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Since induced sputum has become a widely used non-invasive method of recovering cells from the surfaces of the bronchial airways, isolating specific cell populations will be necessary in order to learn more about their specific role in innate immunity and inflammation in the airways. Several studies have demonstrated the ability to conduct ex vivo analyses on sputum cells such as phagocytosis and surface marker measurements, but these have not been performed on isolated cell types.1–3 This study demonstrates the capability to isolate sputum macrophages from human volunteers in order to advance our understanding of macrophage biology in the airways. To this end, techniques that can enrich and isolate cells without significant activation would prove extremely useful. We compared two common methods for isolating and enriching macrophages in sputum: (1) magnetic bead separation; and (2) Percoll gel density gradient centrifugation. Cell purity and markers of cell activation (mRNA tumour necrosis factor α (TNFα)) and interleukin-1β (IL1β)) were measured at various time points in the isolation process.
Nine healthy subjects underwent induced sputum. Sputum collection and sputum processing has been described in detail previously.4 For measuring natural cell activation over time, we incubated the processed sputum cells for 3 h at 37°C and analysed mRNA TNFα and IL1-β at 0 h (baseline) and 3 h. In the positive control experiment we incubated the processed sputum cells with 1 ng/ml LPS (E coli, Sigma). For Percoll (Amersham Biosciences) separation, 600 μl of sputum cell suspension (1×106 cells/ml) was layered over Percoll solution (42%) and centrifuged at 560 g for 10 min. Sputum macrophages were removed and incubated at 37°C for 1, 2 and 3 h, respectively, and a pre-incubation sample was also collected. The macrophages were further pelleted and stored at −70°C. For Dynabead separation, CELLection Pan Mouse IgG Kit (Dynal, Norway) was used for immunomagnetic separation of airway macrophages coated with mouse monoclonal IgG2b HLA-DR antibody (Diatec, Norway). Bead coating and cell isolation was performed according to the protocol from the manufacturer. The isolated cells were incubated at 37°C for 1, 2 and 3 h, respectively, and a pre-incubation sample was also collected. The samples were further pelleted and stored at −70°C. Total RNA was extracted (Qiagen) from all the cell samples and reverse transcription was performed (Superscript III, Invitrogen). We used pre-developed PCR primers and probes for TNFα and the housekeeping gene PGK (Applied Biosystems). Specific primers and probes were designed for IL1β using ProbeLibrary (Exiqon ProbeLibrary). Quantification of mRNA was performed using the ABI Prism 7700 (Applied Biosystems), and the relative standard curve method was used to calculate the relative gene expression.
The results show that the median (range) proportion of macrophages in the pre-isolation sputum sample was 61 (34–70)%. Bead isolation produced 99 (95–99)% macrophage purity compared with 88 (85–94)% with Percoll isolation. mRNA expression of TNFα and IL1β was measured as markers of cell activation in airway macrophages (fig 1) before and after Percoll isolation, Dynabead isolation, no isolation and lipopolysaccharide (1 ng/ml) stimulation (positive control). Levels of mRNA TNFα and IL1β were significantly increased as early as 2 h using Percoll isolation compared with 0 h baseline (p = 0.02) and bead isolation (p<0.01). For bead isolation, mRNA TNFα and IL1β expression were unchanged throughout the isolation period compared with baseline. At 3 h after bead isolation, macrophage mRNA TNFα expression remained near baseline levels whereas Percoll-separated macrophages showed increased activation near positive control (lipopolysaccharide) levels.
The results from this study show that sputum macrophages can be successfully isolated and enriched with a high degree of purity. Furthermore, the magnetic bead isolation technique results in higher macrophage purity and significantly less cell activation than the Percoll isolation technique. As more researchers begin to use individual sputum cell populations to describe airways cellular phenomena, data presented here will provide important technical information to achieve those research aims.
Financial support was kindly parided by the Research Council of Norway, The Working Environmental Fund, Confederation of Norwegion Enterprise and NIH-NHLB1 grant no RO1-HL080337.
Competing interests: None.
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