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Hatanaka, Elaine*†; Shimomi, Flavia Mammy*; Curi, Rui; Campa, Ana*

doi: 10.1097/01.shk.0000238061.69579.a5
Clinical Aspects

Hypertonic sodium chloride (NaCl) solution has been shown to have beneficial effects on patients with hypovolemic shock. Therapy with hypertonic saline seems to neutralize or minimize the deleterious immune responses in these patients. In this study, we addressed how hypertonic NaCl solution affects the release of cytokines by isolated human blood cells. We examined the effect of NaCl on tumor necrosis factor (TNF)-α, interleukin (IL)-8, IL-1β, and IL-1 receptor antagonist released by human neutrophils and mononuclear cells under nonstimulated and LPS-stimulated conditions. Cells isolated from healthy donors were cultured in the presence or absence of lipopolysaccharide (LPS). Cytokine concentrations were measured by enzyme-linked immunosorbent assay in culture supernatants. The addition of NaCl lowered the production of IL-8, TNF-α, and IL-1 receptor antagonist by neutrophils, and IL-8 and IL-1β by mononuclear cells stimulated with LPS. This effect was not observed when NaCl solution was replaced by a solution of potassium iodide in the same conditions. The decreased production of cytokines in the presence of hypertonic saline was not caused by cell death. Our findings support the proposal that the modulation of leukocyte inflammatory response by NaCl occurs by an electrolyte-specific effect in addition to the hyperosmolarity. Furthermore, the results of this study support the therapeutic use of appropriate doses of i.v. NaCl solutions to minimize tissue injury characterized by neutrophil and mononuclear cell infiltration into tissues in post-traumatic patients.

*Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, and Departamento de Fisiologia e Biofísica, Instituto de Ciências Biomédicas, Universidade de São Paulo, Brazil

Received 20 Apr 2006; first review completed 11 May 2006; accepted in final form 14 Jul 2006

Address reprint requests to Elaine Hatanaka, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brasil, CEP 05508-900. E-mail:

This study was supported by the Brazilian agencies Fundação de Amparo à Pesquisa do Estado de São Paulo and the Conselho Nacional de Desenvolvimento Científico e Tecnológico.

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Resuscitation of hemorrhagic shock patients with sodium salt solutions, especially sodium chloride (NaCl), has been used in the treatment of trauma patients because of its beneficial hemodynamic properties, such as rapid expansion of intravascular volume, reduction of endothelial and tissue edema, and improvement of blood viscosity. The use of NaCl hyperosmotic solutions leads to higher survival rates than other sodium salts (e.g., acetate, bicarbonate, and nitrate), chloride salts (e.g., lithium) and nonelectrolytes (e.g., glucose, mannitol, and urea) (1-3).

After intense blood loss, trauma patients frequently show lethal post-traumatic complications, such as acute respiratory distress syndrome, multiple organ failure, and sepsis (4, 5). Hypertonic saline resuscitation decreases susceptibility to sepsis after hemorrhagic shock (6, 7), and recent studies have shed a favorable light on NaCl hyperosmotic solutions as a simple, but effective, tool to modulate leukocyte function after trauma (8-11). Ischemia/reperfusion primes neutrophils and mononuclear cells to produce an exaggerated response to inflammatory stimuli (the so-called 2-hit hypothesis) in post-traumatic patients (12). Prevention of inflammation and immunosuppression has been the main focus of trauma researchers for many years. Recently, hypertonic NaCl resuscitation has attracted attention as a possible therapeutic approach to counteract such deleterious immune responses in trauma patients, especially in relation to neutrophil functions (13-17).

Neutrophils and mononuclear cells can influence the inflammatory process by synthesizing proinflammatory and anti-inflammatory cytokines and growth factors that modulate the inflammatory response (17). Interleukin (IL)-8, tumor necrosis factor (TNF)-α, and IL-1β are proinflammatory cytokines with a central role in the inflammatory processes. Interleukin-8 shows angiogenic, chemotactic, and stimulatory activities toward blood cells, providing the recruitment of a great number of neutrophils and other immune cells to the inflammatory site and promoting neovascularization (18). Tumor necrosis factor-α mediates the systemic effects of inflammation, such as fever, the hepatic release of acute-phase proteins, and hematopoiesis. Tumor necrosis factor-α and IL-1β act on leukocytes and endothelium to induce acute inflammation. An altered production of IL-1 or IL-1 receptor antagonists (IL-1ra) that competitively inhibits binding of IL-1 to cell surface receptors may impair the inflammatory processes (19). The same applies to TNF-α and IL-8 production. It is therefore reasonable to assume that any alteration in cytokine production may strongly affect the progress of the inflammatory response and influence the clinical profile of trauma patients.

Although a clear survival effect of hypertonic NaCl solution has been established in the resuscitation of post-traumatic patients (1, 2), the immunomodulatory effects of hypertonic saline resuscitation in patients sustaining traumatic hemorrhagic shock is unclear. In this study, we examined the effects of NaCl solution on TNF-α, IL-8, IL-1β, and IL-1ra release by human neutrophils and mononuclear cells under nonstimulated and lipopolysaccharide (LPS)-stimulated conditions.

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With the approval of the Ethical Committee of the Pharmaceutical Sciences Faculty-University of São Paulo University (São Paulo, Brazil), 20 healthy volunteers were recruited from the local population. All subjects gave written informed consent. Blood samples were collected, and cell isolation was performed within 1 h of venipuncture.

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Bovine fetal serum, N-2-hydroxyethylpiperazine -N'-2-ethanesulfonic acid, Histopaque, LPS (Escherichia coli 026:B6), NaCl, potassium iodide (KI), penicillin, propidium iodide, RPMI 1640 supplemented with l-glutamine, sodium bicarbonate, and streptomycin were supplied by Sigma Chemical Co. (St Louis, Mo). The reagents, water, and plasticware used in the experiments were all endotoxin free.

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Cell purification, culture, and cytokine determination

Purified human neutrophil (>98%) and mononuclear cell (>98%) preparations were isolated as previously described and used elsewhere (20, 21) from peripheral blood of healthy human donors under endotoxin-free conditions. After purification, neutrophils (2.5 × 106 cells/mL) and mononuclear cells (1.5 × 106 cells/mL) (suspended in RPMI 1640 medium supplemented with 0.3 g/L glutamine, 2.32 g/L N-2-hydroxyethylpiperazine -N'-2-ethanesulfonic acid, 2 g/L sodium bicarbonate, 100 μg/mL streptomycin, 100 UI/mL penicillin, and 10% low endotoxin fetal serum) were counted in a Neubauer chamber and immediately cultured at 37°C and 5% CO2, with and without LPS (1 and 5 μg/mL) and with NaCl or KI solutions in various concentrations (2.5-50 mmol/L). After 2 and 18 h, the supernatants were collected and stored at equal to or less than −40°C before TNF-α, IL-1β, IL-1ra, and IL-8 determination by using enzyme-linked immunosorbent assay ([ELISA] Quantikine, R&D Systems, Minneapolis, Minn). Interleukin-1β, IL-8, and IL-1ra were determined in the supernatant of cells cultured for 18 h, whereas TNF-α was assayed in cells cultured for 4 h in the absence or presence of NaCl or KI (2.5-50 mmol/L). The detection limits of ELISA were from 15 to 2,000 pg/mL for TNF-α; 7.8 to 250 pg/mL for IL-1β; 10 to 2,500 pg/mL for IL-1ra; and 15 to 1,000 pg/mL for IL-8. These limits can be extended if you dilute samples before starting the assay.

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Cell viability

Cells cultured for 4 or 18 h were centrifuged at 1,000g for 15 min at 4°C, and the pellet thus obtained was resuspended in 500 μL phosphate-buffered saline. Afterwards, 50 ìL of propidium iodide solution (50 mg/mL in phosphate-buffered saline) was added, and cell viability was analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Juan, Calif). Propidium iodide is a highly water-soluble fluorescent compound that cannot pass through intact membranes and is generally excluded from viable cells. It binds to DNA by intercalating between the bases with little or no sequence preference. Fluorescence was measured using the FL2 channel (orange-red fluorescence, 585/42 nm). Ten thousand events were analyzed per experiment. Cells with propidium iodide fluorescence were then evaluated using the Cell Quest software (Becton Dickinson) (22).

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Staining DNA using propidium iodide

DNA fragmentation was analyzed by flow cytometry after DNA staining with propidium iodide according to the method described by Nicoletti et al. (23). The presence of detergent in the solution permeabilizes the cells, which promptly incorporate the dye into DNA. Briefly, after the various incubation periods, the cells were centrifuged at 1,000g for 15 min at 4°C. The resulting pellet was carefully resuspended in 300 μL hypotonic solution containing 50 μg/mL propidium iodide, 0.1% sodium citrate, and 0.1% Triton X-100. The cells were then incubated for 2 h at 4°C. Fluorescence was measured and analyzed as described above (22).

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Statistical analysis

The data are expressed as mean ± SD. The statistical analysis consisted of 1-way analysis of variance using the post hoc Student-Newman-Keuls multiple comparison test (INStat; Graph Pad Software, San Diego, Calif). The significance level was set at P < 0.05.

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Sodium chloride, in the range of 2.5 to 50 mmol/L, did not significantly affect the basal production of IL-8, TNF-α, IL-β, and IL-1ra by neutrophils (Fig. 1) and mononuclear cells (Fig. 2). However, NaCl strongly inhibited LPS-mediated IL-8, TNF-α, and IL-1ra release by neutrophils (Fig. 1), and IL-8, IL-β, and IL-1ra liberated by mononuclear cells (Fig. 2). The inhibitory effect was dose dependent for IL-8 and IL-1ra released by neutrophils and IL-8 released by mononuclear cells. At the highest concentration of NaCl (50 mmol/L), the osmolarity of the medium increased by approximately 1.4 times. The osmolarity change varied from 290 to 390 mOsm/L. A rough estimation is that, in our experiments, the addition of 50 mmol/L of NaCl corresponds to the amount of NaCl usually administered to patients, considering, for instance, that in post-traumatic patients, the usually administered i.v. dose of NaCl solution is 4 mL of a solution at 7.5% per kilogram of body mass. Interestingly, when NaCl was replaced by another salt, KI, there was no significant effect on the production of cytokines by basal and LPS-stimulated neutrophils and mononuclear cells in any concentration used (Fig. 2).

Fig. 1

Fig. 1

Fig. 2

Fig. 2

The diminished production of cytokines by neutrophils by the addition of NaCl solution was not caused by cell death because cell membrane integrity loss (necrosis) (Fig. 3) and DNA fragmentation (apoptosis) (Fig. 4) were not modified in basal and LPS-stimulated cells treated with hypertonic saline. The same patterns of response were observed for mononuclear cells (data not shown).

Fig. 3

Fig. 3

Fig. 4

Fig. 4

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Although several authors have recognized the potential use of hypertonic NaCl solutions for the treatment of sepsis or septic shock, there are limited experimental data regarding the clinical use of this solution. Furthermore, clinical studies related to this issue are mainly descriptive. Thus, the decrease in cytokine release described herein may be an additional beneficial effect of hypertonic saline solution on the immune function in resuscitation of post-traumatic patients. From this standpoint, our data are corroborated by recent studies showing that the blockage of surface integrins or selectin molecules expression by hypertonic saline solutions prevents the accumulation of neutrophils in the sites of various inflammation models (24). Hypertonic saline-induced resuscitation from shock markedly reduced lung injury in response to LPS by preventing neutrophil sequestration (25). Hampton et al. (15) reported that hyperosmolarity significantly impairs N-formyl methionyl-leucyl-phenylalanine-stimulated degranulation of neutrophils, as evidenced by reduced release of lysozyme, a granular enzyme. Previous reports have also shown the ability of hypertonic solutions to inhibit a variety of neutrophil functions, including the generation of reactive oxygen species (10, 13-15), expression of adhesion molecules (26), migration (27), and exocytosis (28).

Several hypotheses exist concerning the mechanisms whereby cells sense osmotic changes. First, cells may detect the ionic strength or medium osmolarity. Changes in cytoskeletal architecture lead to cell shrinkage, which activates intracellular signaling pathways (29). An interesting hypothesis is that cells perceive their volume by sensing macromolecular crowding; small changes in cell volume lead to large increases in the thermodynamic activity of macromolecules. One form of crowding leading to such disproportionate increases in activity may be the aggregation of surface receptors, as recently reported by Rosette and Karin (30). They found that osmotic shrinkage of HeLa cells induces clustering of IL-1, epidermal growth factor, and TNF receptors, despite the absence of their ligands. Clustering of receptors is known to be crucial for their signaling cascade activation (30).

According to Ciesla et al. (31) and Rizoli et al. (32), neutrophils respond to a hypertonic environment by reducing the P38 mitogen-activated protein kinase signaling cascade activity. In addition, hypertonic resuscitation prevents systemic oxidative stress by reducing ischemia/reperfusion injury and consequently attenuates distant alveolar macrophage priming, reducing LPS-induced nuclear factor-κB nuclear translocation in alveolar macrophages (17). Thus, it is conceivable that although rapid restoration of plasma volume occurs after resuscitation with hypertonic saline solution, the effective reduction in organ injury and sepsis could be mediated through the reduction of proinflammatory neutrophil and mononuclear cell cytokine release.

Intracellular pH changes may be another mechanism for the diminished production of cytokines induced by NaCl in LPS-stimulated neutrophils. The balance between cytosolic proton loading and extrusion plays an important role for neutrophil function. In fact, microbicidal behavior, cell migration, intracellular oxidant generation, tumor cell cytotoxicity, azurophil granule exocytosis, and neutrophil death are pH dependent (33). Anion and cation exchangers seem to be the major mechanism by which cellular pH is maintained. Several pH-regulating systems have been identified in leukocytes. These include a NA+/H+ antiport, two types of Cl/HCO3 exchangers, and an adenosine 5′ triphosphate-dependent proton pump (34).

We did not observe alterations in cytokine release when NaCl solution was replaced by KI, indicating that the effect observed here was not caused only by an increment in hyperosmolarity, but also to an anion-specificity effect. In this respect, Matsumoto et al. (35) showed suppression of neutrophil chemotactic activity by hyperosmolar NaCl solution, but not by urea. In fact, NaCl hyperosmotic solution raises the survival rates of patients with severe hemorrhagic shock. The same is not observed for other salts such as sodium salts (e.g., acetate, bicarbonate, and nitrate), chloride salts (e.g., lithium), and even for nonelectrolyte solutions (e.g., glucose, mannitol, and urea). Sodium salts raised survival rates (chloride by 100%, acetate by 72%, bicarbonate by 61%, and nitrate by 55%), normalizing the arterial pressure and the acid-base equilibrium. Solutions without sodium-containing chlorides and nonelectrolytes produced low survival rates (glucose and lithium by 5%, mannitol by 11%, Tris by 22%, and urea by 33%), presenting low cardiac output, low mean circulatory filling pressure, and severe metabolic acidosis (3). Apparently, based on clinical evidence, the effect of NaCl to lower the production of cytokines by leukocytes stimulated with LPS is caused by a combined effect of sodium and chloride.

In conclusion, based on our in vitro study, we added additional information that may be potentially important to explain the effects of clinically used hypertonic saline downregulating the proinflammatory response, especially with respect to the production of proinflammatory cytokines produced by human neutrophils and mononuclear cells. Further clinical trials should be carried out to confirm the relative importance of the therapeutic use of adequate doses of i.v. NaCl solutions in post-traumatic patients to minimize tissue injury characterized by neutrophils and mononuclear cell infiltration.

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IL-8; IL-1β; TNF-α; hemorrhagic shock; leukocytes

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