Effect of Alpha-I-Proteinase Inhibitor on Neutrophil Chemotaxis R. A. Stockley, J. Shaw, S. C. Afford, H. M. Morrison, and D. Burnett Lung Immunobiochemical Research Laboratory, Clinical Teaching Block, The General Hospital, Birmingham, United Kingdom
Factors that modulate neutrophil migration into the lung are poorly understood. However, there is evidence that neutrophil activation by formylmethionylleucylphenylalanine (FMLP) depends upon a surface proteinase with chymotrypsin-like activity. This suggests that chymotrypsin inhibitors such as alpha-I-proteinase inhibitor (aIPI) could modify neutrophil migration in response to FMLP. We have studied neutrophil chemotaxis using the multiple blind well assay system. This article presents evidence that alPI is an inhibitor of neutrophil migration in response to FMLP. The effect is related to the inhibitory function of the protein. Alpha-I-antichymotrypsin is more potent than alPI as an inhibitor of this movement, whereas antileukoprotease is less potent. The results suggest that a cell membranebound serine proteinase (perhaps cathepsin G) is necessary for the enhancement of cell movement after receptor binding of FMLP. Oxidized a,PI or a 4,OOO-D peptide cleaved from alPI by porcine pancreatic elastase or human neutrophil elastase are capable of enhancing cell motility. The results suggest that alPI may playa role in cell migration into the lung during acute inflammatory process.
Neutrophil recruitment to the lung and the release of their oxygen radicals and proteolytic enzymes have been implicated in the pathogenesis of many acute and chronic lung diseases. However, the processes involved in neutrophil migration are far from clear. Directed movement of circulating neutrophils can be stimulated by a variety of agents including the peptide FMLP. Binding of FMLP to cell membrane receptors is believed to instigate a "chemotactic" response that is dose dependent. The maximal chemotactic response occurs between 10-8 and 10-7 M FMLP (1), whereas higher concentrations of FMLP (10-6 M) result in degranulation of the neutrophil (2) and superoxide production (3). The sequence of events after FMLP receptor binding is poorly understood, although a cell surface chymotrypsinlike proteinase has been implicated. Internalization of cell surface receptors may depend on such an enzyme (4) and cell migration (5) and superoxide production (6) can be inhibited by chloromethyl ketones or monoclonal antibodies, which inactivate chymotrypsin-like enzymes. Further support for the role of chymotrypsin-like enzymes has been provided by studies with naturally occurring inhibitors. For instance, Key Words: neutrophil chemotaxis, proteolysis, alpha-I-proteinase inhibitor, antileukoprotease, alpha-I-antichymotrypsin (Received in original form July 10, 1989 and in revised form September 28, 1989) Address correspondence to: R. A. Stockley, M.D., D.Sc., ER.C.P., Lung Immunobiochemical Research Laboratory, Clinical Teaching Block, The General Hospital, Steelhouse Lane, Birmingham, B4 6NH, United Kingdom. Abbreviations: alpha-I-antichymotrypsin, alACh; alpha-I-proteinase inhibitor, a,PI; antileukotrotease, ALP; neutrophil elastase, NE; porcine pancreatic elastase, rPE; succinyltrialanyl paranitroanilide, SLAPN. Am. J. Respir. Cell Mol. BioI. Vol. 2. pp. 163-110, 1990
motility of equine neutrophils is reduced by the parasitic proteinase inhibitor taeniaestatin (7). In addition, rat neutrophil migration is reduced by eglin C from the medicinal leech (8), although alpha-I-proteinase inhibitor (a,PI) had little effect (5). However, studies with human neutrophils have shown that alPI can reduce neutrophil chemotaxis using a Boyden chamber technique (9), suggesting that there may be an interspecies difference. More recently, studies have suggested that some molecular forms of alPI may actually stimulate directed movement of the neutrophils. Complexing of the a,PI with neutrophil elastase (NE) generates chemotactic activity (10), and cleavage of the active site with murine macrophage elastase releases a chemotactic peptide (11). Previous studies have shown that both NE-a,PI complexes (12) and cleaved a,PI (13, 14) are present in lung secretions and thus could playa role in further neutrophil recruitment. The evidence suggests that alPI may play a role in the modulation as well as enhancement of neutrophil recruitment to the lung. The purpose of the present studies was to investigate further the role of a,PI on neutrophil chemotaxis. In particular, we wished to partially clarify the mechanism involved in the reduction of chemotaxis by native a,PI. In addition, we wished to determine whether inactivation of the protein by oxidation of the active site or cleavage of the active site using the serine proteinases NE and porcine pancreatic elastase (PPE) could also affect neutrophil recruitment.
Materials and Methods Molecular Forms of alPI a,PI was purified from plasma as described previously (15) and stored at -70 0 C in 0.05 M Tris/HCI buffer (pH 7.4).
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Fresh aliquots were thawed and used for each experiment. The inhibitory activity of the protein was assessed using NE that had been active site titrated (l6). The proteolyzed form of alPI was prepared by 48 h of preincubation with either a 4-fold molar excess of NE or 10fold molar excess of PPE at J]O C in 0.2 M Tris/HCl buffer (pH 8.6) + 0.01 % Triton Xl00. The NE was removed by solid-phase immunoadsorption with monospecific polyclonal antiserum to NE immobilized on Sepharose 4B. The sample was passed down the antibody column, and the fractions were tested with a specific NE ELISA assay to confirm removal of immunoreactive NE. The PPE was separated from alPI (100 p.g) by anion exchange chromatography on DEAE Sephacel at 50 ml/h using a linear salt gradient from 0 to 0.5 M NaCl in 0.05 M Tris/HCl, pH 7.4. Fractions (0.5 ml) were screened for alPI using a specific ELISA with a sheep polyclonal antiserum (17) and PPE activity using the synthetic elastase substrate succinyltrialanyl paranitroanilide (SLAPN). Oxidized alPI was produced by adding 20 p.l of hydrogen peroxide (H20 2 ) (BDH Chemicals) to 2 ml of alPI for 30 min at room temperature. The resulting mixture was placed in dialysis tubing (Sigma Chemical Co., St. Louis, MO) with an exclusion size> 12,000 D and dialyzed against 1 liter of 4-(2-hydroxyethyl)-1-piperazine-ethane sulphonic acid (Hepes)-buffered RPMI-1640 medium (Flow Laboratories, Rickmansworth, Herts, UK) overnight. Similarly, pure active alPI (25 p.g in 2 ml of 0.05 M Tris/HCl buffer, pH 7.4) and the proteolyzed alPI (19.7 p.g in 2 ml of 0.2 M Tris/HCl buffer, pH 8.6) were dialyzed against 1 liter of RPMI Hepes overnight. In addition, several fractions of the proteolyzed alPI from the ion exchange column were pooled (5 ml), placed in a dialysis bag, and dialyzed against 10 ml of RPMI Hepes (2 d at 4 0 C), followed by further dialysis (5 h) against 10 ml of RPMI Hepes. The loss of alPI enzyme inhibitory activity was confirmed by demonstrating no residual ability to inactivate PPE. All pre- and postdialysis samples were assessed for the molecular size of the alPI molecules by SDS-PAGE using a 10% resolving gel on a vertical slab system (Bio-Rad, Richmond, CA). Protein bands were stained using Kenacid blue. The results confirmed that none of the protein was present in its native form (54,000 D) or complexed with enzyme (80,000 D). Neutrophil Migration The assay was based on the multiple blind well assay system (l8). FMLP (Sigma) was diluted in RPMI Hepes to give a final concentration of 10-8 M. The peptide was placed in the blind well of the lower perspex plate and separated from the cells (1.5 X 1(6) in the well in the upper plate by two membranes (a lower 0.45-p.m pore size cellulose acetate Millipore membrane [Millipore Co.] and an upper 2-p.m pore size polycarbonate nucleopore membrane [Appleton Woods, Birmingham, UK]) and sealed with a rubber O-ring. Neutrophils (> 96% pure) were isolated by Percoll gradient separation (19). They were washed in RPMI Hepes and resuspended in RPMI Hepes/BSA buffer (2 p.glml). Plates were left at 37 0 C for 90 min and the Millipore membranes removed and stained as described previously
(2). The experiments were run in triplicate to obtain a single result except where stated. Cell numbers were counted from 5 areas of each membrane selected at random, using a graticule eye piece (x400 magnification). The average value for all three membranes was taken as the final result (within batch coefficient of variation = 5.2 %, n = 10). The effect of different concentrations of the molecular forms of alPI and the other serine proteinase inhibitors was studied in two ways. Firstly, the cells were mixed with increasing concentrations of the proteins 30 min prior to assessing response to 10-8 M FMLP. Secondly, the inhibitor was put in the lower well of the chemotaxis plate in order to assess its effect alone on neutrophil migration. In this way, any reduction in neutrophil response to FMLP could be differentiated from immobilization of the cells as a result of activation by the inhibitor when placed in the upper chamber. In the case of native and oxidized aIPI, this possibility was studied in further detail by checkerboard analysis. PMN Binding of FMLP The effect of alPI on specific FMLP binding to neutrophils was studied as follows. Neutrophils, separated as above, were suspended in RPMI Hepes (39 X 106 /ml). In addition, further cells were isolated using Ficoll Hypaque separation (confirmed to be > 97 % pure) as described previously (20) and suspended in RPMI Hepes (42 X 1Q6/ml). Specific cell binding of FMLP was assessed with tritiated FMLP (60 Ci/mmol) diluted in RPMI to give a final concentration in the reaction mixture of 10-8 M. Nonspecific binding was assessed using the same amount of labeled FMLP with a l,ooo-foid excess of unlabeled FMLP. The experiments were performed at 0 0 C in microfuge tubes to which 100 p.l of a 2:1 mixture of butyl and nonyl phthalate (BDH Chemicals) had been added. The cells (25 p.l; 106 cells) were preincubated for 10 min with active alPI ranging in concentration from 1.35 X 10- 10 to 1.35 X 10-7 M, made up in 0.05 M Tris/HCl buffer, pH 7.4, made up to 100 p.l with RPMI Hepes. The FMLP (25 p.l) was then added, and incubation continued for 30 min. After the incubation periods, the tubes were centrifuged at 8,000 g for 2 min to allow the cells to pass through the phthalate. Some of the supernatant (120 p.l) was removed and added to 2 ml of Optiphase X. The remaining supernatant was discarded, and the tubes, above the oil, were washed (Xl) with 2 % SDS to remove excess counts, taking care not to disturb the cell pellet below the oil. The SDS and oil were then discarded and a further 150 p.l of 2 % SDS was added to lyse the cells and the lysate placed in scintillation vials. The tubes were rinsed with Optiphase X to remove remaining debris and added to the vials with further Optiphase to make a final volume of 2 ml. Vials were counted (4 min) with an LKB Rackbeta liquid scintillation counter. The count, and hence molecules of FMLP bound, was determined from the total counts bound (labeled FMLP experiment) minus the nonspecific binding (labeled and unlabeled FMLP experiment). FMLP binding was compared in the presence and absence of alPI. All experiments were run in duplicate.
Stockley, Shaw, Afford et al.: Neutrophil Activation and Antiproteinases
FMLP-a,PI Interaction In order to investigate the possibility of FMLP binding to aIPI, the 3H-labeled FMLP (10- 8 M) was incubated for 15 min at J"l0 C with 6.5 X 10-7 M pure alPI. The mixture (0.5 ml) was then applied to a 1 X 8 em column containing 5 ml of Sephadex G25 followed by PBS (pH = 7.4) at a flow rate of 1 ml/min. The fractions were collected and assayed for alPI immunologically and for (3H]FMLP by scintillation counting. Comparison with Other Inhibitors The effect of active alPI on neutrophil migration was compared with active al antichymotrypsin (aIACh) purified and assessed as described previously (21). In addition, the results were compared to those obtained with antileukoprotease (ALP) donated by 1. A. Kramps (Leiden), which had also been assessed for its inhibitory activity. Statistical analysis of neutrophil migration was performed on transformed data using analysis of variance with the assumption that variances were not equal.
Results When alPI was placed in the upper chamber ofthe chemotaxis plate, there was a concentration-dependent effect on neutrophil migration of FMLP (10-8 M). In the initial experiments, we found inhibition of cell movement at concentrations greater than 2 Itg/ml (J"l nM). The number of cells migrating in the absence of alPI was 61.2 ± 6.1 cells/field (mean ± SE). The number of cells migrating in the presence of 0.6 Itg/ml (11 nM) was 54.9 ± 9.5 and 57.5 ± 9.5 in the presence of 1.23 Itg/ml alPI (22 nM). However, the cell response fell to Zl.6 ± 0.8 (P < 0.01) at 2.5 Itg/ml (46 nM) and 14.6 ± 12.0 (P < 0.01) at concentrations of 6.3 Itg/ml (117 nM). There was a significant correlation between dose and inhibition (P < 0.01). Experiments performed on two other occasions confirmed that alPI inhibited PMN migration, with the greatest change occurring at the highest concentrations. The results of these three experiments are summarized in Figure 1. Results are shown as percent inhibition of the response of FMLP because chemotactic response for different subjects' PMN showed wide variation as reported previously (2). In a further study on a separate occasion, the FMLP dose-response curve of PMN migration with and without alPI present showed suppression but no shift in the curve, with maximal migration occurring at 10-8 M FMLP in both instances. Furthermore, cell viability was not affected, even at the highest concentrations of alPI (trypan blue exclusion). Further studies of active alPI placed in the upper chamber at a concentration of 3 Itg/ml confirmed the inhibitory effect, with a reduction in response to FMLP of a subsequent neutrophil preparation from 15.9 ± 0.3 to 7.6 ± 0.4 cells/field (P < 0.001). However, 3 Itg of the oxidized alPI (4.8 ± 0.2 cells/field) and 3 Itg/ml of the cleaved alPI produced with NE (5.6 ± 0.1 cells/field) had a similar effect when placed in the upper chamber (P < 0.001) despite loss of proteinase inhibitory function. When the alPI molecular forms were placed in the lower well of the chemotaxis chamber, the oxidized and proteolyzed forms were shown to cause
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 21990
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After extensive dialysis (1 liter dialysate X 24 h), the native and oxidized alPI when mixed with the cells (upper chamber) again reduced the chemotactic response to FMLP. The results are summarized in Figure 3 for protein concentrations of 3 ~g/rn1. Native alPI reduced the response on this occasion from 24.1 ± 1.4 to 12.7 ± 2.1 cells/field (P < 0.05) and the oxidized alPI from 22.9 ± 0.5 to 12.1 ± 2.4 cells/field, although this just failed to reach significance (P = 0.06). In contrast, the dialyzed preparation of profuolyzed alPI no longer inhibited cell motility (control = 24.1 ± 1.4; alPI = 28.1 ± 3.1 cells/field). Dose-response curves confirmed that the dialyzed preparation of oxidized alPI retained its ability to activate cell movement. The number of cells recruited by oxidized alPI in the lower well at 6.7 ~g/rn1 was 37.2 ± 3.4 cells/field (P < 0.01 compared to negative control), rising to 60 ± 11.4 at 15.9 ~g/rn1 (P < 0.01) and falling to 2.9 ± 0.5 at 20.0 ~g/rn1. The maximal response was similar to a positive control with 10-8 M FMLP (59.7 ± 2.3 cells/field), which was run on the same occasion with the same cells. Similar studies with the dialyzed native and proteolyzed alPI showed no effect on cell motility with this cell preparation (5.1 ± 0.8 at 8.5 ~g/rn1; 3.2 ± 0.8 at 19 ~g/rn1; and 2.7 ± 0.7 cells/field at 25.5
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tained no immunoreactive alPI but did contain amidolytic activity against SLAPN, indicating this was the PPE. The second peak contained the immunoreactive alPI but no enzyme activity. When the pooled alPI-positive fractions were subjected to SDS-PAGE, a major protein band of rv49,OOO D was present, together with a small molecular weight band near the leading edge of the electrophoretic front « 5,000 D). The column fractions containing immunoreactive alPI retained their effect on neutrophil motility when tested with a further preparation of PMN as shown in Figure 4 (P < 0.05). In a further setof experiments, dialysis of these fractions (50 to 70, Figure 4) against a small volume of RPMI resulted in appearance of a factor in the dialysis fluid which affected
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Stockley, Shaw, Afford et ai.: Neutrophil Activation and Antiproteinases
167
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cell motility (Figure 5). Continued dialysis resulted in a loss of this factor from the contents of the dialysis bag (Figure 5). These results were associated with the appearance of the low molecular weight protein outside the dialysis bag. This was confirmed by SDS-PAGE as shown in Figure 6. When the dialysis samples were analyzed using the specific alPI ELISA, a positive signal was obtained, confirming that the low molecular weight protein possessed some immunologic identity with the complete alPI molecule. Studies of the effect of alPI on FMLP binding showed an apparent increase in specific binding whether the cells were isolated by Percoll or Ficoll gradient separation. In both experiments, the nonspecific binding accounted for 25 and 24 % of the total binding, respectively. The presence of alPI resulted in a concentration-dependent increase in specific binding compared to cells alone. No effect was seen as
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the concentration of alPI rose from 1.35 x 1(t-1I&J to 1.35 X 10-8 M active protein. However, above this concentration, FMLP binding increased by 40.4 and 59.4% for Pen:oll and Ficoll isolated cells, respectively, at 10-7 M alP}. The results are summarized in Figure 7. After G25 column chromatography of an a,PI-FMLP mixture, the alPI was identified in the void volume of the column. Most of the (3H]FMLP (95%) was found in the same fractions and not retained by the column, suggesting an interaction between the protein and the peptide. The two other proteinase inhibitors, a,AC'b and ALP, also showed concentration-dependent inhibition of chemotaxis. The effect of active alACh was greater and the effect of ALP was less than that seen for aIPI. The results are summarized in Figure 8, comparing the inhibition with the molar concentrations of active amounts of each inhibitor.
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 2 1990
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Factors that modulate neutrophil migration into the lung are poorly understood. However, there is evidence that neutrophil activation by formylmethionylleucylphenylalanine (FMLP) depends upon a surface proteinase with chymotrypsin-like activity. This suggests that chymotrypsin inhibitors such as alpha-I-proteinase inhibitor (aIPI) could modify neutrophil migration in response to FMLP. We have studied neutrophil chemotaxis using the multiple blind well assay system. This article presents evidence that alPI is an inhibitor of neutrophil migration in response to FMLP. The effect is related to the inhibitory function of the protein. Alpha-I-antichymotrypsin is more potent than alPI as an inhibitor of this movement, whereas antileukoprotease is less potent. The results suggest that a cell membranebound serine proteinase (perhaps cathepsin G) is necessary for the enhancement of cell movement after receptor binding of FMLP. Oxidized a,PI or a 4,OOO-D peptide cleaved from alPI by porcine pancreatic elastase or human neutrophil elastase are capable of enhancing cell motility. The results suggest that alPI may playa role in cell migration into the lung during acute inflammatory process.
Neutrophil recruitment to the lung and the release of their oxygen radicals and proteolytic enzymes have been implicated in the pathogenesis of many acute and chronic lung diseases. However, the processes involved in neutrophil migration are far from clear. Directed movement of circulating neutrophils can be stimulated by a variety of agents including the peptide FMLP. Binding of FMLP to cell membrane receptors is believed to instigate a "chemotactic" response that is dose dependent. The maximal chemotactic response occurs between 10-8 and 10-7 M FMLP (1), whereas higher concentrations of FMLP (10-6 M) result in degranulation of the neutrophil (2) and superoxide production (3). The sequence of events after FMLP receptor binding is poorly understood, although a cell surface chymotrypsinlike proteinase has been implicated. Internalization of cell surface receptors may depend on such an enzyme (4) and cell migration (5) and superoxide production (6) can be inhibited by chloromethyl ketones or monoclonal antibodies, which inactivate chymotrypsin-like enzymes. Further support for the role of chymotrypsin-like enzymes has been provided by studies with naturally occurring inhibitors. For instance, Key Words: neutrophil chemotaxis, proteolysis, alpha-I-proteinase inhibitor, antileukoprotease, alpha-I-antichymotrypsin (Received in original form July 10, 1989 and in revised form September 28, 1989) Address correspondence to: R. A. Stockley, M.D., D.Sc., ER.C.P., Lung Immunobiochemical Research Laboratory, Clinical Teaching Block, The General Hospital, Steelhouse Lane, Birmingham, B4 6NH, United Kingdom. Abbreviations: alpha-I-antichymotrypsin, alACh; alpha-I-proteinase inhibitor, a,PI; antileukotrotease, ALP; neutrophil elastase, NE; porcine pancreatic elastase, rPE; succinyltrialanyl paranitroanilide, SLAPN. Am. J. Respir. Cell Mol. BioI. Vol. 2. pp. 163-110, 1990
motility of equine neutrophils is reduced by the parasitic proteinase inhibitor taeniaestatin (7). In addition, rat neutrophil migration is reduced by eglin C from the medicinal leech (8), although alpha-I-proteinase inhibitor (a,PI) had little effect (5). However, studies with human neutrophils have shown that alPI can reduce neutrophil chemotaxis using a Boyden chamber technique (9), suggesting that there may be an interspecies difference. More recently, studies have suggested that some molecular forms of alPI may actually stimulate directed movement of the neutrophils. Complexing of the a,PI with neutrophil elastase (NE) generates chemotactic activity (10), and cleavage of the active site with murine macrophage elastase releases a chemotactic peptide (11). Previous studies have shown that both NE-a,PI complexes (12) and cleaved a,PI (13, 14) are present in lung secretions and thus could playa role in further neutrophil recruitment. The evidence suggests that alPI may play a role in the modulation as well as enhancement of neutrophil recruitment to the lung. The purpose of the present studies was to investigate further the role of a,PI on neutrophil chemotaxis. In particular, we wished to partially clarify the mechanism involved in the reduction of chemotaxis by native a,PI. In addition, we wished to determine whether inactivation of the protein by oxidation of the active site or cleavage of the active site using the serine proteinases NE and porcine pancreatic elastase (PPE) could also affect neutrophil recruitment.
Materials and Methods Molecular Forms of alPI a,PI was purified from plasma as described previously (15) and stored at -70 0 C in 0.05 M Tris/HCI buffer (pH 7.4).
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 21990
Fresh aliquots were thawed and used for each experiment. The inhibitory activity of the protein was assessed using NE that had been active site titrated (l6). The proteolyzed form of alPI was prepared by 48 h of preincubation with either a 4-fold molar excess of NE or 10fold molar excess of PPE at J]O C in 0.2 M Tris/HCl buffer (pH 8.6) + 0.01 % Triton Xl00. The NE was removed by solid-phase immunoadsorption with monospecific polyclonal antiserum to NE immobilized on Sepharose 4B. The sample was passed down the antibody column, and the fractions were tested with a specific NE ELISA assay to confirm removal of immunoreactive NE. The PPE was separated from alPI (100 p.g) by anion exchange chromatography on DEAE Sephacel at 50 ml/h using a linear salt gradient from 0 to 0.5 M NaCl in 0.05 M Tris/HCl, pH 7.4. Fractions (0.5 ml) were screened for alPI using a specific ELISA with a sheep polyclonal antiserum (17) and PPE activity using the synthetic elastase substrate succinyltrialanyl paranitroanilide (SLAPN). Oxidized alPI was produced by adding 20 p.l of hydrogen peroxide (H20 2 ) (BDH Chemicals) to 2 ml of alPI for 30 min at room temperature. The resulting mixture was placed in dialysis tubing (Sigma Chemical Co., St. Louis, MO) with an exclusion size> 12,000 D and dialyzed against 1 liter of 4-(2-hydroxyethyl)-1-piperazine-ethane sulphonic acid (Hepes)-buffered RPMI-1640 medium (Flow Laboratories, Rickmansworth, Herts, UK) overnight. Similarly, pure active alPI (25 p.g in 2 ml of 0.05 M Tris/HCl buffer, pH 7.4) and the proteolyzed alPI (19.7 p.g in 2 ml of 0.2 M Tris/HCl buffer, pH 8.6) were dialyzed against 1 liter of RPMI Hepes overnight. In addition, several fractions of the proteolyzed alPI from the ion exchange column were pooled (5 ml), placed in a dialysis bag, and dialyzed against 10 ml of RPMI Hepes (2 d at 4 0 C), followed by further dialysis (5 h) against 10 ml of RPMI Hepes. The loss of alPI enzyme inhibitory activity was confirmed by demonstrating no residual ability to inactivate PPE. All pre- and postdialysis samples were assessed for the molecular size of the alPI molecules by SDS-PAGE using a 10% resolving gel on a vertical slab system (Bio-Rad, Richmond, CA). Protein bands were stained using Kenacid blue. The results confirmed that none of the protein was present in its native form (54,000 D) or complexed with enzyme (80,000 D). Neutrophil Migration The assay was based on the multiple blind well assay system (l8). FMLP (Sigma) was diluted in RPMI Hepes to give a final concentration of 10-8 M. The peptide was placed in the blind well of the lower perspex plate and separated from the cells (1.5 X 1(6) in the well in the upper plate by two membranes (a lower 0.45-p.m pore size cellulose acetate Millipore membrane [Millipore Co.] and an upper 2-p.m pore size polycarbonate nucleopore membrane [Appleton Woods, Birmingham, UK]) and sealed with a rubber O-ring. Neutrophils (> 96% pure) were isolated by Percoll gradient separation (19). They were washed in RPMI Hepes and resuspended in RPMI Hepes/BSA buffer (2 p.glml). Plates were left at 37 0 C for 90 min and the Millipore membranes removed and stained as described previously
(2). The experiments were run in triplicate to obtain a single result except where stated. Cell numbers were counted from 5 areas of each membrane selected at random, using a graticule eye piece (x400 magnification). The average value for all three membranes was taken as the final result (within batch coefficient of variation = 5.2 %, n = 10). The effect of different concentrations of the molecular forms of alPI and the other serine proteinase inhibitors was studied in two ways. Firstly, the cells were mixed with increasing concentrations of the proteins 30 min prior to assessing response to 10-8 M FMLP. Secondly, the inhibitor was put in the lower well of the chemotaxis plate in order to assess its effect alone on neutrophil migration. In this way, any reduction in neutrophil response to FMLP could be differentiated from immobilization of the cells as a result of activation by the inhibitor when placed in the upper chamber. In the case of native and oxidized aIPI, this possibility was studied in further detail by checkerboard analysis. PMN Binding of FMLP The effect of alPI on specific FMLP binding to neutrophils was studied as follows. Neutrophils, separated as above, were suspended in RPMI Hepes (39 X 106 /ml). In addition, further cells were isolated using Ficoll Hypaque separation (confirmed to be > 97 % pure) as described previously (20) and suspended in RPMI Hepes (42 X 1Q6/ml). Specific cell binding of FMLP was assessed with tritiated FMLP (60 Ci/mmol) diluted in RPMI to give a final concentration in the reaction mixture of 10-8 M. Nonspecific binding was assessed using the same amount of labeled FMLP with a l,ooo-foid excess of unlabeled FMLP. The experiments were performed at 0 0 C in microfuge tubes to which 100 p.l of a 2:1 mixture of butyl and nonyl phthalate (BDH Chemicals) had been added. The cells (25 p.l; 106 cells) were preincubated for 10 min with active alPI ranging in concentration from 1.35 X 10- 10 to 1.35 X 10-7 M, made up in 0.05 M Tris/HCl buffer, pH 7.4, made up to 100 p.l with RPMI Hepes. The FMLP (25 p.l) was then added, and incubation continued for 30 min. After the incubation periods, the tubes were centrifuged at 8,000 g for 2 min to allow the cells to pass through the phthalate. Some of the supernatant (120 p.l) was removed and added to 2 ml of Optiphase X. The remaining supernatant was discarded, and the tubes, above the oil, were washed (Xl) with 2 % SDS to remove excess counts, taking care not to disturb the cell pellet below the oil. The SDS and oil were then discarded and a further 150 p.l of 2 % SDS was added to lyse the cells and the lysate placed in scintillation vials. The tubes were rinsed with Optiphase X to remove remaining debris and added to the vials with further Optiphase to make a final volume of 2 ml. Vials were counted (4 min) with an LKB Rackbeta liquid scintillation counter. The count, and hence molecules of FMLP bound, was determined from the total counts bound (labeled FMLP experiment) minus the nonspecific binding (labeled and unlabeled FMLP experiment). FMLP binding was compared in the presence and absence of alPI. All experiments were run in duplicate.
Stockley, Shaw, Afford et al.: Neutrophil Activation and Antiproteinases
FMLP-a,PI Interaction In order to investigate the possibility of FMLP binding to aIPI, the 3H-labeled FMLP (10- 8 M) was incubated for 15 min at J"l0 C with 6.5 X 10-7 M pure alPI. The mixture (0.5 ml) was then applied to a 1 X 8 em column containing 5 ml of Sephadex G25 followed by PBS (pH = 7.4) at a flow rate of 1 ml/min. The fractions were collected and assayed for alPI immunologically and for (3H]FMLP by scintillation counting. Comparison with Other Inhibitors The effect of active alPI on neutrophil migration was compared with active al antichymotrypsin (aIACh) purified and assessed as described previously (21). In addition, the results were compared to those obtained with antileukoprotease (ALP) donated by 1. A. Kramps (Leiden), which had also been assessed for its inhibitory activity. Statistical analysis of neutrophil migration was performed on transformed data using analysis of variance with the assumption that variances were not equal.
Results When alPI was placed in the upper chamber ofthe chemotaxis plate, there was a concentration-dependent effect on neutrophil migration of FMLP (10-8 M). In the initial experiments, we found inhibition of cell movement at concentrations greater than 2 Itg/ml (J"l nM). The number of cells migrating in the absence of alPI was 61.2 ± 6.1 cells/field (mean ± SE). The number of cells migrating in the presence of 0.6 Itg/ml (11 nM) was 54.9 ± 9.5 and 57.5 ± 9.5 in the presence of 1.23 Itg/ml alPI (22 nM). However, the cell response fell to Zl.6 ± 0.8 (P < 0.01) at 2.5 Itg/ml (46 nM) and 14.6 ± 12.0 (P < 0.01) at concentrations of 6.3 Itg/ml (117 nM). There was a significant correlation between dose and inhibition (P < 0.01). Experiments performed on two other occasions confirmed that alPI inhibited PMN migration, with the greatest change occurring at the highest concentrations. The results of these three experiments are summarized in Figure 1. Results are shown as percent inhibition of the response of FMLP because chemotactic response for different subjects' PMN showed wide variation as reported previously (2). In a further study on a separate occasion, the FMLP dose-response curve of PMN migration with and without alPI present showed suppression but no shift in the curve, with maximal migration occurring at 10-8 M FMLP in both instances. Furthermore, cell viability was not affected, even at the highest concentrations of alPI (trypan blue exclusion). Further studies of active alPI placed in the upper chamber at a concentration of 3 Itg/ml confirmed the inhibitory effect, with a reduction in response to FMLP of a subsequent neutrophil preparation from 15.9 ± 0.3 to 7.6 ± 0.4 cells/field (P < 0.001). However, 3 Itg of the oxidized alPI (4.8 ± 0.2 cells/field) and 3 Itg/ml of the cleaved alPI produced with NE (5.6 ± 0.1 cells/field) had a similar effect when placed in the upper chamber (P < 0.001) despite loss of proteinase inhibitory function. When the alPI molecular forms were placed in the lower well of the chemotaxis chamber, the oxidized and proteolyzed forms were shown to cause
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166
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 21990
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After extensive dialysis (1 liter dialysate X 24 h), the native and oxidized alPI when mixed with the cells (upper chamber) again reduced the chemotactic response to FMLP. The results are summarized in Figure 3 for protein concentrations of 3 ~g/rn1. Native alPI reduced the response on this occasion from 24.1 ± 1.4 to 12.7 ± 2.1 cells/field (P < 0.05) and the oxidized alPI from 22.9 ± 0.5 to 12.1 ± 2.4 cells/field, although this just failed to reach significance (P = 0.06). In contrast, the dialyzed preparation of profuolyzed alPI no longer inhibited cell motility (control = 24.1 ± 1.4; alPI = 28.1 ± 3.1 cells/field). Dose-response curves confirmed that the dialyzed preparation of oxidized alPI retained its ability to activate cell movement. The number of cells recruited by oxidized alPI in the lower well at 6.7 ~g/rn1 was 37.2 ± 3.4 cells/field (P < 0.01 compared to negative control), rising to 60 ± 11.4 at 15.9 ~g/rn1 (P < 0.01) and falling to 2.9 ± 0.5 at 20.0 ~g/rn1. The maximal response was similar to a positive control with 10-8 M FMLP (59.7 ± 2.3 cells/field), which was run on the same occasion with the same cells. Similar studies with the dialyzed native and proteolyzed alPI showed no effect on cell motility with this cell preparation (5.1 ± 0.8 at 8.5 ~g/rn1; 3.2 ± 0.8 at 19 ~g/rn1; and 2.7 ± 0.7 cells/field at 25.5
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tained no immunoreactive alPI but did contain amidolytic activity against SLAPN, indicating this was the PPE. The second peak contained the immunoreactive alPI but no enzyme activity. When the pooled alPI-positive fractions were subjected to SDS-PAGE, a major protein band of rv49,OOO D was present, together with a small molecular weight band near the leading edge of the electrophoretic front « 5,000 D). The column fractions containing immunoreactive alPI retained their effect on neutrophil motility when tested with a further preparation of PMN as shown in Figure 4 (P < 0.05). In a further setof experiments, dialysis of these fractions (50 to 70, Figure 4) against a small volume of RPMI resulted in appearance of a factor in the dialysis fluid which affected
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Figure 5. The effect of dialysis of the cleaved alPI on its ability to mobilize neutrophils. The histograms are mean ± SE for dialysis medium alone (control), the alPI before and after dialysis, together with the first dialysis fluid harvested from outside the dialysis bag.
Stockley, Shaw, Afford et ai.: Neutrophil Activation and Antiproteinases
167
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cell motility (Figure 5). Continued dialysis resulted in a loss of this factor from the contents of the dialysis bag (Figure 5). These results were associated with the appearance of the low molecular weight protein outside the dialysis bag. This was confirmed by SDS-PAGE as shown in Figure 6. When the dialysis samples were analyzed using the specific alPI ELISA, a positive signal was obtained, confirming that the low molecular weight protein possessed some immunologic identity with the complete alPI molecule. Studies of the effect of alPI on FMLP binding showed an apparent increase in specific binding whether the cells were isolated by Percoll or Ficoll gradient separation. In both experiments, the nonspecific binding accounted for 25 and 24 % of the total binding, respectively. The presence of alPI resulted in a concentration-dependent increase in specific binding compared to cells alone. No effect was seen as
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the concentration of alPI rose from 1.35 x 1(t-1I&J to 1.35 X 10-8 M active protein. However, above this concentration, FMLP binding increased by 40.4 and 59.4% for Pen:oll and Ficoll isolated cells, respectively, at 10-7 M alP}. The results are summarized in Figure 7. After G25 column chromatography of an a,PI-FMLP mixture, the alPI was identified in the void volume of the column. Most of the (3H]FMLP (95%) was found in the same fractions and not retained by the column, suggesting an interaction between the protein and the peptide. The two other proteinase inhibitors, a,AC'b and ALP, also showed concentration-dependent inhibition of chemotaxis. The effect of active alACh was greater and the effect of ALP was less than that seen for aIPI. The results are summarized in Figure 8, comparing the inhibition with the molar concentrations of active amounts of each inhibitor.
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 2 1990
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