Detection of LPS in tissues is an integral component of innate immunity that acts to protect against invasion by Gram-negative bacteria. Plasma down-regulates LPS-induced cytokine production from macrophages, thereby limiting systemic inflammation in blood and distant tissues. To identify the protein(s) involved in this process, we used classical biochemical chromatographic techniques to identify fractions of mouse sera that suppress LPS-induced TNF from bone marrow-derived macrophages (BMDMs). Fractionation yielded microgram quantities of a protein that was identified by MS to be hemopexin (Hx). Mouse Hx purified on hemin-agarose beads and rhHx decreased the production of cytokines from BMDMs and peritoneal macrophages induced by LPS. Preincubation of LPS with Hx did not affect the activity of LPS on LAL, whereas preincubation of Hx with macrophages followed by washing resulted in decreased activity of these cells in response to LPS, suggesting that Hx acts on macrophages rather than LPS. Heme-free Hx did not stimulate HO-1 in the macrophages. Purified Hx also decreased TNF and IL-6 from macrophages induced by the synthetic TLR2 agonist Pam3Cys. Our data suggest that Hx, which is an acute-phase protein that increases during inflammation, limits TLR4 and TLR2 agonist-induced macrophage cytokine production directly through a mechanism distinct from HO-1.

SUMMARY Angiogenin (ANG) is a secreted ribonuclease (RNase) with cell type- and context-specific roles in growth, survival, and regeneration. Although these functions require receptor-mediated endocytosis and appropriate subcellular localization, the identity of the cell surface receptor remains undefined. Here, we show that plexin-B2 (PLXNB2) is the functional receptor for ANG in endothelial, cancer, neuronal, and normal hematopoietic and leukemic stem and progenitor cells. Mechanistically, PLXNB2 mediates intracellular RNA processing that contribute to cell growth, survival, and regenerative capabilities of ANG. Antibodies generated against the ANG binding site on PLXNB2 restricts ANG activity in vitro and in vivo, resulting in inhibition of established xenograft tumors, ANG-induced neurogenesis and neuroprotection, levels of pro-self-renewal transcripts in hematopoietic and patient-derived leukemic stem/progenitor cells, and reduced progression of leukemia in vivo. PLXNB2 is therefore required for the physiological and pathological functions of ANG and has significant therapeutic potential in solid and hematopoietic cancers and neurodegenerative diseases.

Detection of microbial components by immune cells via Toll-like receptors (TLRs) with subsequent induction of inflammation is essential for host defense. However, an overactive immune response can cause tissue damage and sepsis. The endogenous molecule hemoglobin and its derivative heme are often released into tissue compartments where there is infection in the presence of degrading blood. We found that hemoglobin synergizes with multiple TLR agonists to induce high levels of tumor necrosis factor and interleukin-6 from macrophages and that this synergy is independent of TLR4 and MyD88. In contrast, heme synergized with some but not all TLR agonists studied. Furthermore, the synergy of both hemoglobin and heme with lipo-polysaccharide was suppressed by hemopexin, a plasma heme-binding protein. These studies suggest that hemoglobin and heme may substantially contribute to microbe-induced inflammation when bacterial or viral infection coexists with blood degradation and that hemopexin may play a role in controlling inflammation in such settings.

Androgen receptor (AR) is a critical effector of prostate cancer (PCa) development and progression. Androgen-dependent PCa rely on the function of AR for growth and progression. Many castration-resistant PCa continue to depend on AR signaling for survival and growth. Ribosomal RNA (rRNA) is essential for both androgen-dependent and castration-resistant growth of PCa cells. During androgen-dependent growth of prostate cells, androgen-AR signaling leads to the accumulation of rRNA. However, the mechanism by which AR regulates rRNA transcription is unknown. We have found that angiogenin (ANG), the 5th member of the vertebrate-specific, secreted ribonuclease superfamily that is upregulated in PCa, mediates androgen-stimulated rRNA transcription in PCa cells. Upon androgen stimulation, ANG undergoes nuclear translocation in androgen-dependent PCa cells where it binds to the ribosomal DNA (rDNA) promoter and stimulates rRNA transcription. ANG antagonists inhibit androgen-induced rRNA transcription and cell proliferation in androgen-dependent PCa cells. ANG also mediates androgen-independent rRNA transcription. It undergoes constitutive nuclear translocation in androgen-insensitive PCa cells, resulting in a constant rRNA overproduction thereby stimulating cell proliferation. ANG overexpression in androgen-dependent PCa cells enables castration-resistant growth of otherwise androgen-dependent cells. Thus, ANG-stimulated rRNA transcription is not only an essential component for androgen-dependent growth of PCa, but also contributes to the transition of PCa from androgen-dependent to castration-resistant growth status.