Objective: To evaluate the role of resident pleural macrophages in the initiation of inflammation.

Methods: We have used a conditional macrophage ablation strategy to determine the role of resident pleural macrophages in the regulation of neutrophil recruitment in a murine model of experimental pleurisy induced by the administration of carrageenan and formalin-fixed Staphylococcus aureus.

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Measurements and Main Results: Conditional macrophage ablation mice express the human diphtheria toxin receptor under the control of the CD11b promoter such that the administration of diphtheria toxin induces ablation of nearly 97% of resident macrophages. Ablation of resident pleural macrophages before the administration of carrageenan or S. aureus dramatically reduced neutrophil influx into the pleural cavity. In the carrageenan model, the reduction in neutrophil infiltration was associated with marked early reduction in the level of macrophage inflammatory protein 2 as well as reduced levels of various cytokines, including tumor necrosis factor α, interleukin 6, and interleukin 10. Adoptive transfer of nontransgenic macrophages partially restored neutrophil infiltration. We also stimulated macrophage-depleted and nondepleted pleural cell populations with carrageenan in vitro and determined the production of chemokines and cytokines. Chemokine and cytokine production was markedly reduced by macrophage depletion, reinforcing the role of resident pleural macrophages in the generation of mediators that initiate acute inflammation.

Conclusion: These studies indicate a critical role for resident pleural macrophages in sensing perturbation to the local microenvironment and orchestrating subsequent neutrophil infiltration.

Keywords: inflammation; macrophage; pleural diseases

The pleural membranes and associated cells are important because they are metabolically active and act as a barrier to invading pathogens by generating an innate and adaptive immunologic response. The pleural cavity is lined with mesothelium and contains resident macrophages (M[straight phi]), mast cells, and lymphocytes (1, 2). During pleural inflammation, it has been reported that mesothelial cells are predominantly responsible for the secretion of C-X-C chemokines, such as interleukin 8 (IL-8), and C-C chemokines, such as macrophage inflammatory protein 1α (MIP-1α) and macrophage chemoattractant protein 1 (MCP-1), which act to recruit neutrophils (polymorphonuclear leukocytes [PMNs]) and mononuclear cells (3-6). In addition, a recent study demonstrated that activated pleural fibroblasts may also be a source of C-X-C and C-C chemokine production (7).

Previous work suggested that the initiation of inflammation is dependent on endogenous IL-6 secretion that subsequently stimulates the additional production of tumor necrosis factor α (TNF-α) and IL-1β from resident pleural cells (8). In contrast, increased IL-1β levels have been reported to precede elevated IL-6 levels (9), thereby suggesting that IL-1β might induce IL-6 production. There is no doubt that TNF-α and IL-1β are key cytokines in the development of pleural inflammation because they act to enhance IL-8 and MCP-1 production from mesothelial cells (3, 5, 10-12). In addition, studies using function-blocking antibodies suggest that activated resident M[straight phi] could be responsible for this TNF-α and IL-1β secretion (10, 12).

Carrageenan-induced pleurisy is a well-established model of acute inflammation (13) and is characterized by a rapid influx of PMNs followed by mononuclear cell infiltration (14, 15). This model is often used to assess the antiinflammatory effects of pharmaceutical agents (16-20) and to assess the in vivo importance of established inflammatory mediators (21-23). Although the neutrophil influx evident in this model is generally used as an experimental readout of acute inflammation, there are data indicating that neutrophils are involved in the release of injurious enzymes and modulation of vascular permeability in carrageenan-mediated pleural inflammation (24, 25).

To date, there has been little study of the role of the resident pleural M[straight phi] in the initiation of inflammation and orchestration of PMN recruitment. Previous work demonstrated a reduced eosinophil influx after administration of LPS to mice that had been previously treated with diphosphonate-containing liposomes to deplete resident pleural M[straight phi] (26). Although this suggests that resident pleural M[straight phi] may play a key role in the initiation of pleural inflammatory responses, there are no definitive data available for PMN infiltration and proinflammatory cytokine production.