A human in vitro granuloma model for the investigation of multinucleated giant cell and granuloma formation

Abstract

A method for the in vitro generation of granulomas and its use in the analysis of the human granulomatous response is summarized. As a target for the cellular response L3 larvae of Nippostrongylus brasiliensis are coincubated with human mononuclear blood cells, and within seven to fourteen days the development of blood monocytes to mature macrophages and to epithelioid cells and multinucleated giant cells (MGC) as typical constituents of granulomas clustered around the nematode is observed. The following review describes the uses and applications of this model for phenotyping, functional, formation and modulating studies of granulomas and MGCs, taking into account its unique features compared to other in vitro models. With respect to MGC formation, procedures are described and examples are given which allow the phenotyping of these cells using immunofluorescence and immunohistological techniques. In addition, the potential of this model for illuminating functional aspects of MGC is described applying an isolation protocol for MGC and a subsequent reverse-transcriptase polymerase chain reaction method for the analysis of single cells. Moreover, the significance and relevance of using this granuloma model is discussed in the follow up analysis of in vivo findings of interleukin-6 expression in MGC of granulomas of patients with sarcoidosis. These in vivo results implicated a role for interleukin-6 in granuloma and MGC development. The in vitro granuloma model was used to investigate potential modulatory effects of this cytokine by analysing the cell numbers and the number of MGC per in vitro granuloma, the size of the MGC formed, the fusion index and the morphology of the in vitro granuloma. The results demonstrated significant modulatory effects of interleukin-6 on the cell number per in vitro granuloma and on the morphology of the cells involved. Conceivably, elevated interleukin-6 levels may modulate granuloma formation with respect to the number of cells involved and in influencing distinct cell populations involved in granuloma formation.