Share this article on:

N-Acetyl-Cysteine Attenuates Endotoxin-Induced Adhesion Molecule Expression in Human Whole Blood

Nandate, Koichiroh MD*; Ogata, Masanori MD; Tamura, Hitomi MD; Kawasaki, Takashi MD; Sata, Takeyoshi MD; Shigematsu, Akio MD

doi: 10.1213/01.ANE.0000148616.24996.E7
Critical Care and Trauma: Research Report

Leukocyte adhesion to endothelial cells plays a pivotal role in the early stage of endotoxin shock. The attenuation of the leukocyte response to endotoxin may contribute to the prevention of further organ dysfunction. Recent evidence implies that N-acetyl-cysteine (NAC) attenuates endotoxin-induced pathophysiological changes. We investigated the effect of NAC on the expression of CD11b and CD62L in endotoxin-stimulated human whole blood. NAC (>10 mM) significantly inhibited the lipopolysaccharide (LPS)-induced upregulation of CD11b in a concentration-dependent manner. However, NAC did not affect the LPS-induced downregulation of CD62L. We also analyzed the effect of NAC on interleukin-8 (IL-8)-induced expression of CD11b in human whole blood. IL-8 (10 ng/mL) significantly upregulated the expression of CD11b, and the IL-8-induced upregulation was significantly attenuated by NAC (>10 mM) in a dose-dependent manner. We conclude that NAC attenuates the increased expression of CD11b in either LPS or IL-8-stimulated human whole blood.

IMPLICATIONS: The regulation of the leukocyte response may be crucial to the prevention of organ dysfunction during endotoxin shock. N-acetyl-cysteine (NAC) was found to attenuate CD11b expression in lipopolysaccharide-stimulated human whole blood. These results suggest the possibility of a therapeutic effect of NAC in endotoxin shock.

*Division of Critical and Emergency Care Medicine and †Department of Anesthesiology, University of Occupational and Environmental Health, Kitakyushu, Japan

Supported, in part, by Grant-in-Aid for Scientific Research (B) 14370499 from the Ministry of Education and Culture of Japan.

Accepted for publication October 8, 2004.

Address correspondence and reprint requests to Masanori Ogata, MD, Department of Anesthesiology, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishiku, Kitakyushu 807-8555, Japan. Address e-mail to mogata@med.uoeh-u.ac.jp.

In severe inflammatory disease or sepsis, adhesion molecules expressed on human polymorphonuclear leukocytes and endothelial cells, as well as excessive production of proinflammatory cytokines such as tumor necrosis factor (TNF)-α or interleukin (IL)-1β, plays an important role in the early phase of sepsis-induced organ dysfunction or multiple organ failure. Adhesion of leukocytes to the endothelium consists of several steps. Initially, L-selectin (CD62L) mediates leukocyte tethering to the vessel wall and rolling over the endothelium. In the next step, leukocytes are activated by stimulants such as N-formyl-methionyl-leucyl-phenylalanine (FMLP), IL-8, or platelet-activating factor. This activation induces up-regulation of β2-integrin (CD11b) expression, shedding of L-selectin (CD62L) on leukocytes and augmented adhesion of leukocytes to the ligands (1,2). Furthermore, CD11b on activated leukocytes adheres to the immunoglobulin superfamily on the endothelium. Finally, leukocytes transmigrate into the intestinal compartment and release oxygen free radicals and cytotoxic cytokines, which cause tissue injury (3). Modulating the expression of adhesion molecules may offer a novel therapeutic method to control the early phase of inflammation and thereby prevent multiple organ dysfunctions.

N-acetyl-cysteine (NAC), a precursor of glutathione and a potent antioxidant, has been reported to reduce lipopolysaccharide (LPS)-induced lethality (4), to improve myocardial function in endotoxin shock (5), and to increase tissue oxygenation in patients with septic shock (6). Previous clinical and experimental studies evaluating the protective effects of NAC on the expression of adhesion molecules have been directed primarily toward endothelial molecules, such as intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 (7,8). However, the effects of NAC on the expression of adhesion molecules in human leukocytes have not been established.

In this study, we investigated the effect of NAC on endotoxin-induced adhesion molecule expression in human whole blood. We showed that NAC significantly inhibits increased expression of CD11b induced with either LPS or IL-8 in a dose-dependent manner.

Back to Top | Article Outline

Methods

Endotoxin (LPS), IL-8, and NAC were purchased from Sigma (Tokyo, Japan). Antibodies were purchased from Becton, Dickinson and Co (Tokyo, Japan).

After IRB approval and informed consent was obtained from 10 healthy volunteers, 10 mL of venous blood was collected from each individual by antecubital venipuncture and stored in citrate-containing tubes (VT-050CWS; Terumo, Tokyo, Japan). The donors were nonsmokers, had no history of allergy or infection, and had never been subjected to immunosuppressive therapy. The volunteers’ white blood cell counts were between 5500 and 8500 cells/μL. Aliquots (0.1 mL) of the whole blood samples were incubated in the absence and presence of NAC at 37°C in 95% air/5% carbon dioxide for 30 min and then stimulated with LPS (10 ng/mL) for 60 min or IL-8 (10 ng/mL) for 90 min at 37°C. Next, the blood cells were incubated for 30 min in the dark with a saturating concentration of fluorescein isothiocyanate (FITC)-labeled monoclonal antibodies directed against CD11b (anti-CD11b-FITC; Becton, Dickinson and Co) or with a saturating concentration of phycoerythrin-labeled monoclonal antibodies directed against CD62L (anti-CD62L-PE; Becton, Dickinson and Co). A lysing medium was added to lyse erythrocytes, and after lysis was complete, the samples were fixed with 0.2% paraformaldehyde and washed twice. All samples were analyzed immediately using flow cytometry and XL software (EPICS-XL; Beckman Coulter Electronics, Krefeld, Germany). Leukocytes were discriminated in terms of forward and side scatter. (Forward scatter is correlated to cell size, and side scatter is related to cell granularity.) The forward scatter threshold was set to exclude cell debris from the measurements. We used FITC-labeled monoclonal anti-human CD14 antibody to discriminate lymphocytes from the other leukocytes (neutrocytes and monocytes) (Fig. 1). For each sample, 10,000 cells were analyzed. The results were described as the mean fluorescence intensity.

Figure 1

Figure 1

All data were presented as median and interquartile range. Statistical analysis was performed with either the Wilcoxon’s signed rank test or the Mann-Whitney test, as appropriate. The Bonferroni correction was applied for multiple comparisons. A P value of <0.05 was considered statistically significant.

Back to Top | Article Outline

Results

Whole blood was stimulated with different concentrations of LPS (0–1000 ng/mL). LPS augmented CD11b expression and downregulated CD62L expression in human whole blood in a dose-dependent manner at concentrations between 0.1 and 10 ng/mL. The change in expression reached a plateau with LPS doses ≥10 ng/mL (Fig. 2, A and B). Therefore, we used LPS at a concentration of 10 ng/mL in the present experiments.

Figure 2

Figure 2

After addition of different concentrations (0–20 mM) of NAC, whole blood was stimulated with LPS (10 ng/mL) (n = 10). Figure 3A shows that LPS significantly augmented the expression of CD11b, as compared with the control, and NAC significantly attenuated the LPS-induced CD11b expression in a dose-dependent manner at concentrations of 10–20 ng/mL, as compared with the control (P < 0.05). At concentrations smaller than 5 ng/mL, NAC had no effect on LPS-induced CD11b expression. Figure 3B shows that LPS significantly downregulated the expression of CD62L, as compared with the control, and that NAC at concentrations of 5–20 ng/mL had no effect on the LPS-induced downregulation of CD62L.

Figure 3

Figure 3

We stimulated whole blood with IL-8 at a concentration of 10 ng/mL, which was reported to be sufficient to induce CD11b expression (9). Figure 4 shows the effect of NAC on the IL-8-induced expression of CD11b. IL-8 significantly augmented the expression of CD11b, as compared with the control, and this effect was attenuated by pretreatment with NAC at concentrations between 10 and 20 mM (P < 0.05). NAC at a concentration of 5 mM did not have a significant effect on the IL-8-induced CD11b expression.

Figure 4

Figure 4

Neither NAC, IL-8, nor LPS had an effect on leukocyte cell viability, as assayed by the exclusion of the vital stain trypan blue.

Back to Top | Article Outline

Discussion

This is the first report showing that NAC dose-dependently attenuated the increased expression of CD11b in human whole blood, but NAC did not inhibit the down regulation of CD62L.

There is only one published report regarding the effect of NAC on adhesion molecules expressed in human leukocytes. Weigand et al. (7) reported that 1 mM of NAC did not significantly affect the expression of CD18 in human whole blood, which is consistent with our results. However, their study demonstrated that 10 mM NAC did not inhibit the expression of CD18 in human whole blood, which is inconsistent with our results. They stimulated human whole blood with FMLP or phorbol-12-myristate-13-acetate, whereas we used LPS; the difference in stimulants might have caused this discrepancy. It should also be noted that 10 mM of NAC tended to suppress the expression of CD18 in their study but was not statistically significant, and the sample size used in their study was small (n = 5). We propose that the small sample size might have masked the statistical significance in their study.

It is important to exclude the possibility that the inhibitory effect of NAC on CD11b expression observed in this study was caused by the cytotoxicity of NAC in human leukocytes. A previous report has shown that NAC at concentrations of <30 mM did not affect the viability of human neutrophils or monocytes (10). We also investigated the cytotoxicity of NAC (5–20 mM), using the trypan blue exclusion test, and found more than 98% cell viability. Therefore, we conclude that the inhibitory effect of NAC is not associated with cell cytotoxicity.

LPS induces many proinflammatory cytokines, such as TNF-α, IL-1β, IL-6, and IL-8. There is increasing evidence that the protective effect of NAC during endotoxin shock is closely associated with the inhibition of proinflammatory cytokines, especially TNF-α. Peristeris et al. (11) showed that the protective effect of NAC against LPS lethality in mice was related to the inhibition of TNF-α. Bakker et al. (12) reported that NAC restored oxygen availability to tissues in the endotoxic dog model and that this was associated with an attenuated release of TNF-α. These results suggested to us the possibility that NAC inhibited the expression of proinflammatory cytokines such as TNF-α, which consequently attenuated the increased expression of CD11b.

We also examined the effect of NAC on the adhesion molecules expressed in human whole blood, stimulated with IL-8. Although IL-8 is a potent chemokine and plays a crucial role in a number of pathological conditions, it displays considerably less proinflammatory action than LPS (13). IL-8 significantly increased the expression of CD11b, which is consistent with previous results (9). The increased expression of CD11b induced with IL-8 was also dose-dependently attenuated by NAC. Our results demonstrated that NAC inhibited the increase of expression of CD11b induced with either LPS or IL-8.

Leukocyte rolling, the initial step in the recruitment of leukocytes to sites of acute inflammation, is followed by leukocyte activation, firm adhesion, and transmigration into the interstitial tissue (3). L-selectin contributes to physiologic leukocyte rolling and is rapidly shed from the surfaces of leukocytes on activation (14,15). Evidence has suggested the shedding of L-selectin as an important regulator of leukocyte rolling velocity (16). NAC did not affect the LPS-induced down-regulation of CD62L in human whole blood, which is consistent with previous data (7). The data we presented suggested that NAC did not affect the leukocyte rolling induced with LPS. However, the physiological function of L-selectin remains unknown (17), and the meaning of the observed effect of NAC on the down-regulation of CD62L must be further investigated.

In conclusion, the present study demonstrated that NAC dose-dependently attenuated increased expression of CD11b induced with LPS or IL-8 in human leukocytes. This result suggests the possibility of a therapeutic effect of NAC to control the early phase of inflammation.

Back to Top | Article Outline

References

1. Parrillo JE. Pathogenetic mechanisms of septic shock. N Engl J Med 1993;328:1471–7.
2. Bone RC. Inhibitors of complement and neutrophils: a critical evaluation of their role in the treatment of sepsis. Crit Care Med 1992;20:891–8.
3. Butcher EC. Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell 1991;67:1033–6.
4. Villa P, Ghezzi P. Effect of N-acetyl-L-cysteine on sepsis in mice. Eur J Pharmacol 1995;292:341–4.
5. Zhang H, Spapen H, Nguyen DN, et al. Protective effects of N-acetyl-L-cysteine in endotoxemia. Am J Physiol 1994;266:H1746–54.
6. Spies CD, Reinhart K, Witt I, et al. Influence of N-acetyl-cysteine on indirect indicators of tissue oxygenation in septic shock patients: results from a prospective, randomized, double-blind study. Crit Care Med 1994;22:1738–46.
7. Weigand MA, Plachky J, Thies JC, et al. N-acetyl-cysteine attenuates the increase in alpha-glutathione S-transferase and circulating ICAM-1 and VCAM-1 after reperfusion in humans undergoing liver transplantation. Transplantation 2001;72:694–8.
8. Schmidt H, Schmidt W, Muller T, et al. N-acetyl-cysteine attenuates endotoxin-induced leukocyte-endothelial cell adhesion and macromolecular leakage in vivo. Crit Care Med 1997;25:858–63.
9. Detmers PA, Lo SK, Olsen-Egbert E, et al. Neutrophil-activating protein 1/interleukin 8 stimulates the binding activity of the leukocyte adhesion receptor CD11b/CD18 on human neutrophils. J Exp Med 1990;171:1155–62.
10. Kharazmi A, Nielsen H, Schiotz PO. N-acetyl-cysteine inhibits human neutrophil and monocyte chemotaxis and oxidative metabolism. Int J Immunopharmacol 1988;10:39–46.
11. Peristeris P, Clark BD, Gatti S, et al. N-acetyl-cysteine and glutathione as inhibitors of tumor necrosis factor production. Cell Immunol 1992;140:390–9.
12. Bakker J, Zhang H, Depierreux M, et al. Effects of N-acetyl-cysteine in endotoxic shock. J Crit Care 1994;9:236–43.
13. Norgauer J, Krutmann J, Dobos GJ, et al. Actin polymerization, calcium-transients, and phospholipid metabolism in human neutrophils after stimulation with interleukin-8 and N-formyl peptide. J Invest Dermatol 1994;102:310–4.
14. Ley K, Bullard DC, Arbones ML, et al. Sequential contribution of L- and P-selectin to leukocyte rolling in vivo. J Exp Med 1995;181:669–75.
15. Kishimoto TK, Jutila MA, Berg EL, Butcher EC. Neutrophil Mac-1 and MEL-14 adhesion proteins inversely regulated by chemotactic factors. Science 1989;245:1238–41.
16. Peschon JJ, Slack JL, Reddy P, et al. An essential role for ectodomain shedding in mammalian development. Science 1998;282:1281–4.
17. Hafezi-Moghadam A, Thomas KL, Prorock AJ, et al. L-selectin shedding regulates leukocyte recruitment. J Exp Med 2001;193:863–72.
© 2005 International Anesthesia Research Society