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. 2008 Jun 1;180(11):7368-75.
doi: 10.4049/jimmunol.180.11.7368.

C5a and TNF-alpha Up-Regulate the Expression of Tissue Factor in Intra-Alveolar Neutrophils of Patients With the Acute Respiratory Distress Syndrome

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Free PMC article

C5a and TNF-alpha Up-Regulate the Expression of Tissue Factor in Intra-Alveolar Neutrophils of Patients With the Acute Respiratory Distress Syndrome

Konstantinos Kambas et al. J Immunol. .
Free PMC article

Abstract

Acute respiratory distress syndrome (ARDS) is characterized by the presence of fibrin-rich inflammatory exudates in the intra-alveolar spaces and the extensive migration of neutrophils into alveoli of the lungs. Tissue factor (TF)-dependent procoagulant properties of bronchoalveaolar lavage fluid (BALF) obtained from ARDS patients favor fibrin deposition, and are likely the result of cross-talk between inflammatory mediators and hemostatic mechanisms. However, the regulation of these interactions remains elusive. Prompted by previous findings suggesting that neutrophils, under certain inflammatory conditions, can express functional TF, we investigated the contribution of intra-alveolar neutrophils to the procoagulant properties of BALF from patients with ARDS. Our results confirm that the procoagulant properties of BALF from ARDS patients are the result of TF induction, and further indicate that BALF neutrophils are a main source of TF in intra-alveolar fluid. We also found that BALF neutrophils in these patients express significantly higher levels of TF than peripheral blood neutrophils. These results suggest that the alveolar microenvironment contributes to TF induction in ARDS. Additional experiments indicated that the ability of BALF to induce TF expression in neutrophils from healthy donors can be abolished by inhibiting C5a or TNF-alpha signaling, suggesting a primary role for these inflammatory mediators in the up-regulation of TF in alveolar neutrophils in ARDS. This cross-talk between inflammatory mediators and the induction of TF expression in intra-alveolar neutrophils may be a potential target for novel therapeutic strategies to limit ARDS-associated disturbances of coagulation.

Figures

FIGURE 1
FIGURE 1
TF-dependent procoagulant properties of BALF and the ability of BALF to induce procoagulant activity in neutrophils as demonstrated by mPT analyses. Average mPT values ± SD of ARDS BALF supernatants (120 μl, bar 3) and ARDS BALF supernatants intermixed with anti-TF mAb (bar 4). Other bars represent average mPT values of supernatants from normal neutrophils (H.N.) incubated with PBS (bar 1), serum from healthy individuals (H.S., bar 2), ARDS BALF (bar 5), and serum from ARDS patients (bar 7). Average mPT values ± SD of supernatants from normal neutrophils (H.N.) incubated ARDS BALF intermixed with anti-TF mAb are represented as bar 6. Mean values shown in this figure were obtained from three independent experiments performed with the use of BALF and sera from all ARDS patients included in the study (n = 7).
FIGURE 2
FIGURE 2
TF expression in BALF cells. A, TF immunostaining of peripheral blood neutrophils (I) and BALF neutrophils (neutrophil purity, 88%) (II) from an ARDS patient (n = 7). B, Multinucleated cells (arrow) strongly positive for TF expression were observed mainly in the BALF of two patients (I). Alveolar macrophages with diffuse cytoplasmic staining (arrow) and large negative atypical pneumocyte type II (small arrow) are surrounded by positive neutrophils (double arrows) (II). C, Representative FACS analysis (n = 4) of TF expression in neutrophils from peripheral blood (green histogram) and BALF fluid (red histogram). Purity of neutrophil populations obtained from BALF was 88%. Isotype negative control is shown as filled purple histogram.
FIGURE 3
FIGURE 3
The 2−DDCT data analysis. Relative quantification of TF and asTF in circulating (n = 7) and BALF purified PMNs (n = 6). Relative expression (indicated by bars) was based on the average DCT values of the target gene (TF or asTF) and GAPDH (DCT of TF BALF-purified neutrophils 5.652 ± 0.85 vs 8.798 ± 0.94 of TF blood PMNs and DCT of asTF BALF-purified neutrophils 11.435 ± 1.10 vs 16.054 ± 1.97 of asTF blood PMN).
FIGURE 4
FIGURE 4
BALF C5a or TNF-α inhibition abolishes TF-dependent procoagulant activity. A, The median and range of concentrations for C5a, TNF-α, and IL-6 (pg/ml) in BALF from ARDS patients (n = 7). B, mPT values of supernatants from neutrophils (H.N.) originating from healthy volunteers and stimulated with ARDS serum (bar 1), ARDS BALF alone (bar 2), ARDS BALF after pretreatment with C5aR antagonist (C5aRA) (bar 3) or with addition of anti-TNF-α Ab (bar 4). Bars represent the average of mPT values ± SD from experiments using BALF and sera from ARDS patients (n = 7). Three independent experiments were performed and representative results are shown. C, TF expression in healthy neutrophils (H.N.) incubated with sera from an ARDS patient (I; Ia: negative control anti-CD19 staining; and Ib: anti-TF staining), ARDS BALF alone (II), ARDS BALF after pretreatment of H.N. with C5aRA (III), or with addition of anti-TNF-α Ab (IV). Images show immunostaining with the use of anti-TF Ab. Binding of anti-TF Ab was visualized by APAAP method (magnification × 1000). Three independent experiments using sera and BALF from ARDS patients were performed (n = 7) and representative examples are shown. D, Representative FACS analysis of H.N. (n = 4) identified by forward- and side-scatter characteristics, before and after stimulation of cells with BALF, as well as after C5aR and TNF-α inhibition studies. E, Representative examples of western blot analyses (n = 7 for each individual experiment). Three independent experiments were performed. Lanes correspond to immunostaining and mPT analyses. Lane I: H.N. incubated with ARDS serum; Lane II: H.N. stimulated with 40 μl BALF; Lane III: H.N. pretreated with C5aRA and stimulated with BALF. Lane IV: H.N. incubated with BALF and anti-TNF-α. Lane V: TF expression from total cell extracts from BALF with a neutrophil purity of 84%.
FIGURE 5
FIGURE 5
Levels of cytokines in BALF. Medians and ranges of concentrations (pg/ml) for G-CSF, GROα, IFN-γ, IL-1β, IL-8, IFN-γ inducible protein of 10kD, MIP-1β, eotaxin, GM-CSF, IL-5, IL-10, MCP-3, MIP-1α, platelet derived growth factor BB, and vascular endothelial growth factor detectable in BALF from ARDS patients are shown (n = 7). Due to high differences in the levels of various cytokines in ARDS BALF, two (right and left) panels separated by a dashed line are presented for better clarity. Values on the left y-axis refer to cytokines in the left panel, whereas values on the right y-axis refer to cytokines in the right panel.

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