Published online before print July 20, 2005
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Vß3 integrins
* Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Denver;
Department of Anesthesiology, Chonnam National University Medical School, Gwangju, Korea;
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia;
Department of Internal Medicine, Chung Ang University College of Medicine, Seoul, Korea; and
¶ Department of Specialty Care Services, The University of Texas Health Center at Tyler
1Correspondence: Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Box C272, 4200 E. Ninth Avenue, Denver, CO 80262. E-mail: edward.abraham{at}uchsc.edu
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, deletion of the kringle domain (KD) from uPA resulted in loss of these potentiating effects. Addition of purified uPA KD to LPS-stimulated neutrophils increased cytokine expression to a degree comparable with that produced by single-chain uPA. Inclusion of the arginine-glycine-aspartic but not the arginine-glycine-glutamic peptide to neutrophil cultures blocked uPA kringle-induced potentiation of proinflammatory responses, demonstrating that interactions between the KD and integrins were involved. Antibodies to V or ß3 integrins or to the combination of Vß3 prevented uPA kringle-induced enhancement of expression of proinflammatory cytokines and also of adhesion of neutrophils to the uPA KD. These results demonstrate that the KD of uPA, through interaction with Vß3 integrins, potentiates neutrophil activation.
Key Words: uPA • IL-1ß • MIP-2 • TNF- • growth factor domain
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B [8
]. Transgenic mice unable to produce uPA were protected from endotoxemia-induced, neutrophil-dependent lung injury. uPA is secreted by many cell populations, including neutrophils and endothelial and epithelial cells, as a single-chain proenzyme, which possesses little or no proteolytic activity [4 , 9 10 11 ]. Single-chain uPA (scuPA) is converted after single cleavage between Lys158 and Ile159 into the proteolytically active dual-chain enzyme, consisting of an NH2-terminal A chain and a proteolytic domain (PD)-containing B chain. uPA is composed of three structurally independent components: a growth factor domain (GFD; amino acids 1–46), a kringle domain (KD; amino acids 47–135), and a PD (amino acids 159–411). The GFD is responsible for the interaction of urokinase with the uPA receptor (uPAR)/CD87 receptor [2 , 9 , 12 ]. The PD includes the catalytically active site of the enzyme. Enzymatic digestion of scuPA yields an amino terminal fragment (ATF), which consists of the GFD and KD, and a low molecular weight fragment [3 , 13 , 14 ]. Single chain and cleaved, two-chain uPA, as well as the ATF of uPA, which contains the GFD, can bind to the uPAR/CD87 receptor [2 , 4 , 9 , 10 , 12 , 15 , 16 ]. uPA lacking the GFD fails to interact with uPAR/CD87 but can bind to other cell-surface receptors, including integrins and those belonging to the low-density lipoprotein receptor (LDLR) family through the KD or PD [17 18 19 20 ].
In recent experiments [8 ], we found that human uPA enhanced activation of murine neutrophils exposed to submaximal stimulatory doses of LPS. As human uPA is unable to bind to murine uPAR [21 ], those studies suggested that receptors other than uPAR were involved in potentiating neutrophil responses. As the uPA GFD is responsible for interactions with uPAR [22 , 23 ], our observations also indicated that other domains of uPA were responsible for the proinflammatory effects of uPA on neutrophils.
In this study, we used purified uPA KD as well as uPA mutants lacking the KD to delineate the nature of proinflammatory interactions between uPA and neutrophils. These experiments showed that the uPA KD is responsible for the ability of uPA to increase cytokine expression by LPS-stimulated neutrophils. In addition, Vß3 integrins, through interactions with the uPA KD, appear to play an important role in the enhancement of neutrophil activation by uPA.
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Materials
Isoflurane was obtained from Abbott Laboratories (Chicago, IL). Escherichia coli 0111:B4 endotoxin was purchased from Sigma Chemical Co. (St. Louis, MO). RPMI 1640/25 mM HEPES/L-glutamine, fetal bovine serum, and penicillin/streptomycin were purchased from Mediatech, Inc. (Herndon, VA). Custom cocktail antibodies and columns for neutrophil isolation were purchased from Stem Cell Technologies (Vancouver, BC). Antibodies to V and ß3 integrins, as well as isotype-specific controls were purchased from Chemicon (Temecula, CA) for human integrins and PharMingen (San Diego, CA) for murine integrins. Arginine-glycine-aspartic (RGD) and arginine-glycine-glutamic (RGE) peptides were purchased from Sigma Chemical Co. Recombinant human scuPA was prepared as described previously, as were the isolated uPA domains and uPA mutants lacking specific domains [18
, 22
, 24
]. The LPS concentration of the stock scuPA as well as all other solutions measured by enzyme-linked immunosorbent assay (ELISA; BioWhittaker, Rockland, ME) was <1 pg/ml.
Isolation of neutrophils
Mouse neutrophils were purified from bone marrow cell suspensions as described previously [8
, 25
, 26
]. Briefly, to obtain the bone marrow cell suspension, the femur and tibia of a mouse were flushed with 5 ml RPMI 1640/penicillin/streptomycin, and the cells passed through a glass wool column and were pelleted by centrifugation at 1000 rpm for 10 min. The cell pellets were resuspended in 0.3% fetal calf serum/phosphate-buffered saline (PBS) and then incubated with 20 µl primary antibodies, specific for cell-surface markers F4/80, CD4, CD45R, CD5, and TER119 for 15 min, rotating at 4°C. This custom cocktail (Stem Cell Technologies) is specific for T and B cells, red blood cells (RBC), monocytes, and macrophages. Antibiotin tetrameric antibody complexes (100 µl) were then added, and the cells incubated for 15 min, rotating at 4°C. Following this, 60 µl colloidal magnetic dextran iron particles were added to the suspension and incubated for 15 min, rotating at 4°C. The entire cell suspension was then placed into a column, surrounded by a magnet. The T cells, B cells, RBC, monocytes, and macrophages were captured in the column, allowing the neutrophils to pass through by negative selection methods.
For human neutrophil isolation, peripheral blood was obtained from healthy volunteers under a protocol approved by the University of Colorado Health Sciences Center Institutional Review Board (Denver). Neutrophils were isolated by plasma-Percoll gradients after dextran sedimentation of erythrocytes, as described previously [27 ].
Neutrophil purity, as determined by Wright’s stained cytospin preparations, was greater than 97%. Cell viability, as determined by trypan blue exclusion, was consistently greater than 98%.
Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)
Quantitative RT-PCR to measure neutrophil cytokine expression was performed as described previously [8
, 28
]. In brief, total cellular RNA was isolated from neutrophils using the Bio-Rad Aqua Pure RNA isolation kit (Bio-Rad, Hercules, CA), as recommended by the manufacturer. Real-time RT-PCR was performed with specific primers and probes corresponding to the proinflammatory cytokine genes interleukin (IL)-1ß, macrophage-inflammatory protein-2 (MIP-2), and tumor necrosis factor (TNF-). For each mRNA detected, a fluorogenic probe and two primers (forward and reverse) for PCR were synthesized (Applied Biosystems, Foster City, CA). The internal oligonucleotide probe was labeled with the fluorescent dyes carboxyfluorescein (FAM) at the 5' end and N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA) at the 3' end.
For human IL-1ß mRNA detection, the forward and reverse primers were 5'-CACGATGCACCTGTACGATCA-3' and 5'-AGACATCACCAAGCTTTTTTGCT-3', respectively. The internal probe was 5'-(FAM)-TGAACTGCACGCTCCGGGACTCA-(TAMRA)-3'. For murine IL-1ß mRNA detection, the forward and reverse primers were 5'-GCTGAAAGCTCTCCACCTCAA-3' and 5'-TCGTTGCTTGGTTCTCCTTGTA-3', respectively. The internal probe was 5'-(FAM)-CAGAATATCAACCAAGTGATATTCTCCATGAGC-(TAMRA)-3'. For human TNF- mRNA detection, the forward and reverse primers were 5'-TCTTCTCGAACCCCGAGTGA-3' and 5'-GGAGCTGCCCCTCAGCTT-3', respectively. The internal probe was 5'-(FAM)-AGCCTGTAGCCCATGTTGTAGCAAACCCT-(TAMRA)-3'. For murine TNF- mRNA detection, the forward and reverse primers were 5'-CTGTAGCCCACGTCGTAGTCAA-3' and 5'-CTCCTGGTATGAGATAGCAAATCG-3', respectively. The internal probe was 5'-(FAM)-TGCCCCGACTACGTGCTCCTCAC-(TAMRA)-3'. The forward and reverse primers for murine MIP-2 were 5'-TGTGACGCCCCCAGGA-3' and 5'-AACTTTTTGACCGCCCTTGAG-3', respectively. The internal probe was 5'-(FAM)-TGCGCCCAGACAGAAGTCATAGCCA-(TAMRA)-3'.
All reagents used in the one-step RT-PCR reactions were purchased from Applied Biosystems. Each one-step RT-PCR reaction contained a total volume of 50 µl. The RT reaction was performed for 30 min at 48°C using MultiScribe RT (Applied Biosystems) at a final concentration of 0.25 U/µl. After the RT step, AmpliTaq Gold polymerase (Applied Biosystems), with a final concentration of 0.025 U/µl, was activated by an increase in temperature to 95°C for 10 min followed by 40 cycles of amplification (95°C for 15 s and 60°C for 1 min) with a Gene Amp 5700 sequence detection system (ABI Prism, Foster City, CA). The amount of cytokine mRNA was determined from a standard curve with tenfold dilutions of known amounts of target RNA for each primer and probe set. RNA amounts were determined using software provided with the Gene Amp 5700 sequence detection system. Quantification was determined by dividing the target amount of each cytokine sample by the amount of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) RNA.
Cytokine ELISA
Immunoreactive murine TNF-, IL-1ß, and MIP-2 and human TNF- and IL-1ß were quantified using commercially available ELISA kits (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions and as described previously [29
].
Neutrophil adhesion
After suspension in RPMI at 106 cells/ml, neutrophils were labeled by incubation with 1.5 µM 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester for 15 min at 37°C in the dark, as described previously [30
]. The cells were then washed three times and resuspended in Dulbecco’s modified Eagle’s medium, supplemented with 2 mM Mg2+. For antibody inhibition experiments, neutrophils were stimulated with LPS (100 ng/ml) for 1 h in the presence of anti-integrin antibodies (V, ß3, or isotype controls) at 10 µg/ml. The neutrophils were then added to 96-well plates (Costar, Cambridge, MA), which had been coated with scuPA, KD, or mutant uPA lacking the KD (
KD uPA). Coating was achieved by adding 100 µl 200 nM scuPA, KD, or KD uPA to each well for 3 h at 37°C, followed by washing the plate and blocking with 0.5% polyvinylpyrrolidone (PVP) for 1 h at room temperature. After a 30-min incubation at 37°C of 2 x 105 cells/well on the coated plates, the fluorescence reading at 492/517 nM of each well was obtained. The nonadherent cells were then removed by gently inverting the plate in PBS, and the fluorescence of each well was read again. Triplicate wells were included for each condition.
Statistical analysis
For each experimental condition, the entire group of animals was prepared and studied at the same time. Data are presented as mean ± SEM for each experimental group. One-way ANOVA, the Tukey-Kramer Multiple Comparisons test (for multiple groups), or Student’s t-test (for comparisons between two groups) were used. P < 0.05 was considered significant.
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KD uPA. As shown in Figure 1
, scuPA significantly increased mRNA levels and secretion of the proinflammatory cytokines TNF-, MIP-2, and IL-1ß in neutrophils cultured with submaximal concentrations of LPS. In contrast, addition of KD uPA to neutrophil cultures had no effect on LPS-induced cytokine expression, even when the concentrations of KD uPA were tenfold greater than those at which scuPA produced potentiating effects. These results suggested that the KD is responsible for the enhancing effects of uPA on neutrophil activation.
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Figure 1. (A) uPA potentiates LPS-induced cytokine expression in neutrophils, which were incubated without (C) or with uPA, 1, 10, or 100 nM, or LPS, 100 ng/ml, alone (uPA1, uPA10, uPA100, LPS, respectively) or with LPS plus uPA, 1, 10, or 100 nM, for 4 h. (B) Deletion of the KD eliminates the ability of uPA to potentiate LPS-induced cytokine expression by neutrophils, which were incubated without (C) or with a KD, 1, 10, 100 nM, or LPS alone (KD1, KD10, KD100, LPS, respectively) or with LPS plus scuPA, 10 nM (uPA10), KD, 1, 10, or 100 nM, for 4 h. (C) The KD of uPA potentiates LPS-induced cytokine expression in neutrophils, which were incubated without (C) or with KD, 1, 10, 100 nM, or LPS alone (KD1, KD10, KD100, LPS, respectively) or with LPS plus KD, 1, 10, or 100 nM, for 4 h. Cytokine mRNA levels were determined by quantitative RT-PCR normalized to GAPDH, and protein levels were measured in the culture supernatants by ELISA. *, P < 0.05, versus control; #, P < 0.05, versus LPS alone. Similar results were found for MIP-2 and IL-1ß.
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KD uPA at the same 10 nM concentration. As shown in Figure 2
, the increase in LPS-induced cytokine expression produced by addition of purified KD to the cultures was as great or even slightly greater than that found after addition of scuPA at the same concentration. In contrast, there was no potentiation achieved in the presence of KD uPA.
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Figure 2. The KD of uPA is required for enhancement of LPS-induced cytokine expression in neutrophils, which were incubated without (C) or with LPS and with scuPA, 10 nM (uPA), the uPA KD, 10 nM (KD), or a KD, 10 nM, for 4 h. Cytokine mRNA levels were determined by quantitative RT-PCR normalized to GAPDH, and protein concentrations were measured in the culture supernatants by ELISA. *, P < 0.05, versus control; #, P < 0.05, versus LPS alone.
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As shown in Figure 3
, addition of the RGD, but not the RGE peptide, blocked KD-induced potentiation of neutrophil responses. The RGD peptide prevented the increase in mRNA levels and protein secretion of IL-1ß, TNF-, and MIP-2 induced by coculture of LPS-stimulated neutrophils with KD.
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Figure 3. Vß3 integrins are involved in potentiation of neutrophil activation by the uPA KD. (A) Neutrophils were incubated without (C), with LPS and the uPA KD, 10 nM (KD), plus RGD or RGE peptide at 50 µM for 4 h. Neutrophils cultured with LPS alone without any KD (LPS) were also included. (B) Antibodies to v or ß3 integrins block uPA KD-induced potentiation of LPS-induced cytokine expression in neutrophils, which were incubated with LPS and KD, 10 nM, plus isotype-specific control antibodies (CAb) or antibodies to v (10 µg/ml), ß3 (10 µg/ml), or both (vß3; 20 µg/ml) for 4 h. Neutrophils not exposed to LPS (C) or cultured with LPS alone without KD (LPS) were also included. Cytokine mRNA levels were determined by quantitative RT-PCR normalized to GAPDH, and protein concentrations were measured in the culture supernatants by ELISA. *, P < 0.05, versus control; #, P < 0.05, versus LPS alone. Similar results were found for MIP-2 and IL-1ß.
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Vß3 integrins is responsible for enhancement of neutrophil activationVß3 and Mß2 integrins are involved in uPA-induced signaling [11
, 39
40
41
42
]. However, interaction of uPA or the uPA KD with these integrins on neutrophils or a role for such interactions in modulating neutrophil functions had not been shown previously.
To investigate the involvement of Vß3 and Mß2 integrins in KD-induced potentiation of neutrophil activation, we added control antibodies or blocking antibodies to V, M, ß3, or ß2 to neutrophil cultures containing LPS and KD. As shown in Figure 3
, the addition of antibodies to Vand ß3 or to V and ß3, but not control antibodies, prevented KD-induced enhancement of proinflammatory cytokine expression by LPS-stimulated neutrophils. There was no effect of antibodies to M and ß2 or to M and ß2 on uPA-potentiated cytokine expression in neutrophils cultured with LPS (data not shown). Of note, the levels of cytokine mRNA or protein in cultures containing antibodies to V, ß3, or both were similar to or even below those produced by neutrophils stimulated with LPS alone.
Interactions between the uPA KD and Vß3 integrins also appeared to be involved in enhancing LPS-induced cytokine production in human neutrophils. As shown in Figure 4
, addition of uPA, KD, but not KD, to human neutrophils cultured with LPS resulted in increased mRNA and protein levels of TNF- and IL-1ß. Such potentiating effects of the uPA KD were blocked when antibodies to Vß3 were added to the cultures.
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Figure 4. The uPA KD and vß3 integrins participate in uPA-induced potentiation of LPS-induced cytokine expression in human neutrophils, which were incubated without (C) or with LPS, and LPS-stimulated neutrophils were also cultured with scuPA, 10 nM (uPA), the uPA KD, 10 nM (KD), or a KD, 10 nM, for 4 h. In addition, neutrophils were cultured with LPS and KD, 10 nM, plus isotype-specific CAb (20 µg/ml) or antibodies to v (10 µg/ml) and ß3 (10 µg/ml). Cytokine mRNA levels were determined by quantitative RT-PCR normalized to GAPDH, and protein concentrations were measured in the culture supernatants by ELISA. *, P < 0.05, versus control; #, P < 0.05, versus LPS alone.
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Vß3 integrins on neutrophils, we investigated neutrophil adhesion to scuPA or uPA KD. Figure 5
demonstrates enhanced adhesion of LPS-stimulated neutrophils to uPA and the uPA KD, but not to a KD. Addition of antibodies to V, ß3, or both, but not control antibodies, decreased neutrophil adhesion on KD-coated plates back to baseline levels. Such results confirm that interactions between neutrophils and the uPA KD are mediated through Vß3 integrins.
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Figure 5. Enhanced adhesion of LPS-stimulated neutrophils to uPA is dependent on the KD and occurs through interactions with V and ß3 integrins. Adhesion of unstimulated and LPS-stimulated neutrophils to wells coated with scuPA (uPA), uPA KD (KD), and a KD is shown, as well as adhesion when neutrophils were incubated with isotype-specific CAb or antibodies to V, ß3, or both before being placed on uPA KD-coated wells. Neutrophils incubated with (LPS) or without (C) LPS and then seeded onto uncoated wells were also included. Each experiment was performed in triplicate. A second experiment produced similar results. * P < 0.05 versus control.
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The involvement of receptors other than the classical uPAR in enhancement of neutrophil activation by the KD of uPA is not surprising, as the KD is not known to associate with uPAR. Recent studies demonstrate that uPA has cell-surface receptors other than uPAR, which are involved in signaling events. For example, blockade of the binding of uPA to uPAR using monoclonal antibodies or depletion of cell-surface uPAR with phosphatidylinositol-specific phospholipase C did not inhibit uPA-induced mitogenic effects on smooth muscle cells [45
]. Putative receptors that are present on neutrophils and have been shown to interact with uPA in other cell types include the LDLR-related protein/2-macroglobulin receptor and integrins, specifically Mß2 and vß3 [11
, 18
, 31
32
33
].
In the present experiments, addition of blocking antibodies to V, ß3, or both prevented the potentiation of proinflammatory cytokine production by LPS and also inhibited binding of LPS-stimulated neutrophils to the uPA KD, indicating that interactions between the uPA KD and Vß3 integrins were responsible for these effects. As the RGD peptide blocks interactions with ß3, but not ß2 integrins, our results showing inhibition of KD-induced potentiation of proinflammatory cytokine production when RGD is present in neutrophil cultures are also consistent with associations between uPA KD and Vß3 integrins in mediating neutrophil activation [35
36
37
38
, 47
, 48
].
Recent studies demonstrate that interactions between uPA and integrins, dependent and independent of uPAR, are involved in the regulation of cell adhesion and migration [2
, 9
, 16
, 34
, 49
]. In addition to interaction of the KD with Vß3, as shown in the present experiments, the KD and PD of uPA, which are distinct from the GFD that binds to uPAR, are recognized by the integrin Mß2 (CD11b/CD18, membrane-activated complex-1) [11
, 50
, 51
]. Blockade of Mß2 suppresses uPA-mediated neutrophil migration and adhesion as well as uPA priming for superoxide anion release [7
]. However, despite studies showing that uPA can bind to integrins, we are unaware of any previous reports that specifically examine the ability of uPA or domains of uPA to modify neutrophil proinflammatory responses, including the expression of proinflammatory cytokines by uPAR-independent processes. In the present experiments, we found that Vß3 but not Mß2 integrins contributed to the enhancement of neutrophil activation induced by the uPA KD.
Previous studies have shown that uPAR participates in host defense against Pseudomonas aeruginosa and pneumococcal pneumonia [1
, 52
]. uPAR–/– mice demonstrate increased mortality as well as bacterial colony-forming units in the lungs after infection compared with wild-type uPAR+/+ mice. It is interesting that the role of uPAR in antibacterial responses appears to be independent of uPA, as neutrophil recruitment in uPA–/– mice in response to P. aeruginosa is indistinguishable from that present in control uPA+/+ mice [52
]. Our data indicate that the KD of uPA, through mechanisms involving Vß3 integrins and not requiring uPAR, plays a central role in modulating proinflammatory effects of uPA in neutrophils stimulated with the bacterial product LPS. Of note, recent studies from our laboratory also show that uPA potentiates Toll-like receptor 2 (TLR2) neutrophil activation induced by exposure to the TLR2 ligand, peptidoglycan (unpublished observations). As the KD does not bind to uPAR, the present findings suggest that proinflammatory properties of uPA may be independent of antibacterial and chemotactic effects that involve uPAR. If this is the case, interruption of KD-integrin interactions may decrease neutrophil-mediated inflammatory injury, such as acute lung injury, without affecting antibacterial host defense. We are presently examining this possibility.
Although the present experiments demonstrate that the uPA KD participates in potentiating LPS-mediated proinflammatory processes, the findings do not exclude the possibility that the proteolytic properties of uPA also contribute to inflammation, particularly under in vivo conditions. In previous studies, we demonstrated that transgenic mice, unable to produce uPA, were protected from endotoxin-induced acute lung injury, which is neutrophil-dependent [8
]. Although elimination in uPA–/– mice of KD-integrin interactions among LPS-stimulated neutrophils in the lungs and other sites may be responsible for the reduction of lung injury, it is possible that prevention of the generation of proinflammatory mediators, such as plasmin, through uPA-dependent proteolysis also contributes to the reduction of tissue injury in this setting. Similarly, although our studies show a primary role for Vß3 integrins in enhancing neutrophil activation under in vitro conditions, uPAR may still be involved in neutrophil-dependent inflammatory processes in vivo through its role in enhancing neutrophil chemotaxis [6
, 15
]. In addition, as uPA binds to uPAR with high affinity, it is possible that association of uPA with uPAR may facilitate the interaction between the uPA kringle and integrins under in vivo situations, when integrins become localized to specialized portions of activated neutrophils during cell adhesion and migration. It is known that uPAR binds to certain integrins, including Vß3, and this association may be promoted by uPA [2
, 9
, 34
, 35
]. Nevertheless, the present studies suggest that therapies directed against the KD of uPA or the Vß3 integrin receptors, to which the uPA KD binds, may be able to diminish the proinflammatory properties of uPA during acute neutrophil-driven inflammatory conditions, such a acute lung injury or sepsis, with concomitant reduction in organ dysfunction and mortality.
Received March 18, 2005; revised May 26, 2005; accepted June 11, 2005.
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