Photochemically immobilized polymer coatings: effects on protein adsorption, cell adhesion, and leukocyte activation.

Amphiphilic chains of 4-benzoylbenzoic acid moieties and polymer were photochemically immobilized onto silicone rubber to ask whether the covalently coupled polymers would passivate the silicone rubber by inhibiting protein adsorption and subsequent cell adhesion and activation. Three groups of polymers were utilized: the hydrophilic synthetic polymers of polyacrylamide, polyethylene glycol, and polyvinylpyrrolidone; the glycosaminoglycan, hyaluronic acid; and poly(glycine-valine-glycine-valine-proline), a polypeptide derived from the sequence of elastin. Each coating variant decreased the adsorption of fibrinogen and immunoglobulin G compared to uncoated silicone rubber. All except the methoxy-polyethylene glycol coating nearly abolished fibroblast growth, but none of the coating variants inhibited monocyte or polymorphonuclear leukocyte adhesion. Interleukin-1beta, interleukin-1 receptor antagonist, and tumor necrosis factor-alpha secretion by leukocytes were not statistically different between any of the coating variants and uncoated silicone rubber. However, the methoxy-polyethylene glycol and elastin-based polypeptide coatings, which supported the highest numbers of adherent monocytes, also elicited the lowest levels of proinflammatory cytokine secretion. When these in vitro data were collectively evaluated, the coating that most effectively passivated silicone rubber was the polypeptide derived from elastin.

Effects of photochemically immobilized polymer coatings on protein adsorption, cell adhesion, and the foreign body reaction to silicone rubber.

Photochemical immobilization technology was utilized to covalently couple polymers to silicone rubber either at multiple points along a polymer backbone or at the endpoint of an amphiphilic chain. The coating variants then were tested in vitro and in vivo for improvement of desired responses compared to uncoated silicone rubber. All coating variants suppressed the adsorption of fibrinogen and immunoglobulin G, and most also inhibited fibroblast growth by 90-99%. None of the coating variants inhibited monocyte or neutrophil adhesion in vitro. However, the surfaces that supported the highest levels of monocyte adhesion also elicited the lowest secretion of pro-inflammatory cytokines. None of the materials elicited a strong inflammatory response or significantly (p< 0.05) reduced the thickness of the fibrous capsule when implanted subcutaneously in rats. Overall, the most passivating coating variant was an endpoint immobilized polypeptide that reduced protein adsorption, inhibited fibroblast growth by 90%, elicited low cytokine secretion from monocytes, and reduced fibrous encapsulation by 33%. In general, although some coating variants modified the adsorption of proteins and the behavior of leukocytes or fibroblasts in vitro, none abolished the development of a fibrous capsule in vivo.

Spatial regulation and surface chemistry control of monocyte/macrophage adhesion and foreign body giant cell formation by photochemically micropatterned surfaces.

A long-standing goal of biomedical device development has been the generation of specific, desired host blood and tissue responses. An approach to meeting this design criteria is precise surface modification that creates micropatterns of distinct physicochemical character to direct cell adhesion and behavior. For this study, poly(ethylene terephthalate) films were coated with poly(benzyl N, N-diethyldithiocarbamate-co-styrene) and sequentially exposed to monomer solutions for photoirradiation. A photomask was placed over different regions to generate micropatterned surfaces with graft polymer stripes of three distinct ionic characters. Human monocytes were cultured on these surfaces to ascertain whether adhesion and fusion of monocytes/macrophages could be controlled. Nonionic polyacrylamide greatly inhibited adhesion and induced clumping of the few monocytes that did adhere. Macrophage adhesion and spreading led to high degrees of interleukin-13 induced foreign body giant cell formation on both the anionic poly(acrylic acid), sodium salt, and benzyl N,N-diethyldithiocarbamate portions of the culture surface. In spite of the highest observed levels of monocyte/macrophage adhesion on cationic poly(dimethylaminopropylacrylamide), methiodide, the adherent cells were not competent to undergo fusion to form foreign body giant cells. These results suggest that inflammatory cell responses may be spatially controlled in a manner that may be ultimately exploited to improve the biocompatibility of medical devices.

Disruption of filamentous actin inhibits human macrophage fusion.

The foreign body reaction to implanted biomaterials, characterized by the presence of macrophages and foreign body giant cells (FBGC), can result in structural and functional failure of the implant. Recently, we have shown that interleukin-4 and interleukin-13 can independently induce human macrophage fusion to form FBGC via a macrophage mannose receptor (MR) -mediated pathway. The MR is believed to mediate both endocytosis of glycoproteins and phagocytosis of microorganisms, which bear terminal mannose, fucose, N-acetylglucosamine, or glucose residues. Polarization of microfilaments to closely apposed macrophage membranes as observed with fluorescence confocal microscopy led us to ask whether MR-mediated fusion occurred via a filamentous actin-dependent pathway. Cytochalasins B and D and latrunculin-A, agents that disrupt microfilaments, inhibited macrophage fusion in a concentration-dependent manner. The concentrations of cytochalasins D and B that inhibited fusion did not significantly decrease macrophage adhesion, spreading, or motility but did inhibit internalization of Candida albicans during interleukin-13-enhanced, MR-mediated phagocytosis. Very low concentrations of cytochalasin B (< 2 microM) induced a slight enhancement of macrophage fusion. Taken together, the results of this study suggest that cytokine-induced, MR-mediated macrophage fusion requires an intact F-actin cytoskeleton and that the mechanism of fusion is similar to phagocytosis.--DeFife, K. M., Jenney, C. R., Colton, E., Anderson, J. M. Disruption of filamentous actin inhibits human macrophage fusion.

Cytoskeletal and adhesive structural polarizations accompany IL-13-induced human macrophage fusion.

During the inflammatory response to an implanted biomaterial, monocytes undergo a striking phenotypic progression of differentiation into macrophages, which may subsequently fuse to form foreign body giant cells (FBGCs). Taking advantage of an in vitro system of cytokine-induced FBGC formation together with the optical slicing capabilities of a confocal microscope, we investigated the cytoskeletal reorganization and adhesive structure development during this dramatic morphological progression. Human monocytes demonstrated diffuse cytoplasmic staining of adhesive structural proteins. Punctate filamentous (F)-actin structures appeared along the ventral cell membrane of macrophages and were identified as the core of podosome adhesive structures by the distinctive ring staining of vinculin, talin, and paxillin around the F-actin. Cytokine-induced FBGCs were characterized by a restriction of podosomes to the extreme periphery of the ventral cell surface. Although macrophages and FBGC contained equivalent amounts of F-actin, significantly more F-actin was located within 1 micron of the ventral plasma membrane in FBGCs compared to macrophages. Taken together, these results provide new information on the dynamic cytoskeletal reorganization and adhesive structure development that occur during phenotypic progression from human monocytes to macrophages to FBGC. Furthermore, they suggest the acquisition of functional specializations on FBGC formation, which may enhance our understanding of chronic inflammatory processes.

Monocyte, macrophage and foreign body giant cell interactions with molecularly engineered surfaces.

To elucidate the mechanisms involved in monocyte/macrophage adhesion and fusion to form foreign body giant cells on molecularly engineered surfaces, we have utilized our in vitro culture system to examine surface chemistry effects, cytoskeletal reorganization and adhesive structure development, and cell receptor-ligand interactions in in vitro foreign body giant cell formation. Utilizing silane-modified surfaces, monocyte/macrophage adhesion was essentially unaffected by surface chemistry, however the density of foreign body giant cells (FBGCs) was correlated with surface carbon content. An exception to the surface-independent macrophage adhesion were the alkyl-silane modified surfaces which exhibited reduced adhesion and FBGC formation. Utilizing confocal immunofluorescent techniques, cytoskeletal reorganization and adhesive structure development in in vitro FBGC formation was studied. Podosomes were identified as the adhesive structures in macrophages and FBGCs based on the presence of characteristic cytoplasmic proteins and F-actin at the ventral cell surface. Focal adhesion kinase (FAK) and focal adhesions were not identified as the adhesive structures in macrophages and FBGCs. In studying the effect of preadsorbed proteins on FBGC formation, fibronectin or vitronectin do not play major roles in initial monocyte/macrophage adhesion, whereas polystyrene surfaces modified with RGD exhibited significant FBGC formation. These studies identify the potential importance of surface chemistry-dependent conformational alterations which may occur in proteins adsorbed to surfaces and their potential involvement in receptor-ligand interactions. Significantly, preadsorption of alpha2-macroglobulin facilitated macrophage fusion and FBGC formation readily on the RGD surface in the absence of any additional serum proteins. As alpha2-macroglobulin receptors are not found on blood monocytes but are expressed only with macrophage development, these results point to a potential interaction between adsorbed 2-macroglobulin and its receptors on macrophages during macrophage development and fusion. These studies identify important surface independent and dependent effects in foreign body reaction development that may be important in the identification of biological design criteria for molecularly engineered surfaces and tissue engineered devices.

Immunocytochemical colocalization of specific immunoglobulin A with sendai virus protein in infected polarized epithelium.

Immunoglobulin (Ig)A provides the initial immune barrier to viruses at mucosal surfaces. Specific IgA interrupts viral replication in polarized epithelium during receptor-mediated transport, probably by binding to newly synthesized viral proteins. Here, we demonstrate by immunoelectron microscopy that specific IgA monoclonal antibodies (mAbs) accumulate within Sendai virus-infected polarized cell monolayers and colocalize with the hemagglutinin- neuraminidase (HN) viral protein in a novel intracellular structure. Neither IgG specific for HN nor irrelevant IgA mAbs colocalize with viral protein. Treatment of cultures with viral-specific IgA but not with viral-specific IgG or irrelevant IgA decreases viral titers. These observations provide definitive ultrastructural evidence of a subcellular compartment in which specific IgA and viral envelope proteins interact, further strengthening our hypothesis of intracellular neutralization of virus by specific IgA antibodies. Our results have important implications for intracellular protein trafficking, viral replication, and viral vaccine development.

Human monocyte/macrophage adhesion, macrophage motility, and IL-4-induced foreign body giant cell formation on silane-modified surfaces in vitro. Student Research Award in the Master's Degree Candidate Category, 24th Annual Meeting of the Society for Biomaterials, San Diego, CA, April 22-26, 1998.

A cytokine-based, in vitro model of foreign body giant cell (FBGC) formation was utilized to examine the effect of biomaterial surface chemistry on the adhesion, motility, and fusion of monocytes and macrophages. Human monocytes were cultured for 10 days on 14 different silane-modified glass surfaces, during which time the cells assumed the macrophage phenotype. The adhesion of monocytes and macrophages during the culture period decreased by an average of approximately 50%, with the majority of cell loss observed during days 1-3. Most important, the adhesion of monocytes and macrophages was surface independent except for two surfaces containing terminal methyl groups, which decreased adhesion levels. Interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were added to the medium to induce FBGC formation and enhance macrophage adhesion, respectively. Surprisingly, GM-CSF decreased long-term monocyte/macrophage adhesion. IL-4-induced FBGC density was strongly influenced by the surface carbon content, as determined by X-ray photoelectron spectroscopy (XPS). In contrast, contact angle and surface energy displayed no correlation with FBGC formation. The motility of adherent macrophages, as measured by time-lapse confocal microscopy, was not affected significantly by differences in surface chemistry or the addition of cytokines. The surface dependence of FBGC formation is hypothesized to be the result of varying levels of silane-derived surface carbon.

Interleukin-13 induces human monocyte/macrophage fusion and macrophage mannose receptor expression.

Inasmuch as we recently demonstrated that IL-4 is a strong inducer of monocyte/macrophage fusion and IL-13 has been observed to mimic many of the biologic effects of IL-4, the ability of IL-13 to promote human macrophage fusion in vitro was tested and compared with IL-4-mediated fusion. IL-13 induced the fusion of monocyte-derived macrophages as potently as IL-4 under identical culture conditions, and resulted in foreign body-type giant cell formation. At optimal concentrations of cytokine added, statistically equal numbers of macrophages participated in IL-13- and IL-4-induced fusion (66.1 +/- 4.6% and 63.9 +/- 4.4%, respectively). However, the effects of IL-13 and IL-4 were not additive or synergistic, and the maximum fusion obtained when both IL-4 and IL-13 were added was 63.8 +/- 3.6%. Only anti-human IL-13 Abs inhibited IL-13-induced foreign body giant cell formation; the fusion-inducing effects of IL-13 continued to be observed in the presence of neutralizing Abs to IL-4 and several other anti-cytokine Abs, including Abs against IFN-gamma, granulocyte-macrophage CSF, IL-3, and TNF-alpha. IL-13 also significantly enhanced the fluorescence intensity detected by anti-human macrophage mannose receptor Abs, indicating that IL-13, like IL-4, up-regulates expression of the receptor that may be an essential participant in macrophage fusion. The results of this study demonstrate that IL-13, like IL-4, is a potent human macrophage fusion factor, and suggest that although IL-13 acts independently of IL-4 to promote foreign body giant cell formation, it may trigger a common mechanism for macrophage fusion.

Protein adsorption on chemically modified surfaces.

Following the implantation of a biomaterial, the first event to occur at the tissue-material interface is protein adsorption. Once proteins have adsorbed to the surface of a material, cells no longer see the material, but only the protein coated surface layer. This adsorbed protein layer can mediate the type of cells that adhere to the surface, which ultimately can determine the type of tissue that develops. In this experiment, glass and polystyrene surfaces were chemically modified forming ionic, non-ionic, hydrophobic, and hydrophilic surfaces. The modified surfaces were incubated in a RPMI diluted serum solution. After incubation, a radioimmunoassay was used to quantify the exposed proteins adsorbed onto the surface of the material. In general, albumin, complement C3 (C3), fibronectin (FN) and vitronectin (VN) competitively adsorbed to the modified surface in a similar fashion, whereas IgG adsorption was the opposite. The hydrophobic surfaces had higher adsorbance of the adhesion proteins (C3, FN and VN) compared to higher adsorption of albumin and IgG onto the hydrophilic surfaces. The surface mobility of the silane modified surfaces also affected the adhesion of proteins. The differences seen in protein adsorption did not directly correlate to monocyte adhesion and Foreign Body Giant Cell (FBGC) development on these surfaces.

Vitamin E as an antioxidant for poly(etherurethane urea): in vivo studies. Student Research Award in the Doctoral Degree Candidate Category, Fifth World Biomaterials Congress (22nd Annual Meeting of the Society for Biomaterials), Toronto, Canada, May 29-June 2, 1996.

Poly(etherurethane) elastomers are useful materials in medical devices because of their mechanical properties and biocompatibility. However, it is necessary to stabilize these elastomers against the oxidation of their ether soft segments. Synthetic antioxidants such as Santowhite and Irganox are often satisfactory; however, particularly for biomedical applications, it was of interest to test the natural antioxidant vitamin E in poly(etherurethane urea) (PEUU) elastomers in vivo. The alpha-tocopherol form of vitamin E was added to PEUU at 5% by weight. Biaxially strained PEUU specimens with and without vitamin E were tested in vivo in the cage implant system. The influence of vitamin E on PEUU biostability was analyzed by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy and scanning electron microscopic (SEM) characterization of the PEUU surface. ATR-FTIR results showed that vitamin E prevented chemical degradation of the PEUU surface up to 5 weeks implantation, and at 10 weeks 82% of the ether remained. In contrast, without an antioxidant, only 18% of the ether remained after 10 weeks. No surface pitting or cracking was observed by SEM on PEUU with vitamin E; PEUU without antioxidant ruptured owing to extensive pitting and cracking. It was concluded that the antioxidant properties of vitamin E prevented oxidation of strained PEUU elastomers in vivo. The influence of vitamin E on PEUU biocompatibility was characterized by exudate leukocyte counts, density of leukocytes adherent to the PEUU, and morphology of adherent leukocytes. These results indicated decreased leukocyte counts in the exudate and less active adherent cells on the PEUU with vitamin E compared to PEUU without antioxidant. A proposed cell-polymer feedback system demonstrates how vitamin E improves both biostability and biocompatibility of PEUU elastomers in vivo.

Interleukin-4-induced macrophage fusion is prevented by inhibitors of mannose receptor activity.

A potential role for the macrophage mannose receptor in human monocyte-derived macrophage fusion was explored by testing the effects of previously described inhibitors of its activity on the formation of interleukin-4-induced foreign body giant cells in vitro Giant cell formation was prevented or reduced in the presence of alpha-man-nan and synthetic neoglycoprotein conjugates according to the following pattern of relative inhibition: mannose-bovine serum albumin (BSA) > N-acetylgucosamine-BSA congruent to glucose-BSA. Laminarin (beta-glucan) or galactose-BSA were not inhibitory. Swainsonine and castanospermine, inhibitors of glycoprotein processing that interfere with the arrival of newly synthesized mannose receptors at the cell surface, also attenuated macrophage fusion and the formation of giant cells, whereas another glycosidase inhibitor, deoxymannojirimycin, was without effect. Mannose receptors were confirmed to be specifically up-regulated by interleukin-4 in this culture system and also demonstrated to be present and concentrated at macrophage fusion interfaces. These data suggest that the macrophage mannose receptor may be an essential participant in the mechanism of interleukin-4-induced macrophage fusion and implicate a novel function for this endocytic/phagocytic receptor in mediating foreign body giant cell formation at sites of chronic inflammation.

Adhesion and cytokine production by monocytes on poly(2-methacryloyloxyethyl phosphorylcholine-co-alkyl methacrylate)-coated polymers.

Human monocytes isolated from peripheral venous blood were assayed for their ability to adhere to various polymers. The culture supernatants were also assayed for the cytokines, interleukin-1 beta (IL-beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha). The polymers evaluated for adherence and cytokine production included Pellethane, polyethylene and poly[n-butyl methacrylate (BMA)] coated with poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-alkyl methacrylate] copolymers. In some experiments the test polymers were adsorbed with fibrinogen or IgG prior to the addition of monocytes. MPC copolymer-coated materials inhibited monocyte and macrophage adhesion after 1 and 8 days of culture relative to corresponding uncoated polymers and tissue culture polystyrene (TCPS). The degree of inhibition by coated Pellethane compared to uncoated Pellethane was the greatest, while inhibition of adhesion by coated poly(BMA) was the least compared to uncoated poly(BMA). However, adhesion was significantly decreased on both coated and uncoated poly(BMA) by day 8. While IL-1 beta, IL-6, and TNF-alpha release was variably influenced by polymer coating, release was consistently inhibited relative to TCPS on day 1. However, cytokine production was not inhibited compared to corresponding uncoated polymers on day 1. With or without protein preadsorption, IL-1 beta release was not detectable in the supernatants of any polymer on day 8, IL-6 production was diminished on day 8, and TNF-alpha production was sustained on day 8. Overall, MPC copolymer-coated and uncoated poly(BMA) were the least stimulating, while TCPS was the most stimulating.(ABSTRACT TRUNCATED AT 250 WORDS)

Human monocyte/macrophage adhesion and cytokine production on surface-modified poly(tetrafluoroethylene/hexafluoropropylene) polymers with and without protein preadsorption.

To study surface property-dependent human monocyte adhesion and cytokine (IL-1 beta, IL-6, TNF-alpha) production, poly(tetrafluoroethylene/hexafluoropropylene) (FEP) polymer was modified to exhibit neutral, anionic, or cationic properties by incorporating amide (CONH2) and/or carboxyl (COOH) or aminoethyl amide [CONH(CH2CH2NH)nCH2CH2NH2] groups on the surface. Monocyte adhesion on surface-modified FEP polymers and cytokines released by monocytes/macrophages (MC/MO) into the culture medium were compared to control tissue culture polystyrene (TCPS) at days 1 and 8. On day 1, the neutral surface FEP polymer with incorporated amide (NH2) groups showed the greatest inhibition of adhesion, 89% (P < .01), and cytokine production (IL-1 beta with 58%, IL-6 with 70%, and TNF-alpha with 39%) compared to control TCPS. In contrast, the highly cationic [CONH(CH2CH2NH)nCH2CH2NH2] surface did not show significant (P > .01) inhibition of monocyte adhesion and cytokine production. When fibrinogen or IgG was preadsorbed to the surface, the inhibitory effects of the neutral surface FEP polymer on monocyte adhesion and cytokine production were not altered. In addition, other surface-modified FEP polymers showed similar inhibition of monocyte adhesion and cytokine production compared to TCPS. Specifically, as the incorporation of carboxyl (COOH) group content increased on FEP polymer surfaces, monocyte adhesion and cytokine production were also increased on day 1 with IgG preadsorption. On day 8, all surface-modified FEP polymers showed significant (P < .01) inhibition of monocyte adhesion when fibrinogen or IgG was preadsorbed. However, without protein (fibrinogen or IgG) preadsorption, monocyte adhesion was not significantly inhibited compared to control TCPS. In addition, cytokine production detected by ELISAs on day 8 showed no detectable levels of IL-1 beta and significantly decreased levels of IL-6 compared to day 1 for all tested polymers, with or without protein preadsorption. Interestingly, the level of TNF-alpha production on day 8 remained high although not as high as on day 1. Based on these results, we suggest that FEP polymers with neutral hydrophilic surface properties may adhere and activate the least number of monocytes, which are important mediators of biocompatibility.

Interaction of amfonelic acid with antipsychotic drugs on dopaminergic neurons.

The effects of two inhibitors of dopamine (DA) reuptake, amfonelic acid and GBR 12909, on the clozapine- and haloperidol-induced increases in DA synthesis, release, and metabolism were investigated in the rat. In the striatum, as well as in the nucleus accumbens, the haloperidol-induced increase in tissue concentrations of dihydroxyphenylacetic acid (DOPAC) or the accumulation of dihydroxyphenylalanine (DOPA) was potentiated or unaltered, respectively, in rats treated with amfonelic acid. In contrast, amfonelic acid attenuated the stimulatory effects of clozapine on DOPAC concentrations and DOPA accumulation in both brain regions. GBR 12909 also differentially affected the haloperidol- and clozapine-induced increases in DOPAC concentrations. However, the clozapine-induced increase in DOPA accumulation in the median eminence was not significantly altered by treatment with amfonelic acid. The haloperidol-induced increase in the extracellular concentrations of DA and DOPAC in the striatum also was potentiated by amfonelic acid, whereas the increase elicited by clozapine was suppressed. The increase in extracellular DA produced by the administration of morphine or the coadministration of ritanserin, a 5-HT2 antagonist, and haloperidol also was potentiated by amfonelic acid. The ability of amfonelic acid to distinguish between the actions of clozapine and haloperidol on nigrostriatal and mesocorticolimbic DA neurons does not appear to be related to differences in the effects of the drugs on DA autoreceptors or 5-HT2 receptors. Moreover, the mechanism through which clozapine activates tuberoinfundibular DA neurons may differ from that which is involved in the activation of nigrostriatal or mesocorticolimbic DA neurons.

Effects of amfonelic acid and GBR 12909 on the haloperidol- and clozapine-induced activation of dopamine neurons.

The purpose of the present study was to establish the extent to which dopamine uptake inhibitors, for example, amfonelic acid (AFA) and GBR 12909, differentially affect the haloperidol- and clozapine-induced activation of dopamine neurons. In the striatum and nucleus accumbens, the haloperidol-induced increases in dopamine synthesis and metabolism, as well as striatal dopamine release, were either potentiated or unaffected by AFA or GBR 12909. In contrast, AFA or GBR 12909 markedly attenuated the clozapine-induced increases in dopamine synthesis, metabolism, and release. However, the clozapine-induced increase in dopamine synthesis within tuberoinfundibular dopamine neurons was not significantly altered by AFA treatment. AFA and GBR 12909, appear to differentially affect the haloperidol- and clozapine-induced activation of nigrostriatal and mesocorticolimbic dopamine neurons. However, the inhibitory effect of AFA on the clozapine-induced activation of dopamine neurons does not extent to the stimulatory effect of clozapine on tuberoinfundibular dopamine neurons.