Neutrophils migrate to CXCL5 via a CXCR2-dependent mechanism

Neutrophils migrate to CXCL5 via a CXCR2-dependent mechanism. immune pathways in chronic inflammation. == Introduction == Chronic inflammation is characterized by unremitting immune responses to persistent microbial contamination or chemical brokers (1). Continued influx of leukocytes and local production of inflammatory mediators are common features at sites of chronic inflammation. Although chemokine gradients play a prominent role in leukocyte migration, the mechanisms responsible for the sustained chemokine production and subsequent influx of neutrophils and monocytes in chronic inflammation are not well defined. Exposure to naturally-occurring hydrocarbon oils is associated with the development of chronic inflammation and a variety of pathological findings in humans and animal models (25). Due to their ability to enhance and sustain inflammation, hydrocarbons are often utilized as adjuvants in vaccines (6,7). Among the most potent hydrocarbons in eliciting chronic inflammation is the medium-length alkane 2,6,10,14 tetramethylpentadecane (TMPD; also known as pristane). A single intraperitoneal dose of TMPD elicits infiltration of neutrophils and monocytes into the peritoneal cavity for several months (8). The chronic inflammatory response promotes the formation of plasmacytomas and lipogranulomas, a form of ectopic lymphoid tissue(5,9). Depending on the genetic background, persistent inflammation in TMPD-treated mice also promotes the development of a plethora of autoimmune AN-2690 manifestations including autoantibodies, glomerulonephritis, arthritis, and pulmonary vasculitis(913). In addition, TMPD augments monoclonal antibody production by hybridoma cells by stimulating IL-6 production (14). Recent studies have begun to unravel the mechanisms responsible for the chronic inflammation induced by TMPD. The response to TMPD is usually orchestrated by major components of the innate immune system. The continued influx of Ly6Chiinflammatory monocytes to the peritoneal cavity requires the presence of type-I interferon (IFN-I) production downstream of Toll-like receptor (TLR)-7 signaling (15). AN-2690 IFN-I activates the production of the monocyte chemoattractants CCL2, CCL7 and CCL12, which collectively recruit monocytes to the site of inflammation in a CC-chemokine receptor 2 (CCR2)-dependent manner (16). The persistent infiltration of neutrophils, on the other hand, remains largely unexplained. In this study, we aimed to define the mechanism of neutrophil recruitment in TMPDinduced chronic inflammation. == Materials and Methods == == Mice == These studies were approved by the Institutional Animal Care and Use Committee. Wild-type C57BL/6, TNF-/, CCR2/and IL-1R/mice (all on a C57BL/6 background), BALB/c, CXCR2/(BALB/c background), C3H/HeJ, C3H/HeOuJ, and CBA/CaJ mice were from Jackson Laboratories (Bar Harbor, ME). FcR-chain/mice AN-2690 were from Taconic (Hudson, NY) and 129/Sv mice were from B&K Universal Limited (Grimston, Aldbrough, England). Mice were maintained in a specific pathogen free (SPF) facility at CALCR the Malcolm Randall VA Medical (Gainesville, FL). MyD88/, AN-2690 ASC/, Nalp3/, caspase-1/, IRAK-1/, IRAK-2/, IRAK-1/IRAK-2/, IRAK-4/, and IRF-7/mice (on a C57BL/6 background) and littermate controls were bred and maintained in a SPF facility at Osaka University. Mice (810-weeks-old) received 0.5 mL intraperitoneal (i.p.) injection of TMPD, pentadecane, n-hexadecane, squalene (Sigma-Aldrich, St. Louis, MO), or mineral oil (Harris Teeter, Matthews, NC). Peripheral blood and peritoneal exudate cells (PECs) were isolated as described (9). For morphological analysis, neutrophils were sorted using phycoerythrin (PE)-conjugated anti-Ly6G and magnetic bead-conjugated anti-PE antibodies (17). Fifty thousand sorted cells were cytocentrifuged onto glass slides (Fisher Scientific, Pittsburgh, PA) and stained using the Hema3 kit (Fisher). For IL-1 and CXCL5 blockade, mice treated with TMPD for 2 weeks were given 200 g of anti-IL-1 neutralizing antibodies, hamster IgG (Biolegend, San Diego, CA), anti-CXCL5 neutralizing antibodies or rat IgG1 isotype control antibodies (R&D Systems, Minneapolis, MN)i.p. and analysis was performed after 24 hrs. == Real-time quantitative PCR.