Cytokine production consequent to T cell–microglia interaction: the PMA/IFNγ-treated U937 cells display similarities to human microglia

Abstract

Cognate interactions between human adult microglia and activated T lymphocytes induce the production of inflammatory cytokines. Since this interaction can occur in a non-antigen-dependent manner, it is relevant to a variety of CNS diseases where activated T cells, regardless of specificities, come into contact with microglia; these disorders include multiple sclerosis, trauma, stroke and Alzheimer's disease. A model cell line would facilitate studies of the engagement between T cells and human adult microglia, since the latter are difficult to obtain in substantial quantity or frequency. This study shows that the PMA/IFNγ-treated U937 cell line shows similarities to microglia in its interaction with activated T lymphocytes, in that the production of tumor necrosis factor (TNF)-α, interleukin (IL)-4, IL-10 and IL-12 is induced. Morphological features and mechanisms of cytokine production resemble those observed in microglia–T cell co-cultures since CTLA-4 and CD40–CD40L blockades reduce TNF-α and IL-10 levels, while anti-CD23 inhibits IL-10 only in U937–T cell interactions. We propose that PMA/IFNγ-treated U937 cells can serve as a model of human adult microglia to study cytokine generation in response to interactions with activated T cells.

Introduction

Microglia are the immune effector cells of the central nervous system (CNS). They serve specific functions in the response of the CNS to injury, in the defense of the CNS against pathogens, and in the removal of tissue debris during normal development. Microglia are often considered as the macrophages of the CNS. In order to achieve their functions, microglia need to be activated. Several mechanisms of microglia activation have been described. Soluble cytokines, such as interferon-γ (IFNγ), produced by T lymphocytes and natural killer (NK) cells, can trigger the microglial production of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β (Meda et al., 1995, Liu et al., 1998). Another mechanism of microglia activation involves the bacterial cell wall component lipopolysaccharide (LPS). In vitro, treatment with a combination of LPS and IFNγ induces the production of reactive nitrogen oxides (NO, ONOO⁻), reactive oxygen intermediates (superoxide anion, H₂O₂), and enzymes, such as lysozyme, cathepsin B/L, and acid hydrolases (Colton et al., 1987, Boje and Arora, 1992, Banati et al., 1993, Liu et al., 1996). This has also been shown in vivo. After injecting a mixture of LPS and IFNγ into the rat hippocampus, microglia are activated as shown by morphological changes and by an increase in IL-1β and iNOS immunostaining (Hartlage-Rubsamen et al., 1999).

Cognate interactions with activated T lymphocytes can also result in microglia activation. We have previously demonstrated that cytokine production, in particular for TNF-α and IL-10, is induced from interactions between human adult microglia and activated T lymphocytes (Chabot et al., 1997, Chabot et al., 1999). This occurs in a non-antigen-specific manner and it is contact dependent, involving molecules such as CD40 and B7 (Chabot et al., 1997, Chabot et al., 1999). Similarly, the ligation of microglial CD40 by CD40L was shown to induce the production of TNF-α (Tan et al., 1999). The contact-dependent T-cell-induced activation of microglia may be an important pathogenic event in multiple sclerosis (MS), since it is believed that the majority of T cells (over 95%) that infiltrate MS lesions are activated, but are non-antigen specific or non-reactive to myelin antigens (Sedgwick et al., 1987, Cross et al., 1990). T cells are also found in the CNS in brain trauma, and in conditions such as Alzheimer's disease and stroke. Thus, the study of microglia–T cell interactions in an antigen-independent context is of great significance. However, microglial cells, particularly from adult human brains at surgical resections, are difficult to obtain in significant quantity and frequency, even though we have acquired extensive experience with their culture in vitro (Yong and Antel, 1997).

The myelomonoblast-like human histiocytic lymphoma cell line U937 (Sundstrom et al., 1976; obtainable from the American Type Culture Collection) is used as a model for human macrophages when treated with phorbol-12-myristate-13-acetate (PMA) (Larrick et al., 1980, Hewison et al., 1992). There is evidence that cellular differentiation of U937 cells into macrophages induced by PMA is the result of growth inhibition (Vrana et al., 1998). In this study, we investigated whether the U937 cells bear similarities to microglia, particularly in their cognate interactions with activated T cells to produce cytokines, and whether the mechanisms involved are similar to those identified in microglia–T cell interactions. If so, then the U937 cell line constitutes a valuable and limitless resource and model to better understand the biochemical and molecular mechanisms that lead to cytokine production in T cell–microglia interaction, an engagement of importance in coordinating inflammation in the CNS.