A Macrophage Activation Switch (MAcS)-Index for Assessment of Monocyte/Macrophage Activation

BACKGROUND: The monocyte/macrophage system plays important roles in host defense, regulation of immune responses, tissue repair, neovascularization, and inflammation. These diverse roles are performed by specific subpopulations of macrophages that are differently activated by surrounding stimuli, simplified by the M1-M2 dichotomy of classically activated (M1), pro-inflammatory cells and alternatively activated (M2), anti-inflammatory cells. Macrophages, however, display a large degree of flexibility and are able to switch between activation states (1)

The hemoglobin scavenger receptor CD163 is expressed exclusively on monocytes and macrophages, and its expression is strongly induced by anti-inflammatory stimuli like IL10 and glucocorticoid, making CD163 an ideal M2 macrophage marker (2)

Furthermore a soluble variant of CD163 (sCD163) is shed from the cell surface to plasma by protease mediated cleavage of the receptor. The shedding of sCD163 is dependent on a pro-inflammatory signal such as toll-like receptor (TLR) activation by LPS (3)

These unique properties led us to investigate whether an index of combined soluble- and monocyte membrane CD163 could be used as a surrogate marker for macrophage activation.

MATERIALS AND METHODS: Blood sample were obtained from 53 patients with malignant hematological disease and 74 healthy individuals. The cellular (mCD163) and soluble (sCD163) CD163 expression were measured by flow cytometry and ELISA respectively. In addition, blood mRNA expression of inflammatory markers (IL-1β, IL-6, IL-8, IL-10, and TNF-α) was determined by RT-qPCR. Normalized values of sCD163 and mCD163 were calculated by dividing each value by the median value of the healthy population. The MAcS-index was then calculated as the ratio between normalized sCD163 and normalized mCD163. A MAcS-index > 1 indicates relative increase in sCD163 as compared to mCD163, suggested to reflect a predominant M1 activation.

RESULTS AND DISCUSSION: The MAcS-index of healthy individuals clustered around 1 (2.5–97.5 percentile: 0.28–3.11), whereas the MAcS-index of the patients varied from 0.06 to 5139, with 4% below the 2.5 % limit of healthy individuals, and 60% above the 97.5 upper limit of healthy individuals. The MAcS-index in infected patients (with assumed M1 activation) was clearly elevated: The index was significantly higher in patients with clinical signs of infection (median: 9.01; range: 1.41–3490) and patients in antibiotic therapy (median: 9.74; range: 0.91–5139) compared to non-infected patients (median: 2.53; range: 0.058–551.5, p<0.05) and non-treated patients (median: 0.97; range: 0.058– 450.8, p<0.0001), respectively. In contrast, patients in glucocorticoid treatment (assumed to drive an M2 activation phenotype) had significantly lower index (median: 1.1; range: 0.058–551) than patients without glucocorticoid treatment (median: 9.4; range: 0.57– 3490), p<0.05. mRNA expression analysis revealed that patients with MAcS-index above upper reference limit expressed higher levels of IL-1β, IL-6, IL-8, and TNF-α compared to patients with MAcS-index within reference range. Patients with malignant myeloma (median: 0.89; range: 0.63–1.51) had significantly lower MAcS-index than patients with AML (median: 20.71; range: 0.57–2214) (p<0.005) and lymphoma (median: 4.34; range 0.058–5139) (p<0.01), suggesting a differentiation towards an anti-inflammatory state.

CONCLUSION: We present a CD163-derived macrophage activation switch (MAcS)- index, which seems able to differentiate between (predominantly) pro-inflammatory and anti-inflammatory macrophage activation. The index needs further validation, however, may be very useful for monitoring diseases with macrophage involvement and response to therapeutic interventions.