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Generation and functional characterisation of canine bone marrow-derived macrophages
Research
in Veterinar?;
Science
1998, 64, 125-132
Generation and functional characterisation of canine bone marrow-derived macrophages A. TIPOLD, Institutes of Veterinary Virology and Animal Neurology, University of Berne, A. ZURBRIGGEN, Institute of Animal Neurology, University of Beme, P. MOORE, VM Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, USA, V. SCHIJNS, Inter-vet, Boxmeer, The Netherlands, T. W. JUNGI*, Institute of Veterinary Virology, University of Berne, Berne, Switzerland
SUMMARY culture of bone marrow cells from the femurs of canine pups at high concentrations of fetal calf serum under non-adherent conditions allowed the proliferation and differentiation of mononuclear phagocyte lineage cells, as evidenced by morphology and CD14 expression. Cells from other lineages progressively diminished in numbers. Cells collected between 12 and 19 days of culture expressed an array of macrophage activities including ingestion of opsonised erythrocytes, generation of superoxide. up-regulation of procoagulant activity and synthesis of tumour necrosis factor (TNF) upon appropriate stimulation. TNF production was enhanced when the cultures were simultaneously stimulated with canine recombinant, or supematant-derived, interferon-y. In contrast, low levels of inducible nitric oxide (NO) synthase were expressed by only a minority of stimulated macrophages, and nitrite could not be detected in the medium. Therefore, canine macrophages generated by this novel culture system resemble human macrophages in their inefficient and restricted generation of NO upon appropriate stimulation. A
MACROPHAGES are important elements in host defence and are homeostatic regulators of a broad array of physiological reactions related to immunity, repair and regeneration (Lewis and McGee 1992). They are also incriminated as mediators of pathologic processes in a variety of conditions. Depending on the environment in which bone marrow-cell-derived cells differentiate, they exert widely different functions, e.g. those mediated by tissue macrophages, Kupffer cells, microglia cells of the brain, or bronchoalveolar macrophages. Depending on the species, macrophages from different compartments are available for study. Bone marrow-derived macrophages (BMM) have mainly been studied in rats and mice (Stewart 198 1, Cooper et al 1984, Keller and Keist 1986). In order to study macrophages in large numbers and uninfluenced by tissuespecific differentiation-promoting conditions, it would also be desirable to have access to BMM in other species, ‘in particular those of veterinary interest. Recent reports from our laboratory showed that it is possible to generate large numbers of functionally active BMM in sheep and cattle (Francey et al 1992a,b, Adler et al 1994, Jungi et al 1996b). In the present study we explored a similar culture system adapted to the generation of canine BMM (Brtigger et al 1992). We show that bone marrow cells from femurs of newborn puppies proliferate and differentiate in culture to exhibiting functional properties of mature BMM, macrophages. Amongst the variety of functions tested we selected erythrophagocytosis via Fc receptor, and the capability to respond to stimuli by eliciting a respiratory burst, two prototypic functions of professional phagocytes. The former function is known to be depressed upon macrophage activation by interferon-y, whereas the latter is enhanced (Jungi et al 1989). Furthermore, a key function of macrophages is * Corresponding author E-mail:
[email protected]
0034.5288/98/020125
+ 08 $18.00/O
their ability to respond to lipopolysaccharide (LPS), e.g. by secretion of cytokines such as tumour necrosis factor (TNF) (Vassalli 1992). LPS is also known to trigger the production of nitric oxide (NO), a mediator thought to be essential for antimicrobial and antitumoural host defence. No generation is catalysed by inducible nitric oxide synthase (iN0.S) (Hibbs et al 1987, Nathan 1992) which is induced by LPS and certain cytokines or combinations of cytokines and bacterial components. However, although readily induced in rodent, bovine and avian macrophages (Sung et al 1991, Nathan 1992, Jungi et al 1996a), induction of strong iNOS expression and NO generation production have not been convincingly demonstrated in vitro, in macrophages of other species such as human, goats, pigs and sheep (Albina 1995, Keller et al 1993, Schneemann et al 1993, Adler et al 1996, Turek et al 1994, Jungi et al 1997). It was therefore of particular interest to determine whether iNOS is inducible in canine BMM by conventional stimuli, such as bacterial components and cytokines.
MATERIALS
AND METHODS
Animals Newborn Swiss beagle puppies were generously provided by Novartis, Basel, Switzerland. Dogs were euthanised with pentobarbiturate, and femurs were collected under sterile conditions. Collection of bone marrow cells The femurs were excised and cut open on both ends of the bone. The bone marrow was flushed out using a syringe and needle with RPM1 1640 under sterile conditions. Cells were washed with RPMI 1640 medium, centrifuged and resuspended at a concentration of 2 x lo5 cells ml-t in culture medium (see below). 0 1998 W. B. Saunders Company Ltd
Bone marrow cell culture The cells from three to four puppies were mixed and cultured together. They were sealed in hydrophobic (teflon) bags as described (Brtigger et al 1992, Francey et al 1992b, Adler et al 1994), and the bags were placed in a humid atmosphere containing 5 per cent CO, in air at 37°C for up to 19 days without change of the medium. This consisted of RPMI 1640 supplemented with 20 per cent foetal calf serum (FCS; Life Technologies, Paisley, UK), penicillin (100 IU ml-l), streptomycin (100 pg ml-t), sodium pyruvate (1 mM), glutamine (2 r&l), MEM vitamin solution (1 per cent v/v; Life Technologies), non-essential amino acid solution (1 per cent v/v; Life Technologies), HEPES (10 n&I), and folic acid (40 mg litre-I). For studies in which NO generation was determined, Iscove’s modified Dulbecco’s minimum essential medium instead of RPMI 1640 was used. To demonstrate functional activity of the mature canine macrophages, they were harvested between day 12 and 19. Cells harvested every two or three days were used for flow cytometry and for morphological characterisation, using Diff-Quik staining of cytospin preparations. To determine the proportions of cell subsets, two samples of 200-300 cells from two independent experiments were counted using light microscopy, and mean values were determined. In other experiments, cells were harvested from teflon bags and subcultured in microtitre plates (6-8 x lo4 macrophages per well). After two hours of adherence, they were exposed either to lipopolysaccharide (LPS; Escherichia coli 055:B5, Sigma Chemical Company, St. Louis, MO, 1 pg ml-t), or to 200 pg ml-t heat-killed (two hour 60°C) Salmonella dublin (strain 24-90) or Listeria monocytogenes (NCTC strain 10,527, serotype 4b), in the presence or absence of various cytokines. These were recombinant canine interferon-y (rcatm-Y), recombinant bovine interleukin- 1B (rboIL- 1) kindly provided by American cyanamid (Princeton, NJ), recombinant human interleukin- 1p (Peprotech, London, UK), recombinant bovine TNF (Novartis), and recombinant human TNF (Eurocetus, Amsterdam, The Netherlands). rcatt+y was derived as an expression product of insect cells (Sf-21 cells) after infection with recombinant baculovirus expressing the canine IFN-y CDNA behind its pl0 promotor. Its biological activity was determined in a classical vesicular stomatitis virus inhibition assay on canine MDCK cells (De Groot and Schijns, unpublished observations). rcaIFN-y was dissolved at 2 x lo4 to 2 x 1O5 antiviral activity units per ml, and loo-fold and lOOOfold dilutions were used in experiments. The other cytokines were tested at 10 and 100 ng ml-t. Flow cytometry The expression of leukocyte surface markers was determined by indirect staining with monoclonal antibodies specific for, or crossreacting with, canine Major Histocompatability Complex Class II (MHC-II) molecules (clone Ca2.1C12), or canine CD14 (clone TUK4, Dako, Glostrup, Denmark). The second antibody was phycoerythrin-labeled goat-anti-mouse IgG F(ab’)z (Jackson Immunoresearch Laboratories, West Grove, PA). Stained cells were analysed in a FAcscan flow cytometer (BectonDickinson, San Jose, CA), using the PC-LYSYS software. Immunocytochemistry Macrophages from teflon bags were subcultured in 24well plates containing a glass coverslip and were stimulated
as described above. After overnight culture, the monolayers were fixed with paraformaldehyde (2 per cent v/v in PBS) for 20 minutes. The cells were then washed twice, followed by Fc receptor blockade with 10 mg ml-) of human IgG for 20 minutes, at which time IgG was replaced by anti-iNos at a dilution of 1:500 (UPI, Lake Placid, NY). Specimens were developed by using the ABC staining system (Immunodiffusion, Geneva, Switzerland). In situ hydridisation A probe specific for canine TNF was cloned after RT-PCR using primers based on sequences conserved between species (Pauli et al 1989, Pauli 1995). As a template, 14day-old canine macrophages harvested from teflon bags and stimulated for six hours with LPSwere used for production of RNA as described (Sambrook et al 1995). Using this method a portion of the canine TNF gene containing 300 base pairs was amplified, cloned and sequenced. The identity of the nucleotide sequence of the clone with canine TNF (Zucker et al 1994) was confirmed by sequence alignment. In situ hybridisation was performed with a riboprobe complementary to mRNA of canine TNF and labelled with digoxigenin (Boehringer-Mannheim, Mannheim, Germany) as described (Zurbriggen et al 1993). The corresponding antisense probe was used as a negative control. Hybridisation was performed at 52°C overnight. The probe was visualised by staining with a digoxigenin-specific phosphatase-conjugated antibody (anti-digoxigenin-AP, Fab fragments: Boehringer-Mannheim). Phagocytosis Phagocytosis of opsonised sheep erythrocytes was measured by a photometric procedure (Jungi 1985, Rtiegg and Jungi 1988). BMM monolayers in 96-well plates were covered with erythrocytes opsonised with affinity-purified sheep erythrocyte-specific antibodies (Diamedix, Miami, FL) and after a 60 minute ingestion period non-attached and attached non-ingested erythrocytes were removed by washing and hypotonic lysis. The monolayer containing ingested red blood cells was lysed with sodium dodecyl suphate, followed by the addition of diamino benzidine and H,O,. The evolving colour reaction, as determined in an ELISA reader at 450 nm, was proportional to the number of ingested erythrocytes. The number of macrophage nuclei was determined in parallel wells by lysing them with the mild detergent, cetavlon, and staining the released nuclei with amidoblack (Nakagawara and Nathan 1983). This allowed ingestion to be expressed as a phagocytic index (ingested red blood cells per nucleus). Superoxide determination Superoxide was determined in BMM subjected to various pretreatments and harvested from teflon bags by chemiluminescence, using lucigenin as an enhancer (Jungi and Peterhans 1988, Rtiegg and Jungi 1988). BMM were resuspended in Hanks’ balanced salt solution at 0.2 x 106 ml-t containing lucigenin (50 pM, Sigma) and dark-adapted in 11 x 47 mm polystyrene tubes (0.5 ml tube-‘). They were stimulated with either phorbol 12-my&ate 13-acetate (PMA; 1O-7 M), or with serum-opsonised zymosan (50 pg ml-‘) and immediately thereafter, the evolving chemiluminescence response was recorded in a LB 950 luminometer (Berthold, Wildbad, Germany).
Canine bone marrow-derived
TABLE 1: Characterisation of bone marrow derived cells different time points after onset of non-adherent culture Morphology* Culture time (days)
Moncqtelike cells w
2 5 7 8 9 12 15
14 15 4 6 3 3 1
* Morphology Diff-Quik
Procoagulant
at
Flow cytometry
Macrophagelike cells (“A) 5 20 80 80 85 90 97
was studied
harvested
Lymphoid and undiff. cells W)
Granulocytes (“M
46 50 10 9 5 2 2
in cytospin
preparations
CD14 positive ceils (%) 6 11 55 51 04 95 93
35 15 6 5 7 5 0 which
were
stained
with
activity
Procoagulant activity (PCA) was measured in BMM harvested from teflon bags and subcultured in microtitre plates in culture medium containing 5 per cent FCS. It was determined by a turbidimetric recalcification time assay, using c&rated human platelet-depleted plasma (Jungi 1990, Briigger et al 1992), and expressed in human thromboplastin units, 1 unit representing recalcification time by 106fold diluted human thromboplastin (Thromborel S, Behring-Werke, Marburg, Germany). Production
of cytokine-enriched
supematants
Mononuclear cells were isolated as described (Wunderli and Felsburg 1989). They were cultured in 25 cm2 tissue culture flasks in the presence of A23187 (0.25 pM) and
FIG 1: Cytospin preparations were stained with Diff-Ouik
of freshly collected canine (A-C) or for surface-expressed
macrophages
127
PMA (5 ng ml-t), at 106 cells ml-l (total volume 5 ml). The supernatant was collected after 48 hours and stored frozen. This supernatant was considered to be rich in cytokines, including interferon-y, as evidenced by its effect on erythrophagocytosis, respiratory burst activity, and MHC classII modulation (see Results section). TNF bioassay BMM were stimulated with 1 pg ml-l LPS for six hours in teflon bags. TNF activity of supernatants from stimulated and control cells was determined by a cytotoxicity assay, using the porcine kidney cell line, PK(15) cells as targets. These are susceptible to TNF from numerous species including the dog (Pauli et al 1994). Cytotoxicity was determined by staining with MTT (Mosmann 1983). TNF specificity was verified with neutralising antibodies specific for human TNF and crossreacting with TNF from several different species (Monosan, Uden, The Netherlands). Griess assays
The content of nitrite, a stable metabolite of NO, was determined in supematants of BMM that had been subjected to various pretreatments for various length of time, using the Griess reaction (Adler et al 1995). In some assays, the total content of nitrate and nitrite was determined by conversion of nitrate into nitrite, followed by the Griess reaction (Jungi et al 1996~). Nitrate was converted into nitrite by treatment of supematants with Pseudomonas oleovorans bacteria which contain a high amount of nitrate reductase, but no nitrite reductase (Granger et al 1991). Treatment was
bone marrow cells (A), and of cells cultured CD14 by immunocytochemistry (D)
for seven
days (B), or 14 days (C, D). They
128
A. Tipold, A. Zurbriggen, P. Moore, V. Schijzs, T. W.Jungi
Fluorescence
intensity
Fig 2: Expression of MHC-class-11 molecules (MHC-II) (upper panel) or CD14 (lower panel) by BMM cultured in the presence (right or absence of IFN-y (feft panel). BMM in teflon bags were exposed for 48 hours to IOCQ-fold diluted recalFN-y (c), 10%fold-diluted y(d) or to four-fold diluted supematant of activated mononuclear cells (e). Histograms of a flow cytometric analysis are shown. resents unstimulated control cells, (b) shows the fluoresence emitted by control cells treated with an isotype control antibody ground fluorescence). The latter did not change by pre-treatment of BMM
performed for 90 minutes at 37°C with a 0.25 per cent v/v content of bacteria, and complete conversion was verified by nitrate-containing control samples.
RESULTS Generation of macrophages in cultures of canine bone marrow cells
Bone marrow cells harvested from femurs of new-born puppies were cultured under non-adherent conditions, in teflon bags, in the presence of high concentrations of FCS. There was a progressive increase in the proportion of CD16positive cells (Table l), and of cells displaying morphological characteristics of monocytes and macrophages, as determined by morphological criteria (Table 1, Fig 1). The cell population developed from a heterogeneic population to a rather homogeneous population of macrophages at day 14 (Fig 1). During a period of one to two weeks, not only the proportion, but also the absolute number of cells showing macrophage characteristics increased. The total cell number harvested at day 12 to day 14 was about equal to that seeded at day 0, and roughly doubled until day 19. Functional properties BMM
of BMM
collected between day 14 and day 15 were subject-
panel) rCitlFN(a) rep(back-
ed to surface marker analysis and to functional tests. The large majority of canine BMM expressed CD14 (Fig 2) and MHC-II on their surface. MHC-II, but not CD14 expression was enhanced by pre-treatment of canine BMM with recamby, or with supematants from mononuclear cells activated with PMA and A23187. Canine BMM ingested IgG-coated sheep erythrocytes (phagocytic index: 16.5ti.3); non-opsonized erythrocytes were taken up to a much lesser degree (1.2a.3). This result suggests that canine BMM express Fc receptors, and that these are functionally linked to phagocytosis. When Bh4h4 were preexposed to supematants of activated mononuclear cells, or to rcarnv-y (lOOO-fold diluted), the phagocytosis index decreased to lO.M.6 or 9.5~331, respectively. Chemiluminescence in the presence of lucigenin indicates the generation of superoxide. Untreated mature canine BMM and BMM pre-exposed for two days to rcan%-y or to supematants of activated mononuclear cells were therefore tested for superoxide production by lucigenin-enhanced chemiluminescence (Fig 3). Both PMA and zymosan induced the production of a modest amount of superoxide. Following treatment with either rcalIFN-7 or lymphocyte supematant containing cytokines, the chemiluminescence signal was increased, particularly when zymosan was used as a stimulus (Fig 3). BMM were subcultured in 96-well plates in which they were treated with LPS. Supematants were collected after 24 hours for determining TNF activity. using a cytotoxicity
Canine bone marrow-derived macrophages
2E6
129
2E6 Control
Supernatant
t
2E6
2E6
lE6
5E5
; 0
.s 4
10
20
30
40
50
60
I 10
0
lE6
I 20
I 30
I 40
I 50
I 60 -
lE6 rcaIFN-y(lOOOx)
rcaIFN-y(lOOx)
5E5< I 10
0
I 20
I 30
I 40
I 50
1 60
5E5)
I
I
0
Time
I 10
20
I 30
I/40
50
60
(minutes)
Fig 3: Chemiluminescence emitted by canine SMM (day 14) stimulated with PMA (solid lines), zymosan (dashed lines) or mock-stimulated (dotted lines), in the presence of lucigenin. BMM had been pre-exposed to reCalFN-y (IOO-fold or lOOO-fold diluted) or to supernatant of activated mononuclear cells (four-fold diluted) for 48 hour, or were untreated control cells. Individual light emission traces are shown
assay capable of detecting TNF of several species including the dog (Pauli et al 1994). Bioactivity of the secreted TNF, as demonstrated by the killing of PK(15) cells, reached half-maximal activity at 300-fold dilution of the supernatant, and it could be specifically neutralised with antiTNF-Specific antibodies (Fig 4). Similarly stimulated cells subcultured on glass slides expressed enhanced levels of mRNA for TNF (Fig 5). We also looked for another indicator of macrophage activation, namely the enhancement of PCA, as determined in a recalcification time assay (Table 2). In this assay, IFN-y alone induced PCAwithin 24 hours, a find100
/
I
I -
I
I 3125 Dilution
I
I 125
I
I 5
I
of supernatant
Fig 4: Production of TNF bioactivity upon stimulation of BMM (day 14) with LPS (1 pg ml-l) for six hours at 37%. Cytotoxicity, as determined by MTT staining of PK(15) target cells, is expressed as percentage of total lysis (mediated by distilled water). Antibody dose-dependent neutralisation of bioactivity demonstrates the Specificity for TNF Key: 0, anti-TNF (200-fold); & anti-TNF (800-fold); 0, anti-TNF (3200-fold)
ing reminiscent of human monocyte-derived macrophages (Schwager and Jungi 1994). Likewise, LPS, heat-killed S dublin and L monocytogenes strongly enhanced PCA, and this effect increased still further by supematants of activated peripheral blood mononuclear cells (Table 2). Production of NO by canine
BMM
BMM subcultured in 96-well plates were exposed to a prototypic Gram-positive organism (L monocytogenes), a Gram-negative organism (S dublin), ,or to LPS, either alone or combined with various cytokines. Cytokines tested included rboIL- 113,rhuIL- 113,rhuTNF, rboTNF, rcaIFN-y and supematant of activated peripheral blood mononuclear cells. These cytokines were also tested alone, in the absence of bacterial stimuli, and in combination with each other. Supernatants were collected 24 or 48 hours later in order to determine their content of nitrite, one of the stable metabolites of NO. None of the treatments induced significantly elevated levels of nitrite. Likewise, the total content of nitrate and nitrite, was not significantly elevated by these procedures. Since macrophage-derived NO is thought to be due to expression of iNOS, BMM exposed to the same stimuli were stained for iNos by immunocytochemistry. Whereas control cells were unstained, there was a weak staining of a minority of cells exposed to S dublin combined with interleukin-1 (Fig 5). A combination of stimuli eliciting a strong iNOS response in mouse and cattle macrophages, L monocytogenes combined with EN-~, was even less active. The cells which produced no detectable NO were indeed activated. This was shown by their increased expression of PCA when compared with unstimulated BMM (Table 3 and data not shown).
A. Tipold, A. Zurbriggen,
P. Moore, V. Schijns, T. W. Jungi
Fig 5: In situ hybridisation for TNF (A and C): positive probe showing enhanced TNF mRNA expression (A), negative control (corresponding antisense probe) (C). Expression of iNOS by unstimulated BMM at day 14 (D), and by BMM exposed to S dub/in and 109 ng ml” rbolL-I @)
DISCUSSION In recent years, mononuclear phagocytes have entered the limelight of immunological research due to their central role in host defence, immunoregulation, regeneration and repair. Depending on environmental factors impacting on mononuclear phagocytes, these cells assume a variety of distinct functional properties. Biochemical investigation of these regulatory processes depends on the availability of high cell numbers. Macrophage type cell lines were first described for the mouse (Mauel and Defendi 1971), then for several other species (Sundstriim and Nilsson, 1976, TABLE 2: Induction bone marrow-derived
of procoagulant macrophages* Stimulus
Co-stimulus (control) rcalFNn/ rcalFN-y Supematant+
-(control)
activity
by activation
LPS
S dub/in
21.80 18.85 22.36 211.70
53.27 99.49 75.82 443.2
of canine L monocytogenes
Concentration
1OO-fold 1 OOO-fold four-fold
4.92 20.49 7.69 70.07
92.16 70.35 46.92 355.00
* Bone marrow cells were harvested from teflon bags at day 19, followed by subcufture in mfcrotitre plates. They were stimulated with the indicated stimuli two hours after onset of microtitre plate culture. Procoagulant activity was determined 24 hours after stimulation T See legend of Fig 2
Ziegler-Heitbrock et al 1988, Glass et al 1989, Haig et al 1991, Sager et al 1997). However, murine macrophage cell lines are exceptional in that cells in exponential growth express functions of differentiated macrophages. In many other situations, proliferating cells fail to express many of the surface markers characteristic for mature macrophages, and their functional activity is highly restricted (Sager et al 1997). In these situations, and in species for which macrophage type cell lines are unavailable, primary macrophages are important tools for cell biological and biochemical studies. There are as yet few reports on macrophage collection and/or generation from the dog (Brtigger et al 1992). We present here a culture system for canine macrophages, starting with bone marrow cells from newborn puppies. It allows the propagation of BMM in high numbers and these may be activated by various stimuli. This renders the cells useful for functional studies on macrophages, e.g. on the interaction with pathogens or as a substrate for biochemical analyses. Moreover, it is expected that having this culture system at our disposal, we shall learn more about the physiology and pathophysiology of macrophages in the dog. Similar culture systems have been described for rnurine BMM and for BMM from sheep and cattle (Francey et al 1992b, Adler et al 1994). However, in sharp contrast to the mouse culture system, the exogenous addition to cultured
Canine bone marrow-derived
macrophages of colony stimulating factors (CSF), e.g. macrophage-CSF (M-CSF), was neither necessary nor sufficient to grow BMM of ruminants and the same applies to dog BMM cultures. The addition of high FCSconcentrations, on the other hand, was a critical requirement (Adler et al 1994, Francey et al 1992a). In contrast to these species, mouse BMM precursors rapidly divide upon exposure to either granulocyte-macrophage-CSF (GM-CSF)or M-CSF, and they are serum-independent. Canine BMM could be propagated at high FCS concentrations and in the absence of exogenous CSF, thereby resembling ruminant BMM. However, whether cell growth and survival rate are proportional to the serum concentrations or whether they can be influenced by exogenous CSFhas not been explored in the dog. In the ruminant BMM culture systems alluded to above, either bone marrow cells of up to nine-months-old sheep (Francey 1993) or fetal bone marrow cells from fetal tibiae (Adler et al 1994) were used, and the resulting macrophages exhibited closely similar functional properties. In an earlier study of this laboratory, biopsy specimens of bone marrow were used to generate macrophages in culture (Briigger et al 1992). We assume therefore that the present canine culture system is not restricted to tibiae of newborn puppies as a starting material, but can also be applied to bone marrow biopsies. It will be interesting to see how alterations in functional and phenotypic properties of cultured macrophages from biopsy material are related to the disease prompting the biopsy. The macrophages generated by the present culture system are at a resting stage, i.e. they may be activated to assume a variety of functions upon appropriate triggering. This is related, in part, to the use of a non-adherent culture system, and to the rigorous exclusion of pyrogens from the culture medium. On the other hand, these cells are responsive to in vitro stimulation, as shown, e.g., by TNF induction, increase in PCA, or by generation of reactive oxygen species upon appropriate triggering. TNF was determined at the mRNA level and by bioassay, and the specificity was proven by antibody neutralisation. The increase in PCA is assumed to reflect an increase in tissue factor expression at the cell surface (Edwards and Rickles 1984, Luther et al 1990). The chemiluminescence method used to demonstrate reactive oxygen species monitors for the presence of superoxide (Minckenberg and Ferber 1984), the direct product of the NAD(P)H-dependent oxidase. It is proposed, therefore, that the cells described here are suitable tools for biochemical studies, investigations on pathogenesis and on host cell-pathogen interaction. This study afforded an opportunity to test the effect of interferon-y, the cytokine regarded to be most important for macrophage activation. We could demonstrate that recaIFNy decreased erythrophagocytosis in a dose-dependent manner, increased zymosan-induced superoxide generation, and up-regulated macrophage MHC-class II molecule expression. Moreover, it induced macrophage PCA. These effects have all been reported in one or more other species (Steeg et al 1982. Adams and Hamilton 1984, Jungi et al 1989, Schwager and Jungi 1994, Adler et al 1995). Similar effects were mediated by supernatants from activated peripheral blood mononuclear cells, which were assumed to contain cytokines, particularly T-cell derived cytokines. That the supernatants had an even stronger effect may have one of the following reasons: (i) the supernatant effects may be due to INF-y at optimal concentrations; (ii) these effects might be due to IFN-Y acting in synergy with other
macrophages
131
cytokines; (iii) these effects might be mediated independently by several cytokines in supematants. When extrapolating from other species, the first hypothesis is the most probable; whether this is also true for the dog can be determined when blocking cytokine-specific antibodies become available. An important antimicrobial effector pathway of activated macrophages is the expression of inducible nitric oxide synthase (iNOS), resulting in the generation of NO. However, although firmly established for rodent macrophages, the role of iNOS induction in macrophages of other species is highly controversial. In human macrophages, for example, NO is generated in low amounts. at best, and activation requirements for iNOS induction appear to be different from those of rodent counterparts (James and Nacy 1993, Keller et al 1993, Schneemann et al 1993, Albina 1995). When surveying the results obtained in other species, it appears that chicken, mouse, rat and cattle have iNOS high responder macrophages, when tested under in vitro conditions. Human, pig, goat, sheep and rabbit macrophages appear to be low-responders, when judged from the amount of nitrite produced in vitro upon maximal stimulation by bacteria and cytokines combined. However, when looking for iNOS expression in activated macrophages, a minority of cells expressing iNOS is clearly detectable by immunocytochemistry, suggesting that this method allowing single-cell analysis is more sensitive than the Griess reaction (Jungi et al 1997). It was therefore of great interest to test whether canine BMM can be triggered to express iNOS. Using a polyclonal antibody specific for murine iNOS and crossreacting with iNos of a large panel of species, iNOS expression was observed in a minority of macrophages in culture, but a significant elevation of nitrite or nitrate levels was not observed in cultures of activiated canine macrophages. Thus, canine macrophages are also low iNOS responders. This emphasises the necessity of investigations in the species of interest rather than deduction of results obtained with laboratory animals. Interestingly, a recent comparison between species with regard to iNOSexpression in vivo, in foci of bacterial infection and in vitro, following stimulation by killed bacteria and cytokines, showed that the difference in iNOS expression between high responder and low responder species is much smaller in vivo, e.g. in the brains or listeric ruminants (Jungi et al 1997). It will be of interest, therefore, to look for iNos expression in lesions of dogs suffering from infections. In conclusion, we show that we can obtain canine BMM using a non-adherent culture system in vitro. These cells are dividing and differentiating in vitro. The differentiated cells perform functions characteristic for macrophages. and express lineage-specific cell surface antigens. Canine macrophages cultured in vitro can be activated by IFN-Ylike those of other species, and they share with human or porcine macrophages their restricted and inefficient expression of iNOS upon activation by conventional bacterial and cytokine stumuli.
ACKNOWLEDGEMENTS This work was supported by grants from the Swiss National Science Fund to AT (grant no. 32-44483.95 and 32-46784.96) and TWJ (grant no. 31-41403-95). The authors thank Professor Marc Vandevelde for useful comments. The generous gift of newborn puppies by Novartis,
I.12
A. Tipold, A. Zurhriggen,
P. Moore, V. Sch@s, T. W. Jungi
Basel, Switzerland, is gratefully acknowledged. The expert technical assistance by Mrs Hedi Pfister and Mrs Marija Brcic is appreciated.
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GRANGER, D. L., HIBBS, J.B. JR. & BROADNAX, L. M. (1991) Urinary nitrate excretion in relation to murine macrophage activation. Influence of dietary Larginine and oral NG-monomethyl-L-arginine. Journal of Immunology 146, 1294-J 302 HAIG, D. M., THOMSON, J. & DAWSON, A. (1991) Reactivity of the workshop monoclonal antibodies with ovine bone marrow cells and bone marrow-derived monocytelmacrophage and mast cell lines. Veterinary Immunology and Immunopathology 27, 135-145 HIBBS. J. B. JR., TAINTOR, R. R. & VAVRIW, 2. (1987) Macrophage cytotoxicity: role for L-arginine deaminase and immune nitrogen oxidation to nitrite. Science 235,473-476 JAMES, S. L. & NACY, C. (1993) Effector functions of activated macrophages against parasites. Current Opinion in Immunology 5, 518-523 JUNGI, T. W. (1985) A rapid and sensitive method allowing photometric determination of erythrophagocytosis by mononuclear phagocytes. Journal of Immunological Methods 82. 141.153 JUNGI, T. W., RUEGG, S. J. & MORELL, A. (1989) Interferon gamma-treated human macrophages display enhanced cytolysis and generation of reactive oxygen metabolites but reduced ingestion upon Fc receptor triggering. Human lmmunolog~~ 24, 77-93
JUNGI. T. W. (I 990) A turbidimetric assay in an ELBA reader for determination of mononuclear phagocyte procoagulant activity. Journal of Immunological Methods 133,21-29 JUNGI, T. W., ADLER, H., ADLER, B., THGNY, M., KRAMPE, M. & PETERHANS, E. (1996a) Inducible nitric oxide synthase of macrophages. Present knowledge and evidence for species-specific regulation. Veterinmy Immunology and Immunopathology
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JUNGI, T. W., FRANCEY, T. & PETERHANS, E. (1996b): Liquid culture of bone marrow macrophages. Chapter 31.8. In Ed I. Lefkovits, Immunology Methods Manual, London, Academic Press. pp 2107-2112 JUNGI, T. W., THGNY, M., BRCIC, M., ADLER, B., PAULI, U. & PETERHANS, E. ( 1996~) Induction of nitric oxide synthase in bovine mononuclear phagocytes is differentiation stage-dependent. Immunobiology 195, 385-400 JUNGI, T. W., PFISTER, H., SAGER, H., FATZER, R., VANDEVELDE, M. & ZURBRIGGEN, A. (1997) Inducible NO synthase expression in brains of List&a monocytogenes-infected ruminants. A comparison between cattle, sheep and goat, and with macrophages stimulated in vitro. Infecfion and Immunity 65,5279-5288 JUNGI, T. W. & PETERHANS, E. (1988) Change in the chemiluminescence reactivity pattern during in vitro differentiation of human monocytes to macrophages. Blut 56.213-220
KELLER, R., KEIST, R., JOLLER, P. & GROSCURTH, P. (1993) Mononuclear phagocytes from human bone marrow progenitor cells; morphology, surface phe notype, and functional properties of resting and activated cells. Clinical md Experimental Immunology 91. 176182 KELLER, R. & KEIST, R. (1986) Induction, maintenance, and reinduction of tumoricidal activity in bone-marrow-derived mononuclear phagocytes hy macrophage-activating lymphokines. Cellulur Immunology 101, 659-666 LEWIS, C. E. & MCGEE, .I. O’D. (1992) The Natural Immune System. The macrophage. Oxford, IRL Press LUTHER, T., FLOSSEL, C.. HIETSCHHOLD, V., KOSLOWSKI. R. & MULLER. M. (1990) Flow cytometric analysis of tissue factor (TF) expression on stimulated monocytes comparison to procoagulant activity of mononuclear blood cells. Blut 61, 375.378 MAUEL, J. & DEFENDI, V. (1971) Infection and transformation of mouse pentoneal macrophages by Simian virus 40. Journal r,fE.t-perimental Medicine 134. 335 MINCKENBERG, I. & FERBER, E. (1984) Lucigenin-dependent chemiluminescence as a new assay for NAD(P)H-oxidase activity in particulate fractions of human polymorphonuclear leukocytes. Journal ojlmmunological Methods 71.6 l-61 MOSMANN, T. R. (1983) Rapid calorimetric assay for cellular growth and survival. Application to proliferation and cytotoxicity assays. Journal of Immunological Methods
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NAKAGAWARA, A. & NATHAN, C. F. (1983) A simple method for counting adherent cells: application to cultured human monocytes, macrophages and multinucleated giant cells. Journal of Immunological Methods 56,261.268 NATHAN, C. (1992) Nitric oxide as a secretory product of mammalian cells. FASEB Journal 6, 305 l-3064 PAULI, U., BEUTLER, B. & PETERHANS, E. (1989) Procine tumor necrosts factor alpha: cloning with the polymerase chain reaction and determination of the nucleotide sequence. Gene 81, 185-191 PAULI, U., BERTONI, G., DUERR, M. & PETERHANS, E. (1994) A bioassay for the detection of tumor necrosis factor from eight different species: evaluation of neutralization rates of a monoclonal antibody against human TNF-alpha. Journal of Immological Methods 171.263-265 PAULI, U. (I 995) TNF - a review. Veterinary Immunology and Immunopatholog~ 47, 187-20 1 RUEGG, S. J. & JUNGI, T. W. (1988) Antibody-mediated erythrolysis and erythrophagocytosis by human monocytes, macrophages and activated macrophages. Evidence for distinction between involvement of high-affinity and low-affinity receptors for IgG by using different erythroid target cells. Immunology 63.5 13.520 SAGER, H., DAVIS, W. C., DOBBELAERE, D. A. E. & JUNGI, T. W. (1997) Macrophage-parasite relationship in theileriosis. Reversible phenotypic and functional dedifferentiation of macrophages infected with Theileria nnnulmu. Journal ofLeukocyte Biology 61,459-468 SAMBROOK, J., FRITSCH, E. F. & MANIATIS, T. (1995) Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laboratory Press SCHNEEMANN, M., SCHOEDON, G., HOFER, S., BLAU, N., GUERRERO, L. 81 SCHAFFNER, A. (1993) Nitric oxide synthase is not a constituent of the antimicrobial armature of human mononuclear phagocytes. Journal of Infrctiom Diseases 167, 1358.1363 SCHWAGER, I. & JUNGI, T. W. (1994) The effect of human recombinant cytokines on the induction of macrophage procoagulant activity. Blood 83, 152.160 STEEG, P. S., MOORE, R. N., JOHNSON, H. M. & OPPENHEIM, J. J. (1982) Regulation of murine macrophage la antigen expression by a lymphokine with unmune interferon activity. Journal ofExperimental Medicine 156, 1780 STEWART, C. C. (1981) Murine mononuclear phagocytes from bone marrow. In Ed D. 0. Adams, et al, Methods of Studying Mononuclear Phagocytes. New York, Academic Press. pp 5-20 SUNDSTRGM, C. & NILSSON. K. (1976) Establishment and characterization of a human histiocytic lymphoma cell line (U-937). International Journal of Cancer 17,565.577 SUNG, Y. J., HOTCHKISS, I. H., AUSTlC, R. E. & DIETERT, R. R. (1991) LArginine-dependent production of a reactive nitrogen intermediate by macrophages of a uricotelic species. Journal ojLeukocytr Biology 50.49-56 TUREK, J. J., SCHOENLEIN, I. A., CLARK, L. K. & VAN ALSTINE, W. G. (1994) Dietary polyunsaturated fatty acid effects on immune cells of the porcine lung. Journal ofLeukocyte Biology 56,599-604 VASSALLI, P. (1992) The pathophysiology of tumor necrosis factors. Annual Reviews in Immunology lo,41 l-452 WUNDERLI, P. S. & FELSBURG, P. J. (1989) An improved method for the isolation of enriched canine peripheral blood mononuclear cell and peripheral blood lymphocte preparations. Veterinaq Immunology and Immunopathology 20, 335344 ZIEGLER-HEITBROCK, H. W. L., THIEL, E., FUTTERER, A., HERZOG, V., WIR’IZ, A. & RIETHMiiLLER, G. (1988) Establishment of a human cell line (Mono Mac 6) with characteristics of mature monocytes. International Journal ojCancer 41,456.461 ZUCKER, K., LU, P., FULLER, L., ASTHANA, D., ESQUENAZI, V. & MILLER, J. ( 1994) Cloning and expression of the CDNA for canine tumor necrosis factoralpha. Lymphokine and Cytokine Research 13, 191-196 ZURBRIGGEN, A., MULLER, C. & VANDEVELDE, M. (1993) In situ hybridization of virulent canine distemper virus in brain tissue using digoxygenin labeled probes. American Journal of Veterinary Science 54, 1457.1461 Accepted
September 4, 1997
in Veterinar?;
Science
1998, 64, 125-132
Generation and functional characterisation of canine bone marrow-derived macrophages A. TIPOLD, Institutes of Veterinary Virology and Animal Neurology, University of Berne, A. ZURBRIGGEN, Institute of Animal Neurology, University of Beme, P. MOORE, VM Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, USA, V. SCHIJNS, Inter-vet, Boxmeer, The Netherlands, T. W. JUNGI*, Institute of Veterinary Virology, University of Berne, Berne, Switzerland
SUMMARY culture of bone marrow cells from the femurs of canine pups at high concentrations of fetal calf serum under non-adherent conditions allowed the proliferation and differentiation of mononuclear phagocyte lineage cells, as evidenced by morphology and CD14 expression. Cells from other lineages progressively diminished in numbers. Cells collected between 12 and 19 days of culture expressed an array of macrophage activities including ingestion of opsonised erythrocytes, generation of superoxide. up-regulation of procoagulant activity and synthesis of tumour necrosis factor (TNF) upon appropriate stimulation. TNF production was enhanced when the cultures were simultaneously stimulated with canine recombinant, or supematant-derived, interferon-y. In contrast, low levels of inducible nitric oxide (NO) synthase were expressed by only a minority of stimulated macrophages, and nitrite could not be detected in the medium. Therefore, canine macrophages generated by this novel culture system resemble human macrophages in their inefficient and restricted generation of NO upon appropriate stimulation. A
MACROPHAGES are important elements in host defence and are homeostatic regulators of a broad array of physiological reactions related to immunity, repair and regeneration (Lewis and McGee 1992). They are also incriminated as mediators of pathologic processes in a variety of conditions. Depending on the environment in which bone marrow-cell-derived cells differentiate, they exert widely different functions, e.g. those mediated by tissue macrophages, Kupffer cells, microglia cells of the brain, or bronchoalveolar macrophages. Depending on the species, macrophages from different compartments are available for study. Bone marrow-derived macrophages (BMM) have mainly been studied in rats and mice (Stewart 198 1, Cooper et al 1984, Keller and Keist 1986). In order to study macrophages in large numbers and uninfluenced by tissuespecific differentiation-promoting conditions, it would also be desirable to have access to BMM in other species, ‘in particular those of veterinary interest. Recent reports from our laboratory showed that it is possible to generate large numbers of functionally active BMM in sheep and cattle (Francey et al 1992a,b, Adler et al 1994, Jungi et al 1996b). In the present study we explored a similar culture system adapted to the generation of canine BMM (Brtigger et al 1992). We show that bone marrow cells from femurs of newborn puppies proliferate and differentiate in culture to exhibiting functional properties of mature BMM, macrophages. Amongst the variety of functions tested we selected erythrophagocytosis via Fc receptor, and the capability to respond to stimuli by eliciting a respiratory burst, two prototypic functions of professional phagocytes. The former function is known to be depressed upon macrophage activation by interferon-y, whereas the latter is enhanced (Jungi et al 1989). Furthermore, a key function of macrophages is * Corresponding author E-mail:
[email protected]
0034.5288/98/020125
+ 08 $18.00/O
their ability to respond to lipopolysaccharide (LPS), e.g. by secretion of cytokines such as tumour necrosis factor (TNF) (Vassalli 1992). LPS is also known to trigger the production of nitric oxide (NO), a mediator thought to be essential for antimicrobial and antitumoural host defence. No generation is catalysed by inducible nitric oxide synthase (iN0.S) (Hibbs et al 1987, Nathan 1992) which is induced by LPS and certain cytokines or combinations of cytokines and bacterial components. However, although readily induced in rodent, bovine and avian macrophages (Sung et al 1991, Nathan 1992, Jungi et al 1996a), induction of strong iNOS expression and NO generation production have not been convincingly demonstrated in vitro, in macrophages of other species such as human, goats, pigs and sheep (Albina 1995, Keller et al 1993, Schneemann et al 1993, Adler et al 1996, Turek et al 1994, Jungi et al 1997). It was therefore of particular interest to determine whether iNOS is inducible in canine BMM by conventional stimuli, such as bacterial components and cytokines.
MATERIALS
AND METHODS
Animals Newborn Swiss beagle puppies were generously provided by Novartis, Basel, Switzerland. Dogs were euthanised with pentobarbiturate, and femurs were collected under sterile conditions. Collection of bone marrow cells The femurs were excised and cut open on both ends of the bone. The bone marrow was flushed out using a syringe and needle with RPM1 1640 under sterile conditions. Cells were washed with RPMI 1640 medium, centrifuged and resuspended at a concentration of 2 x lo5 cells ml-t in culture medium (see below). 0 1998 W. B. Saunders Company Ltd
Bone marrow cell culture The cells from three to four puppies were mixed and cultured together. They were sealed in hydrophobic (teflon) bags as described (Brtigger et al 1992, Francey et al 1992b, Adler et al 1994), and the bags were placed in a humid atmosphere containing 5 per cent CO, in air at 37°C for up to 19 days without change of the medium. This consisted of RPMI 1640 supplemented with 20 per cent foetal calf serum (FCS; Life Technologies, Paisley, UK), penicillin (100 IU ml-l), streptomycin (100 pg ml-t), sodium pyruvate (1 mM), glutamine (2 r&l), MEM vitamin solution (1 per cent v/v; Life Technologies), non-essential amino acid solution (1 per cent v/v; Life Technologies), HEPES (10 n&I), and folic acid (40 mg litre-I). For studies in which NO generation was determined, Iscove’s modified Dulbecco’s minimum essential medium instead of RPMI 1640 was used. To demonstrate functional activity of the mature canine macrophages, they were harvested between day 12 and 19. Cells harvested every two or three days were used for flow cytometry and for morphological characterisation, using Diff-Quik staining of cytospin preparations. To determine the proportions of cell subsets, two samples of 200-300 cells from two independent experiments were counted using light microscopy, and mean values were determined. In other experiments, cells were harvested from teflon bags and subcultured in microtitre plates (6-8 x lo4 macrophages per well). After two hours of adherence, they were exposed either to lipopolysaccharide (LPS; Escherichia coli 055:B5, Sigma Chemical Company, St. Louis, MO, 1 pg ml-t), or to 200 pg ml-t heat-killed (two hour 60°C) Salmonella dublin (strain 24-90) or Listeria monocytogenes (NCTC strain 10,527, serotype 4b), in the presence or absence of various cytokines. These were recombinant canine interferon-y (rcatm-Y), recombinant bovine interleukin- 1B (rboIL- 1) kindly provided by American cyanamid (Princeton, NJ), recombinant human interleukin- 1p (Peprotech, London, UK), recombinant bovine TNF (Novartis), and recombinant human TNF (Eurocetus, Amsterdam, The Netherlands). rcatt+y was derived as an expression product of insect cells (Sf-21 cells) after infection with recombinant baculovirus expressing the canine IFN-y CDNA behind its pl0 promotor. Its biological activity was determined in a classical vesicular stomatitis virus inhibition assay on canine MDCK cells (De Groot and Schijns, unpublished observations). rcaIFN-y was dissolved at 2 x lo4 to 2 x 1O5 antiviral activity units per ml, and loo-fold and lOOOfold dilutions were used in experiments. The other cytokines were tested at 10 and 100 ng ml-t. Flow cytometry The expression of leukocyte surface markers was determined by indirect staining with monoclonal antibodies specific for, or crossreacting with, canine Major Histocompatability Complex Class II (MHC-II) molecules (clone Ca2.1C12), or canine CD14 (clone TUK4, Dako, Glostrup, Denmark). The second antibody was phycoerythrin-labeled goat-anti-mouse IgG F(ab’)z (Jackson Immunoresearch Laboratories, West Grove, PA). Stained cells were analysed in a FAcscan flow cytometer (BectonDickinson, San Jose, CA), using the PC-LYSYS software. Immunocytochemistry Macrophages from teflon bags were subcultured in 24well plates containing a glass coverslip and were stimulated
as described above. After overnight culture, the monolayers were fixed with paraformaldehyde (2 per cent v/v in PBS) for 20 minutes. The cells were then washed twice, followed by Fc receptor blockade with 10 mg ml-) of human IgG for 20 minutes, at which time IgG was replaced by anti-iNos at a dilution of 1:500 (UPI, Lake Placid, NY). Specimens were developed by using the ABC staining system (Immunodiffusion, Geneva, Switzerland). In situ hydridisation A probe specific for canine TNF was cloned after RT-PCR using primers based on sequences conserved between species (Pauli et al 1989, Pauli 1995). As a template, 14day-old canine macrophages harvested from teflon bags and stimulated for six hours with LPSwere used for production of RNA as described (Sambrook et al 1995). Using this method a portion of the canine TNF gene containing 300 base pairs was amplified, cloned and sequenced. The identity of the nucleotide sequence of the clone with canine TNF (Zucker et al 1994) was confirmed by sequence alignment. In situ hybridisation was performed with a riboprobe complementary to mRNA of canine TNF and labelled with digoxigenin (Boehringer-Mannheim, Mannheim, Germany) as described (Zurbriggen et al 1993). The corresponding antisense probe was used as a negative control. Hybridisation was performed at 52°C overnight. The probe was visualised by staining with a digoxigenin-specific phosphatase-conjugated antibody (anti-digoxigenin-AP, Fab fragments: Boehringer-Mannheim). Phagocytosis Phagocytosis of opsonised sheep erythrocytes was measured by a photometric procedure (Jungi 1985, Rtiegg and Jungi 1988). BMM monolayers in 96-well plates were covered with erythrocytes opsonised with affinity-purified sheep erythrocyte-specific antibodies (Diamedix, Miami, FL) and after a 60 minute ingestion period non-attached and attached non-ingested erythrocytes were removed by washing and hypotonic lysis. The monolayer containing ingested red blood cells was lysed with sodium dodecyl suphate, followed by the addition of diamino benzidine and H,O,. The evolving colour reaction, as determined in an ELISA reader at 450 nm, was proportional to the number of ingested erythrocytes. The number of macrophage nuclei was determined in parallel wells by lysing them with the mild detergent, cetavlon, and staining the released nuclei with amidoblack (Nakagawara and Nathan 1983). This allowed ingestion to be expressed as a phagocytic index (ingested red blood cells per nucleus). Superoxide determination Superoxide was determined in BMM subjected to various pretreatments and harvested from teflon bags by chemiluminescence, using lucigenin as an enhancer (Jungi and Peterhans 1988, Rtiegg and Jungi 1988). BMM were resuspended in Hanks’ balanced salt solution at 0.2 x 106 ml-t containing lucigenin (50 pM, Sigma) and dark-adapted in 11 x 47 mm polystyrene tubes (0.5 ml tube-‘). They were stimulated with either phorbol 12-my&ate 13-acetate (PMA; 1O-7 M), or with serum-opsonised zymosan (50 pg ml-‘) and immediately thereafter, the evolving chemiluminescence response was recorded in a LB 950 luminometer (Berthold, Wildbad, Germany).
Canine bone marrow-derived
TABLE 1: Characterisation of bone marrow derived cells different time points after onset of non-adherent culture Morphology* Culture time (days)
Moncqtelike cells w
2 5 7 8 9 12 15
14 15 4 6 3 3 1
* Morphology Diff-Quik
Procoagulant
at
Flow cytometry
Macrophagelike cells (“A) 5 20 80 80 85 90 97
was studied
harvested
Lymphoid and undiff. cells W)
Granulocytes (“M
46 50 10 9 5 2 2
in cytospin
preparations
CD14 positive ceils (%) 6 11 55 51 04 95 93
35 15 6 5 7 5 0 which
were
stained
with
activity
Procoagulant activity (PCA) was measured in BMM harvested from teflon bags and subcultured in microtitre plates in culture medium containing 5 per cent FCS. It was determined by a turbidimetric recalcification time assay, using c&rated human platelet-depleted plasma (Jungi 1990, Briigger et al 1992), and expressed in human thromboplastin units, 1 unit representing recalcification time by 106fold diluted human thromboplastin (Thromborel S, Behring-Werke, Marburg, Germany). Production
of cytokine-enriched
supematants
Mononuclear cells were isolated as described (Wunderli and Felsburg 1989). They were cultured in 25 cm2 tissue culture flasks in the presence of A23187 (0.25 pM) and
FIG 1: Cytospin preparations were stained with Diff-Ouik
of freshly collected canine (A-C) or for surface-expressed
macrophages
127
PMA (5 ng ml-t), at 106 cells ml-l (total volume 5 ml). The supernatant was collected after 48 hours and stored frozen. This supernatant was considered to be rich in cytokines, including interferon-y, as evidenced by its effect on erythrophagocytosis, respiratory burst activity, and MHC classII modulation (see Results section). TNF bioassay BMM were stimulated with 1 pg ml-l LPS for six hours in teflon bags. TNF activity of supernatants from stimulated and control cells was determined by a cytotoxicity assay, using the porcine kidney cell line, PK(15) cells as targets. These are susceptible to TNF from numerous species including the dog (Pauli et al 1994). Cytotoxicity was determined by staining with MTT (Mosmann 1983). TNF specificity was verified with neutralising antibodies specific for human TNF and crossreacting with TNF from several different species (Monosan, Uden, The Netherlands). Griess assays
The content of nitrite, a stable metabolite of NO, was determined in supematants of BMM that had been subjected to various pretreatments for various length of time, using the Griess reaction (Adler et al 1995). In some assays, the total content of nitrate and nitrite was determined by conversion of nitrate into nitrite, followed by the Griess reaction (Jungi et al 1996~). Nitrate was converted into nitrite by treatment of supematants with Pseudomonas oleovorans bacteria which contain a high amount of nitrate reductase, but no nitrite reductase (Granger et al 1991). Treatment was
bone marrow cells (A), and of cells cultured CD14 by immunocytochemistry (D)
for seven
days (B), or 14 days (C, D). They
128
A. Tipold, A. Zurbriggen, P. Moore, V. Schijzs, T. W.Jungi
Fluorescence
intensity
Fig 2: Expression of MHC-class-11 molecules (MHC-II) (upper panel) or CD14 (lower panel) by BMM cultured in the presence (right or absence of IFN-y (feft panel). BMM in teflon bags were exposed for 48 hours to IOCQ-fold diluted recalFN-y (c), 10%fold-diluted y(d) or to four-fold diluted supematant of activated mononuclear cells (e). Histograms of a flow cytometric analysis are shown. resents unstimulated control cells, (b) shows the fluoresence emitted by control cells treated with an isotype control antibody ground fluorescence). The latter did not change by pre-treatment of BMM
performed for 90 minutes at 37°C with a 0.25 per cent v/v content of bacteria, and complete conversion was verified by nitrate-containing control samples.
RESULTS Generation of macrophages in cultures of canine bone marrow cells
Bone marrow cells harvested from femurs of new-born puppies were cultured under non-adherent conditions, in teflon bags, in the presence of high concentrations of FCS. There was a progressive increase in the proportion of CD16positive cells (Table l), and of cells displaying morphological characteristics of monocytes and macrophages, as determined by morphological criteria (Table 1, Fig 1). The cell population developed from a heterogeneic population to a rather homogeneous population of macrophages at day 14 (Fig 1). During a period of one to two weeks, not only the proportion, but also the absolute number of cells showing macrophage characteristics increased. The total cell number harvested at day 12 to day 14 was about equal to that seeded at day 0, and roughly doubled until day 19. Functional properties BMM
of BMM
collected between day 14 and day 15 were subject-
panel) rCitlFN(a) rep(back-
ed to surface marker analysis and to functional tests. The large majority of canine BMM expressed CD14 (Fig 2) and MHC-II on their surface. MHC-II, but not CD14 expression was enhanced by pre-treatment of canine BMM with recamby, or with supematants from mononuclear cells activated with PMA and A23187. Canine BMM ingested IgG-coated sheep erythrocytes (phagocytic index: 16.5ti.3); non-opsonized erythrocytes were taken up to a much lesser degree (1.2a.3). This result suggests that canine BMM express Fc receptors, and that these are functionally linked to phagocytosis. When Bh4h4 were preexposed to supematants of activated mononuclear cells, or to rcarnv-y (lOOO-fold diluted), the phagocytosis index decreased to lO.M.6 or 9.5~331, respectively. Chemiluminescence in the presence of lucigenin indicates the generation of superoxide. Untreated mature canine BMM and BMM pre-exposed for two days to rcan%-y or to supematants of activated mononuclear cells were therefore tested for superoxide production by lucigenin-enhanced chemiluminescence (Fig 3). Both PMA and zymosan induced the production of a modest amount of superoxide. Following treatment with either rcalIFN-7 or lymphocyte supematant containing cytokines, the chemiluminescence signal was increased, particularly when zymosan was used as a stimulus (Fig 3). BMM were subcultured in 96-well plates in which they were treated with LPS. Supematants were collected after 24 hours for determining TNF activity. using a cytotoxicity
Canine bone marrow-derived macrophages
2E6
129
2E6 Control
Supernatant
t
2E6
2E6
lE6
5E5
; 0
.s 4
10
20
30
40
50
60
I 10
0
lE6
I 20
I 30
I 40
I 50
I 60 -
lE6 rcaIFN-y(lOOOx)
rcaIFN-y(lOOx)
5E5< I 10
0
I 20
I 30
I 40
I 50
1 60
5E5)
I
I
0
Time
I 10
20
I 30
I/40
50
60
(minutes)
Fig 3: Chemiluminescence emitted by canine SMM (day 14) stimulated with PMA (solid lines), zymosan (dashed lines) or mock-stimulated (dotted lines), in the presence of lucigenin. BMM had been pre-exposed to reCalFN-y (IOO-fold or lOOO-fold diluted) or to supernatant of activated mononuclear cells (four-fold diluted) for 48 hour, or were untreated control cells. Individual light emission traces are shown
assay capable of detecting TNF of several species including the dog (Pauli et al 1994). Bioactivity of the secreted TNF, as demonstrated by the killing of PK(15) cells, reached half-maximal activity at 300-fold dilution of the supernatant, and it could be specifically neutralised with antiTNF-Specific antibodies (Fig 4). Similarly stimulated cells subcultured on glass slides expressed enhanced levels of mRNA for TNF (Fig 5). We also looked for another indicator of macrophage activation, namely the enhancement of PCA, as determined in a recalcification time assay (Table 2). In this assay, IFN-y alone induced PCAwithin 24 hours, a find100
/
I
I -
I
I 3125 Dilution
I
I 125
I
I 5
I
of supernatant
Fig 4: Production of TNF bioactivity upon stimulation of BMM (day 14) with LPS (1 pg ml-l) for six hours at 37%. Cytotoxicity, as determined by MTT staining of PK(15) target cells, is expressed as percentage of total lysis (mediated by distilled water). Antibody dose-dependent neutralisation of bioactivity demonstrates the Specificity for TNF Key: 0, anti-TNF (200-fold); & anti-TNF (800-fold); 0, anti-TNF (3200-fold)
ing reminiscent of human monocyte-derived macrophages (Schwager and Jungi 1994). Likewise, LPS, heat-killed S dublin and L monocytogenes strongly enhanced PCA, and this effect increased still further by supematants of activated peripheral blood mononuclear cells (Table 2). Production of NO by canine
BMM
BMM subcultured in 96-well plates were exposed to a prototypic Gram-positive organism (L monocytogenes), a Gram-negative organism (S dublin), ,or to LPS, either alone or combined with various cytokines. Cytokines tested included rboIL- 113,rhuIL- 113,rhuTNF, rboTNF, rcaIFN-y and supematant of activated peripheral blood mononuclear cells. These cytokines were also tested alone, in the absence of bacterial stimuli, and in combination with each other. Supernatants were collected 24 or 48 hours later in order to determine their content of nitrite, one of the stable metabolites of NO. None of the treatments induced significantly elevated levels of nitrite. Likewise, the total content of nitrate and nitrite, was not significantly elevated by these procedures. Since macrophage-derived NO is thought to be due to expression of iNOS, BMM exposed to the same stimuli were stained for iNos by immunocytochemistry. Whereas control cells were unstained, there was a weak staining of a minority of cells exposed to S dublin combined with interleukin-1 (Fig 5). A combination of stimuli eliciting a strong iNOS response in mouse and cattle macrophages, L monocytogenes combined with EN-~, was even less active. The cells which produced no detectable NO were indeed activated. This was shown by their increased expression of PCA when compared with unstimulated BMM (Table 3 and data not shown).
A. Tipold, A. Zurbriggen,
P. Moore, V. Schijns, T. W. Jungi
Fig 5: In situ hybridisation for TNF (A and C): positive probe showing enhanced TNF mRNA expression (A), negative control (corresponding antisense probe) (C). Expression of iNOS by unstimulated BMM at day 14 (D), and by BMM exposed to S dub/in and 109 ng ml” rbolL-I @)
DISCUSSION In recent years, mononuclear phagocytes have entered the limelight of immunological research due to their central role in host defence, immunoregulation, regeneration and repair. Depending on environmental factors impacting on mononuclear phagocytes, these cells assume a variety of distinct functional properties. Biochemical investigation of these regulatory processes depends on the availability of high cell numbers. Macrophage type cell lines were first described for the mouse (Mauel and Defendi 1971), then for several other species (Sundstriim and Nilsson, 1976, TABLE 2: Induction bone marrow-derived
of procoagulant macrophages* Stimulus
Co-stimulus (control) rcalFNn/ rcalFN-y Supematant+
-(control)
activity
by activation
LPS
S dub/in
21.80 18.85 22.36 211.70
53.27 99.49 75.82 443.2
of canine L monocytogenes
Concentration
1OO-fold 1 OOO-fold four-fold
4.92 20.49 7.69 70.07
92.16 70.35 46.92 355.00
* Bone marrow cells were harvested from teflon bags at day 19, followed by subcufture in mfcrotitre plates. They were stimulated with the indicated stimuli two hours after onset of microtitre plate culture. Procoagulant activity was determined 24 hours after stimulation T See legend of Fig 2
Ziegler-Heitbrock et al 1988, Glass et al 1989, Haig et al 1991, Sager et al 1997). However, murine macrophage cell lines are exceptional in that cells in exponential growth express functions of differentiated macrophages. In many other situations, proliferating cells fail to express many of the surface markers characteristic for mature macrophages, and their functional activity is highly restricted (Sager et al 1997). In these situations, and in species for which macrophage type cell lines are unavailable, primary macrophages are important tools for cell biological and biochemical studies. There are as yet few reports on macrophage collection and/or generation from the dog (Brtigger et al 1992). We present here a culture system for canine macrophages, starting with bone marrow cells from newborn puppies. It allows the propagation of BMM in high numbers and these may be activated by various stimuli. This renders the cells useful for functional studies on macrophages, e.g. on the interaction with pathogens or as a substrate for biochemical analyses. Moreover, it is expected that having this culture system at our disposal, we shall learn more about the physiology and pathophysiology of macrophages in the dog. Similar culture systems have been described for rnurine BMM and for BMM from sheep and cattle (Francey et al 1992b, Adler et al 1994). However, in sharp contrast to the mouse culture system, the exogenous addition to cultured
Canine bone marrow-derived
macrophages of colony stimulating factors (CSF), e.g. macrophage-CSF (M-CSF), was neither necessary nor sufficient to grow BMM of ruminants and the same applies to dog BMM cultures. The addition of high FCSconcentrations, on the other hand, was a critical requirement (Adler et al 1994, Francey et al 1992a). In contrast to these species, mouse BMM precursors rapidly divide upon exposure to either granulocyte-macrophage-CSF (GM-CSF)or M-CSF, and they are serum-independent. Canine BMM could be propagated at high FCS concentrations and in the absence of exogenous CSF, thereby resembling ruminant BMM. However, whether cell growth and survival rate are proportional to the serum concentrations or whether they can be influenced by exogenous CSFhas not been explored in the dog. In the ruminant BMM culture systems alluded to above, either bone marrow cells of up to nine-months-old sheep (Francey 1993) or fetal bone marrow cells from fetal tibiae (Adler et al 1994) were used, and the resulting macrophages exhibited closely similar functional properties. In an earlier study of this laboratory, biopsy specimens of bone marrow were used to generate macrophages in culture (Briigger et al 1992). We assume therefore that the present canine culture system is not restricted to tibiae of newborn puppies as a starting material, but can also be applied to bone marrow biopsies. It will be interesting to see how alterations in functional and phenotypic properties of cultured macrophages from biopsy material are related to the disease prompting the biopsy. The macrophages generated by the present culture system are at a resting stage, i.e. they may be activated to assume a variety of functions upon appropriate triggering. This is related, in part, to the use of a non-adherent culture system, and to the rigorous exclusion of pyrogens from the culture medium. On the other hand, these cells are responsive to in vitro stimulation, as shown, e.g., by TNF induction, increase in PCA, or by generation of reactive oxygen species upon appropriate triggering. TNF was determined at the mRNA level and by bioassay, and the specificity was proven by antibody neutralisation. The increase in PCA is assumed to reflect an increase in tissue factor expression at the cell surface (Edwards and Rickles 1984, Luther et al 1990). The chemiluminescence method used to demonstrate reactive oxygen species monitors for the presence of superoxide (Minckenberg and Ferber 1984), the direct product of the NAD(P)H-dependent oxidase. It is proposed, therefore, that the cells described here are suitable tools for biochemical studies, investigations on pathogenesis and on host cell-pathogen interaction. This study afforded an opportunity to test the effect of interferon-y, the cytokine regarded to be most important for macrophage activation. We could demonstrate that recaIFNy decreased erythrophagocytosis in a dose-dependent manner, increased zymosan-induced superoxide generation, and up-regulated macrophage MHC-class II molecule expression. Moreover, it induced macrophage PCA. These effects have all been reported in one or more other species (Steeg et al 1982. Adams and Hamilton 1984, Jungi et al 1989, Schwager and Jungi 1994, Adler et al 1995). Similar effects were mediated by supernatants from activated peripheral blood mononuclear cells, which were assumed to contain cytokines, particularly T-cell derived cytokines. That the supernatants had an even stronger effect may have one of the following reasons: (i) the supernatant effects may be due to INF-y at optimal concentrations; (ii) these effects might be due to IFN-Y acting in synergy with other
macrophages
131
cytokines; (iii) these effects might be mediated independently by several cytokines in supematants. When extrapolating from other species, the first hypothesis is the most probable; whether this is also true for the dog can be determined when blocking cytokine-specific antibodies become available. An important antimicrobial effector pathway of activated macrophages is the expression of inducible nitric oxide synthase (iNOS), resulting in the generation of NO. However, although firmly established for rodent macrophages, the role of iNOS induction in macrophages of other species is highly controversial. In human macrophages, for example, NO is generated in low amounts. at best, and activation requirements for iNOS induction appear to be different from those of rodent counterparts (James and Nacy 1993, Keller et al 1993, Schneemann et al 1993, Albina 1995). When surveying the results obtained in other species, it appears that chicken, mouse, rat and cattle have iNOS high responder macrophages, when tested under in vitro conditions. Human, pig, goat, sheep and rabbit macrophages appear to be low-responders, when judged from the amount of nitrite produced in vitro upon maximal stimulation by bacteria and cytokines combined. However, when looking for iNOS expression in activated macrophages, a minority of cells expressing iNOS is clearly detectable by immunocytochemistry, suggesting that this method allowing single-cell analysis is more sensitive than the Griess reaction (Jungi et al 1997). It was therefore of great interest to test whether canine BMM can be triggered to express iNOS. Using a polyclonal antibody specific for murine iNOS and crossreacting with iNos of a large panel of species, iNOS expression was observed in a minority of macrophages in culture, but a significant elevation of nitrite or nitrate levels was not observed in cultures of activiated canine macrophages. Thus, canine macrophages are also low iNOS responders. This emphasises the necessity of investigations in the species of interest rather than deduction of results obtained with laboratory animals. Interestingly, a recent comparison between species with regard to iNOSexpression in vivo, in foci of bacterial infection and in vitro, following stimulation by killed bacteria and cytokines, showed that the difference in iNOS expression between high responder and low responder species is much smaller in vivo, e.g. in the brains or listeric ruminants (Jungi et al 1997). It will be of interest, therefore, to look for iNos expression in lesions of dogs suffering from infections. In conclusion, we show that we can obtain canine BMM using a non-adherent culture system in vitro. These cells are dividing and differentiating in vitro. The differentiated cells perform functions characteristic for macrophages. and express lineage-specific cell surface antigens. Canine macrophages cultured in vitro can be activated by IFN-Ylike those of other species, and they share with human or porcine macrophages their restricted and inefficient expression of iNOS upon activation by conventional bacterial and cytokine stumuli.
ACKNOWLEDGEMENTS This work was supported by grants from the Swiss National Science Fund to AT (grant no. 32-44483.95 and 32-46784.96) and TWJ (grant no. 31-41403-95). The authors thank Professor Marc Vandevelde for useful comments. The generous gift of newborn puppies by Novartis,
I.12
A. Tipold, A. Zurhriggen,
P. Moore, V. Sch@s, T. W. Jungi
Basel, Switzerland, is gratefully acknowledged. The expert technical assistance by Mrs Hedi Pfister and Mrs Marija Brcic is appreciated.
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September 4, 1997