Colocalization of endogenous TNF with a functional intracellular splice form of human TNF receptor type 2

Abstract

Background: Tumor necrosis factor (TNF) is a pleiotropic cytokine involved in a broad spectrum of inflammatory and immune responses including proliferation, differentiation, and cell death. The biological effects of TNF are mediated via two cell surface TNF receptors: p55TNFR (TNFR1; CD120a) and p75TNFR (TNFR2; CD120b). Soluble forms of these two receptors consisting of the extracellular domains are proteolytically cleaved from the membrane and act as inhibitors. A novel p75TNFR isoform generated by the use of an additional transcriptional start site has been described and was termed hicp75TNFR. We focused on the characterization of this new isoform as this protein may be involved in chronic inflammatory processes.

Methods: Cell lines were retroviraly transduced with hp75TNFR isoforms. Subcellular localization and colocalization studies with TNF were performed using fluorescence microscopy including exhaustive photon reassignment software, flow cytometry, and receptosome isolation by magnetic means. Biochemical properties of the hicp75TNFR were determined by affinity chromatography, ELISA, and western blot techniques.

Results: We describe the localization and activation of a differentially spliced and mainly intracellularly expressed isoform of human p75TNFR, termed hicp75TNFR. Expression studies with hicp75TNFR cDNA in different cell types showed the resulting protein mostly retained in the trans-Golgi network and in endosomes and colocalizes with endogenous TNF. Surface expressed hicp75TNFR behaves like hp75TNFR demonstrating susceptibility for TACE-induced shedding and NFkappaB activation after TNF binding.

Conclusion: Our data demonstrate that intracellular hicp75TNFR is not accessible for exogenously provided TNF but colocalizes with endogenously produced TNF. These findings suggest a possible intracellular activation mechanism of hicp75TNFR by endogenous TNF. Subsequent NFkappaB activation might induce anti-apoptotic mechanisms to protect TNF-producing cells from cytotoxic effects of TNF. In addition, the intracellular and not TACE-accessible splice form of the hp75TNFR could serve as a pool of preformed, functional hp75TNFR.

Figure 1

Localization of tagged hp75TNFR and hicp75TNFR molecules in transduced NIH 3T3 cells. Cells transduced either with myc-tagged (A;C) or YFP-tagged (B;D) hp75TNFR (A;B) or hicp75TNFR (C;D) were analyzed microscopically. For detection of YFP the cells were fixed and analyzed. For detection of the myc-tag cells were fixed and permeabilized followed by incubation with a monoclonal mouse anti-human c-myc (9E10) and a secondary anti-mouse FITC antibody. While hp75TNFR staining is mainly found on the plasma membrane, hicp75TNFR staining is perinuclear, punctuated, and distributed throughout the cytoplasm. Nuclei: Dapi (A;C), Hoechst 33342 (B;D).

Figure 2

Localization of hp75TNFR isoforms in transduced NIH 3T3 cells. Cells transduced either with YFP-tagged hp75TNFR (A; C; E; G; I) or YFP-tagged hicp75TNFR (B; D; F; H; J) were costained with ER-Tracker (A; B), Golgi-Tracker (C; D), Mito-Tracker (E; F), Lyso-Tracker (G; H) and ENDO-eCFP (I; J). While hp75TNFR staining is mainly found on the plasma membrane and in the Golgi apparatus, hicp75TNFR shows no plasma membrane staining and colocalizes with the Golgi apparatus and endosomal compartments of the trans-Golgi network.

Figure 3

Cell surface expression of hp75TNFR isoforms on transduced L929 cells. Expression of hp75TNFR isoforms on L929 cells either transduced with control vector (shaded peak), hp75TNFR (black line) or hicp75TNFR (gray line) was analyzed by flow cytometry with (A) or without (B) permeabilization. After permeabilization the same number of epitopes were accessible in both cell lines. Without permeabilization cells expressing hp75TNFR present more epitopes on the cell surface.

Figure 4

Shedded ectodomain of hp75TNFR isoforms. (A)The extracellular domain of both hp75TNFR isoforms is released constitutively (black bars) into the supernatant of L929 cell either transduced with hp75TNFR or hicp75TNFR, respectively. Shedding is increased by rhTNF (6 ng/ml; light gray bars) and attenuated by TAPI (100 nM, dark gray bars). (B)The extracellular domain of hicp75TNFR is bioactive as shown by neutralization of rhTNF in a TNF cytotoxicity assay on L929 cells. Supernatant from L929 cells either transduced with icp75TNFR-YFP (●), or YFP alone (○) were tested. DMEM (▼) served as control medium. All values are given as mean ± S.D. of triplicate cultures. Four independent experiments gave similar results.

Figure 5

Biochemical characterization of hicp75TNFR.(A) The hp75TNFR from NIH3T3 cells either transduced with hp75TNFR or hicp75TNFR, respectively, were immunoprecipitated with monoclonal antibodies against the extracellular domain of hp75TNFR (80M2) and stained after blotting with antibodies to the intracellular domain on hp75TNFR (sc-7862). (B) Affinity purified soluble hicp75TNFR from supernatant of transduced TNFR1/2 knock-out fibroblasts was stained after blotting with a rabbit serum against the extracellular domain of hp75TNFR (80M).

Figure 6

Colocalization of hicp75TNFR with endogenous or exogenous TNF. (A) L929 cells were transduced with hicp75TNFR-YFP and exposed to biotinylated TNF. Receptosomes were isolated after different times, subjected to SDS-PAGE and analyzed by immunostaining of blotted proteins. (B) L929 cells expressing hTNF were transduced with hicp75TNFR and stained with rabbit anti-human TNF antibodies and mouse monoclonal antibody anti-human p75TNFR (80M2).