Serum amyloid P component binds to late apoptotic cells and mediates their uptake by monocyte-derived macrophages.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 48, No. 1, January 2003, pp 248–254 DOI 10.1002/art.10737 © 2003, American College of Rheumatology Serum Amyloid P Component Binds to Late Apoptotic Cells and Mediates Their Uptake by Monocyte-Derived Macrophages Marc Bijl, Gerda Horst, Johan Bijzet, Hendrika Bootsma, Pieter C. Limburg, and Cees G. M. Kallenberg Objective. Some pentraxins, such as C-reactive protein, bind to apoptotic cells and are involved in the clearance of these cells. We undertook this study to determine whether serum amyloid P component (SAP; a pentraxin that, when deficient in mice, results in lupuslike disease) binds to apoptotic cells and to assess the functional consequences of SAP binding for their phagocytosis by macrophages. Methods. Human peripheral blood monocytes were isolated and cultured for 7 days to obtain monocyte-derived macrophages. Jurkat cells were irradiated with ultraviolet B to induce apoptosis. After 4 hours, a mean ⴞ SEM of 54.0 ⴞ 5.1% of these cells stained with annexin V and were propidium iodide negative (early apoptotic [EA] cells). After 24 hours, 77.3 ⴞ 2.7% of cells stained positive with both annexin V and propidium iodide (late apoptotic [LA] cells or secondary necrotic cells). EA and LA cells were incubated with fluorescein isothiocyanate–labeled SAP in the presence or absence of Ca2ⴙ, and binding was measured by flow cytometry. Phagocytosis was tested by incubation of macrophages with EA or LA cells in the presence of normal human serum (NHS) and quantified as a phagocytosis index (PI; number of Jurkat cells internalized by 100 macrophages). Experiments were repeated with SAP-depleted serum and after reconstitution with increasing concentrations of SAP. Results. The majority of LA cells did bind SAP in the presence of Ca2ⴙ, whereas EA cells did not. SAP depletion of NHS resulted in a 50% decrease in the PI for LA cells, and complete restoration of the PI could be demonstrated with SAP reconstitution up to 100 g/ml. SAP depletion had no effect on phagocytosis of EA cells. Conclusion. SAP binds to LA cells and is involved in the phagocytosis of these cells by human monocyte– derived macrophages. This may have consequences for diseases such as systemic lupus erythematosus, in which phagocytosis of apoptotic cells is decreased. Several lines of evidence suggest a role for apoptotic cells in the induction of autoimmunity. First, intracellular autoantigens are exposed at the outer surface of the cell during the apoptotic process. This may explain why autoantibodies can develop against these intracellular antigens (1). Second, injection of large numbers of apoptotic cells in experimental animal models was shown to induce autoimmunity (2). Based on these findings, it has been suggested that self-tolerance is broken by the accumulation of apoptotic cells. Indeed, in systemic lupus erythematosus (SLE), the prototype of systemic autoimmune disease, increased levels of apoptotic lymphocytes have been detected in the peripheral blood (3). This may result from increased production (4) and/or impaired phagocytosis of apoptotic cells (5). Phagocytosis of apoptotic cells is a complex process in which many membrane receptors and serum constituents are involved. Several serum proteins opsonize apoptotic cells, thus facilitating their phagocytosis. Of these serum proteins, the role of complement components has been elucidated best. C1q-deficient mice spontaneously develop antinuclear antibodies and glo- Supported by grant 00-23 from the Jan Kornelis De CockStichting. Marc Bijl, MD, PhD, Gerda Horst, BSc, Johan Bijzet, BSc, Hendrika Bootsma, MD, PhD, Pieter C. Limburg, PhD, Cees G. M. Kallenberg, MD, PhD: University Hospital, Groningen, The Netherlands. Address correspondence and reprint requests to Marc Bijl, MD, PhD, Department of Internal Medicine, Division of Clinical Immunology, University Hospital, PO Box 30.001, 9700 RB Groningen, The Netherlands. E-mail: firstname.lastname@example.org. Submitted for publication February 8, 2002; accepted in revised form September 30, 2002. 248 SAP AND PHAGOCYTOSIS OF APOPTOTIC CELLS merulonephritis (6). Interestingly, a striking feature of glomerulonephritis in these animals was the presence of increased numbers of apoptotic bodies in their glomeruli, suggesting a role for C1q in the clearance of apoptotic cells (6). This hypothesis was supported by findings of additional in vitro studies on phagocytosis of apoptotic cells. Phagocytosis of apoptotic thymocytes by inflammatory macrophages was shown to be significantly reduced in C1q knockout mice (7). In the latter study, the in vitro phagocytic capacity of 3 patients with C1q deficiency was also tested. Indeed, a kinetic defect in the uptake of apoptotic cells was found. C1q-deficient macrophages from these individuals, in autologous serum, showed a significant reduction in phagocytosis compared with controls (7). C1q therefore seems essential for the adequate uptake of apoptotic cells by macrophages. However, other complement components, such as C3 and C4, and the pentraxins C-reactive protein (CRP), serum amyloid P component (SAP), and PTX3 also bind to apoptotic cells and mediate their uptake by macrophages (8,9). The role of SAP in the handling of apoptotic cells has been demonstrated in SAP knockout mice. These animals develop antinuclear antibodies and severe glomerulonephritis, a phenotype resembling human SLE (10). Since SAP binds to apoptotic cells, we evaluated whether this binding has functional consequences for the clearance of apoptotic cells in vitro, using human monocyte–derived macrophages as phagocytic cells. MATERIALS AND METHODS Macrophage culture. Peripheral blood mononuclear cells were isolated by Lymphoprep density gradient centrifugation from citrated blood. Healthy controls served as donors. Cells were suspended in medium containing RPMI, gentamicin, and 2% pooled serum at a concentration of 106/ml. Plastic coverslips (13 mm diameter; Nunc, Roskilde, Denmark) were placed in a 16 mm–diameter 24-well plate (Costar, Schiphol, The Netherlands). In every well, 0.5 ml cell suspension was seeded, and, subsequently, monocytes were allowed to differentiate into macrophages during 7 days at 37°C in a 5% CO2 atmosphere. On day 2 and on day 5, 0.5 ml fresh medium was added to each well. Induction and detection of apoptosis. Jurkat cells suspended in RPMI 1640 supplemented with 13% fetal calf serum, sodium pyruvate, glutamine, gentamicin, fungizone, and ␤-mercaptoethanol (BioWhittaker, Verviers, Belgium) were irradiated with ultraviolet B (UVB; 20W, 170 mJ/cm2) using a TL12 (Philips, Best, The Netherlands) for 15 minutes to induce apoptosis. After incubating for 4 hours or 24 hours at 37°C in a 5% CO2 atmosphere, part of the cells were used for apoptosis detection. For staining, 99 l binding buffer (10 mM HEPES [pH 7.4], 140 mM NaCl, 5 mM CaCl2), 10 l pro- 249 pidium iodide (10 g/ml; Molecular Probes, Leiden, The Netherlands), and 1 l fluorescein isothiocyanate (FITC)– labeled annexin V (Nexins Research, Hoeven, The Netherlands) diluted 1:10 were added. Immediately after incubation for 10 minutes on ice in the dark, immunofluorescence analysis was performed on an Epics-Elite equipped with a gated amplifier (Coulter Electronics, Mijdrecht, The Netherlands). After 4 hours, 54.0 ⫾ 5.1% (mean ⫾ SEM of 3 experiments) of Jurkat cells were positive for annexin V and negative for propidium iodide (early apoptotic [EA] cells), whereas after 24 hours, 77.3 ⫾ 2.7% (3 experiments) of cells stained positive for both annexin V and propidium iodide (late apoptotic [LA] cells). SAP binding to apoptotic cells. SAP was isolated from pooled human serum. SAP obtained (kindly provided by B. P. C. Hazenberg, MD, University Hospital, Groningen, The Netherlands) was 97% pure as confirmed by chromatography. SAP was labeled with FITC (Pierce, Rockford, IL) according to the manufacturer’s instructions. Irradiated Jurkat cells (105) were incubated with FITC-labeled SAP (10 g/ml) in binding buffer with or without 5 mM CaCl2 for 10 minutes on ice in the dark. After incubation, immunofluorescence was analyzed by flow cytometry. Phagocytosis assay. Irradiated Jurkat cells were washed 3 times with RPMI and resuspended in RPMI supplemented with 30% fresh human serum at a concentration of 2 ⫻ 106/ml. Prior experiments had shown increasing uptake of apoptotic cells with increasing serum concentrations, and 30% serum had proved to be a concentration that allowed sufficient quantitation of uptake (data not shown). Jurkat cell suspension was added into fresh 24-well plates (1 ml/well). Coverslips with adherent macrophages were washed with RPMI containing 1% human serum to remove nonadherent cells and were subsequently transferred into the 24-well plates, where cell interaction was allowed for 30 minutes at 37°C in a 5% CO2 atmosphere. Coverslips were washed gently with 0.4% human serum albumin (HSA; Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands) to remove nonbound cells. For uptake of apoptotic Jurkat cells, the presence of fresh human serum was obligatory. Without fresh human serum, no internalization could be detected (data not shown). Experiments were repeated with SAP-depleted serum (see below) and after reconstitution of SAP in increasing concentrations of 1 g/ml, 5 g/ml, 10 g/ml, 50 g/ml, and 100 g/ml. Staining procedure and scoring of binding and phagocytosis. To flatten the macrophages, thereby facilitating the microscopic evaluation of the number of phagocytosed Jurkat cells, the coverslip was mounted to a “spinning pad” consisting of 2 cytospin pads (Shandon, Pittsburgh, PA) glued together (11). From the top pad, 2 squares were cut and coverslips were placed in the squares. Thereafter, 100 l 4% HSA was added to the coverslip, and the “spinning pad” was placed within a 24-well lid for centrifugation at 25g for 10 minutes with the lids mounted at an angle of 100° to the direction of spinning to ensure quick drying of the coverslip. Coverslips were dried under air, fixed for 10 minutes with methanol, and stained in two steps. First, coverslips were stained for 5 minutes with May-Grünwald staining solution (Merck, Darmstadt, Germany) diluted 1:1 in phosphate buffer (0.069M), followed by staining for 15 minutes with filtered Giemsa (Merck) diluted 250 BIJL ET AL Figure 1. Representative figure of annexin V (ANN) and propidium iodide (PI) staining showing serum amyloid P component (SAP) binding to Jurkat cells 4 hours and 24 hours after ultraviolet B (UVB) irradiation. Late apoptotic (LA) cells bind SAP. Jurkat cells were irradiated with UVB. Numbers depict the percentages of cells present in each quadrant. Four hours after irradiation, the majority of cells were early apoptotic (EA; ANN positive and PI negative) (A), whereas 24 hours after irradiation, most cells were LA (ANN positive and PI positive) (B). No binding of SAP to EA cells was found (C). In contrast, the majority (73.7%) of LA cells bound fluorescein isothiocyanate (FITC)–labeled SAP (D). 1:10 in phosphate buffer (0.069M). Excess dye was removed by 2 consecutive incubations in phosphate buffer (0.069M) for 6 minutes. After drying, coverslips were placed upside down with Depex (BDH Chemicals, Poole, UK) on thin microscopic 24 ⫻ 60–mm glasses (Menzel, Braunschweig, Germany). Preparations were scored at 400⫻ magnification under regular light microscopy by 2 independent observers. Binding of apoptotic cells to macrophages was defined as Jurkat cells that were attached to macrophages but not internalized. Binding was scored as a binding index (BI; number of Jurkat cells bound by 100 macrophages). Phagocytosis of apoptotic Jurkat cells was expressed as a phagocytosis index (PI; number of Jurkat cells internalized by 100 macrophages). Both the interobserver and interassay variations were ⬍10% (mean of 10 experiments). All experiments were performed in duplicate. SAP depletion and enzyme-linked immunosorbent assay (ELISA). For SAP depletion, serum of healthy controls (normal human serum [NHS]) was brought on an agarose column enriched with high electroendosmosis agarose (lot no. AG 0493; FMC Bioproducts, Rockland, ME). Efficiency of depletion was tested by ELISA. In this assay, Costar plates (Badhoevedorp, The Netherlands) were coated with monoclonal anti-SAP (Novocastra, Newcastle-upon-Tyne, UK) as catching antibody. Polyclonal anti-SAP (Dakopatts, Glostrup, SAP AND PHAGOCYTOSIS OF APOPTOTIC CELLS 251 Figure 2. Ca2⫹-dependent binding of SAP to LA cells. Incubation of EA cells (A) and LA cells (B) with FITC-labeled SAP was performed in binding buffer without CaCl2 (open pattern) and with CaCl2 added (solid pattern). No binding of SAP is demonstrated in EA cells, irrespective of the presence of Ca2⫹. Adding Ca2⫹ to LA cells demonstrates the presence of two cell populations: 62% of cells bind SAP, while 38% do not. See Figure 1 for definitions. Denmark) was used as detection antibody. Purified SAP was used as a standard for the assay. The mean ⫾ SEM serum level of SAP in healthy controls was 27.7 ⫾ 1.0 g/ml. SAP depletion of ⬎95% was accomplished. To be sure complement levels were not influenced by SAP depletion, we measured levels of C3, C4, and C1q after SAP depletion. These levels were unaltered and were all in the normal range (data not shown). Statistical analysis. Statistics were calculated using GraphPad Prism (version 3.0; GraphPad software, San Diego, CA). Student’s t-test and one-way analysis of variance with Bonferroni’s multiple comparison test were applied. RESULTS In order to analyze SAP binding to apoptotic cells, Jurkat cells were irradiated with UVB to induce apoptosis. After 4 hours, more than one-half of the cells were EA (Figure 1A). SAP did not bind to these EA cells (Figure 1C). At this time point, only a minority of cells were LA. Twenty-four hours after irradiation, two-thirds of the cells were LA (Figure 1B). Most of these cells (67.0 ⫾ 3.5%, mean ⫾ SEM of 3 experiments) did bind SAP (Figure 1D). This binding was shown to be Ca2⫹ dependent, since the absence of Ca2⫹ completely abolished binding of SAP (Figure 2). In addition, we performed phagocytosis experiments in order to evaluate whether the binding of SAP influenced the uptake of apoptotic cells by human monocyte–derived macrophages. Macrophages were in- cubated with EA cells in NHS either containing SAP or ⬎95% SAP depleted. The level of phagocytosis of EA cells by macrophages in SAP-depleted serum was comparable with that when cells were incubated in NHS, reaching mean ⫾ SEM PIs of 44.0 ⫾ 4.0 and 37.0 ⫾ 6.0, respectively (experiments performed in duplicate). Experiments using LA cells showed binding of apoptotic cells to macrophages when incubation had been performed in NHS irrespective of the presence or absence of SAP (mean ⫾ SEM BIs 14.0 ⫾ 3.3 and 13.8 ⫾ 1.9, respectively). However, phagocytosis of LA cells was markedly influenced by the absence of SAP. Using SAP-depleted serum, the mean ⫾ SEM PI for LA cells decreased from 38.3 ⫾ 2.9 to 18.7 ⫾ 1.4 (P ⫽ 0.0038 by Student’s t-test) (Figure 3A). Subsequently, to prove the functional role of SAP in the uptake of LA cells, we repeated the phagocytosis experiments using SAP-depleted serum and reconstituted SAP in increasing concentrations. Complete restoration of the PI was demonstrated with SAP reconstitution up to 100 g/ml (Figure 3B). DISCUSSION In this study, we demonstrate that SAP binds to LA cells (or secondary necrotic cells) in a calciumdependent manner. Furthermore, this is the first study to demonstrate in vitro that this binding has consequences 252 Figure 3. SAP-mediated uptake of LA cells by human monocyte– derived macrophages. LA cells were incubated for 30 minutes with monocyte-derived macrophages to allow their binding and internalization. A, Incubation was performed in the presence of 30% normal human serum (NHS) as well as with SAP-depleted NHS. SAP depletion resulted in a decline in the mean ⫾ SEM phagocytosis index (number of Jurkat cells internalized by 100 macrophages) from 38.3 ⫾ 2.9 to 18.7 ⫾ 1.4 (P ⫽ 0.0038 by Student’s t-test). B, Reconstitution with purified SAP in increasing concentrations normalized the phagocytosis index. SAP-depleted NHS and SAP in concentrations of 1 g/ml, 5 g/ml, and 10 g/ml significantly reduced the phagocytosis index (P ⬍ 0.01, P ⬍ 0.05, P ⬍ 0.01, and P ⬍ 0.05, respectively, versus NHS, by Bonferroni multiple comparison test: P ⫽ 0.0005 versus NHS, by one-way analysis of variance). Data are based on duplicate experiments. Results are shown as the mean and SEM. See Figure 1 for other definitions. for the uptake of LA cells by human monocyte–derived macrophages. Incubation of macrophages with LA Jurkat cells in the absence of SAP reduced their uptake by as much as 50%. BIJL ET AL SAP belongs to the family of pentraxin proteins. These proteins are characterized by cyclic pentameric structure and sequence homology. The interaction of pentraxins with their respective ligands is calcium dependent. The primary function of SAP is the handling of DNA, chromatin, and histones in cell nuclei as well as in solution. As such, SAP seems to prevent the development of autoimmunity, as has recently been reported in SAP-deficient animals (10). SAP-deficient mice spontaneously develop antinuclear antibodies and glomerulonephritis. It has been suggested that Fc␥ receptor type I (Fc␥RI), Fc␥RIIa, and Fc␥RIIIb are receptors for SAP (12). Recently, it has been demonstrated that SAP binds to phosphatidylethanolamine exposed in flip-flopped membranes of apoptotic cells in particular (9). Binding of SAP might result in cell signaling and activation, followed by the internalization of bound cells. In addition, or in conjunction, mouse SAP has been shown to induce the production of colony-stimulating factors by mature macrophages in mice. This mechanism probably indirectly enhances their phagocytic capacity (13). Although SAP is not an acute-phase protein in humans, studies in SAP knockout mice highlighted the potential role of SAP in the etiopathogenesis of human autoimmune disease (10). In the prototype of systemic autoimmune disease, SLE, SAP levels have been measured to analyze whether deficiencies in SAP occur in SLE patients. Levels of SAP were found to be normal in sera from lupus patients, irrespective of disease activity (14). SAP binds to DNA and chromatin and thereby solubilizes native chromatin. It seems straightforward to suggest that SAP plays a role in the clearance of apoptotic cells, since during the process of apoptosis, nuclear constituents such as chromatin are expressed on the cell surface (1). Indeed, in the present study, we show that SAP binding has functional consequences for the clearance of LA cells. We found that SAP modulates the uptake of LA cells by macrophages. Seemingly in contradiction to our results, no accumulation of apoptotic bodies could be demonstrated in SAP-deficient mice (10). Furthermore, the autoantibodies produced were directed against chromatin, histone, and DNA, but not against extractable nuclear antigens (10). Both findings provide evidence against a functional role of SAP in the clearance of apoptotic cells. These discrepancies might be explained by the preserved ability to internalize apoptotic cells in a SAP-deficient milieu. Other serum constituents, such as complement proteins and CRP, are established opsonizing factors for apoptotic cells. For the classical complement proteins, there appears to be a SAP AND PHAGOCYTOSIS OF APOPTOTIC CELLS hierarchy in the clearance of apoptotic cells, with the strongest influence of C1q and a lesser dependence on C4 (7). We show that phagocytosis of apoptotic cells under conditions of SAP depletion remains possible, though at decreased levels. The relative phagocytic defect that occurs in the absence of SAP seems less pronounced than the defect accomplished through C1q deficiency, and probably accounts for the absence of apoptotic bodies in SAP-deficient mice. Our results are consistent with the current concept of the etiopathogenesis of human SLE. SLE is the prototype of a systemic autoimmune disease and is characterized by the presence of antinuclear antibodies, among which those directed to nucleosomes seem to be most relevant. Nucleosomes and other nuclear and cytoplasmic antigens are expressed on the cell surface during the process of apoptosis. In addition, nucleosomes are released. It has been suggested that the occurrence of autoantibodies is induced by the accumulation of apoptotic cells, with persistent presentation of nuclear antigens. Indeed, elevated levels of circulating nucleosomes can be found in plasma of lupus patients (15). Furthermore, increased amounts of apoptotic cells have been demonstrated in the peripheral blood of SLE patients (3). Since the clearance of apoptotic cells normally occurs very rapidly, the presence of apoptotic cells must reflect disturbances in either the production of apoptotic cells or their elimination. Increased susceptibility of peripheral blood lymphocytes to various apoptotic stimuli could be demonstrated in SLE patients and might, in part, explain the increased production of apoptotic cells (4). Clearance of apoptotic cells requires their opsonization with complement components such as iC3b, C1q, and C4 (7,16). A decrease in levels of opsonizing serum complement components, which is a reflection of complement consumption classically seen in SLE patients, might result in a decreased capacity for phagocytosis of apoptotic cells. This, in turn, will result in the persistence of EA cells and, subsequently, LA cells, especially during disease activity in which complement consumption is even more pronounced. We hypothesize that SAP, like surfactant protein A and the Wiskott-Aldrich syndrome protein, functions as a salvage protein offering the immune system a rescue pathway to eliminate LA cells (17,18). SAP binds to these cells and facilitates their uptake by macrophages. In accordance with the concept proposed, a reduced clearance capacity of apoptotic cells in SLE patients was shown by Herrmann et al (5). In that study, the expression of the membrane receptors CD14 and 253 CD36 on freshly isolated peripheral blood monocytes of SLE patients was found to be unaltered compared with that of healthy controls. This finding suggests that the impaired phagocytosis cannot be explained by changes in the membrane expression of CD14 or CD36. Obviously, differences in membrane receptors on other phagocytes as well as differences in the functionality of CD14 or CD36 cannot be excluded. Furthermore, many other membrane receptors on phagocytes, such as the MER receptor and the phosphatidylserine receptor, are involved in the internalization of apoptotic cells, and their role in SLE has not yet been determined. Recently, it was found in lupus-prone MRL and NZB mice that these mouse strains did not show intrinsic defects in phagocytosis of apoptotic cells (19). 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