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

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-α 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.

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“…For TF detection, Western blotting was performed as previously described (6). To detect CTGF production, we used a polyclonal goat anti-human Ab (Santa Cruz Biotechnologies) at a 1/500 dilution, with incubation overnight at 4˚C.…”

“…Relative quantification of TF and CTGF mRNA synthesis Isolation of total RNA and cDNA synthesis were conducted as previously described (6). Real-time RT-PCR for the full-length TF isoform (referred hereafter as TF), CTGF, PAR-1, and C5aR1 (C5a receptor 1) was performed under the conditions described in Supplemental Table II.…”

“…In the case of the BPD infants, further BALF collection was performed on day 12, the earliest day on which two of the BPD infants were extubated. BALF was processed as previously described (6). BALF supernatants from three patients with either BPD or RDS were pooled for further studies, after assessing the potential of all samples to induce collagen production in myofibroblast cultures.…”

“…Peripheral polymorphonuclear cells (PMNs) from infants of the two groups were isolated as previously described (6).…”

“…In addition to activated endothelium and monocytes, neutrophils have been recently shown to express tissue factor (TF) (2-7), the in vivo initiator of coagulation. Importantly, the inflammatory mediators C5a (2-4, 6-8), TNF-a (5,6,9), and IL-1b (10) are all inducers of TF. Furthermore, a recent study has demonstrated a correlation between coagulation and the inflammatory response in patients with acute respiratory distress syndrome (ARDS), indicating that the expression of TF by neutrophils in the alveolar microenvironment is C5a-and TNF-a dependent (6).…”