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Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody (mAb) by Neutralizing TNF Using an In Vitro Bioanalytical Method
Published on: September 16, 2017
Tumour necrosis factor alpha production by polymorphonuclear neutrophils treated with mouse anti-human CD19
1Research Section, Tokyo Metropolitan Red Cross Blood Center, Tokyo, Japan. mo-nishimura@tokyo.bc.jrc.or.jp
Abstract:
Polymorphonuclear neutrophils (PMNs) play pivotal roles as phagocytic cells in immune defence against bacteria and parasites, exerting their effects by production of reactive oxygen species, several cytokines, chemokines and by phagocytotic reaction. In our investigation of properties of activated PMNs, we discovered that one of the two kinds of mouse anti-human CD19 monoclonal antibodies (mAbs) clone SJ25-C1, weakly binds to freshly prepared PMNs. Moreover, the treatment of freshly prepared PMNs with anti-CD19 mAb (clone SJ25-C1) at 37 degrees C for 6 h induces the production and the secretion of tumour necrosis factor alpha (TNF alpha) by PMNs in vitro which was detectable in culture supernatants by bioassay using mouse cell line L929 cells. The concentration of TNF alpha secreted into the culture supernatant of PMNs cultured in the presence of anti-CD19 mAb (clone SJ25-C1) was higher than those of PMNs treated at 37 degrees C for 6 h with various PMN activators, such as anti-CD24 mAb, granulocytes-macrophage colony stimulation factor (GM-CSF) or interferon gamma (IFN gamma). In contrast, another clone of anti-CD19 mAb, HD37, did not bind to freshly prepared PMNs and failed to produce TNF alpha. To confirm that anti-CD19 mAb (clone SJ25-C1)-treated PMNs definitely produce TNF alpha, we measured the levels of intracellular expression of TNF alpha in PMNs permeabilized by saponin. These cells were treated with fluorescence-conjugated mouse anti-human TNF alpha mAb for detection of intracellular TNF alpha expression. Consequently, large amounts of intracellular TNF alpha were detected in PMNs treated with anti-CD19 mAb (clone SJ25-C1) but not in those treated with anti-CD19 mAb (clone HD37).
Insights
Mouse anti-human CD19 monoclonal antibody clone SJ25-C1 activates polymorphonuclear neutrophils (PMNs) to produce and secrete tumor necrosis factor alpha (TNF α) in vitro. This finding reveals a novel function for anti-CD19 antibodies in immune cell activation.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Polymorphonuclear neutrophils (PMNs) are crucial phagocytic immune cells involved in defense against pathogens.
- PMNs exert immune functions through reactive oxygen species, cytokine/chemokine production, and phagocytosis.
- Understanding PMN activation pathways is vital for developing immunotherapies.
Purpose of the Study:
- To investigate the properties of activated PMNs.
- To determine if anti-CD19 monoclonal antibodies (mAbs) can activate PMNs.
- To characterize the specific response induced by anti-CD19 mAb clone SJ25-C1.
Main Methods:
- Treatment of freshly prepared human PMNs with anti-CD19 mAbs (clones SJ25-C1 and HD37).
- Detection of tumor necrosis factor alpha (TNF α) in culture supernatants using bioassay (L929 cell line).
- Measurement of intracellular TNF α expression in permeabilized PMNs via flow cytometry using fluorescence-conjugated anti-TNF α mAb.
Main Results:
- Anti-CD19 mAb clone SJ25-C1 weakly bound to PMNs and induced significant TNF α production and secretion in vitro.
- TNF α levels induced by SJ25-C1 were higher than those induced by other known PMN activators (anti-CD24 mAb, GM-CSF, IFN γ).
- Anti-CD19 mAb clone HD37 did not bind PMNs or induce TNF α production; intracellular TNF α was detected in SJ25-C1 treated PMNs but not in HD37 treated PMNs.
Conclusions:
- Anti-CD19 mAb clone SJ25-C1 is a potent activator of PMNs, inducing TNF α production and secretion.
- This represents a novel mechanism of PMN activation distinct from previously known pathways.
- The findings suggest potential therapeutic applications of specific anti-CD19 mAbs in modulating immune responses.
