Chronic Inflammatory Conditions Enhancing Neutrophil Infiltration under Regulation of the Inflammatory Response:

Neutrophil (polymorphonuclear leukocytes [PMN]) inﬁltration plays a central role in inﬂammation and is also a major cause of tissue damage. Thus, PMN inﬁltration must be tightly controlled. Using zymosan-induced peritonitis as an in vivo PMN inﬁltration model, we show in this study that PMN response and inﬁltration were signiﬁcantly enhanced in mice experiencing various types of systemic inﬂammation, including colitis and diabetes. Adoptive transfer of leukocytes from mice with inﬂammation into healthy recipients or from healthy into inﬂammatory recipients followed by inducing peritonitis demonstrated that both circulating PMN and tissue macrophages were altered under inﬂammatory conditions and that they collectively contributed to enhanced PMN inﬁltration. Detailed analyses of dextran sulfate sodium-elicited colitis revealed that enhancement of PMN inﬁltration and macrophage function occurred only at the postacute/chronic phase of inﬂammation and was associated with markedly increased IL-17A in serum. In vitro and ex vivo treatment of isolated PMN and macrophages conﬁrmed that IL-17A directly modulates these cells and signiﬁcantly enhances their inﬂammatory responses. Neutralization of IL-17A eliminated the enhancement of PMN inﬁltration and IL-6 production and also prevented severe tissue damage in dextran sulfate sodium-treated mice. Thus, IL-17A produced at the chronic stage of colitis serves as an essential feedback signal that enhances PMN inﬁltration and promotes inﬂammation. The Journal of Immunology , 2012, 188: 000–000. 3 ml cold HBSS without Ca 2+ and Mg 2+ followed by analysis with myeloperoxidase (MPO) assay, cell counting, and immunoﬂuorescence labeling using a PE-Cy5–conju- gated anti-mouse Ly-6G (Gr-1) Ab (eBioscience). Macrophages were analyzed using an Alexa Fluor 488-conjugated anti-mouse F4/80 Ab (clone BM8; BioLegend). For leukocyte adoptive transfer experiments, bone marrow cells were obtained by ﬂushing femur bone cavities followed by la- beling with the ﬂuorescent cell tracer CFSE or 1,1’-dihexadecyl-3,3,3 9 ,3 9 tetramethylindocarbocyanine iodide [DiIC 16 (3)] (both from Invitrogen). In some experiments, the bone marrow cells were further separated by a Per-coll (GE Healthcare) density gradient to obtain the population of mature PMN (12). In general, 5 3 10 6 labeled cells ( ∼ 2.5 3 10 6 were Gr-1 + ) were transfused into a recipient mouse via i.v. injection before inducing perito- nitis using zymosan. To determine the role of IL-17A in PMN inﬁltration, ex vivo experiments were performed. In these experiments, freshly isolated bone marrow cells were labeled with CFSE followed by treatment with a recombinant mouse IL-17A (rIL-17A) (2 ng/ml; BioLegend), with or without the presence of a neutralization anti–IL-17A Ab (low endotoxin, azide-free–puriﬁed anti-mouse IL-17A Ab [BioLegend]), or 50 m l serum collected from nontreated mice or mice treated with DSS for 12 d. After incubation for 2 h (37˚C), the cells ( ∼ 2.5 3 10 6 PMN) were combined with equal amount of DiIC 16 (3)-labeled bone marrow PMN before transfer into healthy recipient mice. Zymosan-induced peritonitis was then performed to test the transferred PMN