Human Surfactant Protein D Alters Oxidative Stress and HMGA1 Expression to Induce p53 Apoptotic Pathway in Eosinophil Leukemic Cell Line

Abstract

Surfactant protein D (SP-D), an innate immune molecule, has an indispensable role in host defense and regulation of inflammation. Immune related functions regulated by SP-D include agglutination of pathogens, phagocytosis, oxidative burst, antigen presentation, T lymphocyte proliferation, cytokine secretion, induction of apoptosis and clearance of apoptotic cells. The present study unravels a novel ability of SP-D to reduce the viability of leukemic cells (eosinophilic leukemic cell line, AML14.3D10; acute myeloid leukemia cell line, THP-1; acute lymphoid leukemia cell lines, Jurkat, Raji; and human breast epithelial cell line, MCF-7), and explains the underlying mechanisms. SP-D and a recombinant fragment of human SP-D (rhSP-D) induced G2/M phase cell cycle arrest, and dose and time-dependent apoptosis in the AML14.3D10 eosinophilic leukemia cell line. Levels of various apoptotic markers viz. activated p53, cleaved caspase-9 and PARP, along with G2/M checkpoints (p21 and Tyr15 phosphorylation of cdc2) showed significant increase in these cells. We further attempted to elucidate the underlying mechanisms of rhSP-D induced apoptosis using proteomic analysis. This approach identified large scale molecular changes initiated by SP-D in a human cell for the first time. Among others, the proteomics analysis highlighted a decreased expression of survival related proteins such as HMGA1, overexpression of proteins to protect the cells from oxidative burst, while a drastic decrease in mitochondrial antioxidant defense system. rhSP-D mediated enhanced oxidative burst in AML14.3D10 cells was confirmed, while antioxidant, N-acetyl-L-cysteine, abrogated the rhSP-D induced apoptosis. The rhSP-D mediated reduced viability was specific to the cancer cell lines and viability of human PBMCs from healthy controls was not affected. The study suggests involvement of SP-D in host’s immunosurveillance and therapeutic potential of rhSP-D in the eosinophilic leukemia and cancers of other origins.

Oxidative damage of SP-D abolishes control of eosinophil extracellular DNA trap formation

“…Our data corroborate previous studies in which SP‐D was shown to bind eosinophils in a maltose and Ca ++ ‐dependent manner . SP‐D also inhibited eotaxin‐induced migration, degranulation of eosinophil cationic protein and promoted apoptosis upon IL‐5 priming in vitro …”

“…39 SP-D also inhibited eotaxin-induced migration, degranulation of eosinophil cationic protein 39 and promoted apoptosis upon IL-5 priming in vitro. [40][41][42] SP-D exerts both host-defense and anti-inflammatory functions in the lung. These are not contradictory as they both serve immune protection of the respiratory mucosa through distinct and complementary mechanisms as we and many others have previously shown.…”

Differential Expression of Collectins in Human Placenta and Role in Inflammation during Spontaneous Labor

“…Rest of the SP-D strongly associated with the beads was eluted with 20 mM EDTA in 20 mM Tris-HCl, pH 7.4 at room temperature with mild shaking for 1 h. Supernatant collected after MnCl 2 elution contained SP-D proteins only, while EDTA eluted fractions contained both SP-A and SP-D proteins and they were further separated by gel filtration and analyzed by silver staining ( Fig.S3.i ) and Western blot respectively ( Fig S3.ii ). The purified nSP-D protein was assayed for endotoxin content (given below), its binding and ability to agglutinate A. fumigatus conidia and its potential to enhance killing of fungal conidia by human PBMCs in vitro [31] .…”

Pulmonary surfactant protein SP-D opsonises carbon nanotubes and augments their phagocytosis and subsequent pro-inflammatory immune response

“…17 In this study, we have used recombinant human SP-D expressed in E. coli, which consists of three CRDs, a neck and eight gly-Xaa-Yaa collagen region repeats, where Xaa and Yaa could be any amino acid. 31,46,47 Thus, interaction between SP-D and nanoparticles can have profound and wide-ranging impact on the pulmonary innate and adaptive immune mechanisms. 41 This ligand binding causes opsonic effects, leading to enhanced phagocytosis and clearance mechanisms via superoxidative burst and mostly pro-inflammatory immune response by phagocytic cells.…”