1 Institute of Anatomy and Cell Biology, Philipps University, Robert-Koch-Strasse 6, 35033 Marburg, Germany 2 Present address: Klinik für Allgemein- und Thoraxchirurgie, Sektion Experimentelle Chirurgie, 35385 Giessen, Germany
Correspondence to: O. Stehling; E-mail: stehling{at}mailer.uni-marburg.de
Transmitting editor: T. Hünig
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Abstract |
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Keywords: apoptosis, CD161, flow cytometry, necrosis, transplantation
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Introduction |
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To elucidate the role of mononuclear phagocytes, the destruction of allogeneic organs was investigated in a rat model of acute renal allograft rejection. In this model, the rejection process was accompanied by a substantial infiltration by macrophages (9) and a strong accumulation of monocytes in the blood vessels of the graft (12). T lymphocytes amounted to a maximum of one-third of the infiltrating cells. The accumulation of mononuclear phagocytes and T lymphocytes might be explained by the expression of different CC chemokines in the transplant (13). Interstitial hemorrhage and necrotic patches occurred abruptly between post-operative day 4 and 5 (9). These events might be due to massive endothelial damage or intravascular blood coagulation (12).
Analysis of graft tissue samples and leukocytes from allograft vessels revealed the expression of mRNA for typical products of activated mononuclear phagocytes (12). These include pro-inflammatory cytokines such as IL-1ß, IL-6 and IL-12, and effector molecules such as tumor necrosis factor, inducible NO synthase (iNOS) and tissue factor. Phenotypic analysis was performed by flow cytometry using mAb ED9 for identification of monocytes among the blood mono nuclear cells (14). After allogeneic kidney transplantation, intravascular monocytes expressed surface molecules associated with activation and eventually with cytotoxicity (12,15).
Among these molecules, the NK receptor protein 1 (NKR-P1) is a very sensitive activation marker (16), but its physiological role on monocytes is not well understood. NKR-P1 was first identified in rats (17) by its ability to activate NK cells (18) and is thought to be associated with cell-mediated cytotoxicity. Up to date, six genes of the NKR-P1-family are known, NKR-P1AF (19,20), the products of which are subsumed under the designation CD161. Rat NKR-P1A has been characterized as an activating receptor and rat NKR-P1B as an inhibitory receptor (21).
In the present study, cytotoxicty of in vivo activated monocytes was investigated in vitro using a flow cytometry-based cytotoxicity assay that enables simultaneous analysis of effector and target cells at the single-cell level. The cytotoxic potential of activated monocytes was analyzed using the classical NK-sensitive mouse lymphoma Yac-1 and the NK-insensitive, NO-sensitive mouse mastocytoma P815 as targets. Due to their different sensitivities, both cell lines are useful target cells to give clues as to the killing mechanisms of various effector cells, thus allowing us to standardize comparison of monocytes to NK cells. Yoshida et al. (22,23) demonstrated the usefulness of tumor cell lines for unraveling the role of macrophages during allogeneic skin graft rejection in mice. The present study clearly demonstrated an enhanced monocyte cytotoxicity during allogeneic kidney graft rejection in vitro, suggesting a possible contribution of monocytes to renal allograft damage in vivo.
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Methods |
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Antibodies and secondary reagents
Specificity and source of primary monoclonal mouse anti-rat antibodies are listed in Table 1. RP-1 was a kind gift of F. Sendo (Department of Parasitology, Yamagata University School of Medicine, Yamagata, Japan). Dynabeads M-450 (110.24) coated with pan-anti-mouse IgG were obtained from Dynal (Hamburg, Germany). Unconjugated rabbit anti-mouse Ig (Z 109) was purchased from Dako (Hamburg, Germany).
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Isolation of monocytes and NK cells
Monocytes were harvested by perfusion of the renal or extrapulmonary vasculature as described (12,15,25). The renal vasculature of day-4 graft recipients (LEW to LEW or DA to LEW) was perfused with 150 ml of ice-cold M199/BSA through catheters inserted into the vena cava inferior and the abdominal aorta. Perfusion of the whole extrapulmonary vasculature was performed through cannulae (14G and 17G; Braun, Melsungen, Germany) inserted into the left and right ventricle of the beating heart using ice-cold M199/BSA. Perfusate cells were always chilled on ice and analyzed by flow cytometry, using phycoerythrin (PE)-conjugated mAb ED9 and FITC-conjugated mAb OX-19, OX-33 and 3.2.3. An isotype-matched irrelevant mAb (X40) was always included as a control.
Perfusate cells recovered from individual animals were subjected to Percoll density gradient centrifugation to deplete erythrocytes and granulocytes (12,15,25). Monocytes were further purified by immunomagnetic negative selection as described previously (25). Interphase cells (peripheral blood mononuclear cells) were incubated with a cocktail of mAb R73, OX-19, G53-238, OX-33 and OX-8, and labeled lymphocytes were depleted by pan-anti-mouse IgG Dynabeads M-450. Purity of the remaining monocytes was analyzed by flow cytometry.
After perfusion, spleens were removed and passed through a steel mesh. Spleen homogenates were filtered through a nylon wool column in order to deplete cellular aggregates and were subsequently subjected to Percoll density gradient centrifugation. Interphase cells were incubated with a cocktail of mAb R73, OX-19, G53-238, OX-33, ED9 and OX-43, and labeled leukocytes were depleted by pan-anti-mouse IgG Dynabeads M-450. The composition of the NK cell suspensions was analyzed by flow cytometry.
Cell lines
CRNK-16, an NK cell leukemia line from F344 rats (26), was kindly provided by Dr J. R. Ortaldo (National Cancer Institute, Laboratory of Experimental Immunology, Frederick, MD). Yac-1, a Moloney virus-induced, NK-sensitive mouse lymphoma, and P815-1-1, a NO-sensitive mouse mastocytoma (27), were obtained from the European Collection of Cell Cultures (Salisbury, UK). All cells were grown in polystyrene tissue culture flasks for suspension cells (Sarstedt, Nümbrecht, Germany) using complete RPMI 1640 (RPMI 1640 supplemented with 10% FCS, 1% non-essential amino acids, 2 mM glutamine, 1 mM sodium pyruvate and 50 µM 2-mercaptoethanol).
Immunoprecipitation of CD161
Immunomagnetically purified monocytes, splenic interphase cells or CRNK-16 cells were washed in PBS and resuspended in cold biotinylation buffer (50 mM boric acid and 150 mM sodium chloride, pH 8.0) at a concentration of 10 x 106 cells/ml. Biotin-7-NHS was dissolved in DMSO (99.5% p.a.) and added to the cell suspension at a final concentration of 50 µg/ml. After incubation for 30 min at 4°C on a sample mixer, the cells were washed twice with PBS/1% BSA, counted and centrifuged.
Cell lysis was performed according to Reske et al. (28). Detergent-insoluble material was removed by centrifugation at 2500 g for 10 min and by ultracentrifugation at 200,000 g for 1 h. Cleared supernatants were successively pre-treated by the addition of normal rat serum (100 µl per 1 ml extract) for 1 h, and of Protein A Insoluble and Protein ASepharose Cl-4B for 30 min respectively. For immunoprecipitation, 20 µl of mAb 3.2.3 (1 mg/ml) was coupled to Protein ASepharose, diluted in 80 µl Tris-buffered saline (pH 7.4) containing 0.25% Igepal CA-630 and 0.1% BSA. The mAb-coupled Protein ASepharose was washed and incubated with the pre-cleared cell extracts for 1 h at 4 °C. After washing, retained immune complexes were eluted by boiling in Laemmli sample buffer.
For SDSgel electrophoresis and subsequent blotting, protein samples representing 1.5 x 106 monocytes were applied under reducing conditions. As monocyte samples were contaminated with NK cells, samples of splenic interphases representing corresponding numbers of NK cells were applied for comparison. Splenic interphase samples, comprising 0.15 x 106 NK cells, and CRNK-16 samples, containing 0.15 x 106 cells, were always included as positive controls. SDSgel electrophoresis was carried out according to Laemmlis procedure. Proteins were electrotransferred onto Immobilon-P sheets (Millipore, Bedford, MA) by tank blotting (25 mM Tris, 200 mM glycine, 0.05% SDS and 20% methanol) at 90 mV for 5 h and were visualized with a streptavidinperoxidase chemiluminescence assay (Boehringer, Mannheim).
Cytotoxicity experiments
Before co-incubation with effector cells, Yac-1 or P815-1-1 target cells were labeled with 13.3 and 40 µM FITC respectively, in order to discriminate target and effector cell populations (Fig. 1). Unbound FITC was eliminated by two washes with M199/10% FCS (M199/FCS) and incubation with M199/50% FCS for 10 min.
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After co-incubation, samples were washed twice with M199/BSA/2.7 mM EDTA and stained with 100 µl of the supravital dyes MC 540 (14 µM) or PI (4 µM) for 30 and 3 min respectively. Before PI staining, monocyte samples were incubated with M199/BSA/EDTA for 25 min in order to detach adherent cells. After incubation, cells were washed with M199/BSA/EDTA (without phenol red) and kept on ice until flow cytometry.
Flow cytometry and data analysis
Flow cytometry was performed on a FACScan device (Becton Dickinson, Heidelberg, Germany). Between 7500 and 50,000 events were acquired depending on the target cell number. Cellular debris and erythrocytes were excluded by gating. Cytotoxicity was defined as the cellular damage identified by staining with PI or MC 540 (Fig. 1). For cell size analysis of damaged target cells, target cell subpopulations positive for MC 540 or positive for PI were gated and investigated for their physical parameters. The percentages of small and large damaged target cells were used to further analyze the cytotoxic mechanisms of NK cells and monocytes (29).
Cell-mediated cytotoxicity was expressed by the cytotoxicity coefficient (29).
is a single value that unequivocally describes the cytotoxic activity of effector cells (E) and, moreover, it is absolutely necessary for subtracting the contribution of contaminating NK cells to monocyte cytotoxicity (29).
was deduced by plotting the specific cytotoxicity against the reciprocal value of the proportion of target cells (T) in one sample: 1/[T/(Ec + T)] = (Ec + T)/T = (Ec/T) + 1. This term is an equivalent to the effector:target (E:T) ratio and can also be interpreted as the dilution of target cells by effector cells. Determination of specific cytotoxicity always included corrections for the purity of effector cells (Ec) and for the non-specific cell death of target cells (29).
The term (Ec + T)/T allows plotting the specific cytotoxicity of control samples at the origin of a semilogarithmic coordinate system, showing a linear relation between the specific cytotoxicity of effector cells and the dilution of target cells (see Fig. 2). The slope of a regression line from the origin to the specific cytotoxicity at various target cell dilutions represents the cytotoxicity coefficient , indicating the cytotoxicity of effector cells at a proportion of 10% target cells:
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As monocyte cytotoxicity was markedly lower than that of NK cells, great care was taken to estimate the contributions of NK cell-mediated target cell death (29):
contribution of contaminating NK cells = NK x log [(NK cell contamination/T) + 1].
Subsequently, the contribution of contaminating NK cells was subtracted from the specific cytotoxicity of immunomagnetically purified monocytes. The mean cytotoxicity coefficient of NK cells (NK) was estimated in experiments with splenic NK cells (29) because NK cells were difficult to isolate from renal perfusates. There are no hints that blood NK cell cytotoxicity differs from spleen NK cells (30,31).
Data presentation and statistics
SD were calculated for all mean values. Groups were preferentially compared by non-parametric tests. Multiple comparisons were performed by ANOVA. Where non-parametric tests were not available, parametric tests were used.
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Results |
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For comparison, the whole extrapulmonary vasculature of untreated animals, isograft recipients and allograft recipients was also perfused, resulting in 173.0 ± 23.0 x 106 (n = 14), 208.7 ± 41.2 x 106 (n = 3) and 193.3 ± 27.4 x 106 (n = 4) leukocytes respectively. The percentage of monocytes in extrapulmonary perfusates was 2432%.
After density gradient centrifugation, Pappenheim staining and immunocytology with mAb RP-1 revealed that the proportion of granulocytes among the interphase cells was <1% (data not shown). After immunomagnetic negative selection, final purity was 9296% for renal monocytes and 8793% for extrapulmonary monocytes. NK cells constituted 0.62.7% and lymphocytes 5% of the monocyte preparations.
Monocyte cytotoxicity was compared to NK cell cytotoxicity. NK cells were enriched from spleen homogenates of untreated animals and graft recipients, and were 7085% pure. T and B lymphocytes amounted up to 25%. The number of myeloid cells was negligible.
Monocyte cytotoxicity
Monocyte cytotoxicity was analyzed by flow cytometry using the NK-sensitive target Yac-1 and the NK-insensitive, NO-sensitive target P815. Towards Yac-1 target cells, the cytotoxicity coefficient of allograft monocytes (Fig. 2A and D) was twice as high (P < 0.001) as that of monocytes from isografts (Fig. 2B and E) or untreated kidneys (Fig. 2C and F). PI and MC 540 staining did not differ considerably (P = 0.465; two-way ANOVA) and resulted in a specific cytotoxicity of allograft monocytes of
25% at a proportion of 1% Yac-1 target cells ([Ec/T] + 1 = 100; Fig. 2A and D). MC 540 is supposed to identify apoptotic cells (32), whereas PI is commonly used to identify necrotic cells (33). Thus, the cytotoxic mechanisms of monocytes might include pro-apoptotic and pro-necrotic mechanisms.
Cytotoxicity of activated monocytes from renal allografts towards the NO-sensitive target P815 (Fig. 3A and E) was considerably lower than that towards Yac-1 target cells (P < 0.001; two-way ANOVA with target cell and supravital dye as factors), but it was also independent of the supravital dye used (P = 0.817). At a proportion of 1% P815 target cells. both PI and MC 540 staining resulted in a specific cytotoxicity of 1013%. Thus, the cytotoxicity coefficient was half that observed with Yac-1 target cells.
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NK cell cytotoxicity
NK cell cytotoxicity was determined using splenic NK cells after renal or extramulmonary perfusion, as NK cells could hardly be enriched from renal perfusates due to their low frequency. Splenic NK cells (Table 2) displayed strong cytotoxicity towards the NK-sensitive target Yac-1 and intermediate cytotoxicity towards the NK-resistant target P815. Similar to monocytes, the cytotoxicity coefficient depended on the type of transplantation (P < 0.001) and on the target cells used (P < 0.001). In contrast to monocytes, the proportions of target cell damage differed significantly when identified with PI or MC 540 (P < 0.001; three-way ANOVA).
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Natural cytotoxicity of NK cells towards P815 target cells was considerably lower than towards Yac-1 cells (Table 2). Similar to Yac-1, it depended on the type of transplantation, but, in contrast, MC 540 and PI staining gave comparable results with P815. At a proportion of 1% P815 target cells, the specific cytotoxicity of NK cells from untreated animals and isograft recipients was 1525% (see Table 2). NK cells from allograft recipients showed 4-fold higher cytotoxicity. The lack of differences in MC 540 and PI staining of P815 cells may indicate that NK cells damage Yac-1 and P815 cells by mechanisms different from monocytes.
Target cell size analysis
Cell size analysis supported the results obtained by supravital staining, and further suggested different cytotoxic mechanisms of monocytes and NK cells. Examination of Yac-1 target cell forward scatter after co-incubation with NK cells demonstrated that up to 90% of the PI+ targets were considerably smaller than the PI cells. This is likely due to cell shrinkage as a consequence of apoptotic or secondary necrosis. In contrast, after co-incubation with monocytes, 6070% of the PI-stained target cells had maintained their initial cell size. This might be interpreted as a consequence of primary necrosis induced by monocytes.
Role of extracellular calcium
The well-known essential role of calcium for the cytotoxic activity of NK cells was clearly demonstrated by the flow cytometry-based cytotoxicity assay. The depletion of extracellular calcium by 12.5 mM EDTA resulted in a 90% reduction of the cytotoxicity towards Yac-1 cells (Fig. 4A and B). In contrast, monocyte cytotoxicity was not diminished by EDTA (Fig. 4C and D). Both with and without EDTA, the cytotoxicity coefficient ranged between 15 and 22%, indicating a specific cytotoxicity of 3045% at 1% Yac-1 target cells. Thus, monocyte cytotoxicity did not depend on extracellular calcium.
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Immunoprecipitation with mAb 3.2.3 followed by SDSPAGE under reducing conditions resulted in protein bands with an apparent molecular mass of 30 kDa for purified monocytes, spleen cells and the rat NK cell line CRNK-16 respectively (Fig. 5). These findings are in accordance with the molecular mass of the subunits of CD161 (17). In spite of the fact that NK T cells were not considered when adjusting the splenic NK cell control preparations, monocyte samples produced signals that were much stronger than those of the corresponding spleen cell controls (Fig. 5). The difference among the signal intensities thus indicates the expression of CD161 by activated monocytes.
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Discussion |
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The NK-sensitive mouse lymphoma Yac-1 and the NK-insensitive, NO-sensitive mouse mastocytoma P815 are standardized, commonly used target cells in assays investigating cytotoxicity mediated by NK cells [e.g. (35)] or mononuclear phagocytes [e.g. (22,27,36)]. During renal allograft rejection, the physiological target cells of monocytes might be endothelial cells. However, in contrast to primary cultures of endothelial cells, Yac-1 and P815 cells are well characterized and display a stable phenotype. Thus, they are suitable targets to investigate general aspects of cytotoxic effector functions monocytes acquire during renal graft rejection in vivo. In particular, they allow a detailed comparison to NK cell-mediated cytotoxicity. In perspective, the results may help to analyze the cytotoxic effector functions monocytes may use to damage endothelial cells. Using a similar approach, Yoshida et al. (22,23,37) demonstrated that tumor cell lines are useful targets to investigate cytotoxicity that macrophages acquire during allogeneic skin graft rejection in mice.
To identify the type(s) of cellular damage monocytes might induce in their target cells, the fluorescent dyes MC 540 and PI were used as indicators for apoptosis and necrosis respectively (32,33). Allogeneic kidney transplantation markedly increased the cytotoxicity of both renal monocytes and of monocytes isolated from the whole extrapulmonary vasculature. PI and MC 540 staining led to similar proportions either of labeled Yac-1 target cells or of labeled P815 target cells. More than 60% of the PI+ target cells were large cells of almost normal size, suggesting direct cellular damage by primary necrosis and not by apoptosis (38). This interpretation is supported by studies showing that human macrophages cause more rapid Cr release from target cells than release of [3H]thymidine, indicating an early form of target cell necrosis (39). Thus, activated monocytes might use pro-apoptotic as well as pro-necrotic mechanisms to damage organ grafts.
Little is known about the effector molecules mediating monocyte cytotoxicity. Members of the tumor necrosis factor (TNF) family including TNF, TWEAK and Fas ligand are assumed to initiate apoptosis (1,2,40), and might participate in kidney destruction (12,41,42). The participation of NO as a pro-apoptotic mediator is less likely, at least in the assay used above, because the NO-sensitive P815 target cells were hardly damaged by monocytes. Primary necrosis might be induced by different agents including reactive oxygen intermediates (3), lysosomal enzymes (4) and small cytotoxic peptides (5).
In order to further elucidate cytotoxic mechanisms of monocytes during renal allograft rejection, the role of extracellular calcium and of CD161 was investigated. In contrast to NK cells (43), depletion of extracellular calcium did not diminish monocyte cytotoxicity. This might be a special feature of monocyte cytotoxicity in rats because mouse macrophage cytotoxicity seems to be calcium dependent (37). However, calcium-independent cytotoxicity does not necessarily mean granule-independent cytotoxicity. At least in granulocytes, degranulation does not depend on calcium (44).
In addition, monocyte cytotoxicity in the presence of EDTA indicated that in contrast to NK cells (45), integrin interactions and conjugate formation are not necessary to damage target cells. Integrin binding is dependent on divalent cations (46), but EDTA abrogates conjugate formation between monocytes and target cells (47). In the present study, monocytes killed Yac-1 target cells in the presence of EDTA as well as in its absence. Furthermore, monocyte cytotoxicity in the presence of EDTA also excluded that the data obtained by the flow cytometry-based cytotoxicity assay represented artifacts due to conjugate formation between dying monocytes and living target cells.
Of special interest was the influence of CD161 on monocyte cytotoxicity after ligation with mAb 3.2.3. CD161 consists of two identical disulfide-bonded subunits of 30 kDa and belongs to the type II membrane glycoproteins of the C-type lectin superfamily (18). Homologues of CD161 were found in different species. It is strongly expressed on NK cells, but intermediate expression is also present in T lymphocyte subsets (48). In humans and rats, myeloid cells including granulocytes, monocytes and dendritic cells were also described to weakly express CD161 (17,36,49). The present paper now proved by immunoprecipitation that an authentic member of the CD161 family was expressed on activated intravascular rat monocytes during allogeneic kidney graft rejection. It was crucial to carefully monitor the contribution of NK cells, because flow cytometry indicated that the expression of CD161 on NK cells was >10 times stronger than that on activated renal allograft monocytes.
Recent genetic studies revealed that at least NKR-P1B is expressed on rat monocytes as a member of the CD161 family (50). As mAb 3.2.3 has been shown to react both with the activating receptor NKR-P1A and with the inhibitory receptor NKR-P1B (21), it may, however, not be possible to distinguish whether one or two molecules are detected with the methods used in the present study. In previous studies 3.2.3+ monocytes were also found in rats treated with IFN- (16) and in rats with experimental post-operative peritonitis (51). It is thus suggested that monocytic expression of CD161 is a general phenomenon accompanying monocyte activation.
Despite the fact that up-regulation of CD161 is a very early and sensitive marker for monocyte activation (12,15,16,51), ligation of CD161 by mAb 3.2.3 did not alter monocyte cytotoxicity. A similar lack of effect on cytotoxicity is also described for NK cells (52). This might be explained by antagonistic effects of simultaneous ligation of NKR-P1A and NKR-P1B by mAb 3.2.3 (53). A similar phenomenon might also occur in activated monocytes as, at present, co-expression of NKR-P1A and NKR-P1B cannot be excluded. On the other hand, ligation of CD161 family members induces alterations in cellular physiology and secretion in NK cells and human monocytes (18,49). The present paper now shows that binding of mAb 3.2.3 enhanced monocyte viability.
The markedly enhanced cytotoxicity of monocytes activated in vivo during allogeneic kidney transplantation suggests a considerable role in allograft destruction, eventually affected by extrinsic factors. Elucidating the mechanisms effecting monocyte viability, activation and target cell damage might finally lead to new therapies for organ allograft rejection and other diseases.
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Acknowledgements |
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Abbreviations |
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iNOSinducible NO synthase
MC 540merocyanine 540
M199Medium 199
NKR-P1NK receptor-protein 1
PEphycoerythrin
PIpropidium iodide
sFCSsupplemented FCS
TNFtumor necrosis factor
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References |
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