Glutamine may repress the weak LPS and enhance the strong heat shock induction of monocyte and lymphocyte HSP72 proteins but may not modulate the HSP72 mRNA in patients with sepsis or trauma

Abstract

Objective: We assessed the lipopolysaccharide (LPS) or heat shock (HS) induction of heat shock protein-72 (HSP72) in peripheral blood mononuclear cells (PBMCs) of patients with severe sepsis (SS) or trauma-related systemic inflammatory response syndrome (SIRS), compared to healthy individuals (H); we also investigated any pre- or posttreatment modulating glutamine (Gln) effect.

Methods: SS (11), SIRS (10), and H (19) PBMCs were incubated with 1 μg/mL LPS or 43°HS. Gln 10 mM was either added 1 h before or 1 h after induction or was not added at all. We measured monocyte (m), lymphocyte (l), mRNA HSP72, HSP72 polymorphisms, interleukins (ILs), monocyte chemoattractant protein-1 (MCP-1), and cortisol levels.

Results: Baseline lHSP72 was higher in SS (p < 0.03), and mHSP72 in SIRS (p < 0.02), compared to H. Only HS induced l/mHSP72/mRNA HSP72; LPS induced IL-6, IL-8, IL-10, and MCP-1. Induced mRNA was related to l/mHSP72, and was related negatively to cytokines. Intracellular l/mHSP72/HSP72 mRNA was related to serum ILs, not being influenced by cortisol, illness severity, and HSP72 polymorphisms. Gln did not induce mRNA in any group but modified l/mHSP72 after LPS/HS induction unpredictably.

Conclusions: HSP72 mRNA and l/mHSP72 are higher among critically ill patients, further induced by HS, not by LPS. HSP72 proteins and HSP72 mRNA are related to serum ILs and are negatively related to supernatant cytokines, not being influenced by HSP72 polymorphisms, cortisol, or illness severity. Gln may depress l/mHSP72 after LPS exposure and enhance them after HS induction, but it may not affect early induced HSP72 mRNA.

Figure 1

At 4 hours without any stimulation, baseline mHSP72 was higher in SS and SIRS and lHSP72 in SS compared to H. At 2 hours without any stimulation, mRNA showed a nonsignificant increased trend in SS compared to H.

Figure 2

HS alone or with Gln-induced lHSP72 or mHSP72 compared to LPS in healthy individuals (a-b) in patients with SIRS (c-d) or with severe sepsis (e-f). The box and whisker plots show the median (horizontal line within the box) and the 25th and 75th percentiles (whiskers minimum–maximum values). Solid circles represent outliers and stars extremes.

Figure 3

PBMCs' HSP72 mRNA was significantly induced by HS within 2 hours compared to the LPS induction (a). Dashed lines with p values indicate differences between the two induction modalities in each group. Baseline and various treatment models did not differ among H, SIRS, and SS groups in either HS or LPS induction modes. On the contrary, 24 hr supernatant cytokines were induced by LPS and not by HS, especially among critically ill patients (b). Dashed lines with p values indicate differences between the two induction modes in each group. Cytokines differed among groups in the LPS induction mode. Differences are indicated between groups by #, SIRS versus H, ∗, SS versus H, and ∗∗, SIRS versus SS. Induced mRNA was positively related to the intracellular HSP72 proteins (p < 0.0001). In contrast it was negatively related (p < 0.0001) to IL-6, IL-8, IL-10, TNF, and MCP-1 (c).

Figure 4

In healthy controls only LPS pretreated with Gln suppressed mHSP72 baseline expression (a). Gln given 1 hour before HS further enhanced the induced mHSP72 expression in comparison to Gln given 1 hour after induction (b). In SIRS Gln given 1 hour before LPS further suppressed the already LPS-suppressed mHSP72 baseline expression (c); in the HS induction model, Gln given before HS showed a trend to increase further the already induced lHSP72 expression (d). In septic PBMCs, only Gln given before LPS abolished the LPS induction effect on lHSP72 (e). However, Gln also given before and not after HS further induced lHSP72 baseline expression (f). These differences between HS and LPS and between Gln given before or after induction clearly underline a Gln pretreatment depressive trend to lymphocyte and monocyte HSP72 proteins of PBMCs exposed to LPS but an enhancing trend to those exposed to HS induction effect.

Figure 5

Gln given before or after LPS incubation or HS induction did not modify mRNA in H, SIRS, or SS group (a–f).

Figure 6

In individual patients with severe sepsis, glutamine effects on either LPS modulating or HS-induced HSP72 mRNA fold change (a-b) or lymphocyte or monocyte HSP72 MFI expression (c-d) were unpredictable.

Figure 7

(a) PBMCs' HSP72 mRNA and intracellular lymphocyte and monocyte HSP72 proteins did not differ among haplotypes of the polymorphic rs6457452 and rs1061581 HSP72 SNPs. (b) SS patients had increased serum levels of IL-6, IL-10, INF-γ, and cortisol compared to H; SIRS patients had increased serum levels of IL-6 and IL-10 compared to H; differences between SS and SIRS did not reach statistical significance.