Characterization of the Type II Activated Macrophage and the Regulation of Heparin Binding EGF-like Growth Factor

Abstract

Three populations of activated macrophages were generated in vitro and characterized each. These include the classically (Ca-MΦ), alternatively (AA-MΦ), and type II activated (MΦ-II) macrophages. Here, a side-by-side comparison of the three cell types is presented, focusing primarily on differences between MΦ-II and AA-MΦ, because both have previously been classified as M2 macrophages, distinct from Ca-MΦ. I show that MΦ-II are similar to Ca-MΦ in that MΦ-II and Ca-MΦ, but not AA-MΦ, produce high levels of NO and have low arginase activity. MΦ-II and Ca-MΦ express relatively high levels of CD86, whereas AA-MΦ are virtually devoid of this co-stimulatory molecule. Ca-MΦ and MΦ-II are efficient antigen presenting cells (APCs), whereas AA-MΦ fail to stimulate efficient T-cell proliferation. The differences between Ca-MΦ and MΦ-II are more subtle. Ca-MΦ produce IL-12/23 and give rise to Th1 cells when used as APCs. MΦ-II produce high levels of IL-10 and low IL-12 and thus give rise to Th2 cells secreting IL-4 and IL-10. I identify two new markers for the MΦ-II, sphingosine kinase and LIGHT. Thus, classically, type II, and alternatively activated macrophages represent three distinct populations of cells with different biological functions.

The expression of Heparin-Binding Epidermal Growth Factor-like Growth Factor (HB-EGF) by MΦ-II is then characterized. HB-EGF has previously been associated with a number of physiological and pathological conditions, including tumor growth and angiogenesis. MΦ-II exhibit increased HB-EGF mRNA levels and protein secretion relative to resting and Ca-MΦ. HB-EGF induction is dependent upon the activation of the MAPKs ERK1/2 and p38. The transcription factor, Sp1, was recruited to 3 sites within the first 2kb of the HB-EGF promoter following stimulation, and the site located at -83/-54 was required for HB-EGF promoter activity. These regions of the promoter became more accessible to endonuclease activity following activation, and this accessibility was contingent upon activation of ERK. We show that several conditions which enhanced macrophage IL-10 production also resulted in HB-EGF induction, suggesting that these two genes may be coordinately regulated. These observations indicate that in addition to the secretion of the anti-inflammatory IL-10, another characteristic of MΦ-II is their production of angiogenic HB-EGF.