Early and marked up-regulation of TNF-α in acute respiratory distress syndrome after cardiopulmonary bypass

Abstract

Despite the technique of cardiopulmonary bypass (CPB) improved the development of modern cardiac surgery, many factors during CPB have been reported to induce acute respiratory distress syndrome (ARDS). The present study was to investigate which pro-inflammatory factors involved in the early phase of ARDS. Ten patients underwent valve replacement surgery with or without ARDS were enrolled for analysis of pulmonary function and inflammatory factors release including white blood cell (WBC), neutrophils, CD11b, CD18, interleukin (IL)-8 and tumor necrosis factor-α (TNF-α). The results demonstrated that the ratio of arterial oxygen tension/fraction of inspire oxygen (PaO₂/FiO₂) was greatly reduced in ARDS patients, but only the release of TNF-α was significantly increased, which was reversely correlated to the values of PaO₂/FiO₂. Also, the count of neutrophils adhesive to pulmonary endothelial cells was significantly increased in ARDS patients. Therefore, we concluded that TNF-α was quickly up-regulated and involved in the pathogenesis of CPB-induced ARDS via guiding primed neutrophils to pulmonary interstitium.

Keywords

tumor necrosis factor-α / cardiopulmonary bypass / inflammation / acute respiratory distress syndrome

Introduction

The technique of cardiopulmonary bypass (CPB) improved the development of modern cardiac surgery, but many factors during CPB, either material-dependent (the exposure of blood to nonphysiologic surfaces and conditions) or material-independent (surgical trauma, ischemia/reperfusion injury, changes in body temperature, and release of endotoxin), could induce a complex inflammatory response and ultimately cause acute respiratory distress syndrome (ARDS) [1-5]. Pathogenesis of ARDS includes inflammation characterized by neutrophil accumulation in the lungs during early stages and lymphocyte-induced lung fibrosis during late stages. Neutrophils adhere to the injured capillary endothelium and migrate through the interstitum into the air space. In the air space, cytokines, like interleukin (IL)-1, IL-6, IL-8, IL-10 and tumor necrosis factor-α (TNF-α) secreted by alveolar macrophages, act locally to stimulate chemotaxis and activate neutrophils [6-8]. Despite the extensive work that has been done to characterize the inflammation that occurs in ARDS, the identity of the cellular elements and related inflammatory mediators that initiate or promote pathogenesis are still elusive [9-11].

Reducing the CPB-induced ARDS is of great clinical relevance and believed to be beneficial in improving the prognosis of patients. Therefore, this clinical trial was designed to investigate which pro-inflammatory factors are involved in the early phase of ARDS. The results of this study will provide promising candidate for early diagnosis and treatment of CPB-induced ARDS.

Materials and methods

The study protocol was approved by the Clinical Trial Committee of West China Hospital, Sichuan University (Chinese Clinical Trial Registry No: ChiCTR-ECC-00000317) and was carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association. Before any study-related procedure, patients or authorized legal representatives were required to give written informed consent to participate and were free to withdraw from the study at any time.

Patient population

Patients aged from 18 years to 70 years and scheduled to valve replacement were enrolled in the present study. The exclusive criteria were: (1) pulmonary dysfunction or pulmonary arterial hypertension before surgery; (2) re-do surgery; (3) the classic cardiac function were NYHA (New York Heart Association) III and IV; (4) the patient was unwilling to take part in the study. ARDS was identified according to the definition of the American-European Consensus Conference on ARDS [12] as following: a ratio of the arterial oxygen tension to the fraction of inspired oxygen (PaO₂/FiO₂) was 200 or less; bilateral pulmonary edema was seen on a frontal chest radiograph (infiltrates); and no clinical evidence of left atrial hypertension or a pulmonary-artery wedge pressure of 18 mmHg or less (if measured). Once the ARDS was determined, an additional patient was selected as control based on the following criteria: the patient without ARDS in 24-48 h after same type of surgeon; same type of operation; same gender; the weight and age were within±10% of the ARDS patient’s.

During Mar–Sep 2009, a total of 493 patients underwent valve replacement surgery in West China Hospital, 102 patients were excluded for pulmonary hypertension, pulmonary infection or presence of any other exclusion criterion and 391 patients were included in this study. Five of these patients were diagnosed ARDS and 5 non-ARDS patients were selected as matched-control. The patient characteristics and operative data were shown in Table 1.

Pulmonary function

The arterial oxygen pressure (PaO₂) of blood samples collected before surgery and 4 h after surgery were measured by a portable blood gas analyzer (i-STAT Corporation, Windsor, NJ). The pulmonary function was assessed by PaO₂/FiO₂.

White blood cell and neutrophil counts

Blood samples from central vein were collected before surgery and 4 h after surgery. The white blood cell (WBC) and neutrophil counts were measured by an Auto Hematology Analyzer (BC-3000 Plus, Mindray, Shenzhen, China).

Determination of TNF-α and IL-8 releases in plasma

The plasma of venous blood was separated by centrifugation (2500 rpm × 20 min). The levels of TNF-α and IL-8 were determined by commercial ELISA kits (Bender MedSystems, Vienna, Austria).

Flow cytometry

Neutrophils were isolated from central venous blood by dextran sedimentation and centrifugation on Ficoll-Hypaque density gradient, as previously described [13]. Then neutrophils were washed and suspended in medium (HBSS containing Mg²⁺ and Ca²⁺ plus 0.1% BSA). The flow cytometry analysis of CD11b or CD18 expression in neutrophils was performed after incubation with fluorescein isothiocyanate (FITC)-labeled anti-human CD18 and phycoerythrin (PE)-labeled anti-human CD11b antibodies. CellQuest software was used for acquisition and analysis on a FACSCalibur (BD Biosciences, Heidelberg, Germany). Data were expressed as mean fluorescence intensity (MFI).

Neutrophils adhesion assay

Isolated neutrophils were seeded onto the plate wells and were allowed to adhere to pulmonary endothelia cells for 30 min at 37°C in 5% CO₂. Neutrophils adhesion to pulmonary endothelia cells was examined by phase-contrast microscopy using a Nikon Diaphot microscope equipped with a Nikon Coolpix digital camera. Neutrophils harvested before surgery were used as control. The count of neutrophils adhesive to endothelial cells was measured by planimetry with the use of Image pro plus 5.0 software (Media Cybernetics Inc., Silver Spring, MD) and expressed as a percentage of the control.

Statistical analysis

All values are presented as mean±SD. Comparisons between two groups were analyzed by 2-sided Student t test (SPSS 16.0 software). A simple linear regression analysis was performed to estimate the correlation between IL-8 or TNF-α release and PaO₂/FiO₂. P values<0.05 were considered statistically significant.

Results

After surgery, the PaO₂/FiO₂ in the ARDS group was significantly lower than that in the non-ARDS group (P<0.001, Fig. 1). However, there was no significant difference with regarding to the WBC and neutrophil counts (Fig. 2), MFI of CD11b (Fig. 3A) and CD18 (Fig. 3B). Plasma IL-8 level was also comparable between two groups (Fig. 4A), and the relationship between PaO₂/FiO₂ and IL-8 was not significant (P = 0.150, r = -0.505, Fig. 4B). In contrast, the plasma TNF-α concentration was significantly higher in ARDS patients (P = 0.002, Fig. 4C), and it had a significant negative correlation with the ratio of PaO₂/FiO₂ (P = 0.003, r = -0.855, Fig. 4D). In addition, the count of neutrophils adhesive to endothelial cells of ARDS patients was as high as 3.7-fold of non-ARDS patients (P<0.001, Fig. 5).

Discussion

This study indicated that pro-inflammatory factors, including WBC, neutrophils, CD11b, CD18 and IL-8, were not quickly changed in patients with ARDS, while TNF-α was significantly elevated with the progress of ARDS and associated with increased neutrophil adhesion. The results of our study demonstrated TNF-α may be the initiating mediators involved in CPB-induced ARDS.

In pathological conditions, TNF-α is highly presented in inflamed airways, and acts as a potential activator of neutrophil. Our previous study has proven the release of TNF-α increased in a time-dependent manner from the beginning of extracorporeal circulation and pretreatment with TNF-α antibody inhalation, but not intravenous injection, may inhibit CPB-induced lung injury. The present clinical trial provided additional evidence that TNF-α played an important role at the onset of CPB-induced ARDS.

In this study, only five patients developed ARDS in surgery with CPB in our hospital during Mar–Sep 2009. Such a small sample size may probably amplify systematic bias of the result, but the trend that the counts of neutrophils adhesive to pulmonary endothelial cells would be highly increased in ARDS patients is obvious. The primary function of neutrophils is to host defense by containing and eradicating invaded microbial pathogens. However, under pathologic circumstances, neutrophils are primary perpetrators of inflammatory injury to the lung and other organs [6,14,15]. When pulmonary inflammation occurs, neutrophils will pass through the endothelium, interstitial tissues, and epithelium before ending up in the airspaces. During this time, the unrestrained activation of neutrophils in response to inflammatory stimuli may result in release of cytotoxic compounds that can injure lung tissue [14]. Therefore, the increased neutrophil activity and adhesion to pulmonary endothelium predict the lung inflammation and damage. Neutrophil adhesion to endothelial cells is mediated predominantly through interactions between β₂ integrins (CD11/CD18) on the leukocyte surface and intercellular adhesion molecule 1 (ICAM-1) located on the endothelial surface [16,17]. Neutrophils exposed to chemoattractants up-regulate expression of β₂ integrins, but this upregulation alone does not account for neutrophil/endothelial cell interactions [18]. Instead, the stimulation of neutrophil adhesion appears to be mediated by a change in the activation state of β₂ integrins, rather than simply an increase in their number. This increased avidity of integrins has been shown to be mediated by multiple chemoattractants, including IL8 and TNF-α [17,19]. In the present study, we found that the CD11/CD18 expression and IL-8 release were similar between the ARDS and non-ARDS groups. TNF-α was the only cytokine up-regulated with the development of ARDS. Therefore, it is reasonable to believe TNF-α was responsible (or partially) to the increased neutrophil activity and adhesion in the early phase of ARDS.

In conclusion, we demonstrated that the TNF-α was quickly up-regulated and involved in the pathogenesis of CPB-induced ARDS via guiding primed neutrophils to pulmonary interstitium.

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