Eduardo M. Leano, Department of Aquaculture, SEAFDEC, The Philippines, for reviewing the manuscript. This work was supported by a grant from the Research Fellowship Division, Japan Society for the Promotion of Science, No. 10968.
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References 1 Cines, D. B., Pollak, E. S., Buck, C. A., Loscalzo, J., Zimmerman, G. A., McEver, E. S., Pober, J. S., Wick, T. M., Konkle, B. A., Schwartz, B. S., Barnathan, E. S., McCrae, K. R., Hug, B. A., Schmidt, A. M. & Stern, D. M. (1998). Endothelial cells in physiology and in the pathophysiology of vascular disorders. Journal of the American Society of Hematology 91, 3527–3561. 2 Ainsworth, A. J., Qian, Y., Xue, L. & Hebert, P. (1996). Channel catfish, Ictalurus puntatus Rafinesque, neutrophil adhesion to selected extracellular matrix proteins, lipopolysaccharide, and catfish serum. Developmental & Comparative Immunology 20, 105–114. 3 Qian, Y., Ainsworth, A. J. & Noya, M. (1999). Identification of beta 2 (CD18) molecule in a teleost species, Ictalurus punctatus Rafinesque. Developmental & Comparative Immunology 23, 571–583. 4 Meng, H., Tonnesen, M. G., Marchese, M. J., Clark, R. A., Bahou, W. F. & Gruber, B. L. (1995). Mast cells are potent regulators of endothelial cell adhesion molecule ICAM-1 and VCAM-1 expression. Journal of Cell Physiology 165, 40–53. 5 Reite, O. B. (1998). Mast cells/eosinophilic granule cells of teleostean fish: a review focusing on staining properties and functional response. Fish & Shellfish Immunology 8, 489–513. 6 Matsuyama, T. & Iida, T. (2001). Influence of tilapia mast cell lysate on vascular permeability. Fish & Shellfish Immunology 11, 549–556. 7 Matsuyama, T. & Iida, T. (2000). Primary culture of tilapia capillary endothelial cells. Fish Pathology 35, 163–164. 8 Matsuyama, T. & Iida, T. (1999). Comparison of inflammatory and peripheral neutrophils of carp and tilapia in defense activities. Fish Pathology 34, 45–46. 9 Tonnesen, M. G., Smedly, L. A. & Henson, P. M. (1984). Neutrophil-endothelial cell interactions. Modulation of neutrophil adhesiveness induced by complement fragments C5a and C5a des arg and formyl-methionyl-leucyl-phenylalanine in vitro. Journal of Clinical Investigation 74, 1581–1592. 10 Matsuyama, T., Iida, T. & Endo, M. (1999). Isolation of inflammatory macrophages from swim bladder of tilapia. Fish Pathology 34, 83–84. 11 Matsuyama T. & Iida, T. (1999). Degranulation of eosinophilic granular cells with possible involvement in neutrophil migration to site of inflammation in tilapia. Developmental and Comparative Immunology 23, 451–457. 12 Matsuyama, T. & Iida, T. (2000). In vitro degranulation of tilapia eosinophilic granular cells and the e#ect on neutrophil migration. Fish Pathology 35, 125–129. 13 Ellis, A. E. (1985). Eosinophilic granular cells (EGC) and histamine responses to Aeromonas salmonicida toxins in rainbow trout. Developmental & Comparative Immunology 9, 251–260. 14 Vallejo, A. N. & Ellis, A. E. (1989). Ultrastructural study of the response to eosinophilic granule cells to Aeromonas salmonicida extracellular products and histamine liberators in rainbow trout Salmo gairdneri Richardson. Developmental & Comparative Immunology 13, 133–148. 15 Reite, O. B. (1997). Mast cells/eosinophilic granule cells of salmonids: staining properties and responses to noxious agents. Fish & Shellfish Immunology 7, 567–584. 16 Yee, J. & Christou, N. N. (1994). The local role of tumor necrosis factor in the modulation of neutrophil function at sites of inflammation. Archives of Surgery 129, 1249–1255. 17 Kelly, C. P., Keates, S., Siegenberg, D., Linvsky, J. K., Pothoulakis, C. & Brady, H. R. (1994). IL-8 secretion and neutrophil activation by HT-29 colonic epithelial cells. American Journal of Physiology 267, G991–G997. 18 Dai, Y., Hogate, S. T. & Church, M. K. (1994). Modulation of the chemotactic responsiveness of guinea pig neutrophils to hrIL-8 and fMLP. Journal of Leukocyte Biology 56, 776–783.
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19 Nagamura, Y. & Wakabayashi, H. (1985). Changes in glycogen content of neutrophils in eel, Anguilla japonica by bacterial infection. Fish Pathology 20, 389–397. 20 Park, S. W. & Wakabayashi, H. (1992). Comparison of pronephric and peripheral blood neutrophils of eel, Anguilla japonica, in phagocytic activity. Fish Pathology 27, 149–152. 21 Itou, T., Iida, T. & Kawatsu, H. (1996). Kinetics of oxygen metabolism during respiratory burst in Japanese eel neutrophils. Developmental & Comparative Immunology 20, 323–330. 22 Weber, J. R., Angstwurm, K., Rosenkranz, T., Lindauer, U., Burger, W., Einhaupl, K. M. & Dirnagl, U. (1997). Histamine (H1) receptor antagonist inhibits leukocyte rolling in pial vessels in the early phase of bacterial meningitis in rats. Neuroscience Letters 18, 17–20. 23 Yamaguchi, Y., Matsumura, F., Wang, F. S., Akizuki, E., Liang, J., Matsuda, T., Okabe, K., Ohshiro, H., Horiuchi, T., Yamada, S., Mori, K. & Ogawa, M. (1998). Neutrophil elastase enhances intercellular adhesion molecule-1 expression. Transplantation 65, 1622–1628. 24 Nakagawa, N., Sano, H. & Iwamoto, I. (1995). Substance P induces the expression of intercellular adhesion molecule-1 on vascular endothelial cells and enhances neutrophil transendothelial migration. Peptides 16, 721–725. 25 Wong, D. & Dorovini-Zis, K. (1992). Upregulation of intercellular adhesion molecule-1 (ICAM-1) expression in primary cultures of human brain microvessel endothelial cells by cytokines and lipopolysaccharide. Journal of Neuroimmunology 39, 11–21. 26 Middleton, J., Neil, S., Wintle, J., Clark-Lewis, I., Moore, H., Lam, C., Auer, M., Hub, E. & Rot, A. (1997). Transcytosis and surface presentation of IL-8 venular endothelial cells. Cells 91, 385–395.
Tilapia mast cell lysates enhance neutrophil adhesion to cultured vascular endothelial cells TOMOMASA MATSUYAMA1* AND TAKAJI IIDA2 1
Fish Pathology Division, National Research Institute of Aquaculture, Nansei, Mie 516-0193, Japan 2 Faculty of Agriculture, Miyazaki University, Gakuen Kibanadai-Nishi 1-1, Miyazaki 889-2192, Japan (Received 17 September 2001, accepted 16 November 2001, published electronically 20 February 2002) The possible role of fish mast cells in regulating neutrophil adhesion to vascular endothelial cells was studied using primary cultures of tilapia vascular endothelial cells. The endothelial cell monolayer, which was cultured in 96 well plates, was stimulated for appropriate periods with tilapia mast cell (tMC)-lysates or with Leibovitz-15 (L-15) medium, as a control, and peripheral neutrophils were added into each well after removal of the lysates. After 30 min incubation, cells in the wells were fixed with formalin and non-adherent neutrophils were removed. The cells were stained with Giemsa and neutrophil adhesion was observed microscopically. Although some neutrophils attached to the endothelial cells without stimulation, neutrophil adhesion was enhanced after the incubation of the endothelial cells with tMC-lysates. Neutrophil adhesion was maximal 6 h after the lysate stimulation, with a six-fold increase compared to the control. Neutrophil adhesion also increased when the endothelial cells were stimulated with neutrophil lysates, lipopolysaccharide and zymosan-treated tilapia sera. These results indicate that fish vascular endothelial cells express some neutrophil adhesion molecule(s) after stimulation with various substances. 2002 Published by Elsevier Science Ltd.
Key words:
neutrophil adhesion, vascular endothelial cell, fish mast cell, eosinophilic granule cell, tilapia.
I. Introduction The interaction of circulating neutrophils with vascular endothelial cells is an initial event in acute inflammation. The recruitment of neutrophils from the blood-stream to infected sites not only depends on chemoattractants generated at these sites, but also requires interactions with vascular endothelial cells. During inflammation, neutrophils are tethered to and roll on endothelial cell surfaces. The cells then adhere, and finally migrate out of blood vessels to reach inflammatory sites [1]. Stimulation of channel catfish (Ictalurus punctatus) neutrophils by phorbol dibutyrate intensified their *Corresponding author. E-mail: [email protected]#rc.go.jp 243 1050–4648/02/$-see front matter
2002 Published by Elsevier Science Ltd.
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adhesion to the extracellular matrix [2]. The full-length cDNA beta 2 (CD18) molecule, which is responsible for the tight adhesion of leucocytes, was identified from channel catfish neutrophils [3]. However, adhesion molecules expressed by vascular endothelial cells have not been studied in fish. It is well documented in mammalian species that some of the adhesion molecules, e.g. selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), expressed on endothelial cell surfaces contribute to the adhesion and transmigration of neutrophils [1]. Mammalian mast cells induce expression of adhesion molecules on endothelial cell surfaces by releasing granular components [4]. Mast cell-like cells of fish (fMCs) have been termed eosinophilic granular cells (EGCs) since their granules are eosinophilic unless special fixatives and staining techniques are used [5]. The arrangement of fMCs closely associated with blood vessels suggest an interaction between fMCs and endothelial cells. In fact, we previously reported the vascular permeability promoting e#ect of the tilapia mast cell (tMC) products in the inflamed tilapia [6]. In the present study, we have examined e#ects of tMC-lysates and some other substances on adhesion of tilapia neutrophils to cultured vascular endothelial cells in vitro. II. Materials and Methods FISH
Tilapia (Oreochromis niloticus) weighing 37 to 92 g (average weight S.E., 69·213·8 g) were maintained at 30 C in 180 l tanks in a recirculating water system and were fed daily dry pelleted diet (Chubu Shiryo, Aichi, Japan). ISOLATION AND CULTURE OF TILAPIA VASCULAR ENDOTHELIAL CELLS
Tilapia vascular endothelial cells were isolated from the rete mirabile of swim bladders and cultured in a 96-well flat bottom tissue culture plate (NUNC, New York, U.S.A.) [7]. Endothelial cells were cultured for 10 days and confluent monolayers were used for later analyses. NEUTROPHIL ADHESION ASSAY
Adhesion of neutrophils was examined as follows. Neutrophils were isolated from peripheral blood using a discontinuous density gradient [8]. To induce neutrophilia, fish were injected with formalin-killed Escherichia coli IAM 1239 (1 mg fish 1) into the swim bladder 12 h before the taking of blood. Isolated neutrophils were finally suspended in Leibovitz-15 (L-15) medium containing 10% foetal bovine serum (FBS) to prevent aggregation of the cells (1106 cells ml 1). Cultured endothelial cells were washed twice with L-15 medium and stimulants (or medium alone in control wells) were poured into each well (100 l well 1). After the cells were incubated at 30 C for di#erent time intervals, the stimulants were removed and the endothelial cells were gently washed twice with L-15 medium. The peripheral neutrophils were then added to each well (1105 cells well 1). The cultured endothelial cells of the tilapia readily peeled o# the culture plate by agitation and vortexing of the wash fluid
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altered the distribution of the adherent neutrophils causing them to concentrate in a peripheral ring [9]. To overcome these problems, non-adherent neutrophils were removed after fixing the cells. Briefly, after 30 min incubation at 30 C without agitation, 100 l of 20% formalin in phosphate-bu#ered saline (PBS) was carefully added to each well. The cells were fixed for 30 min. The wells were washed twice with PBS by vortex for 30 s on a plate mixer (Kawakizaki, Tokyo, Japan) to remove non-adherent cells. Neutrophil adhesion was observed microscopically after the staining of cells with Giemsa. Some endothelial cells still peeled o# during the procedure, so only the neutrophils adhering to the endothelial cells were counted. The adhesion activity was expressed as an index of adhesion using the following formula: Index of adhesion=(number of neutrophils adhering to endothelial cells)/ (number of observed endothelial cells)100. Five fish were used for each test. The kinetics of neutrophil adhesion induced by tMC-lysates was measured after cultured endothelial cells had been stimulated with tMC lysates (1105 cells ml 1) or L-15 medium for 30 min, 1 and 2, 4, 6, 8, 12, 16 and 24 h. The tMCs were isolated and pooled from 25 fish, and the tMC lysates were made according to Matsuyama and Iida [6]. Neutrophil adhesion induced by various agents was measured after incubating the endothelial cells for 6 h with the following substances: tMC lysates (1105 cells ml 1), neutrophil lysates (1105 cells ml 1), substance P (100 g/ml 1; Sigma, MI, U.S.A.), lipopolysaccharide from E. coli LPS) (100 g ml 1, Sigma), 10% zymosan-treated tilapia sera (supernatant of normal tilapia sera incubated with zymosan, 1 mg ml 1 for 1 h at 25 C), 10% heat-inactivated tilapia sera, and L-15 medium. Neutrophil lysates were made according to Matsuyama et al. [10]. All reagents were suspended in L-15 medium supplemented with penicillinstreptomycin-fungizone (Bio Whittaker, New York, U.S.A.). The analysis was performed at 30 C in air, and the medium or the stimulants were warmed to 30 C before use. STATISTICS
The results were expressed as the mean ( S.E.). Statistical analysis was performed using Student’s t-test; P<0·05 was considered significant. III. Results CELL COMPOSITION
The isolated and pooled tMCs were 94% pure with 6% neutrophils. The enriched neutrophils isolated from peripheral blood contained neutrophils (61·43·2%), monocytes (6·25·1%), lymphocytes and thrombocytes (32·49·2%) in 15 fish analysed. MORPHOLOGY OF ADHERENT NEUTROPHILS
Neutrophils adhering to vascular endothelial cells became polarized, having a hand mirror shape [Fig. 1(a)]. On the other hand, the neutrophils
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Fig. 1. Neutrophils adhered to vascular endothelial cells (a) or uncovered area in the culture plate (b). These two photographs are of the same well. Arrowheads indicate neutrophils.
attaching to the plastic where endothelial cells had peeled away were oval or rounded [Fig. 1(b)]. Neutrophils adhered tightly to vascular endothelial cells and were not detached by washing. KINETICS OF NEUTROPHIL ADHESION INDUCED BY TMC LYSATES
Some neutrophils attached to endothelial cells without stimulation (Index of adhesion at time 0 was 13·35·4) or after incubation with L-15 medium (minimum was 7·62·1 at 4 h after incubation and maximum was 13·93·9 at 30 min after incubation) (Fig. 2). Incubating the vascular endothelial cells with tMC lysates enhanced the neutrophil adhesion with a peak of adhesion after 6 h incubation: a six-fold increase compared to the stimulation with L-15 medium. Stimulation with the lysates for more than 6 h resulted in a decrease in neutrophil adhesion. After stimulation for 18 and 24 h, there was no significant di#erence between the tMC lysate-stimulated group and the control. NEUTROPHIL ADHESION INDUCED BY VARIOUS AGENTS
Neutrophil adhesion was significantly enhanced by the stimulation of endothelial cells, not only with tMC lysates, but also with neutrophil lysates, LPS and zymosan-treated sera. No significant di#erence was detected with other substances tested (Table 1).
NEUTROPHIL ADHESION TO VASCULAR ENDOTHELIAL CELLS
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80 70
*
Index of adhesion
60
*
50 *
*
40 30 20 10 0
0
1
2
4
6 8 12 18 Time after stimulation (h)
24
Fig. 2. Kinetics of neutrophil adhesion after stimulation with tMC lysates (1105 cells ml 1) () or L-15 medium ( ). n=5, *Significantly di#erent from L-15 mediumstimulated control. Table 1. E#ect of stimulants on neutrophil adhesion Stimulants
Index of adhesion
tMC lysates Neutrophil lysates Substance P LPS 10% zymosan-treated sera 10% heat-inactivated sera L-15 medium No stimulation
48·511·7* 33·68·4* 29·612·2 55·414·6* 49·513·2* 8·01·3 9·71·5 6·61·5
Neutrophil adhesion was measured after incubating the endothelial cells for 6 h with various substances. *Significantly di#erent from L-15 medium control. n=5.
IV. Discussion In this study, a quantitative monolayer adherence assay was used to investigate the adhesion of tilapia neutrophils to cultured vascular endothelial cell monolayers. This is the first report investigating the interaction of fish vascular endothelial cells and neutrophils. When the endothelial cells were stimulated with tMC lysates, the intensity of neutrophil adhesion increased gradually to be six times the control value after 6 h stimulation. In vivo, tMCs are apposed to blood vessels, and rapidly release granular material, which enhances neutrophil migration and the permeability of blood vessels, on injection of non-self materials [6, 11, 12]. Because of the close association of tMCs with vascular endothelial cells, during inflammation, the tMC components probably act on the endothelial cells more e#ectively in vivo than
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in vitro. So, a stronger and prolonged response would be expected in vivo as reported in salmonid experiments [13–15]. The tMC component(s) which acts on vascular endothelial cells, was heat stable as neutrophil adhesion was not diminished by heating the tMC lysates at 100 C for 10 min (data not shown). We reported previously that vascular permeability enhancing components of tMC, which activate cultured vascular endothelial cells to increase the uptake of Ca2+ , were also heat stable [6]. May be the product of tMC enhancing the neutrophil adhesion is the same substance as that promoting the vascular permeability. Neutrophils were spread on the endothelial cells and adhered very tightly. The adhesion molecule integrin (CD18) has been identified on channel catfish neutrophils [3] and the interaction of such adhesion molecule(s) may be involved in the neutrophil adhesion to cultured endothelial cells. The expression of integrin by the neutrophils must be up-regulated before the binding to immunoglobulin superfamily molecules on endothelial cell surfaces. In mammalian species, tumour necrosis factor alpha (TNF-) [16], IL-8 [16] and formyl peptides [18] are known to increase the expression of integrin by neutrophils. In vitro stimulation with phorbol esters also increases the integrin (CD18) expression by channel catfish head kidney neutrophils [3]. The peripheral neutrophils used in this study are expected to be up-regulated in their expression of integrin, following priming by injection of killed E. coli [19–21]. Neutrophil adhesion was also enhanced when the vascular endothelial cells were stimulated with neutrophil lysates, LPS and zymosan-treated sera. In mammals vascular endothelial cells express adhesion molecules after stimulation with various substances. The mast cell granular component TNF- increases the expression of ICAM-1 and VCAM-1 on endothelial cells [4], and histamine enhances the expression of P-selectin [22]. Neutrophil elastase, a granule serine proteinase, enhances ICAM-1 expression [23]. Substance P [24] and LPS [25] are known to induce the expression of ICAM-1 on vascular endothelial cells. Neutrophil adherence was increased after incubating cultured vascular endothelial cells with complement fragments [9]. As observed in mammals, vascular endothelial cells in fish probably regulate the progress of inflammation by reacting to several stimuli. The neutrophils that adhered to the plastic plate at gaps in the monolayer were oval or rounded. On the other hand, those attached to endothelial cells resembled a hand mirror in shape. This indicates that the neutrophils adherent to the endothelial cells were moving on the surface of the monolayer, while the cells adherent to the plastic surface were not. It is probable that stimulated endothelial cells release chemokines (i.e. IL-8) which bind to endothelial cells as reported in mammals [26]. Peripheral neutrophils may adhere to endothelial cells and transmigrate to sites of inflammation by following chemokines presented on the endothelial cell surface. We express our sincere thanks to Dr Eduardo M. Leano, Department of Aquaculture, SEAFDEC, The Philippines, for reviewing the manuscript. This work was supported by a grant from the Research Fellowship Division, Japan Society for the Promotion of Science, No. 10968.
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