RESUMEN
The cytokine-inducing potential of various microspheres were evaluated in a short-time screening assay of lepirudin-anticoagulated human whole blood utilizing the Bio-Plex Human cytokine 27-plex system. The inflammatory cytokines IL-1ß, TNF and IL-6; the anti-inflammatory mediators IL-1ra and IL-10; the chemokines IL-8, MIP-1α and MCP-1; and the growth factor VEGF were induced by polycation (poly-l-lysine or poly(methylene-co-guanidine)) containing microspheres. Alginate microspheres without polycations did not induce the corresponding cytokine panel, nor did soluble alginate. By inhibiting complement C3 using compstatin analog CP20, a total inhibition of complement activation as well as the inflammatory mediators was achieved, indicating that complement activation alone was responsible for the induced cytokines. A strong deposition of C3c on the poly-l-lysine containing surface, while not on the microspheres lacking polycations, also points to the formation of C3 convertase as involved in the biomaterial-induced cytokine induction. These results show that complement is responsible for the induction of cytokines by polycation containing microspheres. We point to complement as an important initiator of inflammatory responses to biomaterials and the lepirudin anticoagulated whole blood assay as an important tool to identify the most tolerable and safe materials for implantation to humans.
Asunto(s)
Activación de Complemento , Citocinas/sangre , Citocinas/inmunología , Guanidinas/inmunología , Poliaminas/inmunología , Polilisina/inmunología , Alginatos/metabolismo , Materiales Biocompatibles/metabolismo , Quimiocina CCL3/sangre , Quimiocina CCL3/inmunología , Activación de Complemento/efectos de los fármacos , Complemento C3/antagonistas & inhibidores , Humanos , Mediadores de Inflamación/sangre , Mediadores de Inflamación/inmunología , Proteína Antagonista del Receptor de Interleucina 1/sangre , Proteína Antagonista del Receptor de Interleucina 1/inmunología , Interleucina-10/sangre , Interleucina-10/inmunología , Interleucina-6/sangre , Interleucina-6/inmunología , Interleucina-8/sangre , Interleucina-8/inmunología , Microesferas , Péptidos Cíclicos/farmacología , Polielectrolitos , Factores de Necrosis Tumoral/sangre , Factores de Necrosis Tumoral/inmunología , Factor A de Crecimiento Endotelial Vascular/sangre , Factor A de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
Alginate microbeads and microcapsules are presently under evaluation for future cell-based therapy. Defining their inflammatory properties with regard to humans is therefore essential. A lepirudine-based human whole blood model was used as an inflammation predictor by measuring complement and leukocyte stimulation. Alginate microbeads were complement-compatible since they did not activate complement as measured by the soluble terminal complement complex (sTCC), Bb or the anaphylatoxins C3a and C5a. In addition, alginate microbeads were free of surface adherent leukocytes. In contrast, microcapsules containing poly-L-lysine (PLL) induced elevated levels of sTCC, Bb, C3a and C5a, surface active C3 convertase and leukocyte adhesion. The soluble PLL induced elevated levels of sTCC and up-regulated leukocyte CD11b expression. PMCG microcapsules containing poly(methylene-co-guanidine) complexed with sodium alginate and cellulose sulfate triggered a fast sTCC response and C3 deposition. The PMCG microcapsules were still less activating than PLL-containing microcapsules as a function of time. The amounts of anaphylatoxins C3a and C5a were diminished by the PMCG microcapsules, whereas leukocyte adherence demonstrated surface activating properties. We propose the whole blood model as an important tool for measuring bioincompatibility of microcapsules and microbeads for future applications as well as determining the mechanisms leading to inflammatory reactions.
Asunto(s)
Alginatos , Materiales Biocompatibles , Sangre , Proteínas del Sistema Complemento/fisiología , Microesferas , Modelos Biológicos , Adhesión Celular , Activación de Complemento , HumanosRESUMEN
The alginate capsule produced by the human pathogen Pseudomonas aeruginosa is composed mainly of mannuronic acid polymers (poly-M) that have immunostimulating properties. Poly-M shares with lipopolysaccharide the ability to stimulate cytokine production from human monocytes in a CD14-dependent manner. In the present study we examined the role of Toll-like receptor (TLR) 2 and TLR4 in responses to poly-M. Blocking antibodies to TLR2 and TLR4 partly inhibited tumor necrosis factor production induced by poly-M in human monocytes, and further inhibition was obtained by combining the antibodies. By transiently transfecting HEK293 cells, we found that membrane CD14 together with either TLR2 or TLR4/MD-2 could mediate activation by poly-M. Transfection of HEK293 cells with TLR2 and fluorescently labeled TLR4 followed by co-patching of TLR2 with an antibody revealed no association of these molecules on the plasma membrane. However, macrophages from the Tlr4 mutant C3H/HeJ mice and TLR4 knockout mice were completely non-responsive to poly-M, whereas the tumor necrosis factor release from TLR2 knockout macrophages was half of that seen with wild type cells. Taken together the results suggest that both TLR2 and TLR4 are involved in cell activation by poly-M and that TLR4 may be required in primary murine macrophages.