Ann Surg Treat Res.  2017 Oct;93(4):217-224. 10.4174/astr.2017.93.4.217.

The inflammatory response of neutrophils in an in vitro model that approximates the postcardiac arrest state

• Cho YD
• Park SJ
• Choi SH
• Yoon YH
• Kim JY
• Lee SW
• Lim CS

• KMID: 2392372
• DOI: http://doi.org/10.4174/astr.2017.93.4.217

Abstract

PURPOSE
Postcardiac arrest syndrome (PCAS) shares many features with sepsis including plasma cytokine elevation with dysregulation of cytokine production, and the presence of endotoxin in plasma. PCAS is closely related to ischemia-reperfusion injury. During ischemia-reperfusion injury, neutrophil, which is the first line of innate immunity, plays a major role. In this study, we investigated the inflammatory response of human neutrophils in an in vitro model which we simulated with hypoxia-normoxia and hypoxia-hyperoxia environments.
METHODS
After separation of neutrophils from the whole blood, they were divided into 3 experimental groups: normoxia-normoxia, hypoxia-normoxia, and hypoxia-hyperoxia groups. The production of Hâ‚‚Oâ‚‚, the expression of Toll-like receptor 4 (TLRâ‚„) receptor, and the extent of apoptosis of the neutrophils were checked.
RESULTS
The in vitro hypoxia-normoxia and -hyperoxia models, which simulated the PCAS, showed initiation of the neutrophils' inflammatory reaction by hypoxia insult. Lipopolysaccharide amplifies such inflammation; therefore, prevention of secondary infection may be critical in postresuscitation patients. Temporary hyperoxia following hypoxic insult showed no difference in inflammatory reaction compared with hypoxia-normoxia. Rather, temporary hyperoxia may suppress or minimize inflammation by attenuation of TLR4 receptor.
CONCLUSION
It is well known that continuous hyperoxygenation after successful cardiac arrest harms patients, but temporary hyperoxygenation with 100% Oâ‚‚ in a clinical situation may be helpful.

Keyword

Neutrophil; Reperfusion injury; Hypoxia; Hyperoxia

MeSH Terms

Anoxia
Apoptosis
Coinfection
Heart Arrest
Humans
Hyperoxia
Immunity, Innate
In Vitro Techniques*
Inflammation
Neutrophils*
Passive Cutaneous Anaphylaxis
Plasma
Reperfusion Injury
Sepsis
Toll-Like Receptor 4
Toll-Like Receptor 4

Figure

• 

  Fig. 1 Culture of neutrophils without lipopolysaccharide under normoxia-normoxia, hypoxia-normoxia, and hypoxia-hyperoxia. Normoxia group: No insult was given to this group. Polymorphonuclear neutrophils (PMN) was cultured and incubated at 37℃ in a 5% CO2 incubator in room air. Normoxia group was control group. Hypoxia-normoxia group: An initial hypoxia insult was administered for 2 hours; then normoxic insult and 2 hours of culture at 37℃ in 5% CO2 incubator followed by incubation in room air for 14 hours. Hypoxia-hyperoxia group: An initial hypoxia insult for 2 hours was given, then hyperoxic insult and 2 hours of culture at 37℃, followed by incubation in room air for 14 hours. Hydrogen peroxide (H2O2) and Toll-like receptor 4 (TLR4) were measured after 4 hours of PMN culture. Apoptosis was measured after 18 hours, including period of first and second insults and culture for 14 hours in room air.

• 

  Fig. 2 Culture of neutrophils with lipopolysaccharide (LPS) under normoxia-normoxia, hypoxia-normoxia, and hypoxia-hyperoxia. Normoxia group: polymorphonuclear neutrophils (PMN) was cultured and incubated with LPS in room air. Normoxia group was control group. Hypoxia-normoxia group: An initial hypoxia insult for 2 hours was given, then normoxic insult and 2 hours of culture at 37℃ followed by incubation in room air for 14 hours with LPS insult. Hypoxia-hyperoxia group: Initial hypoxia insult for 2 hours was given; then hyperoxic insult and 2 hours of culture at 37℃, followed by incubation in room air for 14 hours with LPS insult. hydrogen peroxide (H2O2) and Toll-like receptor 4 (TLR4) receptors were measured after 6 hours of PMN culture. Apoptosis was measured after 18 hours, including period of first and second insults and culture for 14 hours in room air.

• 

  Fig. 3 Effects of hypoxia-normoxia and hypoxia-hyperoxia conditions on inflammatory response of neutrophils. Each value has been calculated as ratio (%) relative to control group. In hypoxia-normoxia group, hydrogen peroxide (H2O2) and Toll-like receptor 4 (TLR4) production were higher and apoptosis was delayed compared to control group. In hypoxia-hyperoxia group, production of H2O2 was increased and apoptosis was delayed compared to control group; however, TLR4 receptor production was restored to control group. Mean ± standard deviation (n = 18). *P < 0.05, paired t-test.

• 

  Fig. 4 Effects of lipopolysaccharide (LPS) insult in neutrophils precultured in normoxia-normoxia, hypoxia-normoxia, and hypoxia-hyperoxia conditions. Each value has been calculated as ratio (%) relative to control group. Compared to control group, hydrogen peroxide (H2O2) production was increased in all groups, apoptosis was delayed in all groups, and Toll-like receptor 4 (TLR4) receptor production was increased in all groups. Mean ± standard deviation (n = 18). *P < 0.05, paired t-test.

• 

  Fig. 5 Comparison of neutrophils' inflammatory response with or without lipopolysaccharide (LPS) insult between hypoxia-normoxia and hypoxia-hyperoxia groups. (A, B) There were no differences in production of hydrogen peroxide (H2O2) between hypoxia-normoxia and hypoxia-hyperoxia groups with or without LPS insult. (A, B) Delay of apoptosis was prominent in hypoxia-normoxia group without LPS insult compared to hypoxia-hyperoxia group with LPS insult. (A, B) Toll-like receptor 4 (TLR4) receptor production was decreased in hypoxia-hyperoxia group with or without LPS insult compared to hypoxia-normoxia group with or without LPS insult. Mean ± standard deviation (n = 18). *P < 0.05, paired t-test.

Reference

1. Negovsky VA. The second step in resuscitation--the treatment of the ‘post-resuscitation disease’. Resuscitation. 1972; 1:1–7.

2. Adrie C, Adib-Conquy M, Laurent I, Monchi M, Vinsonneau C, Fitting C, et al. Successful cardiopulmonary resuscitation after cardiac arrest as a “sepsis-like” syndrome. Circulation. 2002; 106:562–568.

3. Ar'Rajab A, Dawidson I, Fabia R. Reperfusion injury. New Horiz. 1996; 4:224–234.

4. Strieter RM, Kasahara K, Allen RM, Standiford TJ, Rolfe MW, Becker FS, et al. Cytokine-induced neutrophil-derived interleukin-8. Am J Pathol. 1992; 141:397–407.

5. Adrie C, Laurent I, Monchi M, Cariou A, Dhainaou JF, Spaulding C. Postresuscitation disease after cardiac arrest: a sepsis-like syndrome. Curr Opin Crit Care. 2004; 10:208–212.

6. Triantafilou M, Triantafilou K. Lipopolysaccharide recognition: CD14, TLRs and the LPS-activation cluster. Trends Immunol. 2002; 23:301–304.

7. Prabhakar NR, Kumar GK. Oxidative stress in the systemic and cellular responses to intermittent hypoxia. Biol Chem. 2004; 385:217–221.

8. Savill J, Dransfield I, Gregory C, Haslett C. A blast from the past: clearance of apoptotic cells regulates immune responses. Nat Rev Immunol. 2002; 2:965–975.

9. Francois S, El Benna J, Dang PM, Pedruzzi E, Gougerot-Pocidalo MA, Elbim C. Inhibition of neutrophil apoptosis by TLR agonists in whole blood: involvement of the phosphoinositide 3-kinase/Akt and NF-kappaB signaling pathways, leading to increased levels of Mcl-1, A1, and phosphorylated Bad. J Immunol. 2005; 174:3633–3642.

10. Akira S. TLR signaling. Curr Top Microbiol Immunol. 2006; 311:1–16.

11. Eker T, Genc V, Sevim Y, Cumaogullari O, Ozcelik M, Kocaay AF, et al. The effects of ventilation with high density oxygen on the strength of gastrointestinal anastomosis. Ann Surg Treat Res. 2015; 89:17–22.

12. Wood JG, Johnson JS, Mattioli LF, Gonzalez NC. Systemic hypoxia increases leukocyte emigration and vascular permeability in conscious rats. J Appl Physiol (1985). 2000; 89:1561–1568.

13. Haslett C, Savill JS, Whyte MK, Stern M, Dransfield I, Meagher LC. Granulocyte apoptosis and the control of inflammation. Philos Trans R Soc Lond B Biol Sci. 1994; 345:327–333.

14. Mecklenburgh KI, Walmsley SR, Cowburn AS, Wiesener M, Reed BJ, Upton PD, et al. Involvement of a ferroprotein sensor in hypoxia-mediated inhibition of neutrophil apoptosis. Blood. 2002; 100:3008–3016.

15. Walmsley SR, Print C, Farahi N, Peyssonnaux C, Johnson RS, Cramer T, et al. Hypoxia-induced neutrophil survival is mediated by HIF-1alpha-dependent NF-kappaB activity. J Exp Med. 2005; 201:105–115.

16. Yao L, Kan EM, Lu J, Hao A, Dheen ST, Kaur C, et al. Toll-like receptor 4 mediates microglial activation and production of inflammatory mediators in neonatal rat brain following hypoxia: role of TLR₄ in hypoxic microglia. J Neuroinflammation. 2013; 10:23.

17. Kim DH, Billiar TR. Hypoxia activates toll-like receptor 4 signaling in primary mouse hepatocytes through the receptor clustering within lipid rafts. J Korean Surg Soc. 2011; 80:194–203.

18. Hyakkoku K, Hamanaka J, Tsuruma K, Shimazawa M, Tanaka H, Uematsu S, et al. Toll-like receptor 4 (TLR₄), but not TLR3 or TLR9, knock-out mice have neuroprotective effects against focal cerebral ischemia. Neuroscience. 2010; 171:258–267.

19. Suzuki Y, Hattori K, Hamanaka J, Murase T, Egashira Y, Mishiro K, et al. Pharmacological inhibition of TLR4-NOX4 signal protects against neuronal death in transient focal ischemia. Sci Rep. 2012; 2:896.

20. Jian QC, Wu XW, Song HC, Zheng L. The role of toll like receptor-4 signal pathways activation in ischemia-reperfusion injury of island skin flap. Zhonghua Zheng Xing Wai Ke Za Zhi. 2012; 28:444–448.

21. Deitch EA, Sambol JT. The gut-origin hypothesis of MODS. In : Deitch EA, Vincent JL, Windsor A, editors. Sepsis and multiple organ dysfunction. London: Saunders;2002. p. 105–113.

22. Partrick DA, Moore FA, Moore EE, Barnett CC Jr, Silliman CC. Neutrophil priming and activation in the pathogenesis of postinjury multiple organ failure. New Horiz. 1996; 4:194–210.

23. Biffll WL, Moore EE, Moore FA. The-two-hit model of MODS. In : Deitch EA, Vincent JL, Windsor A, editors. Sepsis and multiple organ dysfunction. London: Saunders;2002. p. 127–132.

24. Molloy EJ, O'Neill AJ, Doyle BT, Grantham JJ, Taylor CT, Sheridan-Pereira M, et al. Effects of heat shock and hypoxia on neonatal neutrophil lipopolysaccharide responses: altered apoptosis, Toll-like receptor-4 and CD11b expression compared with adults. Biol Neonate. 2006; 90:34–39.

25. Cavaillon JM, Adib-Conquy M, Fitting C, Adrie C, Payen D. Cytokine cascade in sepsis. Scand J Infect Dis. 2003; 35:535–544.

26. Munford RS, Pugin J. Normal responses to injury prevent systemic inflammation and can be immunosuppressive. Am J Respir Crit Care Med. 2001; 163:316–321.

27. Schmitz K, Jennewein M, Pohlemann T, Seekamp A, Oberringer M. Reoxygenation attenuates the adhesion of neutrophils to microvascular endothelial cells. Angiology. 2011; 62:155–162.