Author(s) : S. Pagliei, P. Ghezzi, C. Bizzarri, V. Sabbatini, G. Frascaroli, S. Sozzani, G. Caselli, R. Bertini, Instituto "Mario Negri" via Eritrea 62, 20157 Milano, Italy..
Summary : Thioredoxin (Trx) is a protein disulfide oxidoreductase which can be secreted and acts as a cytokine. As we recently reported that Trx is chemotactic, we investigated whether it desensitizes monocytes or PMN to other chemokines. Preincubation for 15 min with Trx inhibited the chemotactic response of monocytes to MCP-1, but not to fMLP. This effect was independent of whether Trx was present during the chemotaxis assay or only during the preincubation. Preincubation (5 min) with Trx also inhibited the increase in intracellular Ca2+ induced by MCP-1 in monocytes, but not that induced by fMLP. Preincubation with Trx did not affect the chemotactic response induced in PMN by IL-8. The inhibition of chemotactic and Ca2+ responses to MCP-1 in monocytes was not due to a down-regulation of the MCP-1 receptor, as shown by receptor binding studies. The Ca2+ response to MCP-1 was also inhibited by Trx in a CCR2-transfected cell line. It is suggested that Trx inhibits monocyte responses to chemokines by acting downstream of the chemokine receptors. Since there are high concentrations of circulating Trx in infection and inflammatory diseases, this might act as an inhibitor of monocyte migration in vivo.
Keywords : thioredoxin, monocytes, MCP-1.
Pictures
Figure 1
Trx desensitizes monocytes chemotactic response to MCP-1.
Panel A. Monocytes were preincubated for 15 min with different concentrations
of Trx (closed squares) or vehicle (open bar). Then the cells were put
in the upper compartment of Boyden chambers. MCP-1 (25 ng/ml) was added
to the lower compartment. As negative control, the effect of 30 ng/ml
IL-8 on MCP-1 chemotaxis is also shown (hatched bar).
Panel B. After 15 min preincubation with different concentrations of
Trx (hatched bars) or vehicle (open bar), the cells were washed and put
in the upper compartment of Boyden chambers. MCP-1 was added to the lower
compartment.
Panel C. Trx was boiled for 1 hour or preincubated for 30 min with anti-Trx
or an irrelevant (anti-GST) antibody. Trx (30 or 100 ng/ml) was then tested
for its desensitizing effect on monocyte migration in response to MCP-1
as in panel A. The results are the number of migrated cells in five oil
immersion fields and are the mean ± SD of triplicate samples. *p
< 0.05 versus vehicle group by Student's t test.
Figure 2
Specificity of the Trx effect.
Panel A: Monocytes were preincubated for 15 min with different
concentrations of Trx (closed squares) or vehicle (open bar). Then the
cells were put in the upper compartment of Boyden chambers. FMLP was added
to the lower compartment.
Panel B: Monocytes were preincubated with TNF (30 or 300 ng/ml)
or vehicle, then their chemotactic response to MCP-1 (open bars) was evaluated.
The effect of addition of 300 ng/ml of TNF to both compartments of the
Boyden chamber is also shown (closed bar). The results are the number
of cells migrated in five oil immersion fields and are the mean ±
SD of triplicate samples.
Figure 3
Trx does not desensitize the PMN chemotactic response to IL-8. PMNs
were preincubated for 15 min with different concentrations of Trx (squares)
or vehicle (closed bar).
Then the cells were put in the upper compartment of Boyden chambers.
IL-8 was added to the lower compartment. The results are expressed as
the average number of cells migrated in five oil immersion fields ±
SD of triplicate sample.
Figure 4
Pre-exposure to Trx inhibits the increase of cytosolic Ca2+
induced by MCP-1 in human monocytes.
Tracings indicate levels of [Ca2a]i in single
adherent monocytes where MCP-1 (25 ng/ml) was added after preincubation
with 30 ng/ml Trx (panel A) or an irrelevant protein (50 ng/ml
IL-8; panel B). Panels C and D show the [Ca2+]i
response to fMLP (10- 7M) in monocytes pretreated with Trx
(C) or IL-8 (D). Tracings are representative of 8 to 20 cells. Arrows
indicate the addition of agents to the medium, which caused a spike in
the trace due to exposure to light. The inset shows cumulative data of
8-20 cells analyzed. Cells were considered responsive when the stimulus-induced
increase of [Ca2+]i was more than 30% above baseline
(normalized to 100%). The Ca2+ increase is the mean ±
SEM of responsive cells. Data are expressed as % above basal [Ca2+]i.
* p < 0.05 versus the IL-8-pretreated group by analysis of
variance (ANOVA), Student's multiple comparison t-test, and Dunnett's
multiple range test.
Figure 5
Trx does not affect the MCP-1 receptor binding.
Monocytes were pretreated with Trx (300 ng/ml), LPS (100 ng/ml) or vehicle
for 30 min at 37° C, and then the [125I] MCP-1 binding
assay was done as described in the Methods section. Data are from single
experiments, representative of three. Symbols are the mean of duplicate
determinations. SEM were always < 5% and are not reported.
Figure 6
Trx inhibits the [Ca2+]i increase induced by
MCP-1 in CHO/CCR2 cells.
Tracings indicate levels of [Ca2a]i in CHO/CCR2
cells where MCP-1 (100 ng/ml) was added after 5 min preincubation with
or without 30 ng/ml Trx.
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