RESUMO
Monocytes (MO) migrating into normal, non-inflamed intestinal mucosa undergo a specific differentiation resulting in a non-reactive, tolerogenic intestinal macrophage (IMAC). Recently we demonstrated the differentiation of MO into an intestinal-like macrophage (MAC) phenotype in vitro in a three-dimensional cell culture model (multi-cellular spheroid or MCS model). In the mucosa of patients with inflammatory bowel disease (IBD) in addition to normal IMAC, a reactive MAC population as well as increased levels of monocyte chemoattractant protein 1 (MCP-1) is found. The aim of this study was to investigate the influence of MCP-1 on the differentiation of MO into IMAC. MCS were generated from adenovirally transfected HT-29 cells overexpressing MCP-1, macrophage inflammatory protein 3 alpha (MIP-3alpha) or non-transfected controls and co-cultured with freshly elutriated blood MO. After 7 days of co-culture MCS were harvested, and expression of the surface antigens CD33 and CD14 as well as the intracellular MAC marker CD68 was determined by flow-cytometry or immunohistochemistry. MCP-1 and MIP-3alpha expression by HT-29 cells in the MCS was increased by transfection at the time of MCS formation. In contrast to MIP-3alpha, MCP-1 overexpression induced a massive migration of MO into the three-dimensional aggregates. Differentiation of IMAC was disturbed in MCP-1-transfected MCS compared to experiments with non-transfected control aggregates, or the MIP-3alpha-transfected MCS, as indicated by high CD14 expression of MO/IMAC cultured inside the MCP-1-transfected MCS, as shown by immunohistochemistry and FACS analysis. Neutralization of MCP-1 was followed by an almost complete absence of monocyte migration into the MCS. MCP-1 induced migration of MO into three-dimensional spheroids generated from HT-29 cells and inhibited intestinal-like differentiation of blood MO into IMAC. It may be speculated that MCP-1 could play a role in the disturbed IMAC differentiation in IBD mucosa.
Assuntos
Quimiocina CCL2/metabolismo , Mucosa Intestinal/citologia , Macrófagos/citologia , Monócitos/citologia , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Biomarcadores/análise , Diferenciação Celular/efeitos dos fármacos , Movimento Celular , Quimiocina CCL2/genética , Quimiocina CCL20 , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Depressão Química , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Células HT29 , Humanos , Imuno-Histoquímica , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Receptores de Lipopolissacarídeos/análise , Proteínas Inflamatórias de Macrófagos/genética , Proteínas Inflamatórias de Macrófagos/metabolismo , Macrófagos/imunologia , Monócitos/imunologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Transfecção/métodosRESUMO
A calcium-stimulated protease was purified to apparent homogeneity from the heterocyst-forming cyanobacterium Anabaena variabilis ATCC 29413. As judged from experiments with inhibitors and chromogenic peptide substrates, the enzyme is a serine protease with a substrate specificity like trypsin. Its apparent relative molecular mass is 52,000. Calcium depletion inhibits the enzymic activity by 92%. Half-maximal activity requires about 0.5 microM free Ca2+. The enzyme binds to a hydrophobic column in a calcium-dependent manner, indicating calcium-induced exposure of a hydrophobic domain. The possible role of the protease in heterocyst differentiation is discussed.