The modelling of mononuclear phagocyte—connective tissue adhesion in vitro: application to disclose a specific inhibitory effect of Leishmania infection

Abstract

In this work, we have developed an adhesion assay to study interactions between mononuclear phagocytes and connective tissue in vitro and show its potential use to study diseases caused by intracellular microorganisms. The assay reproduces most of the characteristics of macrophage adhesion to connective tissue in vivo, such as: preferential adhesion to inflamed connective tissue, divalent cation and integrin dependence, and up-regulation upon cell activation. The phagocyte adhesion to connective tissue was inhibited by infection with Leishmania (58+/-22%, p < 0.05) and was not affected by infection with Mycobacterium or by endocytosis of latex beads. Manganese partially reverted the loss in adherence produced by Leishmania infection, indicating that the mechanisms regulating the function of integrins are affected by cell infection with Leishmania. This assay might be a useful tool for the study of the mechanisms by which mononuclear phagocytes play a role in the immune-inflammatory response and in the development of lesions.

LeishmaniaInfection Impairs β1-Integrin Function and Chemokine Receptor Expression in Mononuclear Phagocytes

“…In our previous report, we noticed that the reduction of adhesion observed after phagocyte infection by Leishmania was variable (11). Here, we show that differences in the intensity of infection cause such variation.…”

“…Ten-millimeter-diameter circles were drawn around each section using a nontoxic marker pen (Pap Pen; Daido Sangyo, Japan) to prevent the free flow of the cell suspension over the slides. The purpose of using gelatin-coated glass slides was to minimize the adherence of macrophages to the glass (11).…”

“…Leukocyte-connective tissue adhesion assays were performed according to a previously described method (11 and Mg 2ϩ (250 M) to remove nonadherent cells, and fixed for 30 min in 1% glutaraldehyde. Slides were then stained with hematoxylin and eosin and examined under light microscopy.…”

“…Using this tool, we have shown that different Leishmania species reduce the adherence of phagocytes to the inflamed connective tissue and suggested that mechanisms regulating integrin affinity would be involved in such modulation of cell adhesion by Leishmania (11). Thus, the observed decrease of cell adherence early upon Leishmania infection may account for the first stage of this migratory process.…”

“…We have recently reported the development of an in vitro adhesion assay to study the interactions of mononuclear phagocytes with the connective tissue (11). Using this tool, we have shown that different Leishmania species reduce the adherence of phagocytes to the inflamed connective tissue and suggested that mechanisms regulating integrin affinity would be involved in such modulation of cell adhesion by Leishmania (11).…”