Analysis of sequential aliquots of hypertonic saline solution-induced sputum from clinically stable patients with cystic fibrosis.

STUDY OBJECTIVES: Sputum induction (SI) is a noninvasive tool for sampling inflamed airways. The purpose of this study was to determine the optimal duration of collection in patients with cystic fibrosis (CF). The hypothesis was that the duration of SI collection would quantitatively and qualitatively alter the content of the induced sputum. METHODS: In 10 clinically stable patients with CF (mean +/- SD age, 28 +/- 7 years; mean FEV(1), 2.6 +/- 0.7 L), SI was performed with 3% hypertonic saline solution at five time points over 20 min. RESULTS: SI was well tolerated, with an average maximum fall in FEV(1) of 7 +/- 7%. The sample volumes, urea concentrations, interleukin-8 concentrations, total cell counts, and nonsquamous cell counts remained constant (p > 0.05). The percentage of neutrophils decreased from 89 +/- 5% to 86 +/- 4% (p = 0.03), and the percentage of alveolar macrophages increased 5 +/- 2% to 8 +/- 4% (p < 0.01). The mean quantitative microbiological counts of nonmucoid Pseudomonas aeruginosa and Staphylococcus aureus decreased over the 20-min time period each by half a log (p = 0.05 and p < 0.01, respectively). Surfactant protein-A concentration increased from 1.6 +/- 0.3 to 2.4 +/- 0.4 ng/mL (log(10); p < 0.001). CONCLUSIONS: We conclude that aliquots of induced sputum are similar in clinically stable patients with CF during 4-min intervals, although there is more alveolar sampling after 20 min. When induced-sputum samples are fractionated for research monitoring of inflammatory or microbiologic indexes, power calculations accounting for these variations over time are required.

Surfactant protein A differentially regulates peripheral and inflammatory neutrophil chemotaxis.

Surfactant protein A (SP-A), a pulmonary lectin, plays an important role in regulating innate immune cell function. Besides accelerating pathogen clearance by pulmonary phagocytes, SP-A also stimulates alveolar macrophage chemotaxis and directed actin polymerization. We hypothesized that SP-A would also stimulate neutrophil chemotaxis. With the use of a Boyden chamber assay, we found that SP-A (0.5-25 microg/ml) did not stimulate chemotaxis of rat peripheral neutrophils or inflammatory bronchoalveolar lavage (BAL) neutrophils isolated from LPS-treated lungs. However, SP-A affected neutrophil chemotaxis toward the bacterial peptide formyl-met-leu-phe (fMLP). Surprisingly, the effect was different for the two neutrophil populations: SP-A reduced peripheral neutrophil chemotaxis toward fMLP (49 +/- 5% fMLP alone) and enhanced inflammatory BAL neutrophil chemotaxis (277 +/- 48% fMLP alone). This differential effect was not seen for the homologous proteins mannose binding lectin and complement protein 1q but was recapitulated by type IV collagen. SP-A bound both neutrophil populations comparably and did not alter formyl peptide binding. These data support a role for SP-A in regulating neutrophil migration in pulmonary tissue.

Prognostic value of surfactant proteins A and D in patients with acute lung injury.

OBJECTIVE: The primary objective of this study was to test the hypothesis that in patients intubated for acute lung injury, lower concentrations of surfactant proteins A and D in the pulmonary edema fluid and higher concentrations in the plasma are associated with more severe lung injury and worse clinical outcomes. DESIGN: Observational study. SETTING: Intensive care unit patients in a tertiary university hospital and a university-affiliated city hospital. PATIENTS: Thirty-eight intubated, mechanically ventilated intensive care unit patients with acute lung injury or acute respiratory distress syndrome as defined by the North American European Consensus Conference. INTERVENTIONS: Undiluted pulmonary edema fluid and plasma samples were collected within 24 hrs of endotracheal intubation in all patients. MEASUREMENTS AND MAIN RESULTS: The concentrations of surfactant proteins A and D were measured in pulmonary edema fluid and in plasma. Plasma surfactant protein A, but not surfactant protein D, was higher in patients with fewer days of unassisted ventilation (p = .03) and in patients with an absence of intact alveolar fluid clearance (p =.03). In contrast, pulmonary edema fluid surfactant protein D, but not surfactant protein A, was lower in patients with worse oxygenation, as measured by the alveolar-arterial oxygen difference (p = .01) and was lower in the patients who died (2646 ng/mL) compared with those who survived (5503 ng/mL; p = .02). CONCLUSIONS: These results demonstrate that reduced pulmonary edema fluid surfactant protein D and elevated plasma surfactant protein A concentrations at the onset of acute lung injury may be associated with more severe disease and worse clinical outcome and may serve as valuable biochemical markers of prognosis.

By binding SIRPalpha or calreticulin/CD91, lung collectins act as dual function surveillance molecules to suppress or enhance inflammation.

Surfactant proteins A and D (SP-A and SP-D) are lung collectins composed of two regions, a globular head domain that binds PAMPs and a collagenous tail domain that initiates phagocytosis. We provide evidence that SP-A and SP-D act in a dual manner, to enhance or suppress inflammatory mediator production depending on binding orientation. SP-A and SP-D bind SIRPalpha through their globular heads to initiate a signaling pathway that blocks proinflammatory mediator production. In contrast, their collagenous tails stimulate proinflammatory mediator production through binding to calreticulin/CD91. Together a model is implied in which SP-A and SP-D help maintain a non/anti-inflammatory lung environment by stimulating SIRPalpha on resident cells through their globular heads. However, interaction of these heads with PAMPs on foreign organisms or damaged cells and presentation of the collagenous tails in an aggregated state to calreticulin/CD91, stimulates phagocytosis and proinflammatory responses.

Role of surfactant proteins A, D, and C1q in the clearance of apoptotic cells in vivo and in vitro: calreticulin and CD91 as a common collectin receptor complex.

Removal of cells dying by apoptosis is essential to normal development, maintenance of tissue homeostasis, and resolution of inflammation. Surfactant protein A (SP-A) and surfactant protein D (SP-D) are high abundance pulmonary collectins recently implicated in apoptotic cell clearance in vitro. Other collectins, such as mannose-binding lectin and the collectin-like C1q, have been shown to bind to apoptotic cells and drive ingestion through interaction with calreticulin and CD91 on the phagocyte in vitro. However, only C1q has been shown to enhance apoptotic cell uptake in vivo. We sought to determine the relative importance of SP-A, SP-D, and C1q in pulmonary clearance of apoptotic cells using knockout and overexpressing mice, and to determine the role of calreticulin and CD91 in this process. SP-A, SP-D, and C1q all enhanced apoptotic cell ingestion by resident murine and human alveolar macrophages in vitro. However, only SP-D altered apoptotic cell clearance from the naive murine lung, suggesting that SP-D plays a particularly important role in vivo. Similar to C1q and mannose-binding lectin, SP-A and SP-D bound to apoptotic cells in a localized, patchy pattern and drove apoptotic cell ingestion by phagocytes through a mechanism dependent on calreticulin and CD91. These results suggest that the entire collectin family of innate immune proteins (including C1q) works through a common receptor complex to enhance removal of apoptotic cells, and that collectins are integral, organ-specific components of the clearance machinery.