The 18kDa Translocator Protein (TSPO) is the most commonly used tissue-specific marker of inflammation in positron emission tomography (PET) studies. It is expressed in myeloid cells such as microglia and macrophages, and in rodent myeloid cells expression increases with cellular activation. We assessed the effect of myeloid cell activation on TSPO gene expression in both primary human and rodent microglia and macrophages in vitro, and also measured TSPO radioligand binding with 3H-PBR28 in primary human macrophages. As observed previously, we found that TSPO expression increases (∼9-fold) in rodent-derived macrophages and microglia upon pro-inflammatory stimulation. However, TSPO expression does not increase with classical pro-inflammatory activation in primary human microglia (fold change 0.85 [95% CI 0.58–1.12], p = 0.47). In contrast, pro-inflammatory activation of human monocyte-derived macrophages is associated with a reduction of both TSPO gene expression (fold change 0.60 [95% CI 0.45–0.74], p = 0.02) and TSPO binding site abundance (fold change 0.61 [95% CI 0.49–0.73], p < 0.0001). These findings have important implications for understanding the biology of TSPO in activated macrophages and microglia in humans. They are also clinically relevant for the interpretation of PET studies using TSPO targeting radioligands, as they suggest changes in TSPO expression may reflect microglial and macrophage density rather than activation phenotype.

The translocator protein (TSPO) is a mitochondrial membrane protein, of as yet uncertain function. Its purported high expression on activated macrophages, has lent utility to TSPO targeted molecular imaging in the form of positron emission tomography (PET), as a means to detect and quantify inflammation in vivo. However, existing literature regarding TSPO expression on human activated macrophages is lacking, mostly deriving from brain tissue studies, including studies of brain malignancy, and inflammatory diseases such as multiple sclerosis. Here, we utilized three human sources of monocyte derived macrophages (MDM), from THP-1 monocytes, healthy peripheral blood monocytes and synovial fluid monocytes from patients with rheumatoid arthritis, to undertake a detailed investigation of TSPO expression in activated macrophages. In this work, we demonstrate a consistent down-regulation of TSPO mRNA and protein in macrophages activated to a pro-inflammatory, or ‘M1’ phenotype. Conversely, stimulation of macrophages to an M2 phenotype with IL-4, dexamethasone or TGF-β1 did not alter TSPO expression, regardless of MDM source. The reasons for this are uncertain, but our study findings add some supporting evidence for recent investigations concluding that TSPO may be involved in negative regulation of inflammatory responses in macrophages.

PET radioligands targeted to translocator protein (TSPO) offer a highly sensitive and specific means of imaging joint inflammation in rheumatoid arthritis (RA). Through high expression of TSPO on activated macrophages, TSPO PET has been widely reported in several studies of RA as a means of imaging synovial macrophages in vivo. However, this premise does not take into account the ubiquitous expression of TSPO. This study aimed to investigate TSPO expression in major cellular constituents of RA pannus-monocytes, macrophages, fibroblastlike synoviocytes (FLS cells), and CD4-positive (CD4+) T lymphocytes (T cells)-to more accurately interpret TSPO PET signal from RA synovium. Three RA patients and 3 healthy volunteers underwent PET of both knees using the TSPO radioligandC-PBR28. Through H-PBR28 autoradiography and immunostaining of synovial tissue in 6 RA patients and 6 healthy volunteers, cellular expression of TSPO in synovial tissue was evaluated. TSPO messenger RNA expression andH-PBR28 radioligand binding was assessed using in vitro monocytes, macrophages, FLS cells, and CD4+ T cells. C-PBR28 PET signal was significantly higher in RA joints than in healthy joints (average SUV, 0.82 ± 0.12 vs. 0.03 ± 0.004; < 0.01). Further, H-PBR28-specific binding in synovial tissue was approximately 10-fold higher in RA patients than in healthy controls. Immunofluorescence revealed TSPO expression on macrophages, FLS cells, and CD4+ T cells. The in vitro study demonstrated the highest TSPO messenger RNA expression andH-PBR28-specific binding in activated FLS cells, nonactivated M0 macrophages, and activated M2 reparative macrophages, with the least TSPO expression being in activated and nonactivated CD4+ T cells. To our knowledge, this study was the first evaluation of cellular TSPO expression in synovium, with the highest TSPO expression and PBR28 binding being found on activated synovial FLS cells and M2 macrophages. TSPO-targeted PET may therefore have a unique sensitivity in detecting FLS cells and macrophage-predominant inflammation in RA, with potential utility for assessing treatment response in trials using novel FLS-cell-targeted therapies.