Human truncated thioredoxin (Trx80) as a novel mitogenic cytokine for white blood cells

Thioredoxin (Trx) is a 12 kDa protein present in all species with a well-conserved active site sequence comprising -Cys-Gly-Pro-Cys-, which catalyzes oxido-reductase reactions. Trx regulates the activity of transcription factors and intracellular signalling pathways, and secreted Trx is a co-cytokine with several interleukins. In addition to full-length Trx a 10 kDa C-terminally truncated form of the protein is produced mainly by monocytes. This protein has unique eosinophilic cytotoxicity enhancing effects, although it does not possess the classical oxido-reductase activity of Trx.

We have cloned, overexpressed and purified truncated Trx comprising the 80 or 84 N-terminal amino acids of Trx. By circular dichroic spectroscopy we found that Trx80 had a secondary structure similar to Trx, although Trx80 appeared as a dimer in solution upon Sephadex G-75 chromatography, with a M, of 25, 000. Reducing and non-reducing SDS-gel electrophoresis showed that inter-molecular disulfide bonds did not hold the Trx80 dimer together, but the dimer was more likely formed due to hydrophobic interactions. Trx80 was neither a substrate nor an inhibitor of thioredoxin reductase (TrxR), however Trx was able to reduce disulfides in Trx80.

Since Trx80 has been shown to possess eosinophilic cytotoxicity activity we investigated if Trx80 had effects on other cells in the immune system. Indeed, we found that Trx80 was a mitogenic cytokine for peripheral blood mononuclear cells (P13MC) cultured in both serum-free medium and medium supplemented with 10% FCS. Trx80 stimulated proliferation, measured as thymidine incorporation, of PBMC in culture with a maximum effect at 50-100 nM, which was equivalent to 5 U/ml of IL-2. We purified monocytes and T and B cells from PBMC to evaluate which cell type/types that was the primary target for Trx80. Trx80 stimulated monocytes to proliferate and differentiate in vitro. Monocytes cultured in presence of Trx80 enhanced their expression of the surface antigens CD14, CD40, CD54 and CD86 measured by flow cytometry. Moreover, Trx80 induced a Th1 response in T cells in PBMC cultures. By ELISA and intracellular flow cytometry we found that Trx80 alone stimulated the expression of the Th 1 inducer cytokine IL- 12 from CD40+ monocytes. This effect was in synergy with IL-2. In addition, Trx80 in synergy with IL-2, but not alone, stimulated secretion of IFNgamma. In contrast, Trx80 did not stimulate secretion of the Th2 cytokines IL-4 and IL-5, and did not stimulate purified B or T cells. Hence, we conclude that the primary target cell for Trx80 in PBMC cultures is the monocyte.

We developed monoclonal antibodies, which distinguish between full-length and truncated Trx, in order to investigate the cellular localization of full-length and truncated Trx. There was a striking difference between full- length Trx and truncated Trx both in which cell types that expressed these proteins and in sub-cellular localization. Full-length Trx was mainly found in the cytosol, whereas truncated Trx was mainly localized at the cell membrane. Moreover, full-length Trx was expressed in all cell types tested, but truncated Trx only showed strong expression in cells of monocytic heritage.

Two sandwich ELISA systems based on the above-mentioned antibodies and a polyclonal goat anti-full-length Trx antibody were developed. Using these ELISA systems we found that levels of fall-length and truncated Trx in plasma from healthy blood donors did not correlate. The levels of full-length Trx in 12 donors did not vary to a large extent, while truncated Trx varied between 1-171 ng/ml in the different donors. The median value for full-length Trx was 29 ng/ml and for truncated Trx 21 ng/ml.

Since Trx80 did not possess any activity in reducing insulin-disulfides we examined if the cysteine residues in the Trx active site were essential for the activity of Trx80 as a mitogenic cytokine. Hence, site-directed mutagenesis was performed of Cys31 and Cys34 in Trx80, generating a mutant called Trx8OSGPS, and of the structural cysteine at position 72, generating a mutant named Trx80C72S. These mutants had the same effect as Trx80 in stimulating PBMC proliferation and induction of 11-12 and IFN-gamma.

Finally, since Trx is known to be secreted from cells and levels of Trx are elevated in diseases affecting the immune system, such as HIV and rheumatoid arthritis, we examined if secreted Trx had chemokine activity. We found that Trx was a chemokine for PMNs, T cells and monocytes both in vitro and in vivo with a maximal effect in the same concentrations as wellknown chemokines like RANTES and MCP-1. The activity of Trx as a chemokine depended upon the oxido- reductase activity since a mutant, where the active site cysteines had been replaced with serines, lost chemokine activity. Moreover, Trx did not function via known G-protein coupled chemokine receptors. This was evident since Trx did not increase intracellular calcium levels, and bordetella pertussis toxin that inhibits G-protein coupled chemokine receptors did not inhibit the chemokine activity of Trx.

List of scientific papers

I. Pekkari K, Gurunath R, Arner ES, Holmgren A (2000). "Truncated thioredoxin is a mitogenic cytokine for resting human peripheral blood mononuclear cells and is present in human plasma. " J Biol Chem 275(48): 37474-80
https://pubmed.ncbi.nlm.nih.gov/10982790

II. Sahaf B, Soderberg A, Spyrou G, Barral AM, Pekkari K, Holmgren A, Rosen A (1997). "Thioredoxin expression and localization in human cell lines: detection of full-length and truncated species" Exp Cell Res 236(1): 181-92
https://pubmed.ncbi.nlm.nih.gov/9344598

III. Pekkari K, Avila-Carino J, Bengtsson A, Gurunath R, Scheynius A, Holmgren A (2001). "Truncated thioredoxin (Trx80) induces production of interleukin-12 and enhances CD14 expression in human monocytes. " Blood 97(10): 3184-90
https://pubmed.ncbi.nlm.nih.gov/11342447

IV. Pekkari K, Avila-Carino J, Bengtsson A, Scheynius A, Holmgren A (2001). "Truncated thioredoxin (Trx80) exerts unique cytokine effects via a mechanism independent of thiol oxido-reductase activity." (Manuscript)

V. Bertini R, Howard OM, Dong HF, Oppenheim JJ, Bizzarri C, Sergi R, Caselli G, Pagliei S, Romines B, Wilshire JA, Mengozzi M, Nakamura H, Yodoi J, Pekkari K, Gurunath R, Holmgren A, Herzenberg LA, Herzenberg LA, Ghezzi P (1999). "Thioredoxin, a redox enzyme released in infection and inflammation, is a unique chemoattractant for neutrophils, monocytes, and T" J Exp Med 189(11): 1783-9
https://pubmed.ncbi.nlm.nih.gov/10359582