Biomaterial surface chemistry dictates adherent monocyte/macrophage cytokine expression in vitro.

An in vitro human monocyte culture system was used to determine whether adherent monocyte/macrophage cytokine production was influenced by material surface chemistry. A polyethylene terephthalate (PET) base surface was modified by photograft copolymerization to yield hydrophobic, hydrophilic, anionic and cationic surfaces. Freshly isolated human monocytes were cultured onto the surfaces for periods up to 10 days in the presence or absence of interleukin-4 (IL-4). Semi-quantitative RT-PCR analysis on days 3, 7 and 10 of cell culture revealed that interleukin-10 (IL-10) expression significantly increased in cells adherent to the hydrophilic and anionic surfaces but significantly decreased in the cationic surface adherent monocytes/macrophages. Conversely, interleukin-8 (IL-8) expression was significantly decreased in cells adherent to the hydrophilic and anionic surfaces. Further analysis revealed that the hydrophilic and anionic surfaces inhibited monocyte adhesion and IL-4-mediated macrophage fusion into foreign body giant cells (FBGCs). Therefore, hydrophilic and anionic surfaces promote an anti-inflammatory type of response by dictating selective cytokine production by biomaterial adherent monocytes and macrophages. These studies contribute information necessary to enhance our understanding of biocompatibility to be used to improve the in vivo lifetime of implanted medical devices and prostheses.

Influence of biomaterial surface chemistry on the apoptosis of adherent cells.

A common component of the foreign-body response to implanted materials is the presence of adherent macrophages that fuse to form foreign-body giant cells (FBGCs). These multinucleated cells have been shown to concentrate the phagocytic and degradative properties of macrophages at the implant surface and are responsible for the damage and failure of the implant. Therefore, the modulation of the presence or actions of macrophages and FBGCs at the material-tissue interface is an extensive area of recent investigations. A possible mechanism to achieve this is through the induction of the apoptosis of adherent macrophages, which results in no inflammatory consequence. We hypothesize that the induction of the apoptosis of biomaterial adherent cells can be influenced by the chemistry of the surface of adhesion. Herein, we demonstrate that surfaces displaying hydrophilic and anionic chemistries induce apoptosis of adherent macrophages at a higher magnitude than hydrophobic or cationic surfaces. Additionally, the level of apoptosis for a given surface is inversely related to that surface's ability to promote the fusion of macrophages into FBGCs. This suggests that macrophages fuse into FBGCs to escape apoptosis.

Tears contain the complement regulator CD59 as well as decay-accelerating factor (DAF).

Previous studies have shown that DAF (or CD55), a cell surface inhibitor of autologous C3 activation, is present in tears and that > 90% of the C3 convertase regulatory activity in tear fluid resides in this protein (Lass JH et al., Invest Ophth Vis Sci 1990; 31:1136-48). This study investigated whether (i) the membrane cofactor protein (MCP or CD46), an additional factor that regulates C3 activation, and (ii) the membrane inhibitor of reactive lysis (MIRL or CD59), a cell surface regulator that acts to prevent formation of the membrane attack complex, are also present in tears, and if so, are functional. Two-site immunoradiometric assays showed that MCP is present in tears at low levels (42 + 8 ng/ml, n = 8) while CD59 is present at levels (222 + 78 ng/ml, n = 14) comparable to those of DAF (325 + 289 ng/ml, n = 12). The concentrations of CD59 (i) were increased two-fold or more in closed eye tears, and (ii) were decreased in reflex tears. Western blotting showed that CD59 protein in tears migrates with an apparent mol. wt similar to membrane CD59 protein. Phenyl-Sepharose adsorption and Triton X-114 partitioning of tear CD59 as well as of tear DAF however, showed that both proteins are devoid of GPI anchors. Assays using cobra venom factor-activated human serum and guinea pig erythrocytes showed that CD59 is functionally active in inhibiting autologous C5b-9-mediated lysis and, under constitutive conditions, accounts for > 85% of the C9 inhibitory activity in tear fluid.

Cooperation between decay-accelerating factor and membrane cofactor protein in protecting cells from autologous complement attack.

Decay-accelerating factor (DAF or CD55) and membrane cofactor protein (MCP or CD46) function intrinsically in the membranes of self cells to prevent activation of autologous complement on their surfaces. How these two regulatory proteins cooperate on self-cell surfaces to inhibit autologous complement attack is unknown. In this study, a GPI-anchored form of MCP was generated. The ability of this recombinant protein and that of naturally GPI-anchored DAF to incorporate into cell membranes then was exploited to examine the combined functions of DAF and MCP in regulating complement intermediates assembled from purified alternative pathway components on rabbit erythrocytes. Quantitative studies with complement-coated rabbit erythrocyte intermediates constituted with each protein individually or the two proteins together demonstrated that DAF and MCP synergize the actions of each other in preventing C3b deposition on the cell surface. Further analyses showed that MCP's ability to catalyze the factor I-mediated cleavage of cell-bound C3b is inhibited in the presence of factors B and D and is restored when DAF is incorporated into the cells. Thus, the activities of DAF and MCP, when present together, are greater than the sum of the two proteins individually, and DAF is required for MCP to catalyze the cleavage of cell-bound C3b in the presence of excess factors B and D. These data are relevant to xenotransplantation, pharmacological inhibition of complement in inflammatory diseases, and evasion of tumor cells from humoral immune responses.

Structure/function studies of human decay-accelerating factor.

The decay-accelerating factor (DAF) contains four complement control protein repeats (CCPs) with a single N-linked glycan positioned between CCPs 1 and 2. In previous studies we found that the classical pathway regulatory activity of DAF resides in CCPs 2 and 3 while its alternative pathway regulatory activity resides in CCPs 2, 3 and 4. Molecular modelling of the protein predicted that a positively charged surface area on CCPs 2 and 3 (including KKK125-127) and nearby exposed hydrophobic residues (L147F148) on CCP3 may function as ligand-binding sites. To assess the roles of the N-linked glycan and the above two sets of amino acids in the function of DAF, we mutated N61 to Q, KKK125-127 to TTT and L147F148 to SS. Following expression of the mutated cDNAs in Chinese hamster ovary cells, the glycosylphosphatidylinositol (GPI)-anchored mutant proteins were affinity purified and their functions were assessed. In initial assays, the proteins were incorporated into sheep and rabbit erythrocytes and the effects of the mutations on regulation of classical and alternative C3 convertase activity were quantified by measuring C3b deposition. Since DAF also functions on C5 convertases, comparative haemolytic assays of cells bearing each mutant protein were performed. Finally, to establish if spatial orientation between DAF and the convertases on the cell surface played any role in the observed effects, fluid-phase C3a generation assays were performed. All three assays gave equivalent results and showed that the N-linked glycan of DAF is not involved in its regulatory function; that L147F148 in a hydrophobic area of CCP3 is essential in both classical and alternative pathway C3 convertase regulation; and that KKK125-127 in the positively charged pocket between CCPs 2 and 3 is necessary for the regulatory activity of DAF on the alternative pathway C3 convertase but plays a lesser role in its activity on the classical pathway enzyme.

Localization of classical and alternative pathway regulatory activity within the decay-accelerating factor.

Decay-accelerating factor (DAF) is a cell-associated C regulatory protein that protects host cells from autologous C attack. It functions intrinsically in host cell surface membranes to rapidly dissociate autologous classical and alternative pathway C3 convertases whenever these amplifying enzymes assemble on host cell surfaces. It is composed of four contiguous approximately 70 amino acid long regions termed short consensus repeats (SCRs) that share homology with similar units in other C3 convertase regulatory proteins. It is attached to the cell surface membrane by a glycoinositol phospholipid (GPI) anchor that is added posttranslationally. In this study, we prepared rGPI-anchored DAF proteins devoid of individual SCRs. We then incorporated the GPI-anchored products into sheep erythrocyte (Esh) hemolytic intermediates and examined their abilities to intrinsically regulate classical or alternative pathway activation. We found that classical pathway C3 convertase regulatory function resides within SCR-2 and SCR-3, while alternative pathway C3 convertase regulatory function resides within SCR-2, -3, and -4. Functional comparisons of the variant DAF proteins in fluid phase C3 activation assays established that the differences reflect domain-specific interactions rather than changes in the spatial arrangement of SCRs above the cell surface. In accordance with these findings, we found that variant DAF molecules containing SCR-1, -2, and -3, but not SCR-4, function to selectively inhibit classical pathway activation.