Lysosomal Enzyme Release from Human Neutrophils Adherent to Foreign Material Surfaces: Enhanced Release of Elastase Activity Douglas J. Erfle, MSc,*1 J. Paul Santerre, PhD,† and Rosalind S. Labow, PhD* *Cardiovascular Devices Division and Department of Biochemistry, University of Ottawa Heart Institute, Ottawa, Ontario, Canada; and †Faculty of Dentistry, Department of Biomaterials, University of Toronto, Toronto, Ontario, Canada
11 Neutrophils are the major phagocytic white blood cell present during the acute inflammatory response to cardiovascular medical devices and can become activated to release a wide variety of products that help mediate the overall host response. The purpose of this investigation was to develop an in vitro system to study the release of lysosomal enzymes from neutrophils adherent to biomaterial surfaces. Neutrophils isolated from peripheral human blood were allowed to adhere to different biomaterials and lysosomal enzyme release assessed by monitoring elastase-like activity in the supernatant. The number of adherent neutrophils with intact cytoplasmic membranes was estimated by extracting the cells and quantifying lactate dehydrogenase. Stimulated and non-stimulated neutrophils released significantly different amounts of elastase-like activity depending on the biomaterial surface to which they were adhered. The techniques developed in this study form the basis of an in vitro system for investigating the events associated with neutrophil/biomaterial interactions as well as a method for evaluating the white blood cell response to the materials used in circulatory support devices. Cardiovasc Pathol 1997; 6:333–340 © 1997 by Elsevier Science Inc.
Neutrophils (PMNs), as the major phagocytic white blood cell present during acute inflammation, adhere to foreign material surfaces and become activated to release active oxygen-derived species as well as a variety of enzymes contained within their lysosomal granules (1). PMNs can be activated by both biological and synthetic substances in suspension, but even more so when adherent to a foreign surface (2). One of the critical factors in the success or failure of any cardiovascular medical device is the nature of the material surface that interacts with the tissues and/or fluids of the patient. The materials, which must perform an intended function while exposed to a harsh biological environment represent foreign matter. The intensity and duration of the inflammatory response with the contacting
Manuscript received November 6, 1996; accepted April 7, 1997. 1 Douglas Erfle is currently affiliated with Micrologix Biotec of Vancouver, British Columbia, Canada. Address for correspondence: Rosalind S. Labow, PhD, Cardiovascular Devices Division, University of Ottawa Heart Institute, 1053 Carling Avenue, Ottawa, Ontario, Canada, K1Y 4E9; phone: (613) 761-4010; fax: (613) 761-5035; email: [email protected] Cardiovascular Pathology Vol. 6, No. 6, November/December 1997:333–340 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
surface has been used as a measure of the biocompatibility of the “biomaterial” (3). Although the ability of adherent PMNs to specifically release their lysosomal contents extracellularly in response to non-phagocytosable surfaces (frustrated phagocytosis) has been well established (4), the effect of the chemical properties of the surface on lysosomal enzyme release remains to be defined. Kaplan et al. have shown that PS and poly(urethane) surfaces were able to activate superoxide release from adherent PMNs in the absence of additional stimuli while expanded poly(tetrafluoroethylene) could not (5). Similarly, Falck compared the respiratory burst of PMNs adherent to poly(urethane), poly(vinyl chloride), poly(propylene), and poly(ethylene) surfaces and demonstrated that adherence to these different materials was associated with the production of different amounts of superoxide (6). Differences in the intensity of the respiratory burst of PMNs on different materials may be indicative of their biocompatibility which then may impact on the stability of the foreign material. Phagocyte-derived oxidants are implicated in non-specific destruction of biological tissues (7,8) and have shown the ability to oxidize and degrade biomaterial surfaces in vitro (9,10). 1054-8807/97/$17.00 PII S1054-8807(97)00031-8
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While there is strong evidence for differential respiratory burst responses from PMNs adherent to different biomaterial surfaces, the release of lysosomal hydrolytic enzymes, with equal or greater potential for tissue damage and foreign material degradation, has been less well studied. Azurophilic granules, in addition to myeloperoxidase, contain elastase (11) which represents the majority of proteolytic activity found in PMNs and has been implicated as a mediator of tissue-destructive events in inflammatory diseases. Isolated human PMNs, as well as purified porcine pancreatic elastase, possess the capability to degrade poly(urethane) surfaces (12). The purpose of this investigation was to develop an in vitro system to study adherent PMN activation on different material surfaces with respect to lysosomal enzyme release. The nature of the enzyme (elastase) release in this static in vitro cell system was characterized by simultaneously determining the integrity of the cytoplasmic membranes of adherent cells by measuring the cytoplasmic enzyme, lactate dehydrogenase (LDH). Using these techniques, the interactions between PMNs and poly(ester-urethaneurea), poly(ether-urethane-urea) and PS surfaces were studied. This model system will provide information that may aid in further understanding the complex events surrounding the body’s biological response to engineered cardiovascular medical device implants.
Methods Materials Histopaque 1077 and 1119, phorbol myristate acetate (PMA), LDH assay kit (Procedure No. 500) were obtained from Sigma Chemical Co. (St. Louis, MO). Dulbecco’s phosphate buffered saline (DPBS) and RPMI-1640 tissue culture medium were purchased from Gibco BRL (Burlington, ON, Canada) and used at pH 7.2. Dimethyl sulphoxide (DMSO) and N-2-hydroxyethyl piperazine-N-2-ethanesulphonic acid (HEPES) were purchased from BDH Inc. (Toronto, ON, Canada). Triton X-100 and dimethylacetamide (DMAC) were obtained from Aldrich Chemical (Milwaukee, WI). N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide (elastase substrate 1 (ES1)) was purchased from Calbiochem (San Diego, CA). All general reagents were from Fisher Scientific (Ottawa, ON, Canada).
Isolation of PMNs PMNs were isolated from whole, peripheral human blood using a modification (12) of Boyum (13). Whole blood (80 mL) from healthy volunteers (with approval of the Research Ethics Committee of the Ottawa Civic Hospital) was collected into ethylenediamine tetra-acetic acid (EDTA)containing Vacutainers® (Becton Dickenson). 20 mL of anticoagulated whole blood was layered onto a discontinuous gradient composed of Histopaque 1077 (8 mL) on top of
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Histopaque 1119 (20 mL) in each of four 50 mL polypropylene centrifuge tubes (Falcon 2098). After centrifugation at 700 3 g (1,900 rpm, Model No. RT6000B, Dupont Instruments), the PMN-containing layer was washed free of Histopaque 1119 by centrifuging and resuspending the pellet three times in RPMI-1640 tissue culture medium. Contaminating red blood cells were removed by hypotonic lysis in distilled water and the PMNs resuspended in DPBS and stored in a teflon tube on ice until used. PMNs were always used within 30 minutes from the completion of the isolation procedure. Analysis of PMNs suspensions on a routine basis was performed using an automated cell counter (System 9000, Serono Baker Diagnostics, Allentown, PA). The cell counter results were confirmed independently by manual differential staining performed at the Haematology Laboratory in the Department of Laboratory Medicine at the Ottawa Civic Hospital.
Preparation of Foreign Material Surfaces Two poly(urethane)s were synthesized from toluene diisocyanate (TDI), ethylene diamine (ED), and either poly(caprolactone) (PCL) or poly(tetramethylene oxide) (PTMO). They are referred to as “models” to distinguish them from commercially available poly(urethane)s which are used as a material component in catheters, blood pumps, vascular grafts, breast implants, and artificial skin. The synthesis and characterization of the two model poly(urethane)s have been described previously (14). In accordance with the nomenclature used by Santerre et al. (14), these materials will be referred to as TDI/PCL/ED and TDI/PTMO/ED. The poly(urethane)s were synthesized with a ratio of TDI to PCL to ED of 2.2:1:1.2 or TDI to PTMO to ED of 2:1:1. The solid materials were dissolved in DMAC to yield 2% (w/v) solutions. Any non-soluble material was then removed from the polymer solutions by passing them through a 0.45 mm Teflon® filter (Chromatographic Specialties, C616058). Round glass coverslips (15 mm diameter) (Fisher Scientific, 12-548) were coated with the polymer solution (50 mL) under sterile conditions in a laminar flow hood and dried overnight at 508C. After 24 hours, this was repeated, followed by further drying at 508C under vacuum (2100 kPa) for 48 hours. Prior to use, the polymer-coated slips were “hydrated” by incubating them overnight in sterile DPBS at 378C/5% CO2 and 100% humidity. The hydration step was performed to remove any remaining traces of solvent from the polymers which might affect the cells when added, and also to allow the surface of the poly(urethane)s to equilibrate in an aqueous environment prior to the addition of the cells.
Determination of Elastase Release from PMNs Adherent to Polymer Surfaces All experiments were carried out using standard 24-well tissue culture plates (Falcon, 3047). The bottom surface of each well was either tissue culture-treated PS or a model
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poly(urethane) coated on a glass slip. 1.0 3 106 PMNs, suspended in 1 mL DPBS, were added to the wells of tissue culture plates and incubated at 378C/5% CO2 and 100% humidity for 1 hour. With the plates on an angle, the supernatant and any non-adherent cells were then removed using a sterile pasteur pipet connected to a vacuum aspirator (Millipore, DOA-V184-AA) and discarded. To standardize the flow over the adherent cells when removing the supernatant, the plates were always tilted on the same angle (208).
Cell Activation with the Pharmacologic Agonist, PMA PMA, received as a lyophilized powder, was dissolved in DMSO at a concentration of 1 mg/mL and stored at 2148C. The day of the experiment, the PMA solution was thawed and diluted to 1 3 1027 M with DPBS. Either 750 mL of PMA/DPBS or 750 mL of DPBS alone were added back to the wells and the plates returned to the incubator. At various times (initially, and up to 10 hours for the control PS wells; initially and 3 hours for the two poly(urethane)-coated slips) plates were removed from the incubator, tilted on a 208 angle and the supernatant in each well transferred to a microcentrifuge tube (1.5 mL eppendorf, Brinkman) and stored on ice. The tubes were centrifuged at 18,000 3 g (14,000 rpm, Hermle Z229 Microcentrifuge) for 5 minutes at 48C and the supernatant transferred to a new tube and stored on ice. The elastase activity in each tube was assayed immediately, without being frozen, as described below. The number of cells adherent to the surface of the wells and the poly(urethane)-coated slips at the time of removal of the supernatant was assessed by assaying the cell extract for LDH as described below. Elastase activity in cell culture supernatants was determined by measuring the rate of cleavage of the small synthetic tetrapeptide substrate, N-methoxysuccinyl-Ala-AlaPro-Val-p-nitroanilide (ES1). Since elastase activity was monitored without the use of the enzyme’s “natural” substrate, elastin, any measured value of elastase activity will be referred to as elastase-like activity (ELA). The substrate solution was prepared by dissolving 10 mg of lyophilized ES1 in 2 mL DMSO and adding this solution dropwise to stirring buffer (0.01 M HEPES with 0.1 M sodium chloride, pH 7.4) and the final volume brought to 169 mL. 300 mL of the sample to be assayed were mixed with 500 mL of substrate at 378C and the increase in absorbance at 410 nm followed in a spectrophotometer (Model DU 640, Beckman Instruments). One unit of ELA was defined as that amount of enzyme which resulted in the release of 1 nmol of p-nitroaniline from ES1 per minute at 378C and at an initial substrate concentration of 62.5 mM. Units of ELA were calculated by entering the rate of product formation into the following equation: ∆A ------- min ELA UNIT ( nmol ⁄ min ) = --------------------------------------------–1 ε × ( V CUVETTE )
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DA/minute refers to the change in absorbance at 410 nm per minute. e is the molar absorptivity of p-nitroaniline (8.8 3 1026 nM) at 410 nm and VCUVETTE refers to the total reaction volume in the cuvette (nL).
Preparation of Extracts from Adherent PMNs The number of PMNs adherent to polymer surfaces was estimated by analyzing the LDH that could be extracted from the surface of the polymer after removal of the culture supernatant (initially, and up to 10 hours for PS; initially and after 3 hours for the poly(urethane)-coated slips) at 378C with and without PMA as described above. After removal of the supernatant in each well, ice-cold 1% Triton X-100 in DPBS (300 mL) was added to the tissue culture plate kept on ice, and the contents of each well mixed by repeatedly pipetting up and down. Each well was rinsed twice more with 350 mL of cold 1% Triton X-100/DPBS bringing the extraction volume for each well to 1.0 mL. The pooled extracts were then mixed by vortexing and centrifuged at 18,000 3 g at 48C. The supernatant was assayed for LDH activity as described below. LDH activity in cell extracts was assayed using a modification of the procedure provided by Sigma. This colorimetric assay is based on the LDH-catalyzed conversion of pyruvate to lactate in the presence of NADH (15). Pyruvate is quantified by reaction with 2,4-dinitrophenyl hydrazine (color reagent) which results in the formation of a hydrazone product that absorbs light in the range of 400–500 nm. 50 mL of cell extract were added to 100 mL of pre-warmed pyruvate substrate (0.75 mmol/L) and the mixture incubated at 378C for exactly 30 minutes. 100 mL of color reagent were added and the tube left to stand for 20 minutes at room temperature. Finally, 750 mL of 0.4 N sodium hydroxide were added and the tube left to stand for at least 5 minutes before reading at 450 nm. Samples were diluted to give absorbance values for [PYRUVATE]FINAL that fell within a standard curve generated using known concentrations of pyruvate. Units of LDH activity were calculated by entering these values into the following equation: LDH UNITS = [ PYRUVATE ] INITIAL – [ PYRUVATE ] FINAL -------------------------------------------------------------------------------------------------------- × 30 MINUTES DILUTIONFACTOR One unit of LDH activity was defined as that amount that would catalyze the conversion of one nmol of pyruvate per minute at 378C at an initial pyruvate concentration of 500 mM.
Cell Viability on Biomaterial Surfaces The objective of this study was to determine the amount of ELA released on different biomaterial surfaces which was due to a specific PMN response to foreign materials
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and not due to cell death. To normalize all data, the number of adherent intact cells was quantified. An experiment was performed in which different concentrations of PMNs were added and allowed to incubate for 1 hour on the biomaterial surface. After removal of the supernatant, the adherent cells were extracted with three freeze/thaw cycles in 1% Triton X-100 which was found to release the maximal LDH activity. The total LDH extracted from 1 3 106 PMNs was 41.79 6 1.01 units. A typical standard curve of PMN number versus extractable LDH activity for the numbers of cells ranging from 500,000 to 1,000,000 is shown in Figure 1 for the PS material. There was a positive, linear relationship between the number of PMNs added to the wells. Although not shown in Figure 1, (the study was performed separately for 10,000– 500,000 cells due to the non-linearity of the pyruvate standard curve) the lower detection limit for the amount of LDH activity that could be extracted from the culture surface was approximately 10,000 PMNs (r2 5 0.99638). These experiments confirmed that LDH could be extracted and measured over a wide range of concentrations and validated its use as a means of estimating adherent cell numbers. Tissue culture grade PS was used as the control surface. To measure the specific release of ELA in the supernatant, the time at which minimal cell death had commenced on the control material was determined. Cells were allowed to adhere to the PS surface as described above. The LDH content in the viable cells adherent to the surface of the wells was monitored from 1 to 10 hours after removal of the supernatant from cells treated with 1027 M PMA in DPBS or DPBS
alone. As can be seen in Figure 2, both PMA-stimulated and non-stimulated wells had a relatively constant number of viable, adherent cells for the first 3 hours. After this time, the amount of LDH activity that could be extracted from the surface of wells that contained PMA began to decrease rapidly, indicating that cells had begun to die. By 10 hours, no significant LDH activity could be detected in the wells with PMA. The extractable LDH activity from non-stimulated cells at 10 hours was not significantly different from that at time zero. When LDH activity was used to measure the cell retention and viability on the model poly(urethanes), there was no significant difference in the numbers adhering to PS, TDI/PCL/ED and TDI/PTMO/ED initially (Figure 2). However, at 3 hours following the addition of PMA, there was a significant difference between the LDH activity on the TDI/ PCL/ED and TDI/PTMO/ED relative to the control PS and from the time zero values (Figure 3). The microscopic analysis of adherent PMNs for time points corresponding to Figure 2 is shown in Figure 4. After the 1-hour adherence period, adherent PMNs are still round (panel A). PMNs that were subsequently stimulated with PMA spread over the surface of the well by 3 hours (panel B1) while at the same time point non-stimulated cells retained a more circular shape (panel B2). By 6 hours stimulated cells were less well defined and appeared to be spread quite flat over the surface of the well. This cellular material was present for the remainder of the experiment (panel D1) indicating that the decrease in LDH seen during this time was not due to lifting of intact cells from the surface of the
Figure 1. Estimation of adherent cell number with respect to extractable LDH activity. All points represent the average of quadruplicate measurements 6 SD. The solid line represents the linear regression analysis of the data points. The dotted line represents the 95% confidence interval for the fitted equation.
Figure 2. Kinetic determination of LDH activity related to PMN adherent to poly(styrene). 1 3 106 PMN were added to the wells of poly(styrene) tissue culture plates and incubated for one hour. Open symbols represent data from cells that were stimulated with 1027 M PMA. Closed symbols represent data from cells that were not stimulated.
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well. Non-stimulated cells did not begin to spread noticeably until 9 hours (panel D2) which corresponded to the insignificant decrease in the amount of LDH activity associated with the adherent cells.
Biomaterial Induced Release of ELA The total extractable ELA was determined with the synthetic substrate ES1 in order to relate this to the maximal amount released during activation. The most ELA which could be extracted from 1 3 106 cells was 21.5 6 1.01 units. This value could not be increased by sonication or more than three freeze/thaw cycles. Differences in ELA release from PMNs adherent to the PS and model poly(urethane) surfaces were investigated and the data are presented in Figure 5. Three hours was chosen as the relevant time point to assess PMNs activation using ELA release since there were no significant changes in LDH activity in the PS adherent PMNs and the other surfaces maintained .75% cell viability based on LDH analysis at this time (Figure 3). As well, stimulation of cell response by PMA did not show a significant effect on cell viability prior to 3 hours (see Figure 2). At 3 hours there were significant differences in the ELA activity measured in the PMNs adherent to the differ-
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ent surfaces with and without stimulation with PMA (Figure 5). The amount of ELA in Figure 5 was adjusted for the amount of cell death. The amount of ELA seen in the supernatant of wells containing TDI/PCL/ED-coated slips was higher than those containing TDI/PTMO/ED either in the presence or absence of PMA (Figure 5). Control (PS) wells showed ELA levels significantly higher than both model poly(urethane)s. It should be noted that this relative difference was observed in all experiments, however, the specific amounts of ELA released varied significantly from one donor to another. Since the heterogeneity and biological variability of PMNs from different normal individuals as well as patients, has been demonstrated with many other cellular components (16), this experiment was performed in triplicate using PMNs isolated from the blood of the same donor and represents the averaged data in Figure 5. The maximum amount of ELA released by PMA from PMNs adherent to the biomaterial surfaces was approximately 10-fold less than the maximal activity determined by repeated freeze/thawing described above (i.e., 2.3 and 21.5 units, respectively). However, stimulation with PMA caused the release of 5–10-fold more ELA from the PMNs on the poly(urethane) surfaces, than that released without PMA stimulation, i.e., that due to stimulation by the material surfaces themselves (see Figure 5).
Exposure of ELA in Solution to Different Surfaces
Figure 3. Determination of LDH activity related to viability of adherent PMN. 1 3 106 PMN were added to tissue culture wells and incubated for 1 hour. The wells contained slips coated with TDI/ PCL/ED, TDI/PTMO/ED or no slip (poly(styrene)). The supernatant was removed and Triton X-100 added immediately (open bars) or the wells were refilled with 1027 M PMA for 3 hours followed by Triton X-100 addition (hatched bars). LDH activity in the adherent PMN was then determined. Asterisk indicates the LDH activity is significantly different from time zero, and plus sign indicates significantly different from poly(styrene).
A control experiment was run to determine the effect of the different materials on available ELA in solution over time. This was done to determine if the differences detected for ELA in the supernatant of PMNs (see Figure 5) were due to a difference in the adsorption of enzyme on the various materials rather than a difference in the amount of enzyme released from the cells. Cell extracts were prepared from freshly isolated PMNs without the use of Triton X-100, since in another study Triton X-100 caused chemical degradation of the polymer, TDI/PCL/ED (data not shown). After the third cycle of freeze/thawing without Triton X-100 there was still enough ELA extracted (approximately 2 units/mL) to add to the wells to follow the decrease in ELA. These cell extracts were incubated in tissue culture wells containing polymer surfaces and the ELA in solution was measured over a period of 5 hours. On all three materials, the amount of ELA in solution showed a rapid decrease in the first hour which gradually leveled off (Figure 6). Significant amounts of ELA could still be detected at 5 hours. Cells incubated on the PS well surface itself showed the highest level of ELA remaining in solution at all time points, after 1 minute. The next highest levels were seen in wells containing TDI/ PTMO/ED and finally TDI/PCL/ED. The differences seen between ELA in the presence of the different materials were significant at several time points, but too small to account for the differences in the amount of ELA in the supernatant of PMNs cultured on the different materials (see Figure 5).
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Figure 4. Microscopic analysis of PMN adherent on poly(styrene). Conditions for this experiment were the same as those described in Figure 2. Panel A shows a representative example of cells prior to the addition of PMA/DPBS or pure DPBS. Subsequent panels B1–D2 show stimulated and non-stimulated PMN at 3 (B1,B2), 6 (C1,C2) and 9 hours (D1,D2), respectively. (Original magnification 3200. Bar 5 200 mm.)
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Furthermore, the order of decreasing ELA in solution in the presence of the three surfaces did not correspond to the order of increasing ELA released into the cell supernatant. From Figure 5 the order of ELA measured in solutions following release from PMNs was PS . TDI/PCL/ED . TDI/ PTMO/ED. The order identified from Figure 6 was PS . TDI/PTMO/ED . TDI/PCL/ED.
Discussion By monitoring enzymes found in the cytoplasm (LDH) and inside granules (ELA), the effect of material surfaces on the release of enzymes into the supernatant of activated PMNs was investigated. The number of adherent PMNs with intact cytoplasmic membranes was estimated by determining the LDH content inside the cells, an approach previously used by Cerasoli et al. (17). In these studies as in several others (18–20) tissue culture grade PS was used as the standard surface. However, the cell type and the markers chosen to measure in a particular study will determine whether the PS is a positive or negative control (21). Although PS is specifically treated to enhance cell attachment (i.e., it is usually made hydrophilic), the mechanism of the difference in cell adhesion between treated and untreated PS is not well understood (22). By measuring a marker of PMN activation (ELA release) and cell viability (LDH in remaining adherent PMNs) on standard PS, the importance of the findings on model material surfaces can be evaluated relative to other studies using in vitro cell systems. Three hours was chosen as the time to measure ELA released from the PMNs on all the surfaces, since at 3 hours
Figure 5. Comparison of cumulative ELA release from PMN adherent to model poly(urethane)s after 3 hours. 1 3 106 PMN were added to tissue culture wells and incubated for 1 hour. The wells contained slips coated with TDI/PCL/ED, TDI/PTMO/ED or no slip (poly(styrene)). The supernatant was removed and the wells refilled with 1027 M PMA (open bars) or DPBS (hatched bars).
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no cell death had occurred on the control PS and was only minimal on the poly(urethane)s. The amount of LDH activity in cell extracts showed that the same number of cells adhered to the different polymer surfaces initially. However, at 3 hours after PMA incubation, LDH had decreased significantly on both the TDI/PCL/ED and TDI/PTMO/ED. Therefore, the ELA was normalized in the initial value for live adherent cells for each material (Figure 1). With this correction, these values represented the amount of ELA released into the supernatant during this time as a result of specific lysosomal enzyme release due to PMA and not due to cell death (Figure 5). At later times more ELA release was due to cell death, even on the control PS surfaces (Figure 2). The most interesting and perhaps significant finding of this study was that the differences in the released ELA measured from the PMNs adherent to three different surfaces, were still significant after 3 hours of PMN incubation with PMA agonist (Figure 5). The most ELA was measured in the cell supernatants of stimulated and non-stimulated cells in the PS wells, followed by TDI/PCL/ED and TDI/PTMO/ED (Figure 5). This highlights the importance of the material surface itself with respect to the stimulation of the ELA release. The response of PMNs to activation with a variety of physiologic (N-formylmethionylleucylphenylalanine (FMLP), leukotriene B4) or pharmacologic agonists (phorbol myristate acetate (PMA), calcium ionophore (A23187)) has been measured using many different markers. These include intracellular calcium mobilization, expression of adhesion molecules, the release of arachidonic acid as well as azuro-
Figure 6. Effect of biomaterial surfaces on ELA in solution. Polymer slips were prepared and hydrated. The extracts from a 1 3 106 PMN/mL suspension was prepared and added to wells containing the different test surfaces. Triangles represent the data from poly(styrene) wells. Closed circles represent the data from wells containing TDI/PTMO/ED-coated slips. Open circles represent the data from wells containing TDI/PCL/ED-coated slips.
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philic granule contents which include the respiratory burst products and the lysosomal hydrolases (23–25). It is known that PMA bypasses membrane receptors and acts directly on protein kinase C (26). On the other hand, the adherence activation as measured by the respiratory burst, has been shown to involve adherence receptors such as leukocyte integrins found in the cytoplasmic membrane of the cell (27). Since biomaterials have been shown to cause the release of respiratory burst products even in the absence of secondary stimulation (9,10) it is possible that the mechanism of ELA release, associated with the materials, follows a similar pathway to that of adherence activation. One good example of this is described in a study of the effects of pertussis and cholera toxins on the production of reactive oxygen metabolites in PMNs during cell activation by PMA or quartz particles. The results suggested two mechanisms for the cell activation. The release of reactive oxygen metabolites by PMA activation was not affected by the toxins, whereas it was significantly less by the cell activation with the quartz particles (28). The latter case is analogous to the effect of each material on the PMA induced ELA release. The ELA release caused by PMA was reduced on TDI/PTMO/ED relative to PS and TDI/PCL/ED. A model system has been developed which will make it possible to probe the mechanisms of neutrophil-mediated events at the interface between a cardiovascular medical device and the tissues/fluids surrounding it. Combining neutrophil activation and product release, specifically digestive and tissue remodelling enzymes (biocompatibility) with poly(urethane) degradation (biostability) in a single experimental system will provide a more complete approach to understanding the complex relationship that exists between a biomaterial as a foreign body implant and the human organism.
The authors thank Erin Meek for her technical assistance. These studies were funded in part by a grant from the National Health Research Development Program (NHRDP), Health, Canada. Daniel Duguay prepared the statistics application used in these analyses.
References 1. Shankar R, Greisler HP. Inflammation and biomaterials. In: Greco RS ed. Implantation Biology: The Host Response and Biomedical Devices. Boca Raton, FL: CRC Press, 1994;67–80. 2. Bangalore N, Travis J. Comparison of properties of membrane bound versus soluble forms of human leukocytic elastase and cathepsin G. Biological Chemistry Hoppe-Seyler 1994;375:659–666. 3. Anderson JM. Mechanisms of inflammation and infection with implanted devices. Cardiovasc Pathol 1993;4(3)(Suppl):33–41. 4. Becker EL, Showell HJ, Henson PM, Hsu LS. The ability of chemotactic factors to induce lysosomal enzyme release 1. The characteristics of the release, the importance of surfaces and the relation of enzyme release to chemotactic responsiveness. J Immunol 1974;112(6):2047–2054. 5. Kaplan SS, Basford RE, Jeong MH, Simmons RL. Mechanisms of biomaterial-induced superoxide release by neutrophils. J Biomed Mater Res 1994;28:377–386.
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6. Falck P. Characterization of human neutrophils adherent to organic polymers. Biomaterials 1995;16(1):61–66. 7. Weiss SJ. Tissue destruction by neutrophils. N Engl J Med 1989; 320(6):365–375. 8. Henson PM, Johnston RB Jr. Tissue injury in inflammation: oxidants, proteinases and cationic proteins. J Clin Invest 1987;79:669–674. 9. Sutherland K, Mahoney Jr II, Coury AJ, Eaton JW. Degradation of biomaterials by phagocyte-derived oxidants. J Clin Invest 1993;92: 2360–2367. 10. Williams DF. Some observations on the role of cellular enzymes on the degradation of polymers. In: Syrett BC, Acharya A eds. International Symposium on Corrosion and Degradation of Implant Materials. ASTM STP 684. American Society for Testing and Materials 1979; 61–75. 11. Suchard SJ, Boxer LA. Exocytosis of a subpopulation of specific granules coincides with H2O2 production in adherent human neutrophils. J Immunol 1994;152:290–300. 12. Labow RS, Erfle DJ, Santerre JP. Neutrophil-mediated degradation of segmented polyurethanes. Biomaterials 1995;16:51–59. 13. Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest 1968;21:77–108. 14. Santerre JP, Labow RS, Duguay DG, Erfle DJ, Adams GA. Biodegradation evaluation of polyether and polyester-urethanes with oxidative and hydrolytic enzymes. J Biomed Mater Res 1994;28:1187–1199. 15. Cabaud PG, Wroblewski F. Colorimetric measurement of lactate dehydrogenase activity in body fluids. Amer J Clin Pathol 1958;30:234–236. 16. Wyatt TA, Lincoln TM, Pryzwansky KB. Regulation of human neutrophil degranulation by LY-83583 and L-arginine: role of cGMP-dependent protein kinase. Amer J Physiol 1993;265:(Cell Physiol 34):C201–C211. 17. Cerasoli F Jr, Gee MH, Ishihara Y, et al. Biochemical analysis of the activation of adherent neutrophils in vitro. Tissue & Cell 1988;20(4): 505–517. 18. Bonfield TL, Colton E, Anderson JM. Protein adsorption of biomedical polymers influences activated monocytes to produce fibroblast stimulating factors. J Biomed Mater Res 1992;26:1039–1051. 19. Kaplan SS, Basford RE, Kormos RL, et al. Biomaterial associated impairment of local neutrophil function. ASAIO Trans 1990;36:M172–175. 20. Kaplan SS, Basford RE, Mora E, Jeong MH, Simmons RL. Biomaterial-induced alterations of neutrophil superoxide production. J Biomed Mater Res 1992;26:1039–1051. 21. Skarlatos SI, Rao R, Kruth HS. Accelerated development of human monocyte-macrophages cultured on Plastek-C tissue culture dishes. J Tiss Cult Meth 1992;14:113–118. 22. Underwood PA, Steele JG, Dalton BA. Effects of polystyrene surface chemistry on the biological activity of solid phase fibronectin and vitronectin, analyzed with monoclonal antibodies. J Cell Science 1993; 104:793–803. 23. Rieu P, Porteu F, Bessou G, Lesavre P, Halbwachs-Mecarelli L. Human neutrophils release their major membrane sialoprotein, leukosialin (CD43), during cell activation. Eur J Immun 1992;22:3021–3026. 24. Zhou W, Javors MA, Olson MS. Impaired surface expression of PAF receptors on human neutrophils is dependent upon cell activation. Arch Biochem Biophys 1994;308:439–445. 25. Sandborg RR, Smolen JE. Early biochemical events in leukocyte activation [review]. Lab Invest 1993;59:300–320. 26. Bergstrand H. The generation of reactive oxygen-derived species by phagocytes. In AAs 30. Inflammatory Indices in Chronic Bronchitis. Basel: Birkhauser, Verlag, 1990;199–211. 27. Berton G, Laudanna C, Sorio C, Rossi F. Generation of signals activation neutrophil functions by leukocyte integrins LFA-1 and GP150/ 195 but CR3, are able to stimulate the respiratory burst of human neutrophils. J Cell Biol 1992;116(4):1007–1017. 28. Ruotsalainen M, Savolainen KM. The role of G-proteins in the activation of human leukocytes by particulate stimuli to produce reactive oxygen metabolites. Toxicol 1995;99:67–76.
11 Neutrophils are the major phagocytic white blood cell present during the acute inflammatory response to cardiovascular medical devices and can become activated to release a wide variety of products that help mediate the overall host response. The purpose of this investigation was to develop an in vitro system to study the release of lysosomal enzymes from neutrophils adherent to biomaterial surfaces. Neutrophils isolated from peripheral human blood were allowed to adhere to different biomaterials and lysosomal enzyme release assessed by monitoring elastase-like activity in the supernatant. The number of adherent neutrophils with intact cytoplasmic membranes was estimated by extracting the cells and quantifying lactate dehydrogenase. Stimulated and non-stimulated neutrophils released significantly different amounts of elastase-like activity depending on the biomaterial surface to which they were adhered. The techniques developed in this study form the basis of an in vitro system for investigating the events associated with neutrophil/biomaterial interactions as well as a method for evaluating the white blood cell response to the materials used in circulatory support devices. Cardiovasc Pathol 1997; 6:333–340 © 1997 by Elsevier Science Inc.
Neutrophils (PMNs), as the major phagocytic white blood cell present during acute inflammation, adhere to foreign material surfaces and become activated to release active oxygen-derived species as well as a variety of enzymes contained within their lysosomal granules (1). PMNs can be activated by both biological and synthetic substances in suspension, but even more so when adherent to a foreign surface (2). One of the critical factors in the success or failure of any cardiovascular medical device is the nature of the material surface that interacts with the tissues and/or fluids of the patient. The materials, which must perform an intended function while exposed to a harsh biological environment represent foreign matter. The intensity and duration of the inflammatory response with the contacting
Manuscript received November 6, 1996; accepted April 7, 1997. 1 Douglas Erfle is currently affiliated with Micrologix Biotec of Vancouver, British Columbia, Canada. Address for correspondence: Rosalind S. Labow, PhD, Cardiovascular Devices Division, University of Ottawa Heart Institute, 1053 Carling Avenue, Ottawa, Ontario, Canada, K1Y 4E9; phone: (613) 761-4010; fax: (613) 761-5035; email: [email protected] Cardiovascular Pathology Vol. 6, No. 6, November/December 1997:333–340 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
surface has been used as a measure of the biocompatibility of the “biomaterial” (3). Although the ability of adherent PMNs to specifically release their lysosomal contents extracellularly in response to non-phagocytosable surfaces (frustrated phagocytosis) has been well established (4), the effect of the chemical properties of the surface on lysosomal enzyme release remains to be defined. Kaplan et al. have shown that PS and poly(urethane) surfaces were able to activate superoxide release from adherent PMNs in the absence of additional stimuli while expanded poly(tetrafluoroethylene) could not (5). Similarly, Falck compared the respiratory burst of PMNs adherent to poly(urethane), poly(vinyl chloride), poly(propylene), and poly(ethylene) surfaces and demonstrated that adherence to these different materials was associated with the production of different amounts of superoxide (6). Differences in the intensity of the respiratory burst of PMNs on different materials may be indicative of their biocompatibility which then may impact on the stability of the foreign material. Phagocyte-derived oxidants are implicated in non-specific destruction of biological tissues (7,8) and have shown the ability to oxidize and degrade biomaterial surfaces in vitro (9,10). 1054-8807/97/$17.00 PII S1054-8807(97)00031-8
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While there is strong evidence for differential respiratory burst responses from PMNs adherent to different biomaterial surfaces, the release of lysosomal hydrolytic enzymes, with equal or greater potential for tissue damage and foreign material degradation, has been less well studied. Azurophilic granules, in addition to myeloperoxidase, contain elastase (11) which represents the majority of proteolytic activity found in PMNs and has been implicated as a mediator of tissue-destructive events in inflammatory diseases. Isolated human PMNs, as well as purified porcine pancreatic elastase, possess the capability to degrade poly(urethane) surfaces (12). The purpose of this investigation was to develop an in vitro system to study adherent PMN activation on different material surfaces with respect to lysosomal enzyme release. The nature of the enzyme (elastase) release in this static in vitro cell system was characterized by simultaneously determining the integrity of the cytoplasmic membranes of adherent cells by measuring the cytoplasmic enzyme, lactate dehydrogenase (LDH). Using these techniques, the interactions between PMNs and poly(ester-urethaneurea), poly(ether-urethane-urea) and PS surfaces were studied. This model system will provide information that may aid in further understanding the complex events surrounding the body’s biological response to engineered cardiovascular medical device implants.
Methods Materials Histopaque 1077 and 1119, phorbol myristate acetate (PMA), LDH assay kit (Procedure No. 500) were obtained from Sigma Chemical Co. (St. Louis, MO). Dulbecco’s phosphate buffered saline (DPBS) and RPMI-1640 tissue culture medium were purchased from Gibco BRL (Burlington, ON, Canada) and used at pH 7.2. Dimethyl sulphoxide (DMSO) and N-2-hydroxyethyl piperazine-N-2-ethanesulphonic acid (HEPES) were purchased from BDH Inc. (Toronto, ON, Canada). Triton X-100 and dimethylacetamide (DMAC) were obtained from Aldrich Chemical (Milwaukee, WI). N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide (elastase substrate 1 (ES1)) was purchased from Calbiochem (San Diego, CA). All general reagents were from Fisher Scientific (Ottawa, ON, Canada).
Isolation of PMNs PMNs were isolated from whole, peripheral human blood using a modification (12) of Boyum (13). Whole blood (80 mL) from healthy volunteers (with approval of the Research Ethics Committee of the Ottawa Civic Hospital) was collected into ethylenediamine tetra-acetic acid (EDTA)containing Vacutainers® (Becton Dickenson). 20 mL of anticoagulated whole blood was layered onto a discontinuous gradient composed of Histopaque 1077 (8 mL) on top of
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Histopaque 1119 (20 mL) in each of four 50 mL polypropylene centrifuge tubes (Falcon 2098). After centrifugation at 700 3 g (1,900 rpm, Model No. RT6000B, Dupont Instruments), the PMN-containing layer was washed free of Histopaque 1119 by centrifuging and resuspending the pellet three times in RPMI-1640 tissue culture medium. Contaminating red blood cells were removed by hypotonic lysis in distilled water and the PMNs resuspended in DPBS and stored in a teflon tube on ice until used. PMNs were always used within 30 minutes from the completion of the isolation procedure. Analysis of PMNs suspensions on a routine basis was performed using an automated cell counter (System 9000, Serono Baker Diagnostics, Allentown, PA). The cell counter results were confirmed independently by manual differential staining performed at the Haematology Laboratory in the Department of Laboratory Medicine at the Ottawa Civic Hospital.
Preparation of Foreign Material Surfaces Two poly(urethane)s were synthesized from toluene diisocyanate (TDI), ethylene diamine (ED), and either poly(caprolactone) (PCL) or poly(tetramethylene oxide) (PTMO). They are referred to as “models” to distinguish them from commercially available poly(urethane)s which are used as a material component in catheters, blood pumps, vascular grafts, breast implants, and artificial skin. The synthesis and characterization of the two model poly(urethane)s have been described previously (14). In accordance with the nomenclature used by Santerre et al. (14), these materials will be referred to as TDI/PCL/ED and TDI/PTMO/ED. The poly(urethane)s were synthesized with a ratio of TDI to PCL to ED of 2.2:1:1.2 or TDI to PTMO to ED of 2:1:1. The solid materials were dissolved in DMAC to yield 2% (w/v) solutions. Any non-soluble material was then removed from the polymer solutions by passing them through a 0.45 mm Teflon® filter (Chromatographic Specialties, C616058). Round glass coverslips (15 mm diameter) (Fisher Scientific, 12-548) were coated with the polymer solution (50 mL) under sterile conditions in a laminar flow hood and dried overnight at 508C. After 24 hours, this was repeated, followed by further drying at 508C under vacuum (2100 kPa) for 48 hours. Prior to use, the polymer-coated slips were “hydrated” by incubating them overnight in sterile DPBS at 378C/5% CO2 and 100% humidity. The hydration step was performed to remove any remaining traces of solvent from the polymers which might affect the cells when added, and also to allow the surface of the poly(urethane)s to equilibrate in an aqueous environment prior to the addition of the cells.
Determination of Elastase Release from PMNs Adherent to Polymer Surfaces All experiments were carried out using standard 24-well tissue culture plates (Falcon, 3047). The bottom surface of each well was either tissue culture-treated PS or a model
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poly(urethane) coated on a glass slip. 1.0 3 106 PMNs, suspended in 1 mL DPBS, were added to the wells of tissue culture plates and incubated at 378C/5% CO2 and 100% humidity for 1 hour. With the plates on an angle, the supernatant and any non-adherent cells were then removed using a sterile pasteur pipet connected to a vacuum aspirator (Millipore, DOA-V184-AA) and discarded. To standardize the flow over the adherent cells when removing the supernatant, the plates were always tilted on the same angle (208).
Cell Activation with the Pharmacologic Agonist, PMA PMA, received as a lyophilized powder, was dissolved in DMSO at a concentration of 1 mg/mL and stored at 2148C. The day of the experiment, the PMA solution was thawed and diluted to 1 3 1027 M with DPBS. Either 750 mL of PMA/DPBS or 750 mL of DPBS alone were added back to the wells and the plates returned to the incubator. At various times (initially, and up to 10 hours for the control PS wells; initially and 3 hours for the two poly(urethane)-coated slips) plates were removed from the incubator, tilted on a 208 angle and the supernatant in each well transferred to a microcentrifuge tube (1.5 mL eppendorf, Brinkman) and stored on ice. The tubes were centrifuged at 18,000 3 g (14,000 rpm, Hermle Z229 Microcentrifuge) for 5 minutes at 48C and the supernatant transferred to a new tube and stored on ice. The elastase activity in each tube was assayed immediately, without being frozen, as described below. The number of cells adherent to the surface of the wells and the poly(urethane)-coated slips at the time of removal of the supernatant was assessed by assaying the cell extract for LDH as described below. Elastase activity in cell culture supernatants was determined by measuring the rate of cleavage of the small synthetic tetrapeptide substrate, N-methoxysuccinyl-Ala-AlaPro-Val-p-nitroanilide (ES1). Since elastase activity was monitored without the use of the enzyme’s “natural” substrate, elastin, any measured value of elastase activity will be referred to as elastase-like activity (ELA). The substrate solution was prepared by dissolving 10 mg of lyophilized ES1 in 2 mL DMSO and adding this solution dropwise to stirring buffer (0.01 M HEPES with 0.1 M sodium chloride, pH 7.4) and the final volume brought to 169 mL. 300 mL of the sample to be assayed were mixed with 500 mL of substrate at 378C and the increase in absorbance at 410 nm followed in a spectrophotometer (Model DU 640, Beckman Instruments). One unit of ELA was defined as that amount of enzyme which resulted in the release of 1 nmol of p-nitroaniline from ES1 per minute at 378C and at an initial substrate concentration of 62.5 mM. Units of ELA were calculated by entering the rate of product formation into the following equation: ∆A ------- min ELA UNIT ( nmol ⁄ min ) = --------------------------------------------–1 ε × ( V CUVETTE )
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DA/minute refers to the change in absorbance at 410 nm per minute. e is the molar absorptivity of p-nitroaniline (8.8 3 1026 nM) at 410 nm and VCUVETTE refers to the total reaction volume in the cuvette (nL).
Preparation of Extracts from Adherent PMNs The number of PMNs adherent to polymer surfaces was estimated by analyzing the LDH that could be extracted from the surface of the polymer after removal of the culture supernatant (initially, and up to 10 hours for PS; initially and after 3 hours for the poly(urethane)-coated slips) at 378C with and without PMA as described above. After removal of the supernatant in each well, ice-cold 1% Triton X-100 in DPBS (300 mL) was added to the tissue culture plate kept on ice, and the contents of each well mixed by repeatedly pipetting up and down. Each well was rinsed twice more with 350 mL of cold 1% Triton X-100/DPBS bringing the extraction volume for each well to 1.0 mL. The pooled extracts were then mixed by vortexing and centrifuged at 18,000 3 g at 48C. The supernatant was assayed for LDH activity as described below. LDH activity in cell extracts was assayed using a modification of the procedure provided by Sigma. This colorimetric assay is based on the LDH-catalyzed conversion of pyruvate to lactate in the presence of NADH (15). Pyruvate is quantified by reaction with 2,4-dinitrophenyl hydrazine (color reagent) which results in the formation of a hydrazone product that absorbs light in the range of 400–500 nm. 50 mL of cell extract were added to 100 mL of pre-warmed pyruvate substrate (0.75 mmol/L) and the mixture incubated at 378C for exactly 30 minutes. 100 mL of color reagent were added and the tube left to stand for 20 minutes at room temperature. Finally, 750 mL of 0.4 N sodium hydroxide were added and the tube left to stand for at least 5 minutes before reading at 450 nm. Samples were diluted to give absorbance values for [PYRUVATE]FINAL that fell within a standard curve generated using known concentrations of pyruvate. Units of LDH activity were calculated by entering these values into the following equation: LDH UNITS = [ PYRUVATE ] INITIAL – [ PYRUVATE ] FINAL -------------------------------------------------------------------------------------------------------- × 30 MINUTES DILUTIONFACTOR One unit of LDH activity was defined as that amount that would catalyze the conversion of one nmol of pyruvate per minute at 378C at an initial pyruvate concentration of 500 mM.
Cell Viability on Biomaterial Surfaces The objective of this study was to determine the amount of ELA released on different biomaterial surfaces which was due to a specific PMN response to foreign materials
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and not due to cell death. To normalize all data, the number of adherent intact cells was quantified. An experiment was performed in which different concentrations of PMNs were added and allowed to incubate for 1 hour on the biomaterial surface. After removal of the supernatant, the adherent cells were extracted with three freeze/thaw cycles in 1% Triton X-100 which was found to release the maximal LDH activity. The total LDH extracted from 1 3 106 PMNs was 41.79 6 1.01 units. A typical standard curve of PMN number versus extractable LDH activity for the numbers of cells ranging from 500,000 to 1,000,000 is shown in Figure 1 for the PS material. There was a positive, linear relationship between the number of PMNs added to the wells. Although not shown in Figure 1, (the study was performed separately for 10,000– 500,000 cells due to the non-linearity of the pyruvate standard curve) the lower detection limit for the amount of LDH activity that could be extracted from the culture surface was approximately 10,000 PMNs (r2 5 0.99638). These experiments confirmed that LDH could be extracted and measured over a wide range of concentrations and validated its use as a means of estimating adherent cell numbers. Tissue culture grade PS was used as the control surface. To measure the specific release of ELA in the supernatant, the time at which minimal cell death had commenced on the control material was determined. Cells were allowed to adhere to the PS surface as described above. The LDH content in the viable cells adherent to the surface of the wells was monitored from 1 to 10 hours after removal of the supernatant from cells treated with 1027 M PMA in DPBS or DPBS
alone. As can be seen in Figure 2, both PMA-stimulated and non-stimulated wells had a relatively constant number of viable, adherent cells for the first 3 hours. After this time, the amount of LDH activity that could be extracted from the surface of wells that contained PMA began to decrease rapidly, indicating that cells had begun to die. By 10 hours, no significant LDH activity could be detected in the wells with PMA. The extractable LDH activity from non-stimulated cells at 10 hours was not significantly different from that at time zero. When LDH activity was used to measure the cell retention and viability on the model poly(urethanes), there was no significant difference in the numbers adhering to PS, TDI/PCL/ED and TDI/PTMO/ED initially (Figure 2). However, at 3 hours following the addition of PMA, there was a significant difference between the LDH activity on the TDI/ PCL/ED and TDI/PTMO/ED relative to the control PS and from the time zero values (Figure 3). The microscopic analysis of adherent PMNs for time points corresponding to Figure 2 is shown in Figure 4. After the 1-hour adherence period, adherent PMNs are still round (panel A). PMNs that were subsequently stimulated with PMA spread over the surface of the well by 3 hours (panel B1) while at the same time point non-stimulated cells retained a more circular shape (panel B2). By 6 hours stimulated cells were less well defined and appeared to be spread quite flat over the surface of the well. This cellular material was present for the remainder of the experiment (panel D1) indicating that the decrease in LDH seen during this time was not due to lifting of intact cells from the surface of the
Figure 1. Estimation of adherent cell number with respect to extractable LDH activity. All points represent the average of quadruplicate measurements 6 SD. The solid line represents the linear regression analysis of the data points. The dotted line represents the 95% confidence interval for the fitted equation.
Figure 2. Kinetic determination of LDH activity related to PMN adherent to poly(styrene). 1 3 106 PMN were added to the wells of poly(styrene) tissue culture plates and incubated for one hour. Open symbols represent data from cells that were stimulated with 1027 M PMA. Closed symbols represent data from cells that were not stimulated.
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well. Non-stimulated cells did not begin to spread noticeably until 9 hours (panel D2) which corresponded to the insignificant decrease in the amount of LDH activity associated with the adherent cells.
Biomaterial Induced Release of ELA The total extractable ELA was determined with the synthetic substrate ES1 in order to relate this to the maximal amount released during activation. The most ELA which could be extracted from 1 3 106 cells was 21.5 6 1.01 units. This value could not be increased by sonication or more than three freeze/thaw cycles. Differences in ELA release from PMNs adherent to the PS and model poly(urethane) surfaces were investigated and the data are presented in Figure 5. Three hours was chosen as the relevant time point to assess PMNs activation using ELA release since there were no significant changes in LDH activity in the PS adherent PMNs and the other surfaces maintained .75% cell viability based on LDH analysis at this time (Figure 3). As well, stimulation of cell response by PMA did not show a significant effect on cell viability prior to 3 hours (see Figure 2). At 3 hours there were significant differences in the ELA activity measured in the PMNs adherent to the differ-
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ent surfaces with and without stimulation with PMA (Figure 5). The amount of ELA in Figure 5 was adjusted for the amount of cell death. The amount of ELA seen in the supernatant of wells containing TDI/PCL/ED-coated slips was higher than those containing TDI/PTMO/ED either in the presence or absence of PMA (Figure 5). Control (PS) wells showed ELA levels significantly higher than both model poly(urethane)s. It should be noted that this relative difference was observed in all experiments, however, the specific amounts of ELA released varied significantly from one donor to another. Since the heterogeneity and biological variability of PMNs from different normal individuals as well as patients, has been demonstrated with many other cellular components (16), this experiment was performed in triplicate using PMNs isolated from the blood of the same donor and represents the averaged data in Figure 5. The maximum amount of ELA released by PMA from PMNs adherent to the biomaterial surfaces was approximately 10-fold less than the maximal activity determined by repeated freeze/thawing described above (i.e., 2.3 and 21.5 units, respectively). However, stimulation with PMA caused the release of 5–10-fold more ELA from the PMNs on the poly(urethane) surfaces, than that released without PMA stimulation, i.e., that due to stimulation by the material surfaces themselves (see Figure 5).
Exposure of ELA in Solution to Different Surfaces
Figure 3. Determination of LDH activity related to viability of adherent PMN. 1 3 106 PMN were added to tissue culture wells and incubated for 1 hour. The wells contained slips coated with TDI/ PCL/ED, TDI/PTMO/ED or no slip (poly(styrene)). The supernatant was removed and Triton X-100 added immediately (open bars) or the wells were refilled with 1027 M PMA for 3 hours followed by Triton X-100 addition (hatched bars). LDH activity in the adherent PMN was then determined. Asterisk indicates the LDH activity is significantly different from time zero, and plus sign indicates significantly different from poly(styrene).
A control experiment was run to determine the effect of the different materials on available ELA in solution over time. This was done to determine if the differences detected for ELA in the supernatant of PMNs (see Figure 5) were due to a difference in the adsorption of enzyme on the various materials rather than a difference in the amount of enzyme released from the cells. Cell extracts were prepared from freshly isolated PMNs without the use of Triton X-100, since in another study Triton X-100 caused chemical degradation of the polymer, TDI/PCL/ED (data not shown). After the third cycle of freeze/thawing without Triton X-100 there was still enough ELA extracted (approximately 2 units/mL) to add to the wells to follow the decrease in ELA. These cell extracts were incubated in tissue culture wells containing polymer surfaces and the ELA in solution was measured over a period of 5 hours. On all three materials, the amount of ELA in solution showed a rapid decrease in the first hour which gradually leveled off (Figure 6). Significant amounts of ELA could still be detected at 5 hours. Cells incubated on the PS well surface itself showed the highest level of ELA remaining in solution at all time points, after 1 minute. The next highest levels were seen in wells containing TDI/ PTMO/ED and finally TDI/PCL/ED. The differences seen between ELA in the presence of the different materials were significant at several time points, but too small to account for the differences in the amount of ELA in the supernatant of PMNs cultured on the different materials (see Figure 5).
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Figure 4. Microscopic analysis of PMN adherent on poly(styrene). Conditions for this experiment were the same as those described in Figure 2. Panel A shows a representative example of cells prior to the addition of PMA/DPBS or pure DPBS. Subsequent panels B1–D2 show stimulated and non-stimulated PMN at 3 (B1,B2), 6 (C1,C2) and 9 hours (D1,D2), respectively. (Original magnification 3200. Bar 5 200 mm.)
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Furthermore, the order of decreasing ELA in solution in the presence of the three surfaces did not correspond to the order of increasing ELA released into the cell supernatant. From Figure 5 the order of ELA measured in solutions following release from PMNs was PS . TDI/PCL/ED . TDI/ PTMO/ED. The order identified from Figure 6 was PS . TDI/PTMO/ED . TDI/PCL/ED.
Discussion By monitoring enzymes found in the cytoplasm (LDH) and inside granules (ELA), the effect of material surfaces on the release of enzymes into the supernatant of activated PMNs was investigated. The number of adherent PMNs with intact cytoplasmic membranes was estimated by determining the LDH content inside the cells, an approach previously used by Cerasoli et al. (17). In these studies as in several others (18–20) tissue culture grade PS was used as the standard surface. However, the cell type and the markers chosen to measure in a particular study will determine whether the PS is a positive or negative control (21). Although PS is specifically treated to enhance cell attachment (i.e., it is usually made hydrophilic), the mechanism of the difference in cell adhesion between treated and untreated PS is not well understood (22). By measuring a marker of PMN activation (ELA release) and cell viability (LDH in remaining adherent PMNs) on standard PS, the importance of the findings on model material surfaces can be evaluated relative to other studies using in vitro cell systems. Three hours was chosen as the time to measure ELA released from the PMNs on all the surfaces, since at 3 hours
Figure 5. Comparison of cumulative ELA release from PMN adherent to model poly(urethane)s after 3 hours. 1 3 106 PMN were added to tissue culture wells and incubated for 1 hour. The wells contained slips coated with TDI/PCL/ED, TDI/PTMO/ED or no slip (poly(styrene)). The supernatant was removed and the wells refilled with 1027 M PMA (open bars) or DPBS (hatched bars).
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no cell death had occurred on the control PS and was only minimal on the poly(urethane)s. The amount of LDH activity in cell extracts showed that the same number of cells adhered to the different polymer surfaces initially. However, at 3 hours after PMA incubation, LDH had decreased significantly on both the TDI/PCL/ED and TDI/PTMO/ED. Therefore, the ELA was normalized in the initial value for live adherent cells for each material (Figure 1). With this correction, these values represented the amount of ELA released into the supernatant during this time as a result of specific lysosomal enzyme release due to PMA and not due to cell death (Figure 5). At later times more ELA release was due to cell death, even on the control PS surfaces (Figure 2). The most interesting and perhaps significant finding of this study was that the differences in the released ELA measured from the PMNs adherent to three different surfaces, were still significant after 3 hours of PMN incubation with PMA agonist (Figure 5). The most ELA was measured in the cell supernatants of stimulated and non-stimulated cells in the PS wells, followed by TDI/PCL/ED and TDI/PTMO/ED (Figure 5). This highlights the importance of the material surface itself with respect to the stimulation of the ELA release. The response of PMNs to activation with a variety of physiologic (N-formylmethionylleucylphenylalanine (FMLP), leukotriene B4) or pharmacologic agonists (phorbol myristate acetate (PMA), calcium ionophore (A23187)) has been measured using many different markers. These include intracellular calcium mobilization, expression of adhesion molecules, the release of arachidonic acid as well as azuro-
Figure 6. Effect of biomaterial surfaces on ELA in solution. Polymer slips were prepared and hydrated. The extracts from a 1 3 106 PMN/mL suspension was prepared and added to wells containing the different test surfaces. Triangles represent the data from poly(styrene) wells. Closed circles represent the data from wells containing TDI/PTMO/ED-coated slips. Open circles represent the data from wells containing TDI/PCL/ED-coated slips.
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philic granule contents which include the respiratory burst products and the lysosomal hydrolases (23–25). It is known that PMA bypasses membrane receptors and acts directly on protein kinase C (26). On the other hand, the adherence activation as measured by the respiratory burst, has been shown to involve adherence receptors such as leukocyte integrins found in the cytoplasmic membrane of the cell (27). Since biomaterials have been shown to cause the release of respiratory burst products even in the absence of secondary stimulation (9,10) it is possible that the mechanism of ELA release, associated with the materials, follows a similar pathway to that of adherence activation. One good example of this is described in a study of the effects of pertussis and cholera toxins on the production of reactive oxygen metabolites in PMNs during cell activation by PMA or quartz particles. The results suggested two mechanisms for the cell activation. The release of reactive oxygen metabolites by PMA activation was not affected by the toxins, whereas it was significantly less by the cell activation with the quartz particles (28). The latter case is analogous to the effect of each material on the PMA induced ELA release. The ELA release caused by PMA was reduced on TDI/PTMO/ED relative to PS and TDI/PCL/ED. A model system has been developed which will make it possible to probe the mechanisms of neutrophil-mediated events at the interface between a cardiovascular medical device and the tissues/fluids surrounding it. Combining neutrophil activation and product release, specifically digestive and tissue remodelling enzymes (biocompatibility) with poly(urethane) degradation (biostability) in a single experimental system will provide a more complete approach to understanding the complex relationship that exists between a biomaterial as a foreign body implant and the human organism.
The authors thank Erin Meek for her technical assistance. These studies were funded in part by a grant from the National Health Research Development Program (NHRDP), Health, Canada. Daniel Duguay prepared the statistics application used in these analyses.
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