Heat shock inhibits lipopolysaccharide-induced tissue factor activity in human whole blood

Abstract

Background: During gram-negative sepsis, lipopolysaccharide (LPS) induces tissue factor expression on monocytes. The resulting disseminated intravascular coagulation leads to tissue ischemia and worsens the prognosis of septic patients. There are indications, that fever reduces the mortality of sepsis, the effect on tissue factor activity on monocytes is unknown. Therefore, we investigated whether heat shock modulates LPS-induced tissue factor activity in human blood.

Methods: Whole blood samples and leukocyte suspensions, respectively, from healthy probands (n = 12) were incubated with LPS for 2 hours under heat shock conditions (43 degrees C) or control conditions (37 degrees C), respectively. Subsequent to further 3 hours of incubation at 37 degrees C the clotting time, a measure of tissue factor expression, was determined. Cell integrity was verified by trypan blue exclusion test and FACS analysis.

Results: Incubation of whole blood samples with LPS for 5 hours at normothermia resulted in a significant shortening of clotting time from 357 +/- 108 sec to 82 +/- 8 sec compared to samples incubated without LPS (n = 12; p < 0.05). This LPS effect was mediated by tissue factor, as inhibition with active site-inhibited factor VIIa (ASIS) abolished the effect of LPS on clotting time. Blockade of protein synthesis using cycloheximide demonstrated that LPS exerted its procoagulatory effect via an induction of tissue factor expression. Upon heat shock treatment, the LPS effect was blunted: clotting times were 312 +/- 66 s in absence of LPS and 277 +/- 65 s in presence of LPS (n = 8; p > 0.05). Similarly, heat shock treatment of leukocyte suspensions abolished the LPS-induced tissue factor activity. Clotting time was 73 +/- 31 s, when cells were treated with LPS (100 ng/mL) under normothermic conditions, and 301 +/- 118 s, when treated with LPS (100 ng/mL) and heat shock (n = 8, p < 0.05). Control experiments excluded cell damage as a potential cause of the observed heat shock effect.

Conclusion: Heat shock treatment inhibits LPS-induced tissue factor activity in human whole blood samples and isolated leukocytes.

Figure 1

Effect of heat shock on the LPS-induced shortening of clotting time in whole blood samples. Whole blood samples, supplemented with LPS (100 μg/mL final concentration, LPS) or vehicle (CON), were incubated under heat shock conditions (2 hours at 43°C, 3 hours at 37°C) or normothermia (5 hours at 37°C). Thereafter, samples were recalcified and clotting time was determined. Results are shown as mean and standard deviation of 12 experiments per group. *: p < 0.05.

Figure 2

Effects of protein synthesis inhibition and tissue factor blockade on the LPS-induced shortening of clotting time. Whole blood samples were incubated with LPS (100 μg/mL) or vehicle (CON) in presence and absence of the protein synthesis inhibitor cycloheximide and the inhibitor of tissue factor effects, active site inhibited factor seven (ASIS), respectively. Results are shown as mean and standard deviation of 6 experiments per group. *: p < 0.05

Figure 3

Effects of hyperthermia on LPS-induced tissue factor activity of leukocyte suspensions. LPS (100 ng/mL) or vehicle (CON) was added to freshly isolated leukocytes suspended in PBS, which were then incubated under heat shock conditions or normothermia. Thereafter, platelet poor plasma was added to the samples and clotting time as a measure of tissue factor was determined. Results are shown as mean and standard deviation of 8 experiments per group. *: p < 0.05

Figure 4

FACS analysis showing a contour plot of anti-CD14 fluorescence versus forward scatter. Monocytes are separated from other leukocytes by the more than 100-fold increase in fluorescence.

Figure 5

Distribution of forward and sideward scatter of monocytes treated with and without hyperthermia and LPS. Monocytes were identified by phycoerythrin-labelled anti-CD14 antibodies.