Yuya Yoshioka^1,2, Tatsuaki Mizutani¹, Satoshi Mizuta³, Ayumi Miyamoto^1,2, Satoru Murata¹, Toshiaki Ano^1,2, Hiroshi Ichise¹, Daisuke Morita^1,2, Hiroyuki Yamada⁴, Yoshihiko Hoshino⁵, Tatsuaki Tsuruyama⁶, and Masahiko Sugita^1,2

(1Laboratory of Cell Regulation, Institute for Virus Research, Kyoto University, Kyoto, Japan; 2Laboratory of Cell Regulation and Molecular Network, Graduate School of Biostudies, Kyoto University, Kyoto, Japan; 3Center for Bioinformatics and Molecular Medicine, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan; 4Department of Mycobacterium Reference and Research, Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, Kiyose, Tokyo, Japan; 5Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases, Higashi-Murayama, Tokyo, Japan; and 6Department of Diagnostic Pathology, Kyoto University Hospital, Kyoto, Japan)

Blood Advances 2016 1:184-192; doi: https://doi.org/10.1182/bloodadvances.2016000497

Macrophages have the potential to undergo cellular transformation into epithelioid cells, and their concentric accumulation in tissues results in the development of granulomas.
Although epithelioid cells are an essential and dominant component of granulomas, other cell types have also been detected, which may contribute to the establishment of well-organized granulomas, as observed in human granulomatous diseases.
We have demonstrated that neutrophils may mediate these functions. By taking advantage of the guinea pig pulmonary granuloma model, we obtained a rat monoclonal antibody with unique reactivity to granuloma cells.
This antibody, termed G213, reacted with clusters of neutrophils located in the central area of granulomas, and a biochemical analysis identified the G213-reactive antigen as S100A9, a calcium-binding protein of the S100 family, which was expressed abundantly in neutrophils.
Consistent with the multifaceted functions attributed to S100A9, including its role in neutrophil extravasation and macrophage activation, the blockade of S100A9 functions with the specific inhibitor, tasquinimod, impaired the formation of organized granulomas with neutrophil cores.
These results demonstrate the critical role of neutrophils and the S100A9 protein in granuloma formation. Since intra-granuloma S100A9⁺ neutrophils were also detected in humans, these results indicate the potential of tasquinimod, a new anti-cancer drug candidate, for manipulating human granulomatous diseases.

Figure. S100A9⁺ neutrophils are critical for granuloma formation.

(A) The effect of tasquinimod on granuloma formation. BCG-challenged guinea pigs were either mock-treated or treated with tasquinimod (TasQ), and the lung sections were labeled with G213 mAb, followed by HRP-DAB-based detection. Granulomas containing the central accumulation of S100A9-expressing neutrophils are indicated with arrows. Scale bars, 500 μm.
(B) A proposed model for the neutrophil S100A9-dependent granuloma formation. The neutrophil S100A9 protein is critically involved in multiple steps of granuloma formation. Circulating neutrophils are activated by S100A9 in an autocrine fashion and migrate into insulted tissue. Local accumulation of S100A9⁺ neutrophils contributes to persistent recruitment of macrophages and their transformation into epithelioid cells in a paracrine fashion, resulting in the construction of mature granulomas.