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PHAGOCYTES
From the Joseph J. Jacobs Center for Thrombosis and
Vascular Biology, Department of Molecular Cardiology, The Cleveland
Clinic Foundation, Cleveland, OH, and the Institut Recerca
Oncològica, Departamento Receptores Celulares, Barcelona, Spain.
Plasminogen plays an integral role in the inflammatory response,
and this participation is likely to depend on its interaction with cell
surfaces. It has previously been reported that isolation of
human neutrophils from blood leads to a spontaneous increase in their
plasminogen-binding capacity, and the basis for this up-regulation has
been explored as a model for mechanisms for modulation of plasminogen
receptor expression. Freshly isolated human peripheral blood
neutrophils exhibited relatively low plasminogen binding, but when
cultured for 20 hours, they increased this capacity dramatically, up to
50-fold. This increase was abolished by soybean trypsin inhibitor and
was susceptible to carboxypeptidase B treatment, implicating
proteolysis and exposure of carboxy-terminal lysines in the enhanced
interaction. In support of this hypothesis, treatment of neutrophils
with elastase, cathepsin G, or plasmin increased their plasminogen
binding, and specific inhibitors of elastase and cathepsin G suppressed
the up-regulation that occurred during neutrophil culture. When
neutrophils were stimulated with phorbol ester, their plasminogen
binding increased rapidly, but this increase was insensitive to the
protease inhibitors. These results indicate that plasminogen binding to
neutrophils can be up-regulated by 2 distinct pathways. A major pathway
with the propensity to markedly up-regulate plasminogen binding depends
upon the proteolytic remodeling of the cell surface. In response to
thioglycollate, neutrophils recruited into the peritoneum of mice were
shown to bind more plasminogen than those in peripheral blood,
suggesting that modulation of plasminogen binding by these or other
pathways may also occur in vivo.
(Blood. 2001;97:1070-1078) The migration of leukocytes from the blood to sites
of inflammation is facilitated by proteolytic degradation of the
extracellular matrix.1-3 In addition, proteolytic enzymes
are an important part of the armamentarium of antimicrobial agents
brought by recruited inflammatory cells, in particular neutrophils, to
sites of infection. Nevertheless, the proteolytic activity associated
with inflammatory cells must be tightly regulated to avoid excessive
tissue destruction.2 Plasmin, the active serine protease
generated from plasminogen, has broad proteolytic capabilities. It can
directly degrade multiple matrix proteins,4,5 including
fibronectin,6 laminin,7 and
thrombospondin,8 as well as the major provisional matrix constituent, fibrin. Moreover, plasmin is an activator of several metalloproteinases, yet another class of matrix-degrading enzymes (reviewed in Lijnen9). The importance of plasminogen in the inflammatory response has been underscored in recent studies of mice in
which the plasminogen gene has been inactivated. These animals exhibit
decreased recruitment of cells into their peritoneum in response to a
model inflammatory stimulus, thioglycollate.10
The binding of plasmin(ogen) to cell surfaces is a central mechanism
for mediating its participation in pericellular
proteolysis.11-13 Bound plasminogen is more efficiently
activated to plasmin, and bound plasmin is partially protected from
inactivation by its naturally occurring inhibitors. Several proteins
have been identified as candidate plasminogen receptors, and the
diversity of these proteins,14 coupled with the
extraordinarily high binding capacity of cells for plasminogen,
suggests that no single molecule functions as the plasminogen receptor.
Several of the candidate plasminogen receptors have carboxy-terminal
lysyl residues, which can interact with the lysine-binding sites
associated with the kringle domains of plasminogen. This common
recognition mechanism leads to a similar affinity (KD ~ 1 µM) of plasminogen for different receptor proteins and
renders these interactions susceptible to inhibition by the basic
carboxypeptidases (Cp),15,16 which remove the
carboxy-terminal lysines from proteins, and to lysine analogs, such as
Leukocytes are capable of binding plasminogen,19 and
modulation of this interaction provides a potentially important
mechanism for regulating the proteolytic functions of plasmin during
the inflammatory response. Indeed, regulation of the number of
plasminogen-binding sites on leukocytic cells has been
documented.11,20-22 Agents that induce cellular adhesion,
such as the phorbol ester, phorbol myristate acetate (PMA),
increase the number of plasminogen-binding sites on monocytoid cells by
3- to 17-fold.20 Adhesion of such cells to matrix proteins
down-regulates the plasminogen-binding capacity of the adherent cells
and up-regulates it in the nonadherent cells.20,23
Furthermore, Felez et al20,24 reported that short-term
(overnight) culturing of neutrophils or monocytes leads to a marked
increase in the number of plasminogen-binding sites expressed by these
cells. Such modulation of plasminogen-binding capacity may be
particularly relevant to neutrophil recruitment as early participants
in the inflammatory response. Therefore, in this study, we have sought
to understand the mechanisms underlying this up-regulation of
plasminogen binding to neutrophils. We also have developed preliminary
data to suggest that modulation of plasminogen binding can occur during
an inflammatory response in vivo.
Reagents
Cell preparations and cell lines
The human promyelocytic NB4 cell line was provided by Dr M. Lanotte (Hôpital St. Louis, Paris, France). The human monocytoid U937 cell line was from the American Type Culture Collection (ATCC, Rockville, MD). These cell lines were cultured in RPMI-1640 containing 1 mM sodium pyruvate and 5% to 10% FBS. Ligand binding assays Binding of 125I-plasminogen to neutrophils was performed as previously described.18-20 Briefly, the cells were washed in HBSS containing 25 mM HEPES and resuspended in HBSS-Hepes containing 0.1% bovine serum albumin (BSA) (HBSS-BSA), supplemented with 1.2 mM CaCl2 and 1.6 mM MgSO4. Cells (1 × 107/mL) were incubated with 100 nM 125I-plasminogen in the presence or absence of 100 mM EACA in a total volume of 200 µL at 37°C for 1 hour. Triplicate 50 µL samples were layered over 300 µL of 20% sucrose in HBSS-BSA and centrifuged for 2.5 minutes in a Beckman microfuge (Beckman Instruments, Inc, Fullerton, CA). The tube tips were amputated and counted in a gamma counter. Specific binding was measured as the difference in radioactivity bound in the absence and presence of EACA. We have previously shown that EACA is equivalent to excess nonlabeled plasminogen in defining the nonspecific binding of 125I-plasminogen,19 and approximately 90% of the binding was routinely inhibited by 100 mM EACA. The number of molecules of plasminogen specifically bound were calculated by subtracting the nonspecific binding and using the specific activity of the radiolabeled plasminogen. In selected experiments, a washing step with 50 mM EACA was included to elute plasminogen that may have been bound to the cells in plasma. This procedure led to a slight but statistically insignificant increase in plasminogen binding to cells: 1.61 ± 0.72 vs 2.41 ± 1.05 × 104 plasminogen molecules bound per freshly isolated cell, and 17.79 ± 1.21 vs 18.30 ± 1.50 × 104 plasminogen molecules bound per cultured cell (mean [SEM] of 3 experiments). Therefore, the EACA elution step was not performed routinely.The up-regulation of plasminogen binding was expressed as the fold
increase over the basal binding to freshly isolated neutrophils. The
inhibition of up-regulation was calculated according to the following
formula:
Plasminogen activation by tPA on cells Plasminogen activation studies were carried out in microtiter plates in reaction volumes of 100 µL.26 Briefly, 20 µL tPA (final concentration, 70 pM) was mixed with 40 µL cells (final concentration, 1.5 × 106 cells/mL) and 40 µL of a mixture containing E-plasminogen (final concentration 100 nM) and the chromogenic substrate S-2251 (V-L-K-p-nitroanilide [single-letter amino acid codes], final concentration 0.15 mM). Reactions were performed at 37°C in an assay buffer consisting of Tris-HCl, pH 7.4, containing 1 mg/mL human serum albumin. Absorbance was monitored at 405 nm by means of a Thermomax thermostatted plate reader (Molecular Devices Corp, Stanford, CA). Rates of plasmin generation were calculated as previously described.26Treatment of neutrophils with enzymes and inhibitors The effects of various enzymes on plasminogen binding to neutrophils were assessed by the following protocol. Neutrophils, 500 µL at 2 to 4 × 107/mL in HBSS-BSA, were incubated with the test enzymes, plasmin, elastase, or cathepsin G for 30 to 60 minutes at 37°C. An excess of an inhibitor specific for each enzyme was added: aprotinin (200 kallikrein inhibitory units/mL) as a plasmin/kallikrein inhibitor, MeOSuc-Ala-Ala-Pro-Val-CH2Cl (60 µM) as an elastase inhibitor, and Z-Gly-Leu-Phe-CH2Cl (10 µM) as a cathepsin G inhibitor. The cells were washed 3 times by centrifugation in HBSS-HEPES, and then 125I-plasminogen binding was assayed. With plasmin treatment, 200 mM EACA was included in the initial washing step to help remove cell-bound enzyme. In some of the experiments, enzyme-treated cells were subjected to a subsequent treatment with CpB (5 U/mL, 30 minutes, at 37°C). In these experiments with plasmin-treated cells, the EACA washing step was performed after the CpB treatment since EACA can inhibit CpB. With all enzymes used, their activities were verified with the use of synthetic substrates. We determined the activity of CpB using hippuryl-L-arginine27; of plasmin using the chromogenic substrate S-2251; of elastase using S-2484; and of cathepsin G using succinyl-AAPF-p-nitroanalide. To test the effects of protease inhibitors on the up-regulation of plasminogen binding, the various inhibitors were added to the cultured neutrophils overnight. The cells were then washed 3 times by centrifugation, and their plasminogen binding was assessed.Superoxide anion production by neutrophils The ability of neutrophils to release superoxide anion was determined by measuring the reduction of cytochrome c.28 Neutrophils (2 × 107) were incubated in buffered saline containing 1.3 mg/mL glucose, 0.5 mM CaCl2, 1 mM MgCl2, and 200 µg/mL cytochrome c. Superoxide dismutase (40 µg/mL) was added, and the cells were stimulated with 1 µM PMA for 30 minutes at 37°C in 5% CO2. The neutrophils were removed by centrifugation (at 8,000g for 2 minutes), and activity in the supernatant was measured spectrophotometrically at 550 nm.Neutrophil stimulation with PMA For stimulating neutrophils with PMA, a concentration of 0.1 to 0.5 nM was used. At this concentration of PMA, homotypic aggregation and adhesion of the cells to the reaction tube remained minimal for incubation times of less than 30 minutes. At higher concentrations, cell aggregation occurred that precluded accurate measurements of plasminogen binding. The cells (1 × 107) were suspended in 10 mL Mo-Medium for 30 minutes at 37°C. Viability of the stimulated neutrophils was assessed by trypan blue exclusion, and cell recovery by counting in a hemocytometer. The cells were washed once with HBSS-HEPES before plasminogen binding was measured.Flow cytometry Plasminogen was labeled with Alexa Fluor 488 (Molecular Probes, Eugene, OR) and purified according to the manufacturer's instructions. Dye incorporation was approximately 4 mol/mol plasminogen. The function of fluorescent plasminogen was assessed in terms of its ability to inhibit 125I-plasminogen binding to cells; it competed as well as nonlabeled plasminogen in these assays. For fluorescence-activated cell sorting (FACS) analyses, 106 neutrophils in 500 µL HBSS and 20 µg plasminogen with Alexa Fluor 488 (200 nM), with or without 50 mM EACA, were incubated for 1 hour at 37°C. The binding of the fluorescent plasminogen to these cells was then immediately assessed by means of a FACScan instrument (BD Biosciences, San Jose, CA). FACS data were analyzed by means of the CellQuest software program (version 1.2), and the mean fluorescence intensities derived from this program were used to compare plasminogen binding to the cells.Murine peritoneal inflammation model All animal experiments were performed in accordance with institutionally approved protocols. Thioglycollate was used to induce an inflammatory response as previously described.10 Mice of a C57BL/6: 129 (50%/50%) background were injected intraperitoneally with 0.5 mL of a 4% Brewer thioglycollate solution (Difco Laboratories, Detroit, MI). At 6 hours, the mice were killed by isoflurane inhalation (Abbott Laboratories, North Chicago, IL). The peritoneal cavity was exposed, and the exudate was collected by washing the cavity with sterile saline. The peritoneal lavage fluids from 2 mice were pooled and centrifuged at 200g for 10 minutes. To remove erythrocytes, the cell pellet was subjected to hypotonic lysis. Cells were resuspended in HBSS-HEPES and counted. Neutrophils from untreated mice were isolated from blood obtained by cardiac puncture with heparin used as the anticoagulant. The blood was centrifuged for 15 minutes at 100g. The plasma was removed, and the buffy coats were collected. Contaminating erythrocytes in the buffy coat were subjected to hypotonic lysis, and the leukocytes were resuspended in HBSS-HEPES and counted.Statistical analysis Results are presented as mean ± SEM. Comparisons were made by means of paired Student t tests. A P < .05 was considered to be statistically significant.
Up-regulation of plasminogen binding to neutrophils In previous studies, we reported that removal of leukocytes from their blood environment resulted in a marked increase in their plasminogen-binding capacity.20,24 Since delivery of proteolytic potential from the blood to tissues, either as a releasable pool from within the cell or carried on the cell surface, is a major role for neutrophils in the inflammatory response, the basis for this up-regulation has been examined. Human peripheral blood neutrophils were isolated to a purity of greater than 96% under sterile conditions, and these cells were cultured overnight in media containing 5% FBS. Since our previous studies had demonstrated that changes in plasminogen binding to cells arise from increases in receptor number and not affinity,20,23,29 specific binding (defined as that component of binding inhibitable by EACA) was measured by means of 100 nM 125I-plasminogen for 1 hour at 37°C; ie, approximately 1/10 the Kd under equilibrium conditions. As shown in Figure 1A, plasminogen binding to the cultured neutrophils was dramatically enhanced relative to the freshly isolated cells from the same donor. The increase was over 50-fold in the experiment shown in Figure 1A. Over the course of 19 such experiments, which used the blood of at least 16 different donors, the increase in plasminogen binding averaged 30.9-fold. Freshly isolated neutrophils bound 1.25 ± 0.24 × 104 plasminogen molecules per cell (range, 0.06-3.91), cultured neutrophils bound 38.64 ± 8.96 × 104 plasminogen molecules per cell (range, 7.10-170.43). This up-regulation was not immediate (see Figure 1A); after 6 hours, no increase above the basal level of plasminogen binding to freshly isolated neutrophils was detected. The up-regulation was not associated with a loss in cell viability; trypan blue exclusion of the neutrophils was greater than 95% (range, 95%-100%, n = 19) after overnight culture. Neutrophil functions, as assessed by their capacity to generate O2 (25.2 nmol
for fresh vs 17.6 nmol for cultured) or release of cathepsin G
( A405/minute: 1.6 vs 1.1 × 103) upon
stimulation with PMA (1 to 5 µM), were only slightly diminished by
the 20-hour culture compared with freshly isolated cells. In contrast
to the marked increase in plasminogen binding to cultured cells,
125I-albumin binding, used as a control protein, remained
low (fewer than 1000 molecules bound per cell before or after culture).
In Figure 1A, the cells were maintained in 5% FBS. However, as shown in Figure 1B, the augmentation in plasminogen binding of the cultured neutrophils was independent of the FBS concentration used. The increase
in plasminogen-binding capacity was observed at all concentrations of
FBS tested, including when no FBS was present. In subsequent experiments, the cells were maintained in 5% FBS as this inclusion reduced the tendency of the cells to aggregate spontaneously and to
adhere to the culture flasks.
Carboxy-terminal lysines and up-regulation of plasminogen binding The increase in plasminogen binding to the cultured cells was mediated by its lysine-binding sites since the interaction was inhibited by the carboxy-terminal lysine analog EACA. This recognition specificity raised the possibility that the increase in plasminogen binding depends upon an increased availability of proteins with carboxy-terminal lysine residues on the surface of the cultured cells. To test this hypothesis, the cultured cells were treated with CpB, an enzyme that removes the lysyl and arginyl residues from the carboxy-terminus of proteins and peptides.30,31 As shown in Figure 2, treatment of the cultured cells with CpB decreased their plasminogen binding substantially. The inhibition of up-regulation was greater than 70% at either 10 or 50 U/mL CpB. GEMSA (300 µM), an inhibitor of CpB,32 abrogated the effect, an indication of specificity (Figure 2).
Implication of proteases in the up-regulation of plasminogen binding A ready mechanism by which new carboxy-terminal lysines could be generated is proteolysis at the cell surface. In view of the high serine protease activity associated with neutrophils, we tested whether SBTI would influence the up-regulation of plasminogen binding. As shown in Figure 3, concentrations of SBTI of 100 or 1000 µg/mL markedly suppressed the up-regulation of plasminogen binding. Even 1 µg/mL SBTI suppressed up-regulation by 44%. The specificity of this effect was demonstrated; unrelated proteins, transferrin, or albumin, at 100 µg/mL, failed to influence the increase in plasminogen-binding capacity and did not alter the inhibitory activity of SBTI. The addition of SBTI to the cells after overnight culture did not change their expression of increased plasminogen-binding sites, indicating that the effect of the protease inhibitor was on the up-regulation of binding sites and not on the binding per se. We also considered whether SBTI would influence the basal level of plasminogen binding to freshly isolated neutrophils. When SBTI was included in the blood collection tube and maintained at 1000 µg/mL throughout the isolation of the cells, the basal level of plasminogen binding was not significantly affected: 1.91 ± 0.50 × 104 plasminogen molecules bound per neutrophil when isolated without SBTI vs 1.58 ± 0.41 × 104 when isolated with SBTI (P = .52, n = 8).
Effect of SBTI on plasminogen activation In previous studies, plasminogen binding to cells was shown to enhance plasmin generation by tPA.26,33 With a similar approach, we explored the capacity of freshly isolated or cultured neutrophils to promote plasminogen activation by tPA and the effects of SBTI on this activation. In comparison with freshly isolated neutrophils, a 7.7-fold increase in plasminogen activation rate was observed when neutrophils were cultured overnight (Figure 4). However, as shown in Figure 4, when neutrophils were cultured in the presence of SBTI (1000 µg/mL), plasminogen activation rates were similar to those observed with freshly isolated neutrophils. Since SBTI can inhibit plasmin, carryover of the inhibitor into the plasminogen activation assay was considered. A promyelocytic cell line (NB4) and a monocytoid cell line (U937) were cultured for 24 hours in the presence or absence of SBTI. In contrast to what happened with neutrophil culturing, plasminogen binding on these cell lines was not modulated; thus, changes in plasminogen activation rates would reflect the carryover of SBTI into the plasmin generation assays. Under the same washing conditions as for neutrophils, the rates of plasminogen activation were not modified by the presence of SBTI in the cultures. The promyelocytic NB4 cells generated 0.72 and 0.78 pmol plasmin per second when cultured in the presence or absence of SBTI, respectively; and the monocytoid U937 cells generated 0.45 and 0.51 pmol plasmin per second. Thus, SBTI suppressed the up-regulation of plasminogen activation as well as of plasminogen binding.
Proteases up-regulate plasminogen binding to neutrophils On the basis of the effects of SBTI on both plasminogen binding and activation, we sought to determine if protease treatment of neutrophils could up-regulate their plasminogen-binding capacity. For these experiments, freshly isolated neutrophils were incubated with 50 to 100 nM concentrations of the selected enzyme for 30 to 60 minutes. Inhibitors specific for each enzyme were added, and the cells were washed and then treated or not treated for 1 hour with CpB to determine if changes in plasminogen binding were mediated by exposure of new carboxy-terminal residues. To verify specificity, controls were performed in which each inhibitor was added to the target enzyme prior to its addition to the cells. Incubation of neutrophils with plasmin increased their plasminogen-binding capacity by 2.1-fold (Figure 5A). Aprotinin, an inhibitor of plasmin, abrogated the plasmin-induced increase in plasminogen binding. We also tested 2 major serine proteases of neutrophils, elastase, which cleaves at uncharged or nonaromatic residues,34,35 and cathepsin G, which cleaves primarily at aromatic amino acid residues.36,37 Each of these enzymes generated new plasminogen-binding sites on the neutrophils (Figure 5A). At the concentrations used (selected from dose titration experiments as a concentration of each protease that induced a substantial increment in plasminogen binding), the increase in plasminogen binding was 2.1-fold with elastase and 3.8-fold with cathepsin G. For both enzymes, the increase in plasminogen binding was abrogated by pre-incubation of the enzymes with their respective inhibitors (Figure 5A). For elastase and cathepsin G, as well as for plasmin, the increase in plasminogen binding was statistically significant and was partially sensitive to CpB treatment (Figure 5B). The increase in binding induced by plasmin treatment was 82% inhibited by CpB. The increase in binding induced by cathepsin G was also highly susceptible (78%) to CpB, whereas the increase induced by elastase was less sensitive (47%) but still susceptible (see Figure 5B).
Effect of protease inhibitors on up-regulation of plasminogen binding Having shown that relevant enzymes can increase plasminogen binding to neutrophils and that inhibitors of these enzymes ablated their effects, we assessed whether the same inhibitors would influence the up-regulation of plasminogen binding associated with the culturing of neutrophils. As shown in Figure 6, the inhibitors of the major leukocyte proteases were effective in blocking the up-regulation in plasminogen binding. The chloromethylketone inhibitors of elastase and cathepsin G produced 85% and 86% inhibition, respectively, of the up-regulation and were only slightly less effective than SBTI. MeOSuc-Ala-Ala-Pro-Ala-chloromethylketone (HLE/CMK) is an inhibitor not only of neutrophil elastase but also of the closely related neutrophil enzyme, proteinase 3.38 Accordingly, 2 additional selective elastase inhibitors, MeOSuc-Ala-Ala-Pro-Ala-chloromethylketone, which does not inhibit proteinase 3, and elastatinal were tested. These compounds produced 22% and 56% inhibition of up-regulation, respectively, suggesting that proteinase 3 may also play a role in the up-regulation of plasminogen binding. Not all protease inhibitors affected the up-regulation of plasminogen-binding capacity. EDTA (10 mM) had a minimal effect, and aprotinin produced less than 50% inhibition. Interestingly, o-phenanthroline (500 µM), which inhibits Cp, increased the extent of plasminogen binding by almost 2-fold, suggesting that the Cp enzymes may dampen the extent of up-regulation. Relevant to this observation, CpM, a basic carboxypeptidase, is associated with neutrophils.39
Effect of PMA stimulation on up-regulation of plasminogen binding Stimulation of neutrophils with PMA at concentrations that did not induce homotypic aggregation or adhesion to the culture flask led to a rapid up-regulation of plasminogen binding. As shown in Figure 7, after only 20 minutes, a 4-fold increment in plasminogen binding was observed. Addition of the protease inhibitors SBTI, HLE/CMK, or CG/CMK (results of the latter not shown) did not inhibit the up-regulation of plasminogen binding induced by PMA, indicating that this up-regulation is not dependent on protease action. These results indicate that a second pathway, with no apparent dependency on the major leukocyte proteases, can mediate up-regulation of the plasminogen-binding capacity of neutrophils.
Up-regulation of plasminogen binding to neutrophils in vivo The inflammatory stimulus thioglycollate was used to induce neutrophil recruitment into the peritoneum of mice. As performed in our laboratory10 and consistent with the literature,40,41 neutrophil recruitment into the peritoneum is maximal at 6 hours in this inflammatory model. The peritoneal neutrophil cells were collected at this time by lavage and partially purified, and their plasminogen receptor expression was assessed by FACS with the use of plasminogen labeled with Alexa Fluor 488. The plasminogen binding to these cells was compared with that of neutrophils isolated from blood of mice. As shown in Figure 8, there was a substantial difference in plasminogen binding to the peritoneal vs the blood neutrophils. On the basis of the mean fluorescence intensity values, the peritoneal neutrophils bound 4-fold more plasminogen than the blood neutrophils. Similar results were obtained in a second experiment; the difference in plasminogen binding to the peritoneal vs the blood neutrophils was 3-fold. Within the peritoneal neutrophils, plasminogen binding was heterogeneous, and at least 2 subpopulations, one with lower and one with higher plasminogen-binding capacities, could be distinguished. On average, plasminogen binding to both of these subpopulations was higher than to the blood neutrophil (see Figure 8). These results are compatible with the possibility that up-regulation of plasminogen binding to neutrophils may occur in vivo.
The inflammatory reaction is a complex and multifaceted process,
and pericellular proteolysis is an important element as a mediator not
only of antimicrobial activity but also of the recruitment phase of
this response. Studies in knockout mice have implicated components of
the plasminogen system,42,43 as well as plasminogen itself,10,44 as important contributors to the inflammatory reaction. The ability of plasminogen to mediate efficient fibrinolysis depends upon its binding to fibrin, an interaction that influences its
activation by plasminogen activators and its inhibition by Neutrophils cultured overnight in the presence or absence of FBS dramatically up-regulate (by approximately 30-fold) their expression of plasminogen-binding sites. Concomitantly, their capacity to enhance plasminogen activation by tPA also increased substantially (7.7-fold) with culture. The latter parameter depends upon changes in both the Michaelis constant KM and the catalytic constant kcat24,26 which may account, in part, for the disproportion between the changes in binding and kinetic parameters. Also, tPA and plasminogen share binding sites on certain cells including neutrophils, and the binding of both ligands is modulated by cell culture.24 SBTI, a broad-spectrum serine protease inhibitor, or more specific leukocyte protease inhibitors prevented this up-regulation of plasminogen binding. These observations implicate elastase, cathepsin G, and/or related enzymes, such as the elastaselike protease proteinase 3, in this process. Consistent with these interpretations, treatment of neutrophils with elastase, cathepsin G, as well as plasmin did enhance the plasminogen-binding capacity of the cells. The exogenously added proteases were less effective than culture in up-regulating plasminogen binding. Possible explanations for these differences include the time of exposure (1 hour for the added enzymes vs 20 hours of culture); the combination of proteases to which the cultured cells are exposed; and the imposition of other changes that occur at the cell membrane, which may influence the substrates available. Nevertheless, the increase in plasminogen binding induced by these added enzymes and by the culturing of the cells was associated with a common mechanism, the exposure or generation of new carboxy-terminal basic amino acids, probably lysines, as indicated by the sensitivity of the enhanced interaction to CpB. Previous studies have shown that it is the CpB-sensitive plasminogen-binding sites that enhance plasminogen activation on cell surfaces.18,24 Thus, the protease-dependent up-regulation results in an increase in functionally important plasminogen-binding sites. The suppressive effects of SBTI on plasminogen activation by the cultured neutrophils support this conclusion. The specificity of the enzyme inhibitors used suggests roles for the
major granule proteases of neutrophils, elastase, cathepsin G, and
possibly proteinase 3 in the up-regulation of plasminogen-binding sites. The question of the role of proteinase 3 arises from the lack of
specific inhibitors of this enzyme48 and the uncertainty as
to its susceptibility to HLE/CMK.38,49,50 HNE/CMK, a
specific elastase inhibitor,48 was considerably less
effective than HLE/CMK in blocking up-regulation, compatible with a
role for proteinase 3 in the process. All 3 neutrophil enzymes are
endopeptidases of the serine protease family: elastase cleaves at
uncharged, nonaromatic side chains, A (single-letter amino acid
code), V, L, I; proteinase 3 at A, S, V; and
cathepsin G at aromatic amino acid residues, with only cathepsin G
exhibiting some propensity for the basic amino acids as
well.34-37 Therefore, rather than creating new
carboxy-terminal lysines directly, the neutrophil enzymes are more
likely to influence the exposure and accessibility of pre-existing
carboxy-terminal lysines to plasminogen. Thus, we hypothesize that
these enzymes may enhance plasminogen binding to the cells by
remodeling the cell surface. Numerous cell-surface proteins (eg, tumor
necrosis factor Stimulation of neutrophils with PMA enhanced their plasminogen binding substantially and rapidly. Such stimulation is known to increase expression of both elastase and cathepsin G at the cell surface.1,58 However, this up-regulation was insensitive to the proteinase inhibitors (SBTI, HLE/CMK, CG/CMK) that blocked the augmentation induced by culturing of the cells. The lack of involvement of the proteases in this up-regulation may be a reflection of the very short incubation time (20 minutes) required. Because the up-regulation occurred despite the presence of proteinase inhibitors, the existence of a second and mechanistically distinct pathway for up-regulation of plasminogen-binding sites is suggested: a pathway independent of proteases or, at the least, involving distinct PMA-induced proteases. Exposure of neutrophils to PMA and subsequent stimulation of protein kinase C does change the properties of the cell membrane (ruffling) and the cytoskeleton.63 Therefore, the enhanced plasminogen binding induced by PMA may also be a reflection of remodeling of the cell membrane. In previous studies,20,23 we showed that adhesion and de-adhesion of cells influence the expression of plasminogen-binding sites, which may also be an extension of this concept. While neutrophils have a survival time of 6 to 18 hours in blood, their
survival time in tissues can be extended
substantially.64,65 Therefore, the overnight culture used
in this study to up-regulate plasminogen receptors may have biological
and clinical relevance. We found that, compatible with this
proposition, neutrophils that have migrated into the peritoneum in
response to the inflammatory stimulus thioglycollate exhibited higher
plasminogen binding than neutrophils from peripheral blood. We cannot
exclude that peritoneal cells represent a selected population of
neutrophils with a higher plasminogen-binding capacity, nor can we
conclude that the apparent up-regulation of plasminogen binding to the
peritoneal neutrophils is due to proteolytic modification of their cell
surface. Consistent with these possibilities, Silverstein et
al66 reported that the plasminogen was present on the
surface of peritoneal macrophages of patients with indwelling catheters
and on tissue macrophages from patients with chronic inflammatory
lesions under conditions in which its expression on blood monocytes was
not detected. Also, in a canine model of ischemia-reperfusion injury,
modulation of the surface of the emigrating neutrophils was evidenced
by a loss of integrin
The authors thank the blood donors from the Primary Laboratory Center at the Cleveland Clinic Foundation; Jane Rein, for expert secretarial assistance; and a facility, established with the support of the W. M. Keck Foundation, at the Cleveland Clinic, for FACS analysis.
Submitted December 8, 1999; accepted October 12, 2000.
Supported by National Institutes of Health grant HL17964 (E.F.P.) and, in part, by the Swiss National Science Foundation and the Schweizerische Stiftung für Medizinisch-Biologische Stipendien (T.H.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Edward F. Plow, Joseph J. Jacobs Center for Thrombosis and Vascular Biology, 9500 Euclid Ave, Cleveland, OH 44195; e-mail: plowe{at}ccf.org.
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© 2001 by The American Society of Hematology.
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  R. Das, T. Burke, and E. F. Plow Histone H2B as a functionally important plasminogen receptor on macrophages Blood, November 15, 2007; 110(10): 3763 - 3772. [Abstract] [Full Text] [PDF]
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R. Renckens, J. J. T. H. Roelofs, S. Florquin, A. F. de Vos, J. M. Pater, H. R. Lijnen, P. Carmeliet, C. van 't Veer, and T. van der Poll Endogenous Tissue-Type Plasminogen Activator Is Protective during Escherichia coli-Induced Abdominal Sepsis in Mice J. Immunol., July 15, 2006; 177(2): 1189 - 1196. [Abstract] [Full Text] [PDF]
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V. K. Lishko, V. V. Novokhatny, V. P. Yakubenko, H. V. Skomorovska-Prokvolit, and T. P. Ugarova Characterization of plasminogen as an adhesive ligand for integrins {alpha}M{beta}2 (Mac-1) and {alpha}5{beta}1 (VLA-5) Blood, August 1, 2004; 104(3): 719 - 726. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-aminocaproic acid (EACA).
2-antiplasmin.
2 from the cell
surface and appearance of soluble proteolytic fragments of the
integrin.