Neutrophils have been considered mainly as effector cells of the innate immune response that control bacterial and fungal infections (1
). Recently, however, neutrophils have been recognized as important regulators of adaptive immunity (1
). Neutrophils can interact directly with T cells and, through the production of IFNγ by neutrophils, promote T cell proliferation (3
). In addition, a subset of spleen-resident neutrophils expressing CD15 and CD16 has been shown to induce antibody secretion from marginal-zone B cells in healthy individuals through the production of the cytokines B cell activator of the TNF family (BAFF), a proliferation inducing ligand (APRIL), and IL-21 (2
). Therefore, neutrophils may not only serve to recruit immune cells to sites of inflammation but may also activate them directly through the production of cytokines (5
). In addition to their beneficial role during infections, neutrophils are thought to contribute to the pathogenesis of diseases that involve inflammation. Activated neutrophils release a large number of inflammatory cytokines including IL-1α, IL-1β, IL-6, IL-4, and IL-17 that are linked to allergy, certain cancers, and autoimmune disorders such as systemic lupus erythematosus (1
). Therefore, a greater understanding of neutrophil function in both innate and adaptive immune responses has clinical implications for multiple diseases.
Neutrophils are produced in the bone marrow, are released into the bloodstream and, under normal conditions, survey the body for invading pathogens. In response to pathogens, circulating neutrophils migrate into peripheral tissues to the site of infection where they exert their effector activity (7
). Neutrophils are phenotypically defined as Ly6G+
, regardless of their localization (8
). Tissue-infiltrating neutrophils can be phenotypically differentiated further based on expression levels of the integrin family of cell adhesion molecules [(9
); C.M.C. and L.D.E., personal observations]. Peripheral tissue- resident neutrophils also produce greater amounts of inflammatory cytokines and chemokines that further distinguish them from circulating neutrophils (13
). Therefore, the tissue microenvironment is important for driving the activation of neutrophils that may impact the response of other immune cell types.
Methods have been previously designed for isolating murine neutrophils from blood (14
), bone marrow (16
), peritoneum (15
), and liver (17
). In general, these methods use two different platforms for enrichment of neutrophils: density gradient centrifugation and magnetic beads that label specific cell populations using antibodies against cell surface antigens and then used for isolation either by positive or negative selection. Isolation of neutrophils by positive selection has also been achieved by flow cytometric cell sorting. These methods, tailored for purifying murine neutrophils from the sources described above, are simple and yield excellent cell purities; however, are less effective for isolating neutrophils within secondary lymphoid organs as we present here. Such a method to reliably isolate murine neutrophils from spleen and, in particular, from inflamed spleen where the composition of hematopoietic cells is altered would greatly facilitate the in vitro and in vivo analysis of these innate cells in their effector state during normal and abnormal immune responses. We developed an efficient method to isolate murine neutrophils in inflamed spleen by negative selection for subsequent analysis. We analyzed the steady-state frequencies of neutrophils in the blood, bone marrow, and spleens of the autoimmune-prone B6.Faslpr
/J mouse model. Neutrophils were significantly elevated in the spleens of B6.Faslpr
/J mice compared to healthy control animals. Based on the analysis of other major immune cell types in the spleen, we generated an optimized antibody cocktail (OAC) targeting these immune cell types to isolate splenic neutrophils by magnetic bead negative selection. The purity and viability of neutrophils using this method were highly reproducible and this technique could be used to not only isolate neutrophils from the spleen but also from the bone marrow and blood. Splenic neutrophils isolated with the OAC protocol were not impaired in cell viability or phagocytic activity, indicating that neutrophils were not adversely affected by this isolation procedure. Therefore, the OAC protocol provides a rapid and effective process for isolating spleen-resident neutrophils for further investigation.