The binding of the primary antibodies was detected with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology)

The binding of the primary antibodies was detected with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology). during PcP are not clear; one possible cause is downregulation of the transcription factor PU.1 (11), as it regulates the expression of many macrophage receptors (11,C15). The mechanism of PU.1 downregulation is unknown. We have recently found that myeloid-derived suppressor cells (MDSCs) accumulate in the lungs during PcP (16), and that adoptive transfer of MDSCs from mice with PcP causes lung damage in the recipient mice (16). MDSCs are a heterogeneous population of bone marrow-derived myeloid progenitor cells and immature myeloid cells. In health, these cells quickly differentiate into mature granulocytes, macrophages, or dendritic cells. This differentiation is blocked in certain conditions, such as cancer, various infectious diseases, sepsis, trauma, and some autoimmune diseases (17). MDSCs have the morphology of monocytes or granulocytes; thus, they are classified as monocytic and granulocytic MDSCs. In mice, MDSCs coexpress Gr-1 and CD11b (M-integrin) (18). In humans, MDSCs are HLA-DR? or HLA-DRlow and CD11b+, CD33+, or CD15+ (19). MDSCs are immunosuppressive and ELX-02 sulfate have been shown to suppress the functions of NK cells, T cells, and B cells (20, 21). The suppressive activity of MDSCs appears to be inversely related to the expression of the programmed death 1 protein (PD-1), as MDSCs ELX-02 sulfate from PD-1?/? mice are more immunosuppressive than those from wild-type mice (20). PD-1 (CD279) is a coinhibitory molecule. As with CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) and BTLA (B- and T-lymphocyte attenuator), a major function of PD-1 is to prevent the activated T cells from becoming overzealous, leading to adverse inflammatory responses and organ damage (22,C24). PD-1 is a membrane protein of the CD28 family. It is expressed on the surfaces of many immune cells, including CD4+ T cells, CD8+ T cells, NK T cells, B cells, and monocytes (22, 24, 25). Its ligand PD-L1 (CD274), also called B7 homolog 1 (B7-H1), is a type I transmembrane protein and is constitutively expressed on T cells, B cells, macrophages, and ELX-02 sulfate dendritic cells (22, 24, 25). During persistent antigen exposure, antigen-specific CD8+ T cells may lose their effector functions, such as proliferation and cytokine production; this phenomenon is referred to as CD8+ T-cell exhaustion (26). The PD-1/PD-L1 signaling pathway plays a major role in the generation of exhausted CD8+ T cells ELX-02 sulfate in numerous settings, including cancer and chronic viral infections of human immunodeficiency virus (HIV), hepatitis C virus (HCV), hepatitis B virus (HBV), lymphocytic choriomeningitis virus (LCMV), and simian immunodeficiency virus (SIV) (27,C33). The PD-1/PD-L1 signaling pathway also is involved in immune tolerance, as PD-1?/? knockout leads to autoimmune encephalomyelitis, lupus-like syndrome (34), or dilated cardiomyopathy in mice (35,C37). One intronic single-nucleotide polymorphism of the PD-1 gene is correlated with the development of systemic lupus erythematosus in Europeans and Mexicans (38). The PD-1/PD-L1 signaling pathway also modulates the function of regulatory T cells (Treg), as blockade of the PD-1/PD-L1 pathway abrogates Treg-mediated immune tolerance in mice (39, 40). Although the suppressive effects of MDSCs on T cells have been studied extensively, it is Rabbit polyclonal to Fyn.Fyn a tyrosine kinase of the Src family.Implicated in the control of cell growth.Plays a role in the regulation of intracellular calcium levels.Required in brain development and mature brain function with important roles in the regulation of axon growth, axon guidance, and neurite extension.Blocks axon outgrowth and attraction induced by NTN1 by phosphorylating its receptor DDC.Associates with the p85 subunit of phosphatidylinositol 3-kinase and interacts with the fyn-binding protein.Three alternatively spliced isoforms have been described.Isoform 2 shows a greater ability to mobilize cytoplasmic calcium than isoform 1.Induced expression aids in cellular transformation and xenograft metastasis. unknown whether MDSCs have any adverse effects on macrophages. Since MDSCs express PD-L1 (17, 41, 42) and macrophages have been shown to express PD-1 (43,C47), we tested the hypothesis that MDSCs interact with AMs through PD-1/PD-L1 ligation, causing PU.1 downregulation and defects in phagocytosis during PcP. MATERIALS AND METHODS Animal model of PcP. C57BL/6 mice were obtained from Harlan (Indianapolis, IN). All animals used in this study were female, 18 to 20 g in weight. The study was approved by the Indiana University Animal Care and Use Committee and carried out under the supervision of veterinarians. Immunosuppression of mice was achieved by intraperitoneal injection of 0.3 mg anti-CD4 (L3T4) monoclonal antibody (MAb; clone GK1.5; Harlan, Indianapolis, IN) to each mouse once a week to deplete CD4+ cells until the mice were sacrificed. One week after the initial.