Free-Floating Mesothelial Cells in Pleural Fluid After Lung Surgery

Abstract

Objectives: The mesothelium, the surface layer of the heart, lung, bowel, liver, and tunica vaginalis, is a complex tissue implicated in organ-specific diseases and regenerative biology; however, the mechanism of mesothelial repair after surgical injury is unknown. Previous observations indicated seeding of denuded mesothelium by free-floating mesothelial cells may contribute to mesothelial healing. In this study, we investigated the prevalence of mesothelial cells in pleural fluid during the 7 days following pulmonary surgery.

Study design: Flow cytometry was employed to study pleural fluid of 45 patients after lung resection or transplantation. We used histologically validated mesothelial markers (CD71 and WT1) to estimate the prevalence of mesothelial cells.

Results: The viability of pleural fluid cells approached 100%. Leukocytes and mesothelial cells were identified in the pleural fluid within the first week after surgery. The leukocyte concentration was relatively stable at all time points. In contrast, mesothelial cells, identified by CD71 and WT1 peaked on POD3. The broad expression of CD71 molecule in postoperative pleural fluid suggests that many of the free-floating non-leukocyte cells were activated or proliferative mesothelial cells.

Conclusion: We demonstrated that pleural fluid post lung surgery is a source of mesothelial cells; most of these cells appear to be viable and, as shown by CD71 staining, activated mesothelial cells. The observed peak of mesothelial cells on POD3 is consistent with a potential reparative role of free-floating mesothelial cells after pulmonary surgery.

Keywords: lung healing; lung regeneration; mesothelial cells; pleural fluid; pneumonectomy.

Figure 1

Cellular content of pleural fluid after lung resection (solid line) and lung transplantation (dotted line). When clinically available, samples were obtained from N = 45 patients; N = 37 patients after lung resection; and N = 8 patients after lung transplantation. (A) After red cell lysis, cell concentrations were determined by manual counting with a hematocytometer. (B) Cell viability was determined by light microscopy and trypan blue exclusion (27) as well as stage scan fluorescent microscopy after calcein-AM and ethidium homodimer-1 cell viability staining. Error bars reflect ± 1 SD.

Figure 2

Flow cytometry profile of cells in the pleural fluid. After exclusion of debris and red cells, the remaining cells were studied by flow cytometry. (A) Anti-CD45 staining and side-scatter (SSC) analysis demonstrated a dominant mononuclear population. A representative sample on day 1 after surgery demonstrated 70% mononuclear cells and 5% granulocytes; the remaining 25% of cells were compatible with mesothelial cells. Unstained negative controls were compared to samples stained with detection antibodies (B,C). More detailed size analysis with forward light scatter (FSC) on postoperative day 1, 3, and 7 showed no consistent size relationship between CD45⁺ cells (blue) and CD45⁻ cells (red; the Emphasize Plot Tool, FCS Express, was used for data presentation). Representative histograms are shown.

Figure 3

Fluorescence immunohistochemistry of human pleural mesothelium. Human pleura, obtained from surgical specimens, was stained with fluorochrome-labeled anti-CD71 (A), anti-WT1 (B), and anti-CD45 (C) monoclonal antibodies and counterstained with Hoechst 33342. Both anti-CD71 and anti-WT1 antibodies demonstrated discontinuous staining of the pleural mesothelium. There was no detectable pleural staining with labeled anti-CD45 monoclonal antibody. Bar = 50 um.

Figure 4

Flow cytometry profile of pleural fluid cells after surgical resection. Representative single parameter histograms of CD45⁺ (A), CD71⁺ (B), and WT1⁺ (C) cells at three time points after surgery: postoperative days (POD) 1, 3, and 7 (gray). Negative controls (white) reflect samples without detection antibody.

Figure 5

Time course of pleural fluid cell populations after surgery. Pleural fluid obtained from patients after lung resection (solid line) and lung transplant (dotted line) are shown. (A) Leukocytes, identified by anti-CD45 antibodies, were a relatively consistent percentage of cells during the first week after surgery. (B) In contrast, the percentage of CD71⁺ cells peaked 3 days after surgery. Data based on N = 40 patients. Error bars reflect ± 1 SD.

Figure 6

Flow cytometry of pleural fluid CD45⁻ cells. (A) Representative dual parameter histograms of CD45⁻ cells on postoperative day 3 demonstrated 70% of cells were positive for CD71 and/or WT1; 35% of cells were positive for both CD71 and WT1. (B) Consistent with single parameter staining, the double parameter flow cytometry profiles demonstrated a peak concentration of presumed mesothelial cells on postoperative day 3. N = 21 patients. Error bars reflect ± 1 SD.