Neem leaf glycoprotein facilitates lung carcinoma-associated antigen-specific anti-cancer immune response utilizing macrophage-mediated antigen presentation and induction of type 1 cytokines coupled with nitric oxide production

Abstract

Introduction: Based on earlier observations on unique immunomodulatory and adjuvant functions of neem leaf glycoprotein (NLGP), investigations of this work were designed. NLGP was attempted to be used as an adjuvant for lung carcinoma-associated antigen (LCA) which not only activated macrophages but also induced macrophages to release nitric oxide (NO), a key tumoricidal agent known to regulate T-cell proliferation, cytokine production, cell signaling, and apoptosis. Materials and Methods: Macrophages, generated from peripheral blood mononuclear cells (PBMCs), were pulsed with LCA isolated from lung carcinoma cell line A549, in presence or absence of NLGP for antigen presentation. Intramacrophageal NO was estimated based on Griess reaction. Cytokine levels were estimated by ELISA. Lymphocytic proliferation was checked by MTT assay. Cytotoxic T lymphocytes (CTLs) generated cytotoxicity was tested by LDH assay. Results: NLGP potentiates immune responses during pulsation with LCA by specific lymphocytic proliferation (p<0.001) and generation of CTLs (p<0.001). LCA+NLGP treatment creates a type-1 immune environment by increasing secretion of type-1 cytokines IFN-g and IL-12 (p<0.001) and decrease in type-2 cytokines IL-4 and IL-10 (p<0.001). LCA+NLGP treatment increased the release of type-1 cytokine-dependent NO. In vitro neutralization of IFN-γ/IL-12 results into drastic decrease in NO release from macrophages. Conclusion: Obtained results demonstrated the interdependence of three anti-tumor immune functions, namely, NO production, CTL generation and production of a type-1 immune response mediated through NLGP. NLGP-generated anti-LCA immune response would be an effective strategy to treat lung carcinomas.