Epstein–Barr virus (EBV)-positive T- or NK-cell neoplasms show progressive systemic inflammation and abnormal blood coagulation causing hemophagocytic lymphohistiocytosis (HLH). It was reported that inflammatory cytokines were produced and secreted by EBV-positive neoplastic T- or NK-cells. These cytokines can induce the differentiation of monocytes into macrophages leading to HLH. To clarify which products of EBV-positive neoplastic T- or NK-cells have effects on monocytes, we performed a co-culture assay of monocytes with the supernatants of EBV-positive T- or NK-cell lines. The expression of differentiation markers, the phagocytosis ability, and the mRNA expression of the inflammatory cytokines of THP-1, a monocytic cell line, clearly increased after culturing with the supernatants from EBV-NK-cell lines. Co-culturing with the supernatants promoted the expression of CD80 and CD206 as well as M1 and M2 macrophage markers in human monocytes. Co-culturing with the supernatants of EBV-NK-cell lines significantly enhanced the procoagulant activity and the tissue factor expression of monocytes. Interferon (IFN)-γ was elevated extremely not only in the supernatant of EBV-NK-cell lines but also in the plasma of EBV-positive NK-cell neoplasms patients accompanying HLH. Finally, we confirmed that IFN-γ directly enhanced the differentiation into M1-like macrophages and the procoagulant activity of monocytes. Our findings suggest that IFN-γ may potentially serve as a therapeutic target to regulate HLH in EBV-positive NK-cell neoplasms.

Leukocytes are blood-borne cells derived from the bone marrow that must migrate into tissues to mediate inflammation. Controlling this migration, therefore, represents a critical point for the development of new therapeutic strategies to attenuate inflammation. Traditional endpoint studies have not allowed us to fully dissect the migratory behavior of leukocytes in vivo. Recent advances in imaging technology, however, have provided unprecedented insights into immune cell migration in live animals, greatly enhancing our understanding of the molecular regulation of immune cell trafficking. Here, we present a detailed protocol for in vivo imaging of the joints using a murine type II collagen-induced arthritis (CIA) model. Mice are immunized with type II collagen emulsified in complete Freund's adjuvant, and arthritis progression is monitored by clinical scoring and paw thickness measurements. At the peak of arthritis, leukocytes are visualized in vivo using confocal microscopy in transgenic mice expressing fluorescent proteins or in wild-type mice after intravenous injection of fluorescently labeled antibodies and high-molecular-weight Dextran for vessel staining. Careful surgical exposure of the joint, application of agarose, and proper positioning of the leg allow stable long-term imaging, typically lasting several hours. This approach enables real-time analysis of leukocyte arrest, crawling, and transendothelial migration within inflamed joints. This protocol will be valuable for research groups investigating leukocyte trafficking in arthritis and other inflammatory diseases. It may also serve as a platform for preclinical evaluation of therapies targeting immune cell migration.