Abstract
Sensing of infectious danger by toll-like receptors (TLRs) on macrophages causes not only a reprogramming of the transcriptome but also changes in the cytoskeleton important for cell spreading and motility. Since manual determination of cell contact areas from fluorescence micrographs is very time-consuming and prone to bias, we have developed and tested algorithms for automated measurement of macrophage spreading. The two-step method combines identification of cells by nuclear staining with DAPI and cell surface staining of the integrin CD11b. Automated image analysis correlated very well with manual annotation in resting macrophages and early after stimulation, whereas at later time points the automated cell segmentation algorithm and manual annotation showed slightly larger variation. The method was applied to investigate the impact of genetic or pharmacological inhibition of known TLR signaling components. Deficiency in the adapter protein Myd88 strongly reduced spreading activity at the late time points, but had no impact early after LPS-stimulation. A similar effect was observed upon pharmacological inhibition of MEK1, the kinase activating the mitogen-activated protein kinases (MAPK) ERK1/2, indicating that ERK1/2 mediates Myd88-dependent macrophages spreading. In contrast, macrophages lacking the MAPK p38 were impaired in the initial spreading response but responded normally 8–24 h after stimulation. The dichotomy of p38 and ERK1/2 MAPK effects on early and late macrophage spreading raises the question which of the respective substrate proteins mediate(s) cytoskeletal remodeling and spreading. The automated measurement of cell spreading described here increases the objectivity and greatly reduces the time required for such investigations and is therefore expected to facilitate larger throughput analysis of macrophage spreading, e.g., in siRNA knockdown screens.
Highlights
Macrophages reside in all tissues and play an important role in tissue remodeling and homeostasis by phagocytosis and digestion of dead cells and cellular debris
The major signaling modules activated by toll-like receptors (TLRs) are the IKK complex leading to NFκB translocation to the nucleus, and the cascade of mitogen-activated protein kinases (MAPK)
In our hands, using bone marrow derived mouse macrophages cultured on Permanox chamber slides, the staining of the integrin CD11b with an APC-labeled antibody resulted in more even staining than the lipid-staining molecule PKH and in a better signal-to-noise ratio than the cytosolic dye CFSE
Summary
Macrophages reside in all tissues and play an important role in tissue remodeling and homeostasis by phagocytosis and digestion of dead cells and cellular debris Their second function as sentinels for infectious danger is embodied by the expression of pattern recognition receptors for pathogen-associated molecular patterns. MAPK family members expressed in macrophages are ERK1/2, JNK1/2, and p38 These MAPK control the transcriptional response to TLR ligation through phosphorylation-mediated activation of transcription factors (e.g., AP-1, CREB, and many others; Lang et al, 2006); in addition, the plethora of MAPK substrate proteins are involved in diverse cellular processes including cell motility, adhesion, and phagocytosis (Schmidt et al, 2001; Blander and Medzhitov, 2004; West et al, 2004; Kang et al, 2008)
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