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Mass spectrometry is a powerful proteomic tool enabling researchers to survey the global proteome of a cell. This technique has only recently been employed to investigate cell-material interactions. We had previously identified material scarcity and limited adherent cells as challenges facing mass spectrometric analysis of cell-material interactions. U937 adherent to tissue culture poly(styrene) was used as a model system for identifying proteins expressed by adherent monocytes and analyzed by HPLC coupled offline to MALDI-ToF/ToF (LC-MALDI). We identified 645 proteins from two cation fractions of crude U937 monocyte cell lysate. Forty three proteins of interest from the 645 were chosen based on literature searches for relevance to monocyte-material inflammation and wound healing. Proteins such as 40S ribosomal protein S19 and tyrosyl tRNA synthetase highlight the ability of LC-MALDI to identify proteins relevant to monocyte-material interactions that are currently unexplored. We used PEG-based semi-interpenetrating polymer networks and PEG-only hydrogels to investigate surface dependent effects on the Src family kinase Hck and plasminogen activator inhibitor-2 (PAI-2) using the pyrazolo pyrimidine small molecule inhibitor PP2 and exogenous urokinase plasminogen activator addition, respectively. Hck is well researched in cell adhesion while PAI-2 is virtually unknown in cell-material interactions. U937 on TCPS and PEG-only hydrogels secreted similar levels of inflammatory cytokines and gelatinase MMP-9. MCP-1 secretion from monocytes on PEG-only hydrogels was Hck independent in contrast to Hck dependent MCP-1secretion in U937 on TCPS. Overall, U937 adherent to sIPNs secrete low levels of soluble gelatinase MMP -9, IL-1β, TNF-α, IL-6, and MCP-1 independent of Hck and PAI-2. This work demonstrates significant changes in surface dependent expression of proteins from monocytes adherent to PEG-based materials compared to TCPS.
Mass spectrometry is a powerful proteomic tool enabling researchers to survey the global proteome of a cell. Cell biologists have used this technique for several decades but mass spectrometry has only recently been applied to studying cell-material interactions [1, 2]. Mass spectrometry has the potential to identify proteins expressed by adherent cells such as monocytes that are critical to the monocyte-mediated inflammatory response to biomaterials. Previous analysis of phosphotyrosine-enriched monocyte lysate identified material scarcity and limited adherent cells as unique challenges to studying monocyte-material interactions through mass spectrometry [3, 4]. These issues were addressed in the present study using tissue culture poly(styrene) (TCPS) as a model system to identify proteins expressed by adherent U937 monocytes. TCPS provides a readily available platform to identify and validate proteins expressed by adherent monocytes. TCPS also serves as a reference material with published literature. However, protein expression by monocytes adherent to TCPS may vary significantly from that of monocytes on hydrogels or other tissue engineering constructs. Therefore the proteins identified by mass spectrometry from monocytes adherent to TCPS should serve as a starting point for elucidating the mechanism underlying monocyte-material interactions.
Our lab has developed a semi-interpenetrating polymer network (sIPN) system comprised of peptide-modified gelatin entrapped within photopolymerized poly(ethylene glycol) (PEG) diacrylate [5,6] as a platform to study monocyte-material interactions. The gelatin component of sIPNs can be modified with various extracellular matrix (ECM) protein-derived ligands such as arginine-glycine-aspartic acid (RGD) that has been shown to increase cell adhesion to RGD-grafted biomaterials [7, 8]. The PEG component of the sIPN minimizes protein adsorption [9,10] from serum-containing media thereby enabling isolation of the ligand-specific effects upon monocyte response. sIPNs have previously been characterized [5,6] and investigated for both drug release [5,11] and cell-material interactions including keratinocytes, fibroblasts, and monocytes [12–14]. In addition to minimizing protein adsorption, PEG is known to result in less cell adhesion from a variety of cell types including macrophages [15,16]. We believe comparing protein expression in monocytes adherent to RGD-modified sIPNs to those adherent to PEG-only hydrogels or TCPS will offer unique insight into the mechanism underlying monocyte-material interactions.
Briefly, we analyzed cell lysate from U937 monocytes adherent to TCPS by HPLC coupled offline to MALDI-ToF/ToF (LC-MALDI). We identified 645 proteins from two cation fractions of crude U937 monocyte cell lysate and selected 43 of these proteins as relevant to monocyte-mediated host response to materials. We investigated the effect of hematopoietic cell kinase (Hck), a Src family kinase, and plasminogen activator inhibitor type 2 (PAI-2) on inflammatory cytokine secretion, matrix metalloproteinase (MMP) expression, and the plasminogen system in monocytes adherent to TCPS, RGD-modified sIPNs, or PEG-only hydrogels. Interestingly, monoctye chemotactic protein-1 (MCP-1) secretion showed Src-dependence in monocytes on TCPS but not on PEG-only hydrogels. Secretion of the gelatinase matrix metalloproteinase 9 (MMP-9) from monocytes on PEG-only hydrogels was similar to that seen from monocytes on TCPS. Monocytes on sIPNs secreted very low levels of MMP-9, PAI-2, and MCP-1. These results show significant surface dependent secretion of proteins from monocytes adherent to TCPS and PEG-based matrices. Therefore the use of TCPS as a reference material for biomaterials research should be undertaken cautiously.
Human monocytic cell line CRL-1593.2/U-937 (American Type Culture Collection, ATCC) was used . Reagents were obtained from the following sources: IMUBIND® PAI-2 enzyme-linked immunosorbent assay (ELISA) kit (# 823); IMUBIND® uPA ELISA kit (#894) (American Diagnostica); dimethylsulfoxide (DMSO, ATCC); fetal bovine serum (FBS) (Premium Select, S11550, Atlanta Biologicals); MMP-2 ELISA kit (QIA63), PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), PP3 (4-amino-7-phenylpyrazol[3,4-d]pyrimidine) (Calbiochem); Clonetics reagent pack (trypsin/EDTA, trypsin neutralizing solution, HEPES-buffered saline solution) (Cambrex); HPLC grade acetonitrile (ACN), methanol, 1X Dulbecco’s phosphate buffered saline solution (with Ca2+ and Mg2+, DPBS), 1X phosphate buffered saline solution (PBS), Rosswell Park Memorial Institute (RPMI) 1640 media (CellGro); dimethylformamide (DMF), tetrahydrofuran (THF), 1X phosphate buffered saline solution (PBS) (Fisher); human urine urokinase (uPA, CC4000, 40 % high molecular weight urokinase (54 kDa), 50 % low molecular weight urokinase (33 kDa), 10 % transitory forms, over 100,000 IU/mg), ZipTip® (C18, P10 size), human cytokine LINCOplex kit 4-plex containing IL-1β, IL-6, TNF-α, & MCP-1 (Millipore); bicinchonic acid protein quantification kit and standards, M-PER® mammalian protein extraction reagent, Halt™ Protease Inhibitor Cocktail (Pierce); PolyLC Polysulfoethyl A strong cation exchange column (4.6 mm I.D. × 100 mm, 3 μm resin, 300 pores, PolyLC); trypsin (modified sequence grade) (Promega); RayBio® human MMP-9 ELISA kit (RayBiotech); Quantikine® human uPAR ELISA kit (R&D Systems); N,N-diisopropylethylamine, dithiothreitol (DTT), 4 N HCl dissolved in dioxanes, gelatin from porcine skin (Type A, ~300 bloom), N-hydroxysuccinimide, oxalyl chloride, phorbol 12-myristate 13-acetate (PMA), phosphatase inhibitor cocktail 2, poly(ethylene glycol) 2000, poly(ethylene glycol) diacrylate (Mn 575), pyridine, tert-butyl bromoacetate, trifluoroacetic acid (TFA), trinitrobenzenesulfonic acid (Sigma); Vydac 218TP C18 Reversed-phase high performance liquid chromatography column (4.6 mm I.D. × 250 mm, 5 μm resin, 3 pores, Vydac); lysyl endopeptidase (Lys-C, from Achromobacter lyticus M-497-1, mass spectrometry grade, Wako Chemicals).
U937 were cultured on 75 cm2 TCPS flasks for 24 h with RPMI-1640 with L-Glutamine with 50 ng/ml PMA and 5 % FBS at 37 °C/5 % CO2. Non-adherent and loosely adherent cells were washed off with 1X DPBS. Adherent U937 were harvested with Trypsin/EDTA for 5 min at 37 °C. Trypsin neutralizing solution was added and any remaining adherent U937 were washed free with HBSS prior to centrifugation at 1000 RCF for 10 min. Cells were lysed with 0.5 ml M-PER detergent containing 1X HALT Protease inhibitor and 1X phosphatase inhibitor cocktail 2. Cell lysis was completed with 5 pulses of a wand sonicator at 50 % power prior to clarification at 14,000 RCF for 10 min. Total protein in the cell lysates was quantified using the bicinchonic acid assay. 500 μg of protein was precipitated overnight at −20 °C in 80 % acetone to remove lysis detergent, centrifuged at 5000 RCF for 1 min, and washed three times with acetone. The acetone was allowed to dry prior to resuspension in 50 μl 10 mM DTT/8 M urea. Proteins were reduced at 56 °C for 10 min before urea was diluted with 50 μl Milli-Q. Proteins were digested with 20 μg Lys-C re-suspended in 100 μl 30 mM Tris-HCl pH 8.45 and incubated overnight at 37 °C. Final buffer concentration for digestion was 2 M Urea/2.5 mM DTT/15 mM Tris-HCl with 1:25 w/w Lys-C:protein. Lysate was acidified after digestion with TFA to pH ~ 6.
Digested lysate from U937 on TCPS was separated by strong cation exchange (SCX) on a PolyLC polysulfoethyl A column (4.6 mm I.D. by 100 mm, 3 μm resin, 300 pores). Peptides were bound for 5 min at 1 ml/min in 100 % A (3 mM phosphate (PO43−)/25 % acetonitrile pH 3.5) and were eluted over a 75 min gradient from 100 % A to 35 % A/65 % B (3 mM PO43−/25 % acetonitrile/750 mM KCl pH 3.5). Fractions were collected every minute from 0 to 85 min. Fractions containing peptide were frozen at −80 °C, dried on a vacuum centrifuge to remove acetonitrile, acidified with 0.1 % TFA to pH < 3, and de-salted via C18 pipette tip (Omix). Samples were dried on the vacuum centrifuge and stored at −80 °C until analyzed by LC-MALDI.
Two de-salted cation exchange fractions were re-suspended in ~ 10 μl 0.1 % TFA. Six of the 10 μl was loaded on to a trap column at 150 nl/min and then eluted on to an analytical scale HPLC column (75 μm I.D. × 150 mm, silica resin) and resolved using a 150 min gradient from 98 % A (0.1 % TFA)/2 % B (0.1 % TFA/acetonitrile) to 10 % A/90 % B. Eluent from one to 90 min (up to 40% B) was mixed directly with 50 ng/ml 6 mg/ml α-cyano-4-hydroxycinnamic acid in 50 % acetonitrile and spotted to the stainless steel MALDI target plate. Samples were allowed to dry and analyzed on an Applied Biosystems 4800 MALDI-ToF/ToF mass spectrometer in positive ion mode. The top 20 intensity “y” ions from each spot were selected for MS/MS to obtain sequence data. Mass spectrometer results were then searched against the National Centers for Biotechnology Information database using Mascot (Matrix Science) . Search parameters were as follows: taxonomy was restricted to Homo sapiens; enzyme was Lys-C with 1 missed cleavage site; variable modifications were deamidated (NQ) and oxidation (M); peptide tolerance ± 1.0 Da; MS/MS tolerance ± 0.1 Da; peptide charge 1+ monoisotopic; instrument MALDI-ToF-ToF.
Employing LC-MALDI, we significantly identified 645 proteins from 895 peptides with Mascot ion scores > 26, the threshold for significant database match. The Mascot ion score equals -10 * log10 (P) where P is the probability the observed event is random. Therefore a high Mascot score correlates to a low probability of a random event; for example, peptides detected by LC-MALDI with an ion score of 50 have a 10−5 probability of being a random event (i.e. 99.999 % confidence). 17 peptides had ion scores > 100 and 427 of the 895 peptides reported ion scores > 50. The top 400 peptide hits (ion scores 183.2 to 51.97) were literature searched to determine function. Forty three proteins from the 645 identified proteins were investigated further (Table 1) due to their relevance to the monocyte-mediated host inflammatory response in the presence of TCPS.
Previously established procedures were followed . Briefly, peptides were conjugated via N-hydroxysuccinimide activated esters to one end of difunctionalized PEG 2000. The remaining activated ester was then reacted with lysyl side chains in the gelatin backbone. Byproducts from peptide-PEG conjugation and gelatin modification were removed via ultrafiltration with a 30 kDa nominal molecular weight regenerated cellulose cutoff filter (Millipore YM30 #13742). Synthesis products were analyzed by 1HNMR after each reaction. The extent of gelatin modification was estimated using the trinitrobenzenesulfonic acid (TNBS) colorimetric assay that tests for presence of primary amines . Unmodified gelatin was included as a control for the TNBS assay. RGD modification was 71.4 % ± 1.5 %. Ten weight percent gelatin sIPNs were prepared by dissolving 0.1 g gelatin or RGD-modified gelatin in 1 ml Milli-Q water. Gelatin was dissolved at 60 °C immediately prior to photopolymerization. PEG diacrylate Mn 575 (0.125 g) was weighed into a separate amber vial. MEHQ polymerization inhibitor (400–600 ppm) had been previously removed from the PEG diacrylate by alumina filtration. MEHQ has been observed to impact the response of cells to inflammatory stimuli such as IL-1 . 2,2-Dimethoxy-2-phenylacetophenone (DMPA, 0.02 g) was mixed with 0.5 g PEG diacrylate and heated briefly to dissolve the DMPA crystals. PEG diacrylate was added to the dissolved gelatin, heated ~ 1 min at 60 °C, mixed with 20 μl DMPA, and photopolymerized by light emitting diode at 365 nm, 50 % power, 10 cm height for 3 min (Clearstone Tech CF1000). PEG-only hydrogels (13 wt %) were used as a control material and were synthesized as above for the sIPNs except no gelatin was dissolved in the water. Films were placed in 48-well TCPS plates, sterilized three times with 1 ml 70 % ethanol for 10 min each, washed twice with 0.75 ml 1X PBS for 10 min each, and incubated overnight at room temperature in 0.5 ml antibiotic/antimycotic solution (penicillin/streptomycin/amphotericin B). sIPNs were equilibrated two times in 0.75 ml 1X PBS for 1 h at 37 °C prior to seeding cells.
U937 were seeded at 5.1 × 105 cells/cm2 on TCPS, unmodified sIPNs, RGD-modified sIPNs, and PEG-only hydrogels with 5 % FBS and 50 ng/ml PMA. U937 were pre-treated for 10 min with 10 μM Src family kinase inhibitor PP2 (Hck IC50 = 5 nM; Lck IC50 = 4 nM; Fyn IC50 = 5 nM) [22,23], the inactive analog PP3 , or an equal volume of DMSO for a vehicle control. Separately U937 were treated with 10 IU (100 ng/ml) uPA immediately prior to seeding on films or TCPS. 25 IU uPA (250 ng/ml) was included in two independent replicates for U937 on TCPS or PEG-only hydrogels. 25 IU uPA was not investigated in U937 on sIPNs based on the first replicate showing low MMP and inflammatory cytokine secretion from U937 on the sIPNs. Adherent cells were harvested as previously described. Supernatant was collected from each well, centrifuged at 14,000 RCF for 3 min, and frozen at −80 °C in l00 μl aliquots.
ELISA kits for MMP-2, MMP-9, PAI-2, uPA, & uPAR were performed according to manufacturer’s specifications. ELISAs were performed three times independently (n = 3 of 3 independent replicates). Bio-Plex was performed per manufacturer’s specifications (n = 3 of 3 independent replicates). Briefly, 25 μl sample or standard was mixed with 25 μl assay buffer and incubated with 25 μl antibody beads (Il-1β, IL-6, TNF-α, MCP-1) for 1 h; beads were washed twice and incubated with 25 μl detection antibody for 30 min at room temperature. The beads were then incubated with 25 μl streptavidin-phycoerythrin for 30 min at room temperature, washed two times with wash buffer prior to resuspension in 100 μl sheath fluid for 5 min, and analyzed on a Bio-Plex 200 system. Concentration data was calculated using a 5-parameter logistic curve-fit. ELISA and Bio-Plex data is expressed as mean ± standard deviation of the mean. Data were analyzed via two-way analysis of variance and Tukey post testing (SigmaStat v2.03). The p values were considered to be significant when p < 0.05.
Two cation fractions were pooled, de-salted, and analyzed by LC-MALDI (50 μm ID × 150 mm, 3 μm C18 resin, 200 Å pores). We used a literature search of the top 400 peptide hits to refine the 645 proteins identified by LC-MALDI down to 43 proteins of interest relevant to monocyte-mediated host inflammatory response to materials. Table 2 shows a breakdown of protein function for the 43 proteins of interest identified by LC-MALDI. Cytoskeletal related proteins accounted for the largest group followed by metabolic, cytokine or apoptosis, & signaling proteins. The number of signaling proteins identified demonstrates the sensitivity of LC-MALDI since these proteins are sparingly expressed compared to cytoskeletal proteins. The role of all 43 proteins cannot be directly probed via inhibition, knock down, or antibody blocking without causing undesirable side effects such as cytotoxicity or blocking adhesion. Therefore ten feasible targets that may play a role in the host inflammatory response are briefly summarized here. Two of these ten proteins, 40S ribosomal protein S19 and tyrosyl tRNA synthetase, comprise a growing field of “moonlighting” proteins [25,26] and illustrate the ability of mass spectrometry to identify novel proteins not currently investigated in monocyte-material interactions. 40S ribosomal protein S19 dimerizes upon release from apoptotic cells and triggers monocyte/macrophage chemotaxis while inhibiting neutrophil migration [27,28]. Tyrosyl tRNA synthetase is also released from apoptotic cells and is cleaved by elastases into two chemoattractant fragments. The amino terminal fragment stimulates neutrophil migration while the carboxy terminal fragment triggers monocyte recruitment [29,30]. Splicing factor, arginine/serine-rich 3 was also identified from adherent U937. This splicing factor plays a role in splicing variants of fibronectin-1 and has been linked to wound healing without scarring . Aldose reductase is a lipid metabolic protein that increases resistance to oxidative stress and decreases cytokine and free radical production [32,33]. Nucleolysin TIA-1 isoform p40 can prevent translation of TNF-α and cyclooxygenase-2 messenger RNA and can also form prions that localize to stress granules during oxidative stress . S-100P Ca2+ binding protein increases proliferation, decreases attachment-dependent cell viability , and alters actin polymerization via cofilin phosphorylation . Galectin-1 is an endogenous lectin present at inflammation sites that regulates Fc gamma RI-dependent phagocytosis and inflammatory cytokine secretion [37,38]. Alpha-enolase is a glycolytic enzyme involved in plasminogen regulation in leukocytes and also mediates extracellular proteolytic executor of necrotic cell death [39,40]. We chose the Src family kinase Hck and plasminogen activator inhibitor-2 (PAI-2), for additional study using a small molecule inhibitor and exogenous protein addition, respectively. It is important to note that the role of materials of varying chemical composition on regulating the expression of these proteins is largely unknown. The above summary is mainly based on studies performed on cells in the presence of TCPS.
Src family kinases are well-studied proteins located downstream of integrin receptors and upstream of cytoskeletal proteins such as focal adhesion kinase Pyk2 and phosphatidyl inositol-3 kinase. Regulation of Src family kinases may be a critical divergence point for signaling from integrin-dependent adhesion to ligand-modified materials or other proteins adsorbed to the material surface. In addition, lipopolysaccharide (LPS) treatment of bone marrow derived monocytes with constitutively active Hck increased secretion of MMP-2 and -9 indicating that Hck plays a role in regulation of MMP secretion [41,42]. We have previously observed MMP-2 and -9 secretion from primary blood derived monocytes in response to RGD-sIPNs alone and in co-culture with fibroblasts [13,43]. These gelatinases are of interest since sIPNs are comprised partly of gelatin and may therefore play a role in monocyte-sIPN interactions. Hck and Src family kinases are also known to regulate signaling pathways leading to inducible nitric oxide synthase expression and inflammatory cytokine expression such as IL-6, TNF-α, and MCP-1 [24,25]. Monocytes express multiple forms of Src family kinases including Hck, Fgr, Lyn, Fyn, and Src. This familial redundancy prevents effective investigation via genetic knockouts or siRNA knockdowns as the remaining family members compensate with varying kinetics [41, 44–46]. Inhibition of all Src family kinase members via pyrazolo pyrimidine compounds such as PP2 is an effective investigation approach.
PAI-2 is less well-studied and virtually unknown in cell-material interactions. PAI-2 inhibits both urokinase and tissue type plasminogen activators but inhibits the urokinase type (uPA) more effectively. PAI-2 is a member of the ovalbumin serine protease superfamily and helps regulate the extracellular proteolytic environment. Phorbol myristate acetate is known to increase PAI-2 expression [47,48] but is also necessary to stimulate U937 adhesion. In addition, expression of PAI-2 is known to inhibit migration of inflammatory cells such as monocytes . uPA can also cleave its receptor, urokinase plasminogen activator receptor (uPAR), releasing a chemotactic fragment that binds soluble uPA and prevents cellular recognition of uPA and covalent binding of uPA to vitronectin, which stabilizes its extracellular proteolytic activity. Matrix metalloproteinases can also cleave uPAR  resulting in migration via G protein-coupled receptor . uPA/plasmin can also activate pro-MMP-2 and pro-MMP-9, which may then regulate the plasminogen activation system via inactivation of PAI-2 and uPA or even plasminogen. However, most research investigating the interaction of MMPs and uPA/uPAR has focused on tissue invasion of cancer cells and not on the role of PAI-2 and the plasminogen system in cell-material interactions. In addition, inflammatory cytokines such as IL-1β, IL-6, and TNF-α have been found to influence expression levels of PAI-2 [52,53].
Src family kinase inhibitor PP2 and PP3 showed no effect on MMP-2 secretion on any of the four surfaces tested (S Fig. S1). However, MMP-2 from U937 on unmodified sIPNs and RGD-modified sIPNs were lower than both PMA− (i.e. non-adherent U937) and LPS-treated controls. MMP-9 secretion from U937 on TCPS and PEG-only hydrogels was significantly higher than that for U937 on sIPNs (Fig 1) . MMP-9 from LPS-treated and U937 on TCPS were above detection limits for the assay. Surface effects appeared to dominate Src family kinase inhibition by PP2 as TCPS elicited the highest secretion of MMP-9 followed by PEG, which is consistent with previous observations . The low MMP-2 &-9 gelatinase response from U937 on sIPNs was unexpected since sIPNs are comprised partly of gelatin. Primary monocytes adherent to RGD-modified sIPNs have been observed to secrete elevated levels of MMP-9 at 96 h but not from monocytes on unmodified sIPNs possibly indicating a cell source-specific difference . Matrix-associated proteolytic activity or specific MMP inhibitors like TIMPs may also account for the low MMP-2 and MMP-9 levels observed from U937 on sIPNs. Overall, U937 adherent to sIPNs showed no Hck dependent secretion of MMP-2 (S Fig S1), MMP-9 (Fig 1), or uPA/PAI-2/uPAR (S Figs S2, S3, & S4, respectively). The MMP/plasminogen systems are closely linked sharing feedback loops therefore these systems appear to be Src family kinase independent in U937 on TCPS, sIPNs, and PEG-only hydrogels.
IL-1β levels were low for all treatments and surfaces including LPS (S Table S1). PMA is known to stimulate inflammatory cytokine secretion , but all conditions were treated with PMA except the non-adherent control (PMA−). The low levels of cytokines may be due to sampling only at 24 h or the anti-inflammatory effect of DMSO, which was used to solubilize PP2 and PP3, on adherent U937 as reflected by the differences in MCP-1 and TNF-α concentrations from this study (Figs 2, ,3,3, ,8,8, & 9) [56,57]. Unmodified sIPNs elicited slightly higher levels of TNF-α secretion from adherent U937 than TCPS or RGD-modified sIPNs (p < 0.001 and = 0.004, respectively, Fig 2). However, only U937 treated with PP3 on unmodified sIPNs was significantly different when compared against other surfaces within treatment group (p = 0.007). TNF-α and IL-1β are secreted via similar mechanisms and triggered by similar pathways [58,59]. Hence relatively low levels of TNF-α correlate well with low levels of IL-1β. Nearly all culture conditions (i.e. surface and PP2/PP3 treatment) were significantly lower than LPS treated U937 on TCPS but were not different than non-adherent PMA− U937 (Fig 2). Unmodified and RGD-modified sIPNs elicited less MCP-1 secretion from adherent U937 at 24 h than TCPS (p = 0.013 & 0.014, respectively) and PEG-only hydrogels (p < 0.001, Fig 3). PP2 treatment of U937 adherent to TCPS demonstrated the Src-dependent nature of MCP-1 release (p = 0.053). Since the sIPNs elicited a very minor MCP-1 response from U937, the effect of PP2 treatment, if any, was not apparent on these surfaces. The Src-dependence of MCP-1 was not apparent in U937 on PEG hydrogels, which may indicate PEG hydrogels stimulate MCP-1 secretion through a different pathway perhaps via C3-mediated mechanism. IL-6 secretion from U937 on TCPS, sIPNs, and PEG is comparable to non-adherent PMA− U937. IL-6 secretion from U937 on TCPS, unmodified sIPNs, and RGD-modified sIPNs was below detection threshold for two of three independent replicates (S Table S2). However, the slight increase in IL-6 on PEG hydrogels is minor compared to LPS treated U937 (485 ± 52.4 pg/ml). PP2’s parent compound, PP1, decreased IL-6 secretion from Kupfer cells in response to hypoxic shock . However, the low levels of IL-6 observed from U937 adherent to TCPS, sIPNs, and PEG hydrogels indicate that IL-6 may not be a major effector resulting from PMA stimulation or adhesion to unmodified sIPNs, RGD-modified sIPNs, or PEG-only hydrogels.
Overall, sIPNs do not appear to elicit an inflammatory cytokine response from adherent U937 at 24 h. This lack of inflammatory cytokine secretion is independent of RGD peptide modification. With the exception of decreased MCP-1 secretion from U937 on TCPS in response to Src family kinase inhibition, little difference in cytokine secretion was observed from U937 on TCPS or PEG-only hydrogels. TCPS is a widely used reference surface in biological cell-based research yet the present work shows the connection between TCPS and material-modulated cell response should be made cautiously.
U937 on TCPS secrete more MMP-2 independent of exogenous uPA than on unmodified sIPNs, RGD-modified sIPNs, or PEG-only hydrogels (S Fig S5). No significant treatment effect was observed on any surface. The levels of MMP-2 secreted are similar to those found in the supernatant from the Hck study indicating the DMSO used to solubilize the Src family kinase inhibitor does not have any effect upon MMP-2 secretion. In addition, MMP-2 levels from U937 are consistent with that from primary monocytes on unmodified and RGD-modified sIPNs . U937 on unmodified and RGD-modified sIPNs secreted very low levels of MMP-9 (Fig 4). MMP-9 secreted from U937 on TCPS and PEG-only hydrogels was above the detection limits of the ELISA kit. The addition of 10 IU uPA (~100 ng/ml), which is known to directly activate MMP-9 , had no effect upon MMP-9 levels from U937 on sIPNs, TCPS, or PEG-only hydrogels. PMA has been observed to stimulate a 50-fold molar excess of PAI-2, which inhibits uPA, in U937 monocytes . Thus the exogenous uPA might have been inactivated by the PMA-dependent increase in PAI-2..
PAI-2 secretion from U937 adherent to TCPS, sIPNs, and PEG-only hydrogels was surface dependent. U937 adherent to unmodified or RGD-modified sIPNs secreted significantly less PAI-2 than U937 on TCPS or PEG-only hydrogels and U937 treated with LPS (Fig 5). Exogenous addition of uPA at 10 IU (100 ng/ml) or 25 IU (250 ng/ml) did not effect PAI-2 secretion from U937 on TCPS or PEG-only hydrogels. U937 on RGD-modified sIPNs or unmodified sIPNs were not treated with 25 IU uPA based on the first replicate that showed low levels of PAI-2 from U937 on sIPNs. PMA is known to increase PAI-2 secretion from U937 [47, 48], but the low levels of PAI-2 secreted from U937 on sIPNs indicates the surface may play a role in down regulating PAI-2 secretion or that the proteolytic activity of the plasminogen system is matrix associated.
Increased levels of uPA were detected in wells treated with exogenous uPA for all surfaces tested (p < 0.001, Fig 6). Less than 5 ng/ml uPA can be detected 24 h after addition of 10 IU (100 ng/ml) or 25 IU (250 ng/ml) uPA. However, no decrease in secreted PAI-2 was observed after 10 IU or 25 IU uPA addition (Fig 5). This lack of uPA treatment effect may indicate that some of the PAI-2/uPA system is associated with the matrix or the adherent cell surface.
uPAR is a glycophosphatidylinositol-linked cell surface receptor that lacks both a transmembrane and cytoplasmic domain. This glycophosphatidylinositol linkage can be cleaved creating soluble uPAR that can act as a chemotactic signal recruiting monocytes to sites of inflammation and ECM remodeling [50, 51]. Soluble uPAR was lower in supernatant from U937 on unmodified or RGD-modified sIPNs than those from TCPS or PEG-only hydrogels (p < 0.001, Fig 7) Addition of 10 IU exogenous uPA significantly lowered soluble uPAR in supernatant from U937 on TCPS and PEG-only hydrogels. 25 IU exogenous uPA also resulted in lower soluble uPAR from U937 on PEG-only hydrogels compared to U937 on PEG-only hydrogels without uPA addition (p = 0.026). Qualitatively, addition of 25 IU uPA to U937 adherent to TCPS and PEG-only hydrogels had a similar effect on soluble uPAR as 10 IU (Fig 7). Soluble uPAR has been observed to inhibit uPA/uPAR signaling though the exact mechanism is not known [62,63]. Therefore the exogenous uPA may have bound soluble uPAR rather than inhibiting PAI-2. Since uPAR can interact with both integrin receptors on the cell surface and bind directly to vitronectin adsorbed to the biomaterial surface, this receptor may play an important, but currently unexplored, role in host-material interactions.
IL-1β secretion from U937 adherent to TCPS, unmodified sIPNs, and RGD-modified sIPNs was below detection limits for two of the three independent replicates (S Table S3). U937 on PEG-only hydrogels did appear to secrete slightly higher levels of IL-1β. This overall lack of IL-1β from uPA treated adherent monocytes is consistent with Lee and colleague’s findings that neither 5 μg/ml tPA nor 100 IU uPA elicited IL-1β secretion from rat cortical astrocytes even though both tPA and uPA elicited strong cytokine and chemokine responses [64–66]. TNF-α secretion from U937 adherent to PEG-only hydrogels showed a consistent and dramatic increase over U937 on TCPS or sIPNs (p < 0.001, Fig 8). There was no difference among TNF-α secretion from U937 on TCPS, unmodified or RGD-modified sIPNs (Fig 8). The higher levels of TNF-α from U937 on PEG-only hydrogels coincides with increased levels of IL-1β, which are secreted via similar pathways.
U937 on TCPS or PEG-only hydrogels secreted significantly more MCP-1 than those on unmodified or RGD-modified sIPNs (p< 0.001) independent of exogenous uPA (Fig 9). The addition of 10 or 25 IU exogenous uPA decreased MCP-1 secretion from U937 on TCPS or PEG-only hydrogels, respectively (p = 0.002 and 0.025, respectively). MCP-1 secreted from U937 on TCPS or PEG-only hydrogels was significantly lower than that from LPS-treated U937 (193,844 ± 237,097 pg/ml). MCP-1 is a major chemoattractant recruiting additional monocytes to the host-material interface [67,68] High levels of MCP-1 are not surprising from U937 on PEG-only hydrogels as the higher levels of IL-1β and TNF-α indicate an inflammatory response to PEG-only hydrogels. However, the high level of MCP-1 from U937 on TCPS is intriguing since IL-1β and TNF-α levels are similar to that seen from U937 on sIPNs and overall are very low. Addition of exogenous uPA (100 IU) to rat cortical astrocytes stimulated an increase in MCP-1 and TNF-α over controls , but since TNF-α levels overall were low, the effect of uPA is likely cell specific. IL-1 or TNF-α can induce MCP-1; however, IL-1 and TNF-α were low in supernatant from U937 on TCPS indicating MCP-1 secretion is independent of IL-1 and TNF-α but is surface dependent. MCP-1 can increase integrin αM and αX (CD11b & c, respectively) receptors, uPA, uPAR, and MMP expression and secretion [68,69]. Therefore the increase in MCP-1 but not TNF-α by U937 on TCPS may be a specific effect induced by serum protein adsorption specific to TCPS. More research into the relationship between uPA, MCP-1, and other inflammatory cytokines such as IL-1 and TNF-α is warranted to elucidate the feedback loops regulating the monocyte response to TCPS, sIPNs, and PEG-only hydrogels.
This work represents the first we could find investigating the role of PAI-2 in monocyte-material interactions. Manipulating the expression of PAI-2 poses several unique challenges. PAI-2 is expressed intracellularly and is redox sensitive [70,71] therefore small interfering RNA knockdown or knock out may also impact intracellular processes. This work shows that addition of exogenous uPA reduced levels of soluble uPAR more than PAI-2. The low levels of PAI-2 and MMP-9 detected from monocytes on sIPNs may indicate expression of these proteins is associated with the matrix or cell surface and should be investigated in future research into the role of the plasminogen system in monocyte-material interactions. MMP, uPAR, and uPA activation and regulation are closely linked and may help elucidate the mechanism of the monocyte-mediated inflammatory response to implanted materials.
The sIPNs contained an identical amount of PEG as the PEG-only hydrogels. The addition of gelatin, independent of peptide modification, appears to be responsible for the significant decrease in MMP-9, PAI-2, and MCP-1 detected in this work. The use of ELISA detects soluble proteins in the culture supernatant therefore the presence of these proteins associated with the matrix cannot be dismissed. Regardless, the addition of gelatin had significant surface dependent effects on soluble protein expression. Whether the gelatin exerted this decrease directly through cell interactions or through an indirect route such as altered mechanical properties remains to be determined. Surface dependent changes in protein expression of other proteins identified by LC-MALDI from U937 adherent to TCPS such as 40S ribosomal S19 and tyrosyl tRNA synthetase may offer additional insight into the mechanism of monocyte-material interactions.
PAI-2 and Hck are known to play important roles in pathways relevant to the monoctye-mediated host inflammatory response to implanted materials. MCP-1 showed Hck-dependent secretion in monocytes adherent to TCPS but not PEG-only hydrogels at 24 h. Monocytes on sIPNs secreted very low levels of MMP-9 and PAI-2 compared to those on PEG-only hydrogels, which secreted similar levels as on TCPS. The addition of gelatin effects the surface dependent decrease in MMP-9 and PAI-2 since the sIPNs contained the same amount of PEG as PEG-only hydrogels. This work shows the use of TCPS as a reference surface for biomaterial research should be approached with caution.
This work was funded in part by NIH R01 HL77825.
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