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Generation of primary monocyte-like cultures from rainbow trout head kidney leukocytes
Developmental and Comparative Immunology 25 (2001) 447±459 www.elsevier.com/locate/devcompimm
Generation of primary monocyte-like cultures from rainbow trout head kidney leukocytes James L. Stafford a, Pamela E. McLauchlan b,c, Christopher J. Secombes b, Anthony E. Ellis c, Miodrag Belosevic a,d,* a
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada, T6G 2E9 b Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen, AB24 2TZ, UK c FRS Marine Laboratory, PO Box 101, Victoria Road, Aberdeen AB11 9DB, UK d Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada, T6G 2E9 Received 7 December 2000; revised 28 February 2001; accepted 2 March 2001
Abstract Trout primary kidney monocyte-like cultures (T-PKM) were generated by incubating head kidney leukocytes in the presence of cell-conditioned medium (CCM). This technique was adapted from procedures that were previously used to cultivate in vitro-derived kidney macrophages (IVDKM) from the gold®sh. Flow cytometric analysis of the initial T-PKM cultures, identi®ed three cell sub-populations, but only one of these sub-populations survived extensive cultivation periods (i.e. .8 days) in the presence of CCM. Functionally, reactive oxygen intermediate (ROI) production was detected following stimulation of T-PKM with PMA. However, these cells failed to produce reactive nitrogen intermediates (RNI) in response to immunological stimuli. In contrast, gold®sh IVDKM were capable of producing both ROI and RNI. Using the dihydrorhodamine (DHR) assay and ¯ow cytometry, we identi®ed two ROI-producing sub-populations in gold®sh IVDKM but only a single ROIproducing sub-population was present after extended cultivation of T-PKM. This T-PKM sub-population was subsequently sorted using the ¯ow cytometer and shown to possess monocyte-like morphology by microscopic and cytometric analysis. Thus, acquisition of antimicrobial functions following cultivation of kidney leukocytes of rainbow trout and gold®sh is markedly different, and may be due to the failure of trout monocyte-like cells to undergo a ®nal differentiation step in vitro. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Monocytes; Macrophages; Flow cytometry; Dihydrorhodamine; Respiratory burst; Nitric oxide; Fish
1. Introduction Mammalian macrophage hematopoiesis is initiated in response to exogenous macrophage colony stimulating factor (M-CSF or CSF-1) [1±4] and other growth factors such as IL-6 and GM-CSF [5±7]. In * Corresponding author. Tel.: 11-780-492-1266; fax: 11-780492-9234. E-mail address: [email protected] (M. Belosevic).
contrast, ®sh macrophages produce endogenous growth factors that regulate their differentiation and proliferation [8,9]. While much is known about the macrophage proliferation and differentiation events in mammals, relatively little is know about these events in lower vertebrates such as ®sh. The ability to culture ®sh macrophages over extended periods is necessary for the understanding of ®sh macrophage hematopoiesis and their antimicrobial functions. Recently, we developed a culture
0145-305X/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 0145-305 X(01)00 015-5
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system for obtaining high yields of macrophages from the hematopoietic tissue (head kidney) of gold®sh (Carassius auratus) [8±10]. This method involved the isolation of kidney leukocytes using a 51% Percoll gradient, and incubation of these cells in the presence of cell conditioned-medium (CCM). These CCMs contain macrophage growth factors (MGFs) which were shown previously to be secreted by both gold®sh kidney leukocyte and a gold®sh macrophage cell line (GMCL) [8,9]. The endogenously produced MGFs were also shown to support proliferation and differentiation of in vitro-derived kidney macrophages (IVDKM) [10]. After 8±12 days of cultivation, gold®sh IVDKM cultures exhibit three distinct macrophage sub-populations; represented by macrophage progenitors, monocyte-, and macrophage-like cells (designated R1, R3, and R2, respectively) [9±11]. Functionally, gold®sh IVDKM produce both reactive oxygen (ROI) and reactive nitrogen (RNI) intermediates in response to immunological stimuli [10,12±14]. In this report, we show that the techniques used to generate gold®sh IVDKM can be successfully adapted to obtain monocyte-like cultures from rainbow trout. Cultivation of rainbow trout head kidney leukocytes, in the presence of cell-conditioned medium (CCM), resulted in the enrichment of functional monocyte-like cells. Designated as T-PKM, the trout monocyte-like cells were characterized based on their response to CCMs, ¯ow cytometric pro®les, microscopic analysis, myeloperoxidase staining, and generation of antimicrobial functions.
2. Materials and methods 2.1. Fish Gold®sh (C. auratus) were purchased from either Ozark Fisheries Inc (Southland, MI) or Grassy Forks Fisheries (Martinsville, IN). Rainbow trout (Oncorhynchus mykiss) WALBUM were obtained from a local ®sh hatchery supplier. The animals were maintained at the Aquatic Facility of the Department of Biological Sciences, University of Alberta. The gold®sh were maintained at 208C and rainbow trout at 158 in a ¯ow-through water system on a simulated natural photoperiod (Edmonton, Alberta). Fish were fed to satiation daily with trout pellets, and acclimated to
this environment for at least 3 weeks prior to use in experiments. 2.2. Medium The medium used to culture the gold®sh IVDKM and T-PKM has been previously described [9]. Brie¯y, to make 2 l of incomplete medium, the following chemicals were added to 700 ml of MilliQ water; 7.0 g HEPES (Sigma), 0.688 g KH2PO4 (BDH), 0.570 g K2HPO4 (BDH), 0.75 g NaOH (Fisher), 0.34 g NaHCO3 (BDH), 0.584g l-glutamine (Sigma) and 0.01 g insulin (Sigma). The following solutions were then added, 1 l of a 50:50 (v/v) mixture of Leibovitz's-15 medium and Dulbecco's Modi®ed Eagle Medium (Gibco), 80 ml of 10 £ HBSS, 25 ml each of MEM amino acid solution (50 £ ), MEM nonessential amino acid solution (100 £ ) and sodium pyruvate (100 mM) (Gibco), 20 ml MEM vitamin solution (100 £ ; Gibco), 20 ml of nucleic acid precursor solution (containing 0.067 g adenosine, 0.061 g cytidine, 0.034 g hypoxanthene, 0.061 g thymidine and 0.061 g uridine/100 ml water) and 7 ml of 2-bmercaptoethanol (Sigma). After the addition of all reagents, medium was balanced to pH 7.2 with 1 N NaOH and adjusted to a ®nal volume of 2 l with MilliQ water and ®lter-sterilized with a 0.22 mm ®lter (Millipore). Complete medium contained 100 mg/ml gentamicin, 100 U/ml penicillin, 100 mg/ml streptomycin, 10% newborn calf serum (Hyclone), and 5% carp serum. 2.3. Isolation of gold®sh leukocytes and generation of in vitro-derived gold®sh kidney macrophages (IVDKM) Isolation of gold®sh kidney leukocytes and the generation of IVDKM were done as previously described [13]. Brie¯y, ®sh were anesthetized with MS222 (Syndel), killed by cervical dislocation, and the kidneys aseptically removed and placed in a Petri dish containing incomplete medium. Kidneys were gently passed through sterile mesh screens and the screens were rinsed with 12.5 ml of homogenization buffer (incomplete medium containing 50 mg/ml gentamicin, 100 U/ml penicillin, 100 mg/ml streptomycin, and 50 U/ml heparin). The cell suspension was layered on 51% Percoll (Pharmacia) and centrifuged at 400 g for 25 min. Cells at the medium 51%
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Percoll interface were removed and washed twice in incomplete medium and centrifuged at 200 g for 10 min. Viable leukocytes were enumerated using a haemocytometer after staining with trypan blue (Gibco). Generation of IVDKM was performed by seeding 15±20 £ 10 6 kidney leukocytes in 20 ml of complete medium supplemented with 25% cell conditioned medium (CCM) [9]. Cells were incubated at 208C and supplemented after 5 days with fresh complete medium. Cultures, 8±12 days old, were harvested, enumerated, and used as a source of macrophages for the assessment of antimicrobial functions (i.e. RNI and ROI production). 2.4. Isolation of rainbow trout head kidney leukocytes and the generation of trout-primary kidney monocyte cultures (T-PKM) Trout were anesthetized with MS222, killed by cervical dislocation, and the head kidneys were aseptically removed. Head-kidney leukocyte suspensions were obtained using a 51% Percoll gradient [9,15]. Brie¯y, kidneys were gently passed through sterile mesh screens and the screens were rinsed with 12.5 ml of homogenization buffer (incomplete medium containing 50 mg/ ml gentamicin, 100 U/ml penicillin, 100 mg/ml streptomycin, and 50 U/ml heparin). The cell suspensions were layered onto 51% Percoll (Pharmacia) and centrifuged at 400 g for 25 min. Cells at the medium Percoll interface were removed, washed twice in serum-free medium and centrifuged at 200 g for 10 min. Viable leukocytes were enumerated using a haemocytometer after staining with trypan blue (Gibco). Trout kidney leukocytes isolated by 51% Percoll were used for the generation of T-PKM cultures. The generation of T-PKM was performed using similar protocols for the establishment of IVDKM [9]. Brie¯y, trout kidney leukocytes (10 £ 10 6 cells) isolated using 51% Percoll were seeded into 25 cm 2 ¯asks and incubated at 208C. Microscopic and ¯ow cytometric analysis was performed on alternate days (see below). Initial cultivation attempts were performed to obtain cell-conditioned medium (CCM) which was used for the establishment of future cultures [9]. Brie¯y, kidney leukocytes isolated by
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51% Percoll (as described above), were placed into 25 cm 2 tissue culture ¯asks (10 £ 10 6 cells) and cultured for 8±12 days. The CCM was then collected from 8±12 day old T-PKM by centrifuging the cells at 200 g for 10 min. The CCM was subsequently ®ltersterilized (0.22 mm ®lter) and stored at 48C. This procedure has proven successful for the development and maintenance of gold®sh IVDKM [8,9] and suggests that trout macrophages also produce endogenous growth factors required for proliferation and differentiation [10]. To test for CCM activity, leukocytes were seeded into 25 cm 2 ¯asks as described above and incubated in the absence (complete medium only) or the presence of CCM (25% v/v). Cultures were analyzed by ¯ow cytometry after 4, 8, and 12 days of cultivation. 2.5. Flow cytometric analysis of gold®sh and rainbow trout macrophage sub-populations IVDKM and T-PKM were analyzed using a FACS Calibur ¯ow cytometer equipped with a cell sorter (Becton Dickinson). Forward scatter (FSC; size) and side scatter (SSC; internal complexity) parameters were considered for examination of isolated ®sh leukocytes. Fluorescence (FL1) was used for DHR experiments (see below). All ¯ow cytometry was performed on cells suspended in phosphate-buffered saline (PBS) and 10 000 events were routinely collected during analysis. The following ¯ow cytometer settings were used for the analysis of IVDKM and T-PKM, respectively; forward side scatterphotodiode set to E-1, E00, AmpGain set to 9.33, 1.05; and side scatter±photomultiplier voltage set to 455 V, 450 V, AmpGain set to 1.00, 1.00. 2.6. Production of mitogen-stimulated gold®sh and trout kidney leukocyte conditioned supernatants Mitogen-stimulated leukocyte supernatants were generated using protocols described previously [12]. Brie¯y, kidney leukocytes were isolated from 20 gold®sh or six trout, pooled, enumerated, and seeded in 75 cm 2 tissue culture ¯asks at a concentration of 4 £ 10 6 cells/ml. Kidney leukocytes were incubated overnight in medium containing 2.5% carp serum and 10% FCS at
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208C. The following morning the mixed leukocyte cultures were stimulated with 10 mg/ml Concanavalin A (Con A, Boehringer Mannheim), 10 ng/ml phorbol myristate acetate (PMA, Sigma), and 100 ng/ml calcium ionophore A23187 (Sigma). Cultures were incubated in the presence of mitogens for 6 h, after which the mitogens and serum were removed by washing the cells with three exchanges of 25 ml Hanks balanced salt solution (HBSS). The remaining cells were given fresh incomplete medium and incubated for 72 h at 208C. After 72 h, the supernatants from the ¯asks were pooled, ®lter sterilized, and stored at 2208C. Homologous MAFs were used to induce both ROI and RNI responses of IVDKM and T-PKM. 2.7. Detection of gold®sh IVDKM and T-PKM respiratory burst activity using DHR The respiratory burst activity of IVDKM and TPKM was studied using a modi®ed procedure used for the analysis of mammalian neutrophil respiratory burst activity [16±18]. DHR (Molecular Probes) was dissolved in DMSO at a concentration of 29 mM (stock solution), and stored in 30 ml aliquots at 2808C. Samples of IVDKM or T-PKM cultures (days 8±12) were (2.5 £ 10 5 cells/500 ml) seeded into 6 ml polystyrene tubes (Falcon) and allowed to equilibrate for 1 h at 228C prior to determination of the respiratory burst response. Following the 1 h incubation, tubes were centrifuged at 200 g for 10 min. Supernatants were removed and the cells were re-suspended in 2 ml sterile PBS. The wash step was repeated once more to remove all traces of serum. After the ®nal wash, cells were loaded with 100 ml DHR (10 mM ®nal concentration in PBS) and incubated for 5 min at 228C. The respiratory burst response was triggered by adding 400 ml PMA (100 ng ®nal concentration in PBS) and the cells were incubated for an additional 30 min to allow for the oxidation of DHR to the ¯uorescent compound Rho 123. Control tubes were loaded with DHR but were treated for 30 min with sterile PBS only. A positive control was included by incubating DHR-loaded cells with 1 mM hydrogen peroxide (Merck) and samples incubated at 48C (reaction inhibition step)were used as the negative control. After the 30 min priming step, samples were immediately
analyzed using a ¯ow cytometer. Instrumental settings for forward and side scatter were the same as described above, and the assessment of green ¯uorescence (Rho 123) was determined (FL1) using histograms and a logarithmic scale. Cells treated with DHR only (no triggering) were used to calibrate the FL1. Each analysis was performed on 10 4 events and analyzed using the LYSIS II software. The degree of respiratory burst activity was assessed from the mean ¯uorescence intensity values obtained from the histogram plots. For detection of functional populations, regional analysis was performed on histograms and cytograms using the LYSIS II software. 2.8. Nitric oxide bioassay Gold®sh IVDKM or T-PKM were seeded (5 £ 10 4 cells/ml) into 96 well half-area culture plates (Costar) in complete medium, and stimulated with homologous MAF (1:4 and 1:16 ®nal concentrations). Lipopolysaccharide (1±25 mg/ml ®nal concentration) was added to each treatment and the NO produced by the stimulated cells was analyzed by sampling the supernatants after 72 h incubation for the presence of nitrite, using the Griess reaction [9±12]. Brie¯y, 75 ml of supernatants were removed from individual wells and placed in a separate 96 well microtitre plate. One hundred ml of 1% sulphanilamide in 2.5% phosphoric acid was added to each sample followed by 100 ml of 0.1% N-naphthyl-ethylenediamine in 2.5% phosphoric acid. The optical density of each well was determined using an automated plate reader (Biotek) at 540 nm. The approximate concentration of nitrite in the samples was determined from a standard curve generated using known concentrations of sodium nitrite. 2.9. Statistics One- and two-way analysis of variance using SuperAnova software (Abacus) for the Power Macintosh was used for statistical analysis of the data. A least square means was used for determining signi®cance between control and experimental groups. A probability level of P , 0.05 was considered signi®cant.
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3. Results 3.1. Cultivation of rainbow trout head kidney leukocytes and the development of trout-primary kidney monocyte-like cultures (T-PKM) We previously demonstrated that cultivation of gold®sh leukocytes in the presence of CCM, which contains proliferation and differentiation factors, resulted in the development of stable macrophage cultures (IVDKM) [8,9]. A similar cultivation technique was applied for the development of T-PKM cultures. Trout head kidney leukocytes cultured in the presence of a trout CCM were monitored overtime by ¯ow cytometry (Fig. 1). This approach resulted in the identi®cation of three sub-populations of cells based on FSC and SSC parameters. These cell sub-populations were gated according to foci of cells that were identi®ed using density plot analysis and were designated as R1, R2, and R3 (Fig. 1, see trout ¯ow cytometry template). At least 20 individual ®sh kidney leukocyte preparations were examined, and in all cases these three sub-populations of cells were observed. The differences between the gold®sh IVDKM and the T-PKM ¯ow cytometry gates represents the differences in size and internal complexity of cultured kidney leukocytes from these different ®sh species. An example of the changes in ¯ow cytometric pro®les of trout leukocytes over extended cultivation periods is shown in Fig. 1. This representative trout leukocyte culture demonstrates the standard ¯ow cytometric gating pattern observed after cultivation of the cells in the presence of 25% CCM. In all ®sh examined, the %R3 population increased after extended cultivation (i.e. 8±12 days), while the other sub-populations did not. Initially, day 0 to day 4, the majority of events gated to the R1 and R2 regions. Subsequent cultivation demonstrated a steady decline of the R1-gated events, no change in the R2gated events and a gradual increase in the R3-gated events. This trend for the consistent enrichment of the R3 sub-population was observed for all trout leukocyte cultures obtained from different ®sh (Table 1). The R3 cells from T-PKM cultures were subsequently sorted using the ¯ow cytometer and cytospin smears were made of pre-sort and sorted cells (Fig. 2). The sorted R3 cells were large round cells that often
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had eccentrically placed kidney-shaped nuclei.(Fig. 2(b)). In addition, these cells demonstrated dark and diffuse staining for myeloperoxidase (Fig. 2(b)) Based on these morphological and cytochemical characteristics, the R3 sub-population was classi®ed as monocyte-like cells. These monocyte-like cells were also the largest cells within T-PKM based on FSC, SSC, and microscopic analysis, ranging in size from 10 to 15 mm. Morphologically, the other cell types in TPKM (pre-sort) were small, round, and contained very little cytoplasm (similar to R1 cells of the IVDKM [9±12]), or resembled lymphocytes with large round nuclei and very little cytoplasm (Fig. 2(a)). Some of the cells in the R2 region may be promonocytes due to their size and dark-blue staining cytoplasm which is characteristic of immature cell types. Interestingly, a mature macrophage-like subpopulation in T-PKM was not observed in the smears. In contrast, gold®sh IVDKM cultures comprise cells with typical monocyte-like morphology (i.e. round cells with kidney-shaped nuclei) and macrophagelike morphology (i.e. large irregularly shaped cells with extensive vacuolisation) [9,10]. Flow cytometric analysis of T-PKM cultures failed to demonstrate a macrophage-like cell population (Fig. 2(a)). To con®rm the presence of an R3-enriching factor within trout CCM, trout leukocytes were cultured in the presence of complete medium alone (no CCM) or were cultured in complete medium supplemented with 25% CCM. Two separate CCMs were tested (designated CCM1 and CCM2). T-PKM cultures were subsequently analyzed after 4, 8, and 12 days using ¯ow cytometry. Clearly, the cultivation of T-PKM in the presence of two different CCMs resulted in an enrichment of the R3-cell sub-population during the ®rst 12 days of cultivation (Fig. 3). 3.2. Identi®cation of ROI-producing sub-populations in ®sh leukocyte cultures (gold®sh IVDKM and TPKM) 3.2.1. IVDKM The respiratory burst response of gold®sh macrophages was assessed by ¯ow cytometry using the ¯uorescent probe DHR. IVDKM were loaded with DHR (as described above) and the respiratory burst was triggered by the addition of PMA. Cells not exposed to PMA were used to calibrate the ¯ow
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Fig. 1. Cultivation of trout head kidney leukocytes and the generation of trout primary kidney macrophage (T-PKM) cultures. Rainbow trout head kidney leukocytes were isolated as described in the materials and methods. Kidney leukocytes at the 51% Percoll interface were seeded into 25 cm 2 (10 £ 10 6/¯ask) and grown in the presence of 25% cell-conditioned medium (CCM). Sub-samples were removed from the culture ¯asks on the indicated days and analyzed by ¯ow cytometry. Note an increase in the R3-type cell number after extended cultivation periods in the presence of CCM. Data is a representative ®sh out of 20 different rainbow trout cultures that were generated.
560±2380 (5.6±23.8%) 270±840 (2.7±8.4%) 3370±7750 (33.7±77.5%) 680 (6.8%) 680 (6.8%) 6260 (62.6%) 560 (5.6%) 430 (4.3%) 7750 (77.5%) R1 R2 R3
2290 (22.9%) 580 (5.8%) 4290 (42.9%)
2380 (23.8%) 840 (8.4%) 5010 (50.1%)
2090 (20.9%) 270 (2.7%) 3370 (33.7%)
810 (8.1%) 510 (5.1%) 5590 (55.9%)
1460 ^ 350 (14.6 ^ 3.5%) 550 ^ 80 (5.5 ^ 0.8%) 5380 ^ 630(53.8 ^ 6.3%)
Range Mean (^SEM) Fish 6 Fish 5 Fish 4 Fish 3 Fish 2 Fish 1
Gated cells (total: 10 000 events collected)
Table 1 Proportion of R1, R2, and R3-type cells in trout primary monocyte-like cultures. Trout head kidney leukocytes were isolated using a 51% Percoll gradient and the viable leukocytes were cultured as indicated in the Materials and Methods section. Using the standard gates derived from analysis of trout kidney leukocyte cultures, the proportions of cells within each region was determined after 8±12 days in culture. R1, R2 and R3 represent the gating regions of the ¯ow cytometer based on size and internal complexity of leukocytes
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cytometer for ¯uorescence intensity (FL1). For example, DHR loaded cells were analyzed and the histogram plot was adjusted so that DHR loading was expressed as low ¯uorescence (approximately 10 1 on the log scale). As shown in Fig. 4(b) (left panel), IVDKM that were loaded with DHR had a pronounced peak on the FL1 histogram plot. After triggering with PMA, there was a shift in the ¯uorescence intensity (Fig. 4(c), left panel) due to the release of respiratory burst products. DHR allowed for the analysis of the respiratory burst activity of different sub-populations within the heterogeneous IVDKM cultures comprised of R1-, R2-, and R3-type macrophages. The sub-populations responsible for the oxidation of DHR to Rho 123 (i.e. shift in ¯uorescence intensity) were identi®ed as the R2- and R3type cells (Fig. 4(d)). The R1-type cells did not have a detectable respiratory burst response. The respiratory burst activity was expressed as mean ¯uorescence intensity per cell in the individual sub-populations. For all gold®sh cultures examined, the monocytelike (R3) cells had a higher respiratory burst activity compared to the macrophage-like (R2) cells (P , 0.05) (Fig. 4). 3.2.2. T-PKM DHR and ¯ow cytometry were also used to identify the functional population within T-PKM cultures (Fig. 4; right panel). Days 8±12 T-PKM were used for analysis (Fig. 4(a), right panel). Cells were loaded as described above and these DHR-loaded cells were used to calibrate FL1 (same as above for IVDKM) (Fig. 4(b), right panel). Triggering with 100 ng PMA caused a pronounced shift in the ¯uorescence histogram (Fig. 4(c), right panel). The population of cells responsible for this shift were identi®ed as the R3-type monocytes (Fig. 4(d), right panel). Interestingly, this was the same population that survived extensive culture periods in the presence of 25% CCM (see Figs. 1 and 3). 3.3. Production of RNI by gold®sh IVDKM and TPKM Stimulation of 8±12 day old gold®sh IVDKM and T-PKM demonstrated that only IVDKM were capable of producing RNI following immunological stimuli (Fig. 5). A similar effect was seen when diluted
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Fig. 2. Flow cytometric analysis of sorted R3-type cells after 8 days of cultivation in the presence of CCM. T-PKM cultures were established as indicated in the Materials and Methods section. The R3-type cells that were enriched for over extended cultivation periods were subsequently sorted using the ¯ow cytometer. Cytologic appearance of the cells was characterized by staining cells in cytocentrifuge smears (Cytospin II, Shandon Instruments) with a modi®ed Wright's stain (Fisher). Three predominant cell types were observed in the pre-sort pro®le (R1, R2, and R3) (A). The R1 cells were the smallest and had a high nucleus:cytoplasm ratio (Scale bar 10 mm). R2 cells were medium in size and were typically round cells with large round nuclei and blue-staining cytoplasm. R3 cells were round with kidney-shaped shaped nuclei. These cells displayed characteristic monocyte-like morphology and were the cell type identi®ed following ¯ow cytometric sorting (B1). These cells were also positive for myeloperoxidase (Sigma) and demonstrated a dark diffuse staining pattern (B2).
MAF (1:16) in conjunction with LPS (1±25 mg/ml) were used (data not shown). Flow cytometric analysis of gold®sh IVDKM and T-PKM, using identical instrument settings, clearly demonstrated that the TPKM cultures did not have a sub-population of cells equivalent to mature gold®sh macrophages (i.e. R2). The ROI-producing sub-population in T-PKM cultures (R3) gated more closely to the monocytelike cells found in gold®sh IVDKM (R3). 4. Discussion We previously showed that the cultivation of gold®sh kidney leukocytes in the presence of CCM resulted in the generation of stable macrophage cultures [8±10]. Gold®sh IVDKM cultures contain three distinct sub-populations of macrophages. These sub-populations represent cells that are devel-
opmentally arrested at different stages of macrophage differentiation (i.e. early progenitors, monocytes, and macrophages, designated R1, R3 and R2, respectively). In contrast, the cultivation of rainbow trout head kidney leukocytes, in the presence of CCM, generated a single population of functional cells which demonstrated monocyte-like properties (designated R3). The increased %R3 (monocyte-like cells) during extended cultivation periods, in the presence of CCM, suggests that there is at least one MGF present in trout-derived CCM. The majority of smaller cells present in early cultures of T-PKM, were most likely lymphocyte-like cells, which have been previously reported [19]. However, some of these cells may also represent pro-monocyte-like cells (i.e. R2 in TPKM), which in the presence of appropriate factors found in CCM, differentiate into the maturer monocyte-like cells (R3). Therefore, within the hematopoietic tissue of the trout kidney, monocytes
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Fig. 3. Demonstration of an R3-enriching factor presence in trout-derived CCM. Rainbow trout head kidney leukocytes were isolated as described in the Materials and Methods section. Leukocytes were seeded into 25 cm 2 and cultured over 12 days in the presence and absence of 25% (v/v) trout-derived CCM. On days 4, 8, and 12, samples were removed from the culture ¯asks and examined by ¯ow cytometry. Using the trout ¯ow cytometer template, the number of cells per gate was determined out of 10 000 collected events. Two different CCMs were tested (designated CCM 1 and CCM 2). Data from two independent experiments, (1) and (2), are shown.
differentiate from pro-monocytes and these monocytes likely enter the circulation to become tissue macrophages. This pathway of differentiation is similar to that of mammalian macrophages. In contrast, gold®sh macrophage differentiate fully in vitro, and do not appear to require additional tissue `cues' before they acquire the ability to generate RNI in response to immunological stimuli [8±10]. T-PKM also exhibited different functional properties when compared to gold®sh IVDKM. Gold®sh IVDKM produce both ROI and RNI in response to MAF and/or LPS [12±14]. Recently, we characterized a gold®sh macrophage activation factor, by demonstrating that cleavage products of transferrin are important for the induction of RNI production by
these cells [20]. We suggested that one of the necessary events for NO production by IVDKM involved the increased differentiation of monocyte-like cells (R3) into mature macrophage-like cells (R2) [21± 26], which have been previously shown to be the RNI-producing cell sub-population in the IVDKM cultures [10]. In contrast, the T-PKM did not produce RNI but did generate ROI as demonstrated using the DHR assay. A single monocyte-like sub-population (R3) was responsible for the ROI response in TPKM cultures, while two ROI-producing sub-populations were present in gold®sh IVDKM (i.e. monocyte (R3)- and macrophage (R2)-like cells). In mammals, cultured monocytes undergo a variety of changes that may be similar to those that
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Fig. 5. Nitric oxide (NO) production by gold®sh IVDKM and T-PKM. Gold®sh and trout kidney leukocytes were cultured in the presence of 25% CCM as described in the Materials and Methods section. After 8±12 days, the cells were harvested and seeded into 96-well half area tissue culture plates (5 £ 10 4 cells/well). Cells were stimulated with LPS (1±25 mg/ml; black bars) and/or MAF (1:4 and 1:16 ®nal concentration; shaded bars) and incubated for 72 h at 228C before determination of NO response. Note that gold®sh IVDKM and T-PKM were treated with homologous MAFs. Each bar represents the mean ^ SEM of triplicate cultures and the data are from a representative experiment out of three that were performed.
occur after migration into the tissue from the peripheral blood [27±29]. In addition, cultured human monocytes are believed to resemble human resident tissue macrophages [28,29]. Therefore,
cultivation of monocytes over several days results in their differentiation into the maturer macrophage phenotype. The differentiation of the monocytes in culture is concomitant with changes in the
Fig. 4. DHR analysis of functional sub-populations found in heterogeneous leukocyte cultures (i.e. IVDKM and T-PKM). Cultured ®sh macrophages: gold®sh (left panel); trout (right panel) were loaded with DHR as indicated in the Materials and Methods section. Control, non-stimulated gold®sh (left panel, B) and trout (right panel, B) DHR-loaded cells exhibited ¯uorescence intensity (FL1) of less than 10 1.5 (no ROI production). After PMA treatment, both IVDKM and T-PKM produced ROI, indicated by a signi®cant increase in ¯uorescence intensity (C). Region analysis identi®ed the functional populations in gold®sh and trout cultures (D). Note that the shift in DHR ¯uorescence intensity of IVDKM was evident for two sub-populations (R3 and R2), while T-PKM culture demonstrated only one ROI-producing population (R3).
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generation of both ROI and RNI [30]. Freshly isolated human monocytes have a high capacity to generate ROI, and this response declines after 4 days in culture [30]. Conversely, the production of RNI is not detectable in freshly isolated monocytes but after 4±5 days of cultivation, they acquire the ability to produce RNI[30]. A similar process may be important for the induction of the RNI response by gold®sh macrophages. Gold®sh monocyte-like cells (R3) do not produce RNI but the mature macrophage-like cells (R2) are capable of producing NO in response to MAF and/or LPS[10]. It has also been demonstrated that gold®sh monocytes can be induced to differentiate into macrophage-like cells in response to activating stimuli (i.e. MAF or LPS) and CCM [10]. This differentiation step may be a necessary event for RNI production by gold®sh macrophages. In contrast, the inability of T-PKM cells to produce RNI may be due to an absence of a mature macrophage-like sub-population in the cultures. The reasons for the absence of this population in T-PKM cultures is not known, but may involve the absence of necessary factor(s) required for the functional differentiation of monocytes into mature macrophages in vitro. Another major difference between T-PKM and IVDKM cultures is that the progenitor-like cells (R1 sub-population) do not survive extended cultivation periods in T-PKM cultures. These progenitor cells are required for both the development of monocyteand macrophage-like sub-populations, which has been previously demonstrated for gold®sh IVDKM and gold®sh macrophage cell line [9±12]. The absence of a mature macrophage sub-population in T-PKM may explain the inability of T-PKM to produce RNI in response to immunological stimuli. During an in¯ammatory response in trout, kidney-derived monocytes are recruited to the in¯ammatory site [31] and, in tissues, presumably differentiate into mature macrophages that can produce RNI, as iNOS expression has been detected in trout kidney [32]. However, the ability of kidney leukocytes to produce RNI appears to be rapidly down-regulated in vitro culture [33]. Thus, trout kidney macrophages and T-PKM, unlike gold®sh macrophages, may require the continual presence of other appropriate tissue cues for differentiation into fully functional mature macrophages, with the ability to produce RNI.
In conclusion, we showed that enriched primary monocyte-like cultures could be derived from trout head kidney leukocyte cultures, using techniques developed for the establishment of gold®sh IVDKM. However, these cells require as yet unknown signals to differentiate into fully mature macrophages with the ability to produce RNI. The enrichment for a functional sub-population of monocyte-like cells by the addition of homologous CCM, suggests that there is at least one growth factor present in trout CCM. The use of these cultivation techniques will be invaluable for the enrichment of functional monocyte-like cells from trout head kidney leukocytes. These cells can subsequently be used in bioassays that measure cellmediated immune responses of rainbow trout exposed to a variety of agents (i.e. environmental contaminants, pathogens, or immunization) and for the further characterization of trout MGFs responsible for trout monocyte/macrophage development. Acknowledgements This work was supported by Natural Sciences and Engineering Council of Canada (NSERC) and Alberta Heritage Foundation for Medical Research (AHFMR) to M.B. J.L.S. was supported by a PhD scholarship from NSERC. P.E.M. was funded by EC FAIR CT98-4087 and a travel grant provided by the University of Aberdeen Development Trust and The Royal Society. References [1] Virelizier JL, Arenzana-Seisdedos F. Immunological functions of macrophages and their regulation by interferons. Med Biol 1985;63:149±59. [2] Loms Ziegler-heitbrock HW. The biology of the monocyte system. Eur J Cell Biol 1989;49:1. [3] Stewart CC, Reidy MC, Stewart SJ. The proliferation and differentiation of macrophages. Immun Series 1994;60:3±27. [4] Rutherford MS, Witsell A, Schook LB. Mechanisms generating functionally heterogeneous macrophages: chaos revisited. J Leuk Biol 1993;53:602±18. [5] Donahue RE, Seehra J, Metzger M, Lefebvre D, Rock B, Carbone S, Nathan DG, Garnick M, Sehgal PK, Laston D, LaVallie E, McCoy J, Schendel PF, Norton C, Turner K, Yang Y-C, Clark SC. Human IL-3 and GM-CSF act synergistically in stimulating hematopoiesis in primates. Science 1988;241:1820±3.
J.L. Stafford et al. / Developmental and Comparative Immunology 25 (2001) 447±459 [6] Falk LA, Vogel S. Granulocyte-macrophage colony stimulating factor (GM-CSF) and macrophage colony stimulating factor (CSF-1) synergize to stimulate progenitor cells with high proliferative potential. J Leuk Biol 1988;44:455±64. [7] Jansen J, Kluin-Nelemans JC, van Damme J, Wientjens GJHM, Willemze R, Fibbe WE. Interleukin 6 is a permissive factor for monocytic colony formation by human hematopoietic progenitor cells. J Exp Med 1992;175:1151±4. [8] Wang R, Neumann NF, Shen Q, Belosevic M. Establishment and characterization of macrophage cell line from the gold®sh. Fish Shell®sh Immunol 1995;5:329±46. [9] Neumann NF, Barreda D, Belosevic M. Production of a macrophage growth factor(s) by a gold®sh macrophage cell line and macrophages derived from gold®sh kidney leukocytes. Dev Comp Immunol 1998;4:417±32. [10] Neumann NF, Barreda D, Belosevic M. Generation and functional analysis of distinct macrophage sub-populations from gold®sh (Carassius auratus L.) kidney leukocyte cultures. Fish and Shell®sh Immunol 2000;10:1±20. [11] Barreda DR, Neumann NF, Belosevic M. Flow cytometric analysis of PKH26-labeled gold®sh kidney-derived macrophages. Dev Comp Immunol 2000;24:395±406. [12] Neumann NF, Belosevic M. Deactivation of primed respiratory burst response of gold®sh macrophages by leukocytederived macrophage activating factor(s). Dev Comp Immunol 1996;20:427±39. [13] Neumann NF, Fagan D, Belosevic M. Macrophage activating factor(s) secreted by mitogen stimulated gold®sh kidney leukocytes synergize with bacterial lipopolysaccharide to induce nitric oxide production in teleost macrophages. Dev Comp Immunol 1995;19:473±82. [14] Neumann NF, Stafford JL, Belosevic M. Biochemical and functional characterization of macrophage stimulating factors secreted by mitogen-induced gold®sh kidney leukocytes. Fish and Shell®sh Immunol 2000;10:167±86. [15] Secombes CJ. Isolation of salmonid macrophages and analysis of their killing activity. In: Stolen JS, Fletcher TC, Anderson DP, Robertson BS, van Muiswinkel WB, editors. Techniques in ®sh immunology, vol. 1. New Jersey: SOS Publications, 1992. pp. 137±154. [16] Emmendorffer A, Hecht M, Lohmann Matthes ML, Roesler J. A fast and easy method to determine the production of reactive oxygen intermediates by human and murine phagocytes using dihydrorhodamine 123. J Immunol Methods 1990;131:269. [17] Rothe G, Emmendorffer A, Oser A, Roesler J, Valet G. Flow cytometric measurement of the respiratory burst activity of phagocytes using dihydrorhodamine 123. J Immunol Methods 1994;138:133. [18] Richardson MO, Ayliffe MJ, Helbert M, Davies EG. A simple ¯ow cytometric assay using dihydrorhodamine for the measurement of the neutrophil respiratory burst in whole blood: comparison with the quantitative nitrobluetetrazolium test. J Immunol Methods 1998;219:187±93. [19] Chilmonczyk S, Monge D. Flow cytometry as a tool for
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assessment of the ®sh cellular immune responses to pathogens. Fish and Shell®sh Immunol 1999;9:319±33. Stafford JL, Neumann NF, Belosevic M. Products of proteolytic cleavage of transferrin induce nitric oxide response of gold®sh macrophages. Dev Comp Immunol 2001;25:101±15. Andreesen R, Osterholz J, Bodemann H, Bross KJ, Costabl U, Lohr GW. Expression of transferrin receptors and intracellular ferritin during terminal differentiation of human monocytes. Blut 1984;49:195±202. Hirata T, Bitterman PB, Mornex J-F, Crystal RG. Expression of the transferrin receptor gene during the process of mononuclear phagocyte maturation. J Immunol 1986;4:1339±45. Hamilton TA, Weiel JE, Adams DO. Expression of the transferrin receptor in murine peritoneal macrophages is modulated in the different stages of activation. J Immunol 1984;132(50):2285±90. Evans WH, Wilson SM, Bednarek JM, Peterson EA, Knight RD, Mage MG, McHugh L. Evidence for a factor in normal human serum that induces human neutrophilic granulocyte end-stage maturation in vitro. Leukemia Research 1989;13(8):673±82. Breitman TR, Collins SJ, Keene BR. Replacement of serum by insulin and transferrin supports growth and differentiation of the human promyelocytic cell line, HL-60. Experimental Cell Research 1980;126(2):494±8. Schmidt JA, Marshall J, Hayman MJ, Ponka P, Beug H. Control of erythroid differentiation: possible role of the transferrin cycle. Cell 1986;46(1):41±51. Becker S. In¯uence of interferon on human monocyte to macrophage development. Cell Immunol 1984;84:145. Johnson WD, Mei Jr B, Cohn A. The separation, long-term cultivation and maturation of the human monocyte. J Exp Med 1977;146:1613. Zuckerman SH, Ackerman SK, Douglas SD. Long-term peripheral blood monocyte-macrophage cell cultures. Immunology 1979;38:401. Martin JHJ, Edwards SW. Changes in mechanisms of monocyte/macrophage-mediated cytotoxicity during culture. J Immunol 1993;150:3478±86. Laing KJ, Hardy LJ, Aartsen W, Grabowski PS, Secombes CJ. Expression of an inducible nitric oxide synthase gene in rainbow trout Oncorhynchus mykiss. Dev Comp Immunol 1999;23:71±85. Afonso A, Lousada S, Sliva J, Ellis AE, Silva MT. Neutrophil and macrophage responses to in¯ammation in the peritoneal cavity of rainbow trout Oncorhynchus mykiss. A light and electron microscopic cytochemical study. Dis Aq Org 1998;34:27±37. Campos-Perez JJ, Ward M, Grabowski RS, Ellis AE, Secombes CJ. The gills are an important site of iNOS expression in rainbow trout Oncorhynchu mykiss after challenge with Gram-negative pathogen Renibacterium salmonarium. Immunology 2000;99:153±61.
Generation of primary monocyte-like cultures from rainbow trout head kidney leukocytes James L. Stafford a, Pamela E. McLauchlan b,c, Christopher J. Secombes b, Anthony E. Ellis c, Miodrag Belosevic a,d,* a
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada, T6G 2E9 b Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen, AB24 2TZ, UK c FRS Marine Laboratory, PO Box 101, Victoria Road, Aberdeen AB11 9DB, UK d Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada, T6G 2E9 Received 7 December 2000; revised 28 February 2001; accepted 2 March 2001
Abstract Trout primary kidney monocyte-like cultures (T-PKM) were generated by incubating head kidney leukocytes in the presence of cell-conditioned medium (CCM). This technique was adapted from procedures that were previously used to cultivate in vitro-derived kidney macrophages (IVDKM) from the gold®sh. Flow cytometric analysis of the initial T-PKM cultures, identi®ed three cell sub-populations, but only one of these sub-populations survived extensive cultivation periods (i.e. .8 days) in the presence of CCM. Functionally, reactive oxygen intermediate (ROI) production was detected following stimulation of T-PKM with PMA. However, these cells failed to produce reactive nitrogen intermediates (RNI) in response to immunological stimuli. In contrast, gold®sh IVDKM were capable of producing both ROI and RNI. Using the dihydrorhodamine (DHR) assay and ¯ow cytometry, we identi®ed two ROI-producing sub-populations in gold®sh IVDKM but only a single ROIproducing sub-population was present after extended cultivation of T-PKM. This T-PKM sub-population was subsequently sorted using the ¯ow cytometer and shown to possess monocyte-like morphology by microscopic and cytometric analysis. Thus, acquisition of antimicrobial functions following cultivation of kidney leukocytes of rainbow trout and gold®sh is markedly different, and may be due to the failure of trout monocyte-like cells to undergo a ®nal differentiation step in vitro. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Monocytes; Macrophages; Flow cytometry; Dihydrorhodamine; Respiratory burst; Nitric oxide; Fish
1. Introduction Mammalian macrophage hematopoiesis is initiated in response to exogenous macrophage colony stimulating factor (M-CSF or CSF-1) [1±4] and other growth factors such as IL-6 and GM-CSF [5±7]. In * Corresponding author. Tel.: 11-780-492-1266; fax: 11-780492-9234. E-mail address: [email protected] (M. Belosevic).
contrast, ®sh macrophages produce endogenous growth factors that regulate their differentiation and proliferation [8,9]. While much is known about the macrophage proliferation and differentiation events in mammals, relatively little is know about these events in lower vertebrates such as ®sh. The ability to culture ®sh macrophages over extended periods is necessary for the understanding of ®sh macrophage hematopoiesis and their antimicrobial functions. Recently, we developed a culture
0145-305X/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 0145-305 X(01)00 015-5
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system for obtaining high yields of macrophages from the hematopoietic tissue (head kidney) of gold®sh (Carassius auratus) [8±10]. This method involved the isolation of kidney leukocytes using a 51% Percoll gradient, and incubation of these cells in the presence of cell conditioned-medium (CCM). These CCMs contain macrophage growth factors (MGFs) which were shown previously to be secreted by both gold®sh kidney leukocyte and a gold®sh macrophage cell line (GMCL) [8,9]. The endogenously produced MGFs were also shown to support proliferation and differentiation of in vitro-derived kidney macrophages (IVDKM) [10]. After 8±12 days of cultivation, gold®sh IVDKM cultures exhibit three distinct macrophage sub-populations; represented by macrophage progenitors, monocyte-, and macrophage-like cells (designated R1, R3, and R2, respectively) [9±11]. Functionally, gold®sh IVDKM produce both reactive oxygen (ROI) and reactive nitrogen (RNI) intermediates in response to immunological stimuli [10,12±14]. In this report, we show that the techniques used to generate gold®sh IVDKM can be successfully adapted to obtain monocyte-like cultures from rainbow trout. Cultivation of rainbow trout head kidney leukocytes, in the presence of cell-conditioned medium (CCM), resulted in the enrichment of functional monocyte-like cells. Designated as T-PKM, the trout monocyte-like cells were characterized based on their response to CCMs, ¯ow cytometric pro®les, microscopic analysis, myeloperoxidase staining, and generation of antimicrobial functions.
2. Materials and methods 2.1. Fish Gold®sh (C. auratus) were purchased from either Ozark Fisheries Inc (Southland, MI) or Grassy Forks Fisheries (Martinsville, IN). Rainbow trout (Oncorhynchus mykiss) WALBUM were obtained from a local ®sh hatchery supplier. The animals were maintained at the Aquatic Facility of the Department of Biological Sciences, University of Alberta. The gold®sh were maintained at 208C and rainbow trout at 158 in a ¯ow-through water system on a simulated natural photoperiod (Edmonton, Alberta). Fish were fed to satiation daily with trout pellets, and acclimated to
this environment for at least 3 weeks prior to use in experiments. 2.2. Medium The medium used to culture the gold®sh IVDKM and T-PKM has been previously described [9]. Brie¯y, to make 2 l of incomplete medium, the following chemicals were added to 700 ml of MilliQ water; 7.0 g HEPES (Sigma), 0.688 g KH2PO4 (BDH), 0.570 g K2HPO4 (BDH), 0.75 g NaOH (Fisher), 0.34 g NaHCO3 (BDH), 0.584g l-glutamine (Sigma) and 0.01 g insulin (Sigma). The following solutions were then added, 1 l of a 50:50 (v/v) mixture of Leibovitz's-15 medium and Dulbecco's Modi®ed Eagle Medium (Gibco), 80 ml of 10 £ HBSS, 25 ml each of MEM amino acid solution (50 £ ), MEM nonessential amino acid solution (100 £ ) and sodium pyruvate (100 mM) (Gibco), 20 ml MEM vitamin solution (100 £ ; Gibco), 20 ml of nucleic acid precursor solution (containing 0.067 g adenosine, 0.061 g cytidine, 0.034 g hypoxanthene, 0.061 g thymidine and 0.061 g uridine/100 ml water) and 7 ml of 2-bmercaptoethanol (Sigma). After the addition of all reagents, medium was balanced to pH 7.2 with 1 N NaOH and adjusted to a ®nal volume of 2 l with MilliQ water and ®lter-sterilized with a 0.22 mm ®lter (Millipore). Complete medium contained 100 mg/ml gentamicin, 100 U/ml penicillin, 100 mg/ml streptomycin, 10% newborn calf serum (Hyclone), and 5% carp serum. 2.3. Isolation of gold®sh leukocytes and generation of in vitro-derived gold®sh kidney macrophages (IVDKM) Isolation of gold®sh kidney leukocytes and the generation of IVDKM were done as previously described [13]. Brie¯y, ®sh were anesthetized with MS222 (Syndel), killed by cervical dislocation, and the kidneys aseptically removed and placed in a Petri dish containing incomplete medium. Kidneys were gently passed through sterile mesh screens and the screens were rinsed with 12.5 ml of homogenization buffer (incomplete medium containing 50 mg/ml gentamicin, 100 U/ml penicillin, 100 mg/ml streptomycin, and 50 U/ml heparin). The cell suspension was layered on 51% Percoll (Pharmacia) and centrifuged at 400 g for 25 min. Cells at the medium 51%
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Percoll interface were removed and washed twice in incomplete medium and centrifuged at 200 g for 10 min. Viable leukocytes were enumerated using a haemocytometer after staining with trypan blue (Gibco). Generation of IVDKM was performed by seeding 15±20 £ 10 6 kidney leukocytes in 20 ml of complete medium supplemented with 25% cell conditioned medium (CCM) [9]. Cells were incubated at 208C and supplemented after 5 days with fresh complete medium. Cultures, 8±12 days old, were harvested, enumerated, and used as a source of macrophages for the assessment of antimicrobial functions (i.e. RNI and ROI production). 2.4. Isolation of rainbow trout head kidney leukocytes and the generation of trout-primary kidney monocyte cultures (T-PKM) Trout were anesthetized with MS222, killed by cervical dislocation, and the head kidneys were aseptically removed. Head-kidney leukocyte suspensions were obtained using a 51% Percoll gradient [9,15]. Brie¯y, kidneys were gently passed through sterile mesh screens and the screens were rinsed with 12.5 ml of homogenization buffer (incomplete medium containing 50 mg/ ml gentamicin, 100 U/ml penicillin, 100 mg/ml streptomycin, and 50 U/ml heparin). The cell suspensions were layered onto 51% Percoll (Pharmacia) and centrifuged at 400 g for 25 min. Cells at the medium Percoll interface were removed, washed twice in serum-free medium and centrifuged at 200 g for 10 min. Viable leukocytes were enumerated using a haemocytometer after staining with trypan blue (Gibco). Trout kidney leukocytes isolated by 51% Percoll were used for the generation of T-PKM cultures. The generation of T-PKM was performed using similar protocols for the establishment of IVDKM [9]. Brie¯y, trout kidney leukocytes (10 £ 10 6 cells) isolated using 51% Percoll were seeded into 25 cm 2 ¯asks and incubated at 208C. Microscopic and ¯ow cytometric analysis was performed on alternate days (see below). Initial cultivation attempts were performed to obtain cell-conditioned medium (CCM) which was used for the establishment of future cultures [9]. Brie¯y, kidney leukocytes isolated by
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51% Percoll (as described above), were placed into 25 cm 2 tissue culture ¯asks (10 £ 10 6 cells) and cultured for 8±12 days. The CCM was then collected from 8±12 day old T-PKM by centrifuging the cells at 200 g for 10 min. The CCM was subsequently ®ltersterilized (0.22 mm ®lter) and stored at 48C. This procedure has proven successful for the development and maintenance of gold®sh IVDKM [8,9] and suggests that trout macrophages also produce endogenous growth factors required for proliferation and differentiation [10]. To test for CCM activity, leukocytes were seeded into 25 cm 2 ¯asks as described above and incubated in the absence (complete medium only) or the presence of CCM (25% v/v). Cultures were analyzed by ¯ow cytometry after 4, 8, and 12 days of cultivation. 2.5. Flow cytometric analysis of gold®sh and rainbow trout macrophage sub-populations IVDKM and T-PKM were analyzed using a FACS Calibur ¯ow cytometer equipped with a cell sorter (Becton Dickinson). Forward scatter (FSC; size) and side scatter (SSC; internal complexity) parameters were considered for examination of isolated ®sh leukocytes. Fluorescence (FL1) was used for DHR experiments (see below). All ¯ow cytometry was performed on cells suspended in phosphate-buffered saline (PBS) and 10 000 events were routinely collected during analysis. The following ¯ow cytometer settings were used for the analysis of IVDKM and T-PKM, respectively; forward side scatterphotodiode set to E-1, E00, AmpGain set to 9.33, 1.05; and side scatter±photomultiplier voltage set to 455 V, 450 V, AmpGain set to 1.00, 1.00. 2.6. Production of mitogen-stimulated gold®sh and trout kidney leukocyte conditioned supernatants Mitogen-stimulated leukocyte supernatants were generated using protocols described previously [12]. Brie¯y, kidney leukocytes were isolated from 20 gold®sh or six trout, pooled, enumerated, and seeded in 75 cm 2 tissue culture ¯asks at a concentration of 4 £ 10 6 cells/ml. Kidney leukocytes were incubated overnight in medium containing 2.5% carp serum and 10% FCS at
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208C. The following morning the mixed leukocyte cultures were stimulated with 10 mg/ml Concanavalin A (Con A, Boehringer Mannheim), 10 ng/ml phorbol myristate acetate (PMA, Sigma), and 100 ng/ml calcium ionophore A23187 (Sigma). Cultures were incubated in the presence of mitogens for 6 h, after which the mitogens and serum were removed by washing the cells with three exchanges of 25 ml Hanks balanced salt solution (HBSS). The remaining cells were given fresh incomplete medium and incubated for 72 h at 208C. After 72 h, the supernatants from the ¯asks were pooled, ®lter sterilized, and stored at 2208C. Homologous MAFs were used to induce both ROI and RNI responses of IVDKM and T-PKM. 2.7. Detection of gold®sh IVDKM and T-PKM respiratory burst activity using DHR The respiratory burst activity of IVDKM and TPKM was studied using a modi®ed procedure used for the analysis of mammalian neutrophil respiratory burst activity [16±18]. DHR (Molecular Probes) was dissolved in DMSO at a concentration of 29 mM (stock solution), and stored in 30 ml aliquots at 2808C. Samples of IVDKM or T-PKM cultures (days 8±12) were (2.5 £ 10 5 cells/500 ml) seeded into 6 ml polystyrene tubes (Falcon) and allowed to equilibrate for 1 h at 228C prior to determination of the respiratory burst response. Following the 1 h incubation, tubes were centrifuged at 200 g for 10 min. Supernatants were removed and the cells were re-suspended in 2 ml sterile PBS. The wash step was repeated once more to remove all traces of serum. After the ®nal wash, cells were loaded with 100 ml DHR (10 mM ®nal concentration in PBS) and incubated for 5 min at 228C. The respiratory burst response was triggered by adding 400 ml PMA (100 ng ®nal concentration in PBS) and the cells were incubated for an additional 30 min to allow for the oxidation of DHR to the ¯uorescent compound Rho 123. Control tubes were loaded with DHR but were treated for 30 min with sterile PBS only. A positive control was included by incubating DHR-loaded cells with 1 mM hydrogen peroxide (Merck) and samples incubated at 48C (reaction inhibition step)were used as the negative control. After the 30 min priming step, samples were immediately
analyzed using a ¯ow cytometer. Instrumental settings for forward and side scatter were the same as described above, and the assessment of green ¯uorescence (Rho 123) was determined (FL1) using histograms and a logarithmic scale. Cells treated with DHR only (no triggering) were used to calibrate the FL1. Each analysis was performed on 10 4 events and analyzed using the LYSIS II software. The degree of respiratory burst activity was assessed from the mean ¯uorescence intensity values obtained from the histogram plots. For detection of functional populations, regional analysis was performed on histograms and cytograms using the LYSIS II software. 2.8. Nitric oxide bioassay Gold®sh IVDKM or T-PKM were seeded (5 £ 10 4 cells/ml) into 96 well half-area culture plates (Costar) in complete medium, and stimulated with homologous MAF (1:4 and 1:16 ®nal concentrations). Lipopolysaccharide (1±25 mg/ml ®nal concentration) was added to each treatment and the NO produced by the stimulated cells was analyzed by sampling the supernatants after 72 h incubation for the presence of nitrite, using the Griess reaction [9±12]. Brie¯y, 75 ml of supernatants were removed from individual wells and placed in a separate 96 well microtitre plate. One hundred ml of 1% sulphanilamide in 2.5% phosphoric acid was added to each sample followed by 100 ml of 0.1% N-naphthyl-ethylenediamine in 2.5% phosphoric acid. The optical density of each well was determined using an automated plate reader (Biotek) at 540 nm. The approximate concentration of nitrite in the samples was determined from a standard curve generated using known concentrations of sodium nitrite. 2.9. Statistics One- and two-way analysis of variance using SuperAnova software (Abacus) for the Power Macintosh was used for statistical analysis of the data. A least square means was used for determining signi®cance between control and experimental groups. A probability level of P , 0.05 was considered signi®cant.
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3. Results 3.1. Cultivation of rainbow trout head kidney leukocytes and the development of trout-primary kidney monocyte-like cultures (T-PKM) We previously demonstrated that cultivation of gold®sh leukocytes in the presence of CCM, which contains proliferation and differentiation factors, resulted in the development of stable macrophage cultures (IVDKM) [8,9]. A similar cultivation technique was applied for the development of T-PKM cultures. Trout head kidney leukocytes cultured in the presence of a trout CCM were monitored overtime by ¯ow cytometry (Fig. 1). This approach resulted in the identi®cation of three sub-populations of cells based on FSC and SSC parameters. These cell sub-populations were gated according to foci of cells that were identi®ed using density plot analysis and were designated as R1, R2, and R3 (Fig. 1, see trout ¯ow cytometry template). At least 20 individual ®sh kidney leukocyte preparations were examined, and in all cases these three sub-populations of cells were observed. The differences between the gold®sh IVDKM and the T-PKM ¯ow cytometry gates represents the differences in size and internal complexity of cultured kidney leukocytes from these different ®sh species. An example of the changes in ¯ow cytometric pro®les of trout leukocytes over extended cultivation periods is shown in Fig. 1. This representative trout leukocyte culture demonstrates the standard ¯ow cytometric gating pattern observed after cultivation of the cells in the presence of 25% CCM. In all ®sh examined, the %R3 population increased after extended cultivation (i.e. 8±12 days), while the other sub-populations did not. Initially, day 0 to day 4, the majority of events gated to the R1 and R2 regions. Subsequent cultivation demonstrated a steady decline of the R1-gated events, no change in the R2gated events and a gradual increase in the R3-gated events. This trend for the consistent enrichment of the R3 sub-population was observed for all trout leukocyte cultures obtained from different ®sh (Table 1). The R3 cells from T-PKM cultures were subsequently sorted using the ¯ow cytometer and cytospin smears were made of pre-sort and sorted cells (Fig. 2). The sorted R3 cells were large round cells that often
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had eccentrically placed kidney-shaped nuclei.(Fig. 2(b)). In addition, these cells demonstrated dark and diffuse staining for myeloperoxidase (Fig. 2(b)) Based on these morphological and cytochemical characteristics, the R3 sub-population was classi®ed as monocyte-like cells. These monocyte-like cells were also the largest cells within T-PKM based on FSC, SSC, and microscopic analysis, ranging in size from 10 to 15 mm. Morphologically, the other cell types in TPKM (pre-sort) were small, round, and contained very little cytoplasm (similar to R1 cells of the IVDKM [9±12]), or resembled lymphocytes with large round nuclei and very little cytoplasm (Fig. 2(a)). Some of the cells in the R2 region may be promonocytes due to their size and dark-blue staining cytoplasm which is characteristic of immature cell types. Interestingly, a mature macrophage-like subpopulation in T-PKM was not observed in the smears. In contrast, gold®sh IVDKM cultures comprise cells with typical monocyte-like morphology (i.e. round cells with kidney-shaped nuclei) and macrophagelike morphology (i.e. large irregularly shaped cells with extensive vacuolisation) [9,10]. Flow cytometric analysis of T-PKM cultures failed to demonstrate a macrophage-like cell population (Fig. 2(a)). To con®rm the presence of an R3-enriching factor within trout CCM, trout leukocytes were cultured in the presence of complete medium alone (no CCM) or were cultured in complete medium supplemented with 25% CCM. Two separate CCMs were tested (designated CCM1 and CCM2). T-PKM cultures were subsequently analyzed after 4, 8, and 12 days using ¯ow cytometry. Clearly, the cultivation of T-PKM in the presence of two different CCMs resulted in an enrichment of the R3-cell sub-population during the ®rst 12 days of cultivation (Fig. 3). 3.2. Identi®cation of ROI-producing sub-populations in ®sh leukocyte cultures (gold®sh IVDKM and TPKM) 3.2.1. IVDKM The respiratory burst response of gold®sh macrophages was assessed by ¯ow cytometry using the ¯uorescent probe DHR. IVDKM were loaded with DHR (as described above) and the respiratory burst was triggered by the addition of PMA. Cells not exposed to PMA were used to calibrate the ¯ow
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Fig. 1. Cultivation of trout head kidney leukocytes and the generation of trout primary kidney macrophage (T-PKM) cultures. Rainbow trout head kidney leukocytes were isolated as described in the materials and methods. Kidney leukocytes at the 51% Percoll interface were seeded into 25 cm 2 (10 £ 10 6/¯ask) and grown in the presence of 25% cell-conditioned medium (CCM). Sub-samples were removed from the culture ¯asks on the indicated days and analyzed by ¯ow cytometry. Note an increase in the R3-type cell number after extended cultivation periods in the presence of CCM. Data is a representative ®sh out of 20 different rainbow trout cultures that were generated.
560±2380 (5.6±23.8%) 270±840 (2.7±8.4%) 3370±7750 (33.7±77.5%) 680 (6.8%) 680 (6.8%) 6260 (62.6%) 560 (5.6%) 430 (4.3%) 7750 (77.5%) R1 R2 R3
2290 (22.9%) 580 (5.8%) 4290 (42.9%)
2380 (23.8%) 840 (8.4%) 5010 (50.1%)
2090 (20.9%) 270 (2.7%) 3370 (33.7%)
810 (8.1%) 510 (5.1%) 5590 (55.9%)
1460 ^ 350 (14.6 ^ 3.5%) 550 ^ 80 (5.5 ^ 0.8%) 5380 ^ 630(53.8 ^ 6.3%)
Range Mean (^SEM) Fish 6 Fish 5 Fish 4 Fish 3 Fish 2 Fish 1
Gated cells (total: 10 000 events collected)
Table 1 Proportion of R1, R2, and R3-type cells in trout primary monocyte-like cultures. Trout head kidney leukocytes were isolated using a 51% Percoll gradient and the viable leukocytes were cultured as indicated in the Materials and Methods section. Using the standard gates derived from analysis of trout kidney leukocyte cultures, the proportions of cells within each region was determined after 8±12 days in culture. R1, R2 and R3 represent the gating regions of the ¯ow cytometer based on size and internal complexity of leukocytes
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453
cytometer for ¯uorescence intensity (FL1). For example, DHR loaded cells were analyzed and the histogram plot was adjusted so that DHR loading was expressed as low ¯uorescence (approximately 10 1 on the log scale). As shown in Fig. 4(b) (left panel), IVDKM that were loaded with DHR had a pronounced peak on the FL1 histogram plot. After triggering with PMA, there was a shift in the ¯uorescence intensity (Fig. 4(c), left panel) due to the release of respiratory burst products. DHR allowed for the analysis of the respiratory burst activity of different sub-populations within the heterogeneous IVDKM cultures comprised of R1-, R2-, and R3-type macrophages. The sub-populations responsible for the oxidation of DHR to Rho 123 (i.e. shift in ¯uorescence intensity) were identi®ed as the R2- and R3type cells (Fig. 4(d)). The R1-type cells did not have a detectable respiratory burst response. The respiratory burst activity was expressed as mean ¯uorescence intensity per cell in the individual sub-populations. For all gold®sh cultures examined, the monocytelike (R3) cells had a higher respiratory burst activity compared to the macrophage-like (R2) cells (P , 0.05) (Fig. 4). 3.2.2. T-PKM DHR and ¯ow cytometry were also used to identify the functional population within T-PKM cultures (Fig. 4; right panel). Days 8±12 T-PKM were used for analysis (Fig. 4(a), right panel). Cells were loaded as described above and these DHR-loaded cells were used to calibrate FL1 (same as above for IVDKM) (Fig. 4(b), right panel). Triggering with 100 ng PMA caused a pronounced shift in the ¯uorescence histogram (Fig. 4(c), right panel). The population of cells responsible for this shift were identi®ed as the R3-type monocytes (Fig. 4(d), right panel). Interestingly, this was the same population that survived extensive culture periods in the presence of 25% CCM (see Figs. 1 and 3). 3.3. Production of RNI by gold®sh IVDKM and TPKM Stimulation of 8±12 day old gold®sh IVDKM and T-PKM demonstrated that only IVDKM were capable of producing RNI following immunological stimuli (Fig. 5). A similar effect was seen when diluted
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Fig. 2. Flow cytometric analysis of sorted R3-type cells after 8 days of cultivation in the presence of CCM. T-PKM cultures were established as indicated in the Materials and Methods section. The R3-type cells that were enriched for over extended cultivation periods were subsequently sorted using the ¯ow cytometer. Cytologic appearance of the cells was characterized by staining cells in cytocentrifuge smears (Cytospin II, Shandon Instruments) with a modi®ed Wright's stain (Fisher). Three predominant cell types were observed in the pre-sort pro®le (R1, R2, and R3) (A). The R1 cells were the smallest and had a high nucleus:cytoplasm ratio (Scale bar 10 mm). R2 cells were medium in size and were typically round cells with large round nuclei and blue-staining cytoplasm. R3 cells were round with kidney-shaped shaped nuclei. These cells displayed characteristic monocyte-like morphology and were the cell type identi®ed following ¯ow cytometric sorting (B1). These cells were also positive for myeloperoxidase (Sigma) and demonstrated a dark diffuse staining pattern (B2).
MAF (1:16) in conjunction with LPS (1±25 mg/ml) were used (data not shown). Flow cytometric analysis of gold®sh IVDKM and T-PKM, using identical instrument settings, clearly demonstrated that the TPKM cultures did not have a sub-population of cells equivalent to mature gold®sh macrophages (i.e. R2). The ROI-producing sub-population in T-PKM cultures (R3) gated more closely to the monocytelike cells found in gold®sh IVDKM (R3). 4. Discussion We previously showed that the cultivation of gold®sh kidney leukocytes in the presence of CCM resulted in the generation of stable macrophage cultures [8±10]. Gold®sh IVDKM cultures contain three distinct sub-populations of macrophages. These sub-populations represent cells that are devel-
opmentally arrested at different stages of macrophage differentiation (i.e. early progenitors, monocytes, and macrophages, designated R1, R3 and R2, respectively). In contrast, the cultivation of rainbow trout head kidney leukocytes, in the presence of CCM, generated a single population of functional cells which demonstrated monocyte-like properties (designated R3). The increased %R3 (monocyte-like cells) during extended cultivation periods, in the presence of CCM, suggests that there is at least one MGF present in trout-derived CCM. The majority of smaller cells present in early cultures of T-PKM, were most likely lymphocyte-like cells, which have been previously reported [19]. However, some of these cells may also represent pro-monocyte-like cells (i.e. R2 in TPKM), which in the presence of appropriate factors found in CCM, differentiate into the maturer monocyte-like cells (R3). Therefore, within the hematopoietic tissue of the trout kidney, monocytes
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Fig. 3. Demonstration of an R3-enriching factor presence in trout-derived CCM. Rainbow trout head kidney leukocytes were isolated as described in the Materials and Methods section. Leukocytes were seeded into 25 cm 2 and cultured over 12 days in the presence and absence of 25% (v/v) trout-derived CCM. On days 4, 8, and 12, samples were removed from the culture ¯asks and examined by ¯ow cytometry. Using the trout ¯ow cytometer template, the number of cells per gate was determined out of 10 000 collected events. Two different CCMs were tested (designated CCM 1 and CCM 2). Data from two independent experiments, (1) and (2), are shown.
differentiate from pro-monocytes and these monocytes likely enter the circulation to become tissue macrophages. This pathway of differentiation is similar to that of mammalian macrophages. In contrast, gold®sh macrophage differentiate fully in vitro, and do not appear to require additional tissue `cues' before they acquire the ability to generate RNI in response to immunological stimuli [8±10]. T-PKM also exhibited different functional properties when compared to gold®sh IVDKM. Gold®sh IVDKM produce both ROI and RNI in response to MAF and/or LPS [12±14]. Recently, we characterized a gold®sh macrophage activation factor, by demonstrating that cleavage products of transferrin are important for the induction of RNI production by
these cells [20]. We suggested that one of the necessary events for NO production by IVDKM involved the increased differentiation of monocyte-like cells (R3) into mature macrophage-like cells (R2) [21± 26], which have been previously shown to be the RNI-producing cell sub-population in the IVDKM cultures [10]. In contrast, the T-PKM did not produce RNI but did generate ROI as demonstrated using the DHR assay. A single monocyte-like sub-population (R3) was responsible for the ROI response in TPKM cultures, while two ROI-producing sub-populations were present in gold®sh IVDKM (i.e. monocyte (R3)- and macrophage (R2)-like cells). In mammals, cultured monocytes undergo a variety of changes that may be similar to those that
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Fig. 5. Nitric oxide (NO) production by gold®sh IVDKM and T-PKM. Gold®sh and trout kidney leukocytes were cultured in the presence of 25% CCM as described in the Materials and Methods section. After 8±12 days, the cells were harvested and seeded into 96-well half area tissue culture plates (5 £ 10 4 cells/well). Cells were stimulated with LPS (1±25 mg/ml; black bars) and/or MAF (1:4 and 1:16 ®nal concentration; shaded bars) and incubated for 72 h at 228C before determination of NO response. Note that gold®sh IVDKM and T-PKM were treated with homologous MAFs. Each bar represents the mean ^ SEM of triplicate cultures and the data are from a representative experiment out of three that were performed.
occur after migration into the tissue from the peripheral blood [27±29]. In addition, cultured human monocytes are believed to resemble human resident tissue macrophages [28,29]. Therefore,
cultivation of monocytes over several days results in their differentiation into the maturer macrophage phenotype. The differentiation of the monocytes in culture is concomitant with changes in the
Fig. 4. DHR analysis of functional sub-populations found in heterogeneous leukocyte cultures (i.e. IVDKM and T-PKM). Cultured ®sh macrophages: gold®sh (left panel); trout (right panel) were loaded with DHR as indicated in the Materials and Methods section. Control, non-stimulated gold®sh (left panel, B) and trout (right panel, B) DHR-loaded cells exhibited ¯uorescence intensity (FL1) of less than 10 1.5 (no ROI production). After PMA treatment, both IVDKM and T-PKM produced ROI, indicated by a signi®cant increase in ¯uorescence intensity (C). Region analysis identi®ed the functional populations in gold®sh and trout cultures (D). Note that the shift in DHR ¯uorescence intensity of IVDKM was evident for two sub-populations (R3 and R2), while T-PKM culture demonstrated only one ROI-producing population (R3).
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generation of both ROI and RNI [30]. Freshly isolated human monocytes have a high capacity to generate ROI, and this response declines after 4 days in culture [30]. Conversely, the production of RNI is not detectable in freshly isolated monocytes but after 4±5 days of cultivation, they acquire the ability to produce RNI[30]. A similar process may be important for the induction of the RNI response by gold®sh macrophages. Gold®sh monocyte-like cells (R3) do not produce RNI but the mature macrophage-like cells (R2) are capable of producing NO in response to MAF and/or LPS[10]. It has also been demonstrated that gold®sh monocytes can be induced to differentiate into macrophage-like cells in response to activating stimuli (i.e. MAF or LPS) and CCM [10]. This differentiation step may be a necessary event for RNI production by gold®sh macrophages. In contrast, the inability of T-PKM cells to produce RNI may be due to an absence of a mature macrophage-like sub-population in the cultures. The reasons for the absence of this population in T-PKM cultures is not known, but may involve the absence of necessary factor(s) required for the functional differentiation of monocytes into mature macrophages in vitro. Another major difference between T-PKM and IVDKM cultures is that the progenitor-like cells (R1 sub-population) do not survive extended cultivation periods in T-PKM cultures. These progenitor cells are required for both the development of monocyteand macrophage-like sub-populations, which has been previously demonstrated for gold®sh IVDKM and gold®sh macrophage cell line [9±12]. The absence of a mature macrophage sub-population in T-PKM may explain the inability of T-PKM to produce RNI in response to immunological stimuli. During an in¯ammatory response in trout, kidney-derived monocytes are recruited to the in¯ammatory site [31] and, in tissues, presumably differentiate into mature macrophages that can produce RNI, as iNOS expression has been detected in trout kidney [32]. However, the ability of kidney leukocytes to produce RNI appears to be rapidly down-regulated in vitro culture [33]. Thus, trout kidney macrophages and T-PKM, unlike gold®sh macrophages, may require the continual presence of other appropriate tissue cues for differentiation into fully functional mature macrophages, with the ability to produce RNI.
In conclusion, we showed that enriched primary monocyte-like cultures could be derived from trout head kidney leukocyte cultures, using techniques developed for the establishment of gold®sh IVDKM. However, these cells require as yet unknown signals to differentiate into fully mature macrophages with the ability to produce RNI. The enrichment for a functional sub-population of monocyte-like cells by the addition of homologous CCM, suggests that there is at least one growth factor present in trout CCM. The use of these cultivation techniques will be invaluable for the enrichment of functional monocyte-like cells from trout head kidney leukocytes. These cells can subsequently be used in bioassays that measure cellmediated immune responses of rainbow trout exposed to a variety of agents (i.e. environmental contaminants, pathogens, or immunization) and for the further characterization of trout MGFs responsible for trout monocyte/macrophage development. Acknowledgements This work was supported by Natural Sciences and Engineering Council of Canada (NSERC) and Alberta Heritage Foundation for Medical Research (AHFMR) to M.B. J.L.S. was supported by a PhD scholarship from NSERC. P.E.M. was funded by EC FAIR CT98-4087 and a travel grant provided by the University of Aberdeen Development Trust and The Royal Society. References [1] Virelizier JL, Arenzana-Seisdedos F. Immunological functions of macrophages and their regulation by interferons. Med Biol 1985;63:149±59. [2] Loms Ziegler-heitbrock HW. The biology of the monocyte system. Eur J Cell Biol 1989;49:1. [3] Stewart CC, Reidy MC, Stewart SJ. The proliferation and differentiation of macrophages. Immun Series 1994;60:3±27. [4] Rutherford MS, Witsell A, Schook LB. Mechanisms generating functionally heterogeneous macrophages: chaos revisited. J Leuk Biol 1993;53:602±18. [5] Donahue RE, Seehra J, Metzger M, Lefebvre D, Rock B, Carbone S, Nathan DG, Garnick M, Sehgal PK, Laston D, LaVallie E, McCoy J, Schendel PF, Norton C, Turner K, Yang Y-C, Clark SC. Human IL-3 and GM-CSF act synergistically in stimulating hematopoiesis in primates. Science 1988;241:1820±3.
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