Surfactant protein SP-D modulates activity of immune cells: proteomic profiling of its interaction with eosinophilic cells

Abstract

Surfactant protein D (SP-D), a C-type lectin, is known to protect against lung infection, allergy and inflammation. Its recombinant truncated form comprising homotrimeric neck and CRD region (rhSP-D) has been shown to bring down specific IgE levels, eosinophilia and restore Th2–Th1 homeostasis in murine models of lung hypersensitivity. SP-D knockout mice show intrinsic hypereosinophilia and airway hyper-responsiveness that can be alleviated by rhSP-D. The rhSP-D can bind activated eosinophils, inhibit chemotaxis and degranulation, and selectively induce oxidative burst and apoptosis in sensitized eosinophils. A global proteomics study of rhSP-D-treated eosinophilic cell line AML14.3D10 identified large-scale molecular changes associated with oxidative burst, cell stress and survival-related proteins potentially responsible for apoptosis induction. The data also suggested an involvement of RNA binding- and RNA splicing-related proteins. Thus, the proteomics approach yielded a catalog of differentially expressed proteins that may be protein signatures defining mechanisms of SP-D-mediated maintenance of homeostasis during allergy.

Keywords::

• allergy
• apoptosis
• eosinophilia
• heterogeneous nuclear ribonucleoproteins
• p53 pathway
• proteomics
• SP-D
• surfactant

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Key issues

• The role of surfactant protein D (SP-D) in immunosurveillance and immunomodulation is well documented in pulmonary allergy and asthma with clearance of eosinophilia as one of the potent anti-allergy mechanisms.

• A direct carbohydrate recognition domain-mediated interaction of SP-D with human eosinophils resulted in inhibition of eosinophil chemotaxis and degranulation, and selective induction of oxidative burst and apoptosis in the eosinophils from allergic patients, IL-5-primed eosinophils from healthy donors and eosinophilic leukemic cell line, acute myeloid leukemia 14.3D10 (AML14.3D10).

• The recombinant trimeric carbohydrate recognition domain region of SP-D (rhSP-D)-induced G2/M phase cell cycle arrest, p53-mediated and dose and time-dependent apoptosis in the eosinophilic cell line.

• The underlying molecular mechanism of rhSP-D-mediated modulation of activated eosinophils was investigated by proteomics approach using AML14.3D10 cells. The differentially expressed proteins of AML14.3D10 cell line on treatment with rhSP-D belonged to the following major functional categories: oxidoreductases; chaperones; inflammation and survival; ubiquitin–proteasome pathways; energy metabolism; transcription and translation; RNA binding and metabolism; cytoskeleton; vesicle fusion; synthesis and trafficking; metabolic enzymes and others.

• The study showed that the increase in oxidative burst induced by rhSP-D was likely to precede the induction of apoptosis. The localized production of reactive oxygen species triggered by rhSP-D was counteracted by the action of cellular-reducing agents and coordinated induction of proteins with radical-scavenging properties and protective activities.

• In contrast, the mitochondrial antioxidant defense system was compromised in rhSP-D-treated cells. There was a decreased expression of Ubiquinol–cytochrome c reductase, Peroxiredoxin 3 isoform b and manganese superoxide dismutase. This may result in mitochondrial dysfunction and trigger the intrinsic pathway of apoptosis and ultimately cell death.

• The striking common feature between rhSP-D-modified protein profile and rhSP-D-binding proteins was the presence of a range of RNA binding and RNA splicing-related proteins. Although speculative, it is likely that SP-D may affect specific changes in mRNA processing and, most probably, the rates of translation and/or protein degradation.

• The differential expression of proteins indicated rhSP-D-mediated susceptibility of cells to decreased survival: Aryl-hydrocarbon receptor-interacting protein analogous to a related domain of immunophilin proteins was upregulated, suggesting increased susceptibility of treated eosinophilic cells to immunosuppressants. Heterogeneous nuclear ribonucleoprotein K, specifically upregulated in response to DNA damage, was increased in rhSP-D-treated cells. Calreticulin precursor variant overexpression has been shown to increase Ca²⁺ fluxes across the endoplasmic reticulum but decreases mitochondrial Ca²⁺ and membrane potential, which is proposed to damage mitochondria.

Notes