9 research outputs found
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
Requirements for the different cysteines in the chemotactic and desensitizing activity of human thioredoxin
: Thioredoxin (Trx) is a protein disulfide oxidoreductase that can be secreted and act as a chemoattractant for leukocytes. Like chemokines, it causes desensitization of monocytes against its chemotactic activity and that of monocyte chemoattractant protein-1 (MCP-1). To investigate the role of the redox properties of Trx, and particularly of some of its five cysteines, in its chemotactic and desensitizing action, we tested different mutants, including Trx80, a truncated form, and various mutants lacking specific cysteines: Trx C62S/C73S and the redox-inactive mutant Trx C32S/C35S. Of the mutants, only Trx80 maintained the chemotactic activity of wild-type Trx toward both monocytes and polymorphonuclear neutrophils, all of them desensitized monocytes against wild-type Trx or MCP-1, but not chemotactic peptide formyl-methionyl-leucil peptide. These data indicate that different redox-active cysteines are important for Trx chemotactic action, whereas its desensitizing action does not have these requirements, suggesting a redox-independent mechanism