PAI-1 inhibits neutrophil efferocytosis Young-Jun Park*†, Gang Liu*†‡, Emmanuel F. Lorne*§, Xia Zhao*, Jing Wang*, Yuko Tsuruta*, Jaroslaw Zmijewski*, and Edward Abraham*‡ *Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294; and §Pole Anesthe´sie Re´animation, Centre Hospitalier Universitaire and Institut National de la Sante´ et de la Recherche Me´dicale, ERI-12, Amiens, France Edited by Charles A. Dinarello, University of Colorado Health Sciences Center, Denver, CO, and approved June 3, 2008 (received for review February 19, 2008)
Phagocytosis of apoptotic cells, also called efferocytosis, is an essential feature of immune responses and critical for the resolution of inflammation. Plasma and tissue levels of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, are elevated in inflammatory conditions, including sepsis and acute lung injury, in which activated neutrophils accumulate in tissues and contribute to organ dysfunction. In this study, we explored the potential involvement of PAI-1 in modulating neutrophil efferocytosis. We found enhanced phagocytosis of viable PAI-1 deficient (PAI-1ⴚ/ⴚ) and of wild-type neutrophils treated with anti-PAI-1 antibodies. PAI-1 levels were decreased on the surface of apoptotic neutrophils and the enhanced phagocytosis of apoptotic wild-type neutrophils or of viable PAI-1ⴚ/ⴚ neutrophils was diminished by preincubation with PAI-1. The increased phagocytosis associated with PAI-1 deficiency or blockade depended on both the lipoprotein receptor-related protein (LRP) and its ligand, calreticulin (CRT), because the LRP-mediated increase in phagocytosis of viable neutrophils induced by blockade of CD 47 was abrogated by PAI-1. CRT levels are increased on viable PAI-1ⴚ/ⴚ neutrophils. While CRT colocalizes with PAI-1 on viable neutrophils, markedly diminished colocalization of PAI-1 and CRT was present on apoptotic neutrophils. Our data therefore indicate that PAI-1 serves as a novel ‘‘don’t eat me’’ signal for viable and apoptotic neutrophils. calreticulin 兩 phagocytosis 兩 plasminogen activator inhibitor-1
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hagocytosis of apoptotic cells, also known as efferocytosis, occupies an important role in normal tissue homeostasis and regulation of immune responses (1–3). Rapid clearance of dying cells prevents leakage of potentially cytotoxic or antigenic contents into the extracellular milieu and the resultant initiation of inflammatory processes and tissue injury (4). Defects or delays in apoptotic corpse elimination have been closely associated with autoimmune and inflammatory diseases, such as systemic lupus erythematosus and acute lung injury (5–7). Apoptotic cells are marked by a number of alterations in various cell surface molecules. One of the major changes on the cell surface during apoptosis is appearance of phosphatidylserine (PtdSer) (5). PtdSer is recognized and bound by PtdSer receptors through opsonins, a group of soluble molecules in the extracellular milieu, including milk-fat globule EGF-factor 8 (MFG-E8), growth arrestspecific gene 6 (Gas6), and thrombospondin, that serve as bridges between PtdSer and cell surface receptors on phagocytes, such as integrins and Mer family type I receptor tyrosine kinases (8–10). Calreticulin (CRT) has recently been found to mediate efferocytosis through binding to the low-density lipoprotein related protein (LRP) on phagocytes (1, 11). However, in contrast to PtdSer, CRT is also expressed on viable cells, which raises the question of how living cells can resist inadvertent uptake. At least a partial response to this dilemma relates to CD47, an integrin associated protein that binds to SIRP␣, inducing its phosphorylation in phagocytes and subsequent inhibition of phagocytosis (1). In addition, another integrin associated protein, CD31, has been shown to block viable cell ingestion (12). At present, CD31 and CD47 are the only ‘‘don’t eat me’’ signals described for viable cells. PAI-1 is a member of the serine protease inhibitor (SERPIN) superfamily (13). The primary molecules to which PAI-1 binds are 11784 –11789 兩 PNAS 兩 August 19, 2008 兩 vol. 105 兩 no. 33
urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), fibrin, vitronectin, and members of the low density lipoprotein receptor (LRP) family (13, 14). Elevated serum and tissue levels of PAI-1 have been found in a number of inflammatory diseases, including myocardial infarction, sepsis, and acute lung injury, with increased circulating PAI-1 levels being associated with unfavorable outcomes (15–18). Previous studies suggested that microvascular thrombosis associated with inhibition of fibrinolytic processes by PAI-1 and the role of PAI-1 as a chemotactic factor promoting the migration of lymphocytes and neutrophils into inflammatory sites contributed to its inflammatory effects (19–21). In addition, PAI-1 also appears to have intrinsic proinflammatory properties by potentiating Toll-like receptor 4 (TLR4) mediated activation of neutrophils (22). Considering the critical role that phagocytosis of neutrophils and other activated cell populations occupies in the resolution of inflammation and the significant contribution of PAI-1 to inflammatory progression, we examined the possibility that PAI-1 might modulate neutrophil efferocytosis. Quite unexpectedly, we found that PAI-1 functions as a novel don’t eat me signal and is required for viable neutrophils to avoid ingestion. PAI-1 can also function to inhibit the engulfment of apoptotic neutrophils by macrophages. Results PAI-1 Deficiency Is Associated with Enhanced Phagocytosis of Viable Neutrophils. We first examined uptake of viable and apoptotic
mouse neutrophils by mouse peritoneal macrophages. As shown in Fig. 1A, there was a significantly higher phagocytic index for apoptotic neutrophils compared with that found for viable neutrophils. However, viable neutrophils from transgenic mice lacking PAI-1, i.e., PAI-1⫺/⫺, also demonstrate increased ingestion by macrophages to levels comparable with those present for apoptotic wild-type (WT) cells (Fig. 1B). Viable PAI-1⫺/⫺ neutrophils have the same levels of staining for annexin V as do viable WT neutrophils [supporting information (SI) Fig. S1A], indicating that the increase in phagocytosis is not caused by altered cell viability, but indeed is due to PAI-1 deficiency. We next determined whether blockade of PAI-1 on the neutrophil surface affects the phagocytosis of viable neutrophils in a manner similar to that seen with PAI knockout cells. As shown in Fig. 1C, preincubation of viable WT neutrophils with rabbit anti-PAI-1 antibodies resulted in significantly increased phagocytosis. To confirm this effect, we used mouse anti-PAI-1 monoclonal antibodies and found that preincubation of viable WT neutrophils with mouse anti-PAI-1 monocloAuthor contributions: Y.-J.P., G.L., and E.A. designed research; Y.-J.P., G.L., E.F.L., X.Z., J.W., Y.T., and J.Z. performed research; Y.-J.P., G.L., and E.A. analyzed data; and G.L. and E.A. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. †Y.-J.P.
and G.L. contributed equally to this work.
‡To
whom correspondence may be addressed. E-mail:
[email protected] or eabraham@ uab.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/ 0801394105/DCSupplemental. © 2008 by The National Academy of Sciences of the USA
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Fig. 1. PAI-1 deficiency is associated with enhanced phagocytosis of viable neutrophils that is abolished by exogenous PAI-1. (A) Phagocytosis of apoptotic neutrophils is significantly increased compared with that of viable cells. Phagocytic indices are expressed as the percentage of macrophages containing at least one ingested neutrophil. Mean ⫾ SD. **, P ⬍ 0.01 compared with viable neutrophils. (B) PAI-1 deficiency is associated with increased phagocytosis of viable neutrophils. Neutrophils were purified from PAI-1⫺/⫺ mice or control C57BL/6 mice. Mean ⫾ SD. **, P ⬍ 0.01 compared with WT group. (C) Blockade of PAI-1 produces enhanced neutrophil engulfment. Viable WT and PAI-1⫺/⫺ neutrophils were incubated without or with 1 g/ml rabbit anti-PAI-1 antibodies or 1 g/ml rabbit IgG before efferocytosis assays were performed. Mean ⫾ SD. ***, P ⬍ 0.001 compared with no treatment (control) or treatment with rabbit IgG. (D) Exogenous PAI-1 decreases phagocytosis of viable PAI-1⫺/⫺ neutrophils. Viable WT and PAI-1⫺/⫺ neutrophils were incubated with 0, 0.01, 0.1, 0.5 or 1 g/ml mouse recombinant PAI-1 before efferocytosis assays were performed. Mean ⫾ SD, ***, P ⬍ 0.001 compared with treatment with BSA. (E) Phagocytosis of viable PAI-1⫺/⫺ neutrophils in vivo is significantly increased. 10 ⫻ 106 viable WT or PAI-1⫺/⫺ neutrophils were intratrachaelly injected into WT mice. Mean ⫾ SEM, n ⫽ 5. ***, P ⬍ 0.001 compared with WT neutrophils. (F) Preincubation of PAI-1⫺/⫺ neutrophils with PAI-1 abrogated their increased uptake in vivo. PAI-1⫺/⫺ neutrophils were preincubated with mouse albumin or PAI-1 for 30 min before intratracheal injection. Mean ⫾ SEM, n ⫽ 5. **, P ⬍ 0.01 compared with albumin group.
nal antibodies also enhanced their engulfment by macrophages (Fig. S1B). There was no change in phagocytosis of viable PAI-1⫺/⫺ neutrophils after incubation with anti-PAI-1 antibodies (Fig. 1C), further establishing that the increase in phagocytosis of viable WT neutrophils after treatment with anti-PAI-1 antibodies is PAI-1dependent. The increased phagocytosis of viable PAI-1⫺/⫺ neutrophils suggests a role for cell surface associated PAI-1 in the regulation of efferocytosis. To examine this issue, we incubated viable PAI-1⫺/⫺ neutrophils with exogenous PAI-1 protein and then determined whether such pretreatment affected the phagocytic index. As shown in Fig. 1D, uptake of viable PAI-1⫺/⫺ neutrophils was dosedependently inhibited by exposure to PAI-1 and was decreased to basal levels after incubation with 1 g/ml PAI-1. Of note, similar or greater concentrations of PAI-1 are found in serum and tissues of patients with inflammatory conditions, such as acute lung injury (15–18). These data suggest that binding of PAI-1 to viable PAI1⫺/⫺ neutrophils blocks their phagocytosis. To determine whether PAI-1 deficiency leads to increased Park et al.
phagocytosis of neutrophils by macrophages under in vivo conditions, viable WT or PAI-1⫺/⫺ neutrophils were intratracheally injected into WT mice. As shown in Fig. 1E, there was a significant increase in the phagocytosis of PAI-1⫺/⫺ neutrophils by alveolar macrophages compared with that found after administration of WT cells into the lungs. Preincubation of PAI-1⫺/⫺ neutrophils with PAI-1 protein, but not with mouse albumin, before intratracheal administration decreased the phagocytic index to levels similar to those seen with injection of WT neutrophils (Fig. 1F). LRP Participates in PAI-1 Associated Modulation of Neutrophil Phagocytosis. LRP is a major receptor for PAI-1 and participates in
efferocytosis through binding to CRT, a major ‘‘eat me’’ signal on target cells (1). We therefore examined whether LRP was involved in the PAI-1 associated modulation of neutrophil phagocytosis. As shown in Fig. 2A, pretreatment of macrophages with anti-LRP antibodies or the LRP specific blocking peptide, RAP, inhibited the enhanced phagocytosis of viable PAI-1⫺/⫺ neutrophils. The enhanced phagocytosis of viable WT neutrophils that occurs after PNAS 兩 August 19, 2008 兩 vol. 105 兩 no. 33 兩 11785
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Fig. 2. LRP participates in PAI-1 associated modulation of neutrophil phagocytosis, whereas PAI-1 inhibits LRP-mediated phagocytosis produced by CD-47 blockade. (A) The enhanced phagocytosis of PAI-1⫺/⫺ neutrophils is decreased by LRP blockade. Viable PAI⫺/⫺ neutrophils were pretreated without or with 1 g/ml PAI-1 protein or BSA, then cultured with macrophages untreated or pretreated with 1 g/ml anti-LRP, isotype IgG, RAP, or BSA. Mean ⫾ SD. ***, P ⬍ 0.001 compared with treatment with IgG or BSA. (B) PAI inhibits the enhancement in neutrophil phagocytosis induced by blockade of CD47. Viable WT neutrophils were pretreated without or with 1 g/ml isotype IgG, 1 g/ml mouse anti-CD47 antibody, or 1 g/ml mouse anti-CD47 antibody plus 1 g/ml rabbit anti-PAI-1 antibodies, 1 g/ml BSA, or 1 g/ml mouse anti-CRT antibody, then cultured with WT macrophages. Mean ⫾ SD. ***, P ⬍ 0.001 compared with treatment with IgG. ### P ⬍ 0.001 compared with treatment with BSA.
incubation with anti-PAI-1 antibodies was reversed by pretreatment of macrophages with anti-LRP antibodies or RAP (Fig. S2). CD47 is a major don’t eat me signal on the surface of viable cells (1). Loss of CD47 induces the uptake of viable cells through mechanisms that involve interaction of LRP and CRT (1). Because of the involvement of LRP in modulating PAI-1 associated effects on phagocytosis, we determined whether PAI-1 is able to affect the increase in phagocytosis that normally occurs after CD47 blockade. As shown in Fig. 2B, exposure to PAI-1 inhibited the enhanced neutrophil engulfment produced by CD47 blockade, indicating that PAI-1 can modulate LRP/CRT-dependent efferocytosis. AntiCRT antibodies also abrogated the increase in phagocytosis produced by CD47 blockade, consistent with previous studies (1). Suppression of Phagocytosis by PAI-1 Does Not Require ProteaseInhibiting Nor LRP-Binding Activity, but Does Involve Binding to Vitronectin. Although the above experiments indicated that LRP is
involved in PAI-1 mediated reduction of neutrophil phagocytosis, they did not establish whether direct interaction of PAI-1 with LRP was required for this effect. To examine this question, and the requirement for PAI-1’s protease inhibitory activity, in modulating phagocytosis of viable neutrophils, we preincubated viable neutrophils with the PAI-1 mutants R76E, which is unable to bind to LRP, or R346A, which is incapable of inhibiting protease activity (23), and then determined the phagocytic index. Both of these PAI-1 mutants were as effective as wild-type PAI-1 in inhibiting the enhanced phagocytosis of PAI-1⫺/⫺ neutrophils or of WT neutro11786 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0801394105
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Fig. 3. Suppression of phagocytosis by PAI-1 does not require proteaseinhibiting nor LRP-binding activity, but does need vitronectin-binding activity. (A) The protease-inhibiting or LRP-binding activities of PAI-1 are not required to inhibit phagocytosis of viable PAI-1⫺/⫺ neutrophils. Viable PAI-1⫺/⫺ neutrophils were pretreated without or with 1 g/ml WT mouse PAI-1, PAI1(R76E), PAI-1(R346A), or BSA. Mean ⫾ SD. ***, P ⬍ 0.001 compared with treatment with BSA. (B) The Vn-binding activity of PAI-1 is required to inhibit phagocytosis of viable PAI-1⫺/⫺ neutrophils. Experiments were performed as in A except that cells were treated with 1 g/ml PAI-1 (R101A), PAI-1 (Q123K), and PAI-1 (R101A/Q123K).
phils preincubated with anti-CD47 antibodies (Fig. 3A and Fig. S3). These results suggest that LRP binding and protease inhibition are not essential for the ability of PAI-1 to block macrophage uptake of viable neutrophils. In vivo, most circulating PAI-1 is bound to vitronectin (Vn) (24–26). We therefore examined whether Vn participates in modulating the effects of PAI-1 on phagocytosis. As shown in Fig. 3B, the Vn-binding deficient mutants, PAI-1 (R101A), PAI-1 (Q123K), and PAI-1 (R101A/Q123K) all lost the ability to inhibit the enhanced phagocytosis of PAI-1⫺/⫺ neutrophils, indicating that binding of PAI-1 to Vn is necessary for the inhibitory effects of PAI-1 on efferocytosis. PAI-1 Levels Are Decreased on the Surface of Apoptotic Neutrophils, Whereas Restoration of Surface PAI-1 Inhibits Phagocytosis of Apoptotic Neutrophils. As our experiments demonstrated that PAI-1 is
a potent don’t eat me signal for viable neutrophils, it was also possible that PAI-1 might participate in modulating phagocytosis of apoptotic cells. In initial studies, we performed confocal microscopy to examine the distribution of PAI-1 on viable and apoptotic neutrophils. As shown in Fig. 4A, large amounts of PAI-1 are present on the membrane of viable neutrophils. In contrast, the levels of PAI-1 on the apoptotic neutrophils are dramatically decreased. These results were confirmed by flow cytometry analysis of neutrophil associated PAI-1 staining (Fig. 4B). These data suggest that changes in the amount of PAI-1 on the apoptotic cell surface may contribute to the enhanced engulfment of apoptotic cells by phagocytes. Because there are diminished amounts of PAI-1 on apoptotic neutrophils, it was possible that restoration of membrane associated PAI-1 would inhibit phagocytosis of apoptotic neutrophils. To Park et al.
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examine this issue, apoptotic neutrophils were preincubated with PAI-1. As shown in Fig. 4C, exposure of apoptotic neutrophils to PAI-1 significantly decreased their uptake by macrophages. Consistent with previous studies, blockade of LRP on the engulfing cells with either anti-LRP antibodies or RAP also diminished the phagocytosis of apoptotic neutrophils. To demonstrate that PAI-1 does bind to apoptotic neutrophils, we labeled PAI-1 protein with fluorescent dye. As shown in Fig. 4D. the annexin V positive (apoptotic) neutrophils demonstrated increased PAI-1 binding after incubation with PAI-1. In contrast, there was no increase in binding of a control protein, mouse albumin, to the surface of apoptotic neutrophils. Incubation of viable neutrophils with labeled PAI-1 did not demonstrate any increase in PAI-1 binding, suggesting that saturating amounts of PAI-1 are present on the surface on viable cells. Similar to findings with viable neutrophils, LRP binding and protease inhibiting activities of PAI-1 are not required for its inhibitory activities on neutrophil phagocytosis, as shown by decrease in the phagocytic index by PAI-1 mutants unable to bind LRP (i.e., R76E) or lacking protease activity (i.e., R346A) (Fig. S4). CRT Is Involved in PAI-1 Associated Modulation of Neutrophil Phagocytosis. Our experiments demonstrated that even though LRP is
involved in increased phagocytosis of viable PAI-1⫺/⫺ cells, LRPbinding activity is not required for PAI-1 to inhibit this increase in phagocytosis. We therefore decided to examine whether PAI-1 exerts its anti-phagocytic effects through modifying expression of the LRP ligand, CRT. We found that CRT levels on the surface of viable PAI-1⫺/⫺ neutrophils are increased compared with those on WT cells (Fig. 5A). Furthermore, incubation of viable PAI-1⫺/⫺ neutrophils with PAI-1 protein decreased surface CRT to levels similar to those on WT cells (Fig. 5A). These data suggest that elevated levels of CRT may contribute, at least in part, to the increase in phagocytosis of viable PAI-1⫺/⫺ neutrophils. We also examined whether PAI-1 regulates levels of the don’t eat me signal Park et al.
CD47 on neutrophils. In contrast to the relationship between PAI-1 and CRT expression, there was no difference in CD47 expression on the surface of viable WT and PAI-1⫺/⫺ neutrophils (Fig. 5B). Although we found that CRT is increased on the surface of PAI-1⫺/⫺ neutrophils and that neutrophil exposure to PAI-1 decreases CRT levels on PAI-1⫺/⫺ cells, these alterations in CRT expression are relatively modest and may be insufficient mechanisms for the anti-phagocytic effects of PAI-1. Therefore, we examined the distribution of PAI-1 and CRT on the surfaces of viable and apoptotic neutrophils. We found that PAI-1 colocalizes with CRT on viable cells, suggesting that PAI-1 could affect recognition and/or binding of CRT by its receptor, LRP (Fig. 5C). PAI-1 levels were dramatically decreased on the surface of apoptotic neutrophils (Fig. 4 A and B and 5C), but were still visible after increasing the pinhole of the green fluorescence. In contrast to staining of living cells, the patterns for both CRT and PAI-1 became punctuate on apoptotic neutrophils (Fig. 5C). In addition, whereas there was colocalization of PAI-1 and CRT on viable cells, there was very little overlap of PAI-1 and CRT staining on apoptotic neutrophils. Discussion In the present study, we found that PAI-1 has a novel role in the phagocytosis of neutrophils, acting as a don’t eat me signal for viable and apoptotic cells. Because apoptotic cell clearance is critical to the resolution of inflammation, our data suggest that PAI-1 may exacerbate inflammatory responses by blocking the clearance of apoptotic neutrophils, thereby allowing such neutrophils to become necrotic and to release their intracellular contents into the extracellular milieu. Our findings, together with previous studies demonstrating that PAI-1 enhances LPS induced neutrophil activation (22) and promotes the migration and infiltration of lymphocytes and neutrophils into inflammatory sites (19–21, 27), demonstrate that PAI-1 has multiple proinflammatory roles that PNAS 兩 August 19, 2008 兩 vol. 105 兩 no. 33 兩 11787
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Fig. 4. PAI-1 levels are decreased on the surface of apoptotic neutrophils, whereas restoration of PAI-1 inhibits phagocytosis of apoptotic neutrophils. (A) Viable WT neutrophils were plated on cover slides and apoptosis induced by incubation at 43°C for 45 min, followed by 37°C for 3h. The cells were fixed, then stained with rabbit anti-PAI-1 antibodies followed by secondary antibody conjugated with Alexor 488. Confocal images were obtained for viable and apoptotic cells under the same settings. (B) Viable and apoptotic neutrophils were stained with rabbit anti-PAI-1 antibodies or rabbit IgG, followed by secondary antibody conjugated with Alexor 488. (C) PAI-1 inhibits phagocytosis of apoptotic neutrophils. Apoptotic WT neutrophils were pretreated without or with 1 g/ml PAI-1 then cultured with WT untreated macrophages or macrophages pretreated with 1 g/ml anti-LRP, isotype IgG, RAP, or BSA. Mean ⫾ SD. ***, P ⬍ 0.001 compared with treatment with IgG or BSA. (D) PAI-1 binds to the surface of apoptotic neutrophils. Apoptotic neutrophils were incubated with Chromeo 642 fluorescent dye-labled mouse albumin or PAI-1 for 1h and FITC-Annexin V for 15 min.
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Fig. 5. CRT is involved in PAI-1-regulated phagocytosis. (A) CRT is increased on PAI-1⫺/⫺ neutrophils. Viable WT and PAI-1⫺/⫺ neutrophils that had been preincubated without or with PAI-1 protein were fixed, stained with mouse anti-CRT antibodies or isotype specific IgG, and flow cytometry performed. (B) CD47 is unchanged on PAI-1⫺/⫺ neutrophils. Experiments were performed as in A except staining with anti-CD47 antibody. (C) CRT and PAI-1 colocalize on viable neutrophils, but not on apoptotic cells. Viable or apoptotic WT were fixed and stained with rabbit anti-PAI-1 and mouse anti-CRT antibodies followed by incubation with secondary antibodies.
are independent of its participation in coagulation and microvascular thrombosis. We found that the enhanced neutrophil phagocytosis associated with PAI-1 deficiency can be abrogated by blocking LRP on macrophages with anti-LRP antibodies or the LRP-specific blocking peptide RAP, indicating that LRP participates in PAI-1 mediated modulation of efferocytosis. In addition, the increase in neutrophil phagocytosis produced by blockade of CD47, which has been demonstrated to be LRP-dependent, is prevented by exposure to PAI-1. These data support a role for LRP in PAI-1-regulated phagocytosis. However, the LRP-binding deficient PAI-1 mutant, R76E, inhibits the phagocytosis of viable PAI-1⫺/⫺ neutrophils to a similar extent as does wild-type PAI-1, indicating that binding between LRP and PAI-1 is not required for the inhibitory effects of PAI-1 on neutrophil uptake. Previous studies have shown that LRP mediates viable cell phagocytosis through binding to CRT on the target cell surface (1). Thus, blocking either LRP on the engulfing cells or CRT on the target cells can have similar effects in reducing efferocytosis. Although the PAI-1 R76E mutant cannot directly bind to LRP on the target cell surface, it can still potentially affect the recognition and binding of CRT on the target cells by phagocyte associated LRP through interactions with uPAR, integrins, membrane associated vitronectin, and/or other yet-to-beidentified ligands on the target cell surface. Indeed, PAI-1 mutants that are unable to bind to vitronectin, unlike wild type PAI-1, have no effect on the increased phagocytosis of PAI-1⫺/⫺ neutrophils, indicating that interactions between vitronectin and PAI-1 are required to mediate the inhibitory effects of PAI-1 on neutrophil engulfment by macrophages. These results suggest that vitronectin may participate in regulating interactions between PAI-1, CRT, and LRP in a manner similar to that described for its role in modulating clearance of thrombin and PAI-1 complexes by LRP (28). 11788 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0801394105
We found that PAI-1 colocalizes with CRT on the surface of viable, but not apoptotic neutrophils, providing a potential mechanism involving PAI-1 mediated inhibition of CRT-LRP interactions through which PAI-1 could regulate CRT-mediated ingestion of viable cells and contribute to the resistance of living cells to inadvertent ingestion. In addition, CRT is increased on viable PAI-1 (⫺/⫺) neutrophils, suggesting an additional mechanism for the enhanced phagocytosis of viable PAI-1 (⫺/⫺) cells. Of note, incubation of PAI-1⫺/⫺ neutrophils with PAI-1 resulted in return of the increased CRT concentrations on the cell membrane to baseline levels, indicating that the inhibitory effects of PAI-1 on neutrophil phagocytosis may be indirect, and occur, in part, through regulating levels of CRT on viable neutrophils. We found that PAI-1 levels on the surface of apoptotic neutrophils are dramatically decreased. If the mechanism through which PAI-1 acts as a don’t eat me signal is by regulating interactions of CRT with LRP, then the loss of membrane associated PAI-1 in apoptotic cells would lead to enhanced CRT mediated uptake of these cells. Support for this hypothesis comes from the finding that the colocalization of PAI-1 and CRT found on viable neutrophils was disrupted on the apoptotic cell surface, indicating that even the low levels of PAI-1 on apoptotic neutrophils are unable to prevent recognition and binding of CRT by LRP, thus contributing to the increase in the phagocytosis of apoptotic cells. Together, these data suggest that there may be more than one mechanism by which PAI-1 can inhibit phagocytosis of apoptotic neutrophils. Tissue and circulating PAI-1 levels remain elevated for prolonged periods in acute inflammatory conditions, such as sepsis or acute lung injury, that are associated with massive accumulation of activated neutrophils into the lungs and other organs (15, 16). Therefore, modulation of PAI-1 expression on activated neutrophils may be of clinical benefit through enhancing neutrophil clearance and resolution of inflammation in injured organs. Park et al.
Materials. Custom mixture antibodies for neutrophil isolation were from StemCell Technologies. Annexin V-FITC and propidium iodide were from R&D. Rabbit anti-PAI-1 (H-135, sc-8979, recognizes an epitope corresponding to amino acids 24 –158 of human PAI-1), mouse anti-PAI-1 (C-9, sc-5297), and mouse anti-CD47 antibodies were from Santa Cruz. Mouse anti-calreticulin antibody was from Abcam. Mouse anti-LRP antibody was from American Diagnostica. Alexa Fluor 488-conjugated goat anti-rabbit and Alexa Fluor 555-conjugated goat antimouse antibodies were from Invitrogen. Chromeo 647 protein fluorescent labeling kit was from Active Motif. Wild type PAI-1, PAI-1(R76E), and PAI-1 (R346A) proteins were gifts from Dr. Victoria Ploplis. LRP specific blocking peptide RAP was a gift from Dr. Dudley Strickland. Mice. PAI-1 knockout mice backcrossed with C57/BL6 mice for at least nine generations and age-and sex-matched control mice C57/BL6 were from Jackson Laboratory (Bar Harbor, ME). 8 –10-week-old animals were used for experiments. All animal protocols were approved by the UAB institutional review committee. Isolation and Culture of Bone Marrow Neutrophils and Peritoneal Macrophages. Isolation of bone marrow neutrophils was as described (22, 29). Peritoneal macrophages were isolated 5 days after i.p. injection of 4% thioglycollate (Sigma). The macrophages were washed and plated in 96-well plates at 0.2 ⫻ 106 cells per well. After incubation for 2 h at 37°C, the wells were washed three times to remove nonadherent cells. Induction and Assessment of Neutrophil Apoptosis. Apoptosis of neutrophils was induced by heating to 43°C for 45 min, followed by incubation at 37°C in 5% CO2 for 3 h. This methodology routinely yielded populations that included 40 –70% cells positively staining with annexin V with ⬍10% positive for propidium iodide (PI).
In Vitro Efferocytosis Assay. Phagocytosis of neutrophils was determined by adding 1 ⫻ 106 viable or apoptotic neutrophils suspended in 200 l Opti-MEM to 96-well plates containing adherent macrophage monolayers and then incubating at 37°C for 90 min. Mouse serum 5% was included during the coincubations. After coincubation of macrophages and neutrophils, the plates were washed three times with ice-cold PBS and trypsinized. The detached cells were collected
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and cytospin slides prepared and stained with HEMA 3 . Phagocytosis was evaluated by two independent blinded observers counting 200 –300 macrophages per slide from triplicate experiments. Results for phagocytosis index are expressed as the percentage of macrophages containing at least one ingested neutrophil.
In Vivo Efferocytosis Assay. 10 ⫻ 106 viable WT or PAI-1⫺/⫺ neutrophils were resuspended in 50 l PBS and intratracheally injected into isofluorane anesthetized mice. After 90 min, the mice were killed and bronchoalveolar lavage performed with 3 ml PBS. Cytospin slides were prepared using 250 l bronchoalveolar lavage fluid. To determine the effect of PAI-1 protein on phagocytosis of PAI-1⫺/⫺ neutrophils in vivo, 10 ⫻ 106 viable PAI-1⫺/⫺ neutrophils were preincubated with 2 g mouse albumin or PAI-1 protein for 30 min in 50 l PBS before being injected intratracheally. Flow Cytometry. Neutrophils were incubated with anti-PAI-1 antibody (1 g/ml) or anti-CRT antibody for 1 h on ice. The cells were then fixed in PBS containing 4% paraformaldehyde and 3% sucrose for 10 min. After three washes with PBS, neutrophils were incubated with Alexa Fluor 488-conjugated goat anti-rabbit antibody for 30 min. To determine binding of PAI-1 to neutrophils, PAI-1 protein or mouse albumin was labeled with Chromeo 647. Apoptotic neutrophils were incubated with 1 g/ml labeled PAI-1 or mouse albumin for 1h and FITC-Annexin V for 15 min, then analyzed by two-color flow cytometry. Immunofluorescent Confocal Microscopy. To induce apoptosis, viable neutrophils plated on cover slides were incubated at 43°C for 45 min, followed by 37°C for 3h. The cells were then fixed and stained with anti-PAI-1 or CRT antibodies overnight, and then incubated with secondary antibodies conjugated with Alexor 488 or Alexor 594 for 1 h. Confocal images were taken for viable and apoptotic cells under the same settings. Statistical Analysis. For each experiment, neutrophils were isolated and pooled from 3 to 4 mice. One way ANOVA followed by the Tukey-Kramer multiple comparisons test (for multiple groups), or Student’s t test (for comparisons between two groups) was used. P ⬍ 0.05 was considered to be statistically significant. ACKNOWLEDGMENTS. This work was supported in part by National Institutes of Health Grants HL62221, HL76206, and HL068743 to E.A.
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PNAS 兩 August 19, 2008 兩 vol. 105 兩 no. 33 兩 11789
CELL BIOLOGY
Materials and Methods
