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Deborah J. Nelson Lab
University of Chicago
Dept. of Neurobiology,
 Pharmacology & Physiology
947 East 58th Street
Abbott Hall 500, MC 0926
Chicago, IL 60637 (map)
tel 773.702.6795
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Role of Cl- channels in sculpting the innate immune response

 
   Regulated exocytosis in non-excitable cells has several characteristics in common with excitable tissues yet remains poorly investigated. The activated macrophage secretes a large array of small and macromolecular products including cytokines, chemokines, growth factors, and oxidative metabolites.  This complex secretory system is contained in cytoplasmic vesicles of varying sizes.   Many of these products are exocytosed by vesicular fusion with the plasma membrane, yet the components of the secretory apparatus and the identity of the cargo in each vesicle population is not well understood. We have found that secretion in macrophages is controlled by both Ca2+ and GTP which may act independently or synergistically in the regulation of secretion 1.  Secretory granules maintain a low intragranular pH and it is becoming increasingly recognized that this phenomenon is important to secretion. Cl- entry across the granule membrane is thought to be required to shunt H+ influx via the V-ATPase, thus preventing the build-up of a large transgranular membrane potential. Recently, we demonstrated that murine alveolar macrophages (AMs) but not neutrophils employ the CFTR (Cystic Fibrosis Transmembrane conductance Regulator) Cl- channel as a major shunt mechanism in lysosomal as well as phagosomal acidification 2. Data obtained in our recent experiments demonstrate that isolated AMs from CFTR knockout and mutant mice appear defective in regulated secretion.  Experiments currently ongoing in the laboratory are directed at the determining the importance of CFTR expression to regulated secretion in the AM. It is our working hypothesis that the secretion of macromolecular products is defective in CFTR knockout and mutant mice as a direct result of aberrant vesicle acidification.   We are currently using a combination of cytokine profiling, single-cell capacitance measurements of cell surface area, and both live cell and TIRF microscopy to identify the cargo in specific populations of vesicles in activated alveolar macrophages.

 

    Macrophages and neutrophils are key cells of the innate immune system. Blood monocytes infiltrate different tissues and then differentiate into tissue-specific macrophages that perform vital host defense functions while neutrophils (PMNs) are recruited from the blood to sites of infection. Mature macrophages from distinct sources exhibit significant variation in molecular and cellular properties as well as gene expression profiles specific for their host tissue, while maintaining a common set of core functions 3.  One such cell type is the alveolar macrophage (AM) that resides in the terminal airway alveoli of the lung from where they recruit PMNs that respond chemotactically to microbial insult.  PMNs are generally considered the dominant component of innate immunity because of their sheer numerical superiority.  While less numerous than the mobile PMNs, AMs live longer and are a more potent source of the cytokines that orchestrate the immune response to bacterial pathogens. Ultimately, macrophages are also responsible for clearing apototic PMNs from infection sites, also by phagocytosis 4, 5.  Delayed removal of the dying cells results in chronic inflammation with resultant tissue damage as seen in several chronic inflammatory lung diseases including cystic fibrosis (CF) (for review see Vandiver et al. 6).

 

    Professional phagocytes have specialized pathways that ensure efficient killing of pathogens in phagosomes7. A common element in these pathways is organellar acidification that facilitates the optimal functioning of various degradative enzymes, particularly in phagosomes 8.  Indeed, low pH is required in several organelles for diverse functions in many cell types: e.g. maturation of secretory products and their final secretion by the exocytotic pathway, dissociation and recycling in the endosomal pathway. Generation of low organellar pH is primarily driven by the V-ATPases, proton pumps that use cytoplasmic ATP to load H+ into the organelle 9, 10. Alongside the pumps are various channels that shunt the transmembrane potential generated by movement of protons; in different organelles these comprise H+ channels, K+ channels and Cl- channels. Without these shunt pathways acidification is limited and organelle function is compromised 11. Nevertheless, the contribution of these pathways to maintenance of intraorganellar pH is poorly studied. Cl- channels are central to the function of several intracellular organelles 12 and recently we showed definitively that the specialized Cl- channel CFTR is important in lysosomal and phagosomal acidification in murine and human AMs.  In parallel, others have shown that CFTR is expressed on secretory granules in neutrophils and may be involved in human neutrophil phagocytic function 3. These studies prompted the current emphasis of experiments in the laboratory that seek to define the role of CFTR and other Cl- channels in the behavior of innate immune cells.

  

REFERENCES

 

1.         Di, A., Krupa, B. & Nelson, D.J. Calcium-G protein interactions in the regulation of macrophage secretion. The Journal of biological chemistry 276, 37124-37132 (2001).

2.         Di, A. et al. CFTR regulates phagosome acidification in macrophages and alters bactericidal activity. Nat Cell Biol 8, 933-944 (2006).

3.         Li, J. et al. cDNA microarray analysis reveals fundamental differences in the expression profiles of primary human monocytes, monocyte-derived macrophages, and alveolar macrophages. Journal of leukocyte biology 81, 328-335 (2007).

4.         Krysko, D.V. et al. Macrophages use different internalization mechanisms to clear apoptotic and necrotic cells. Cell Death Differ 13, 2011-2022 (2006).

5.         Krysko, D.V., D'Herde, K. & Vandenabeele, P. Clearance of apoptotic and necrotic cells and its immunological consequences. Apoptosis 11, 1709-1726 (2006).

6.         Vandivier, R.W., Henson, P.M. & Douglas, I.S. Burying the dead: the impact of failed apoptotic cell removal (efferocytosis) on chronic inflammatory lung disease. Chest 129, 1673-1682 (2006).

7.         Haas, A. The phagosome: compartment with a license to kill. Traffic (Copenhagen, Denmark) 8, 311-330 (2007).

8.         Steinberg, B.E., Touret, N., Vargas-Caballero, M. & Grinstein, S. In situ measurement of the electrical potential across the phagosomal membrane using FRET and its contribution to the proton-motive force. Proceedings of the National Academy of Sciences of the United States of America 104, 9523-9528 (2007).

9.         Demaurex, N. pH Homeostasis of cellular organelles. News Physiol Sci 17, 1-5 (2002).

10.       Grabe, M. & Oster, G. Regulation of organelle acidity. The Journal of general physiology 117, 329-344 (2001).

11.       Weisz, O.A. Organelle acidification and disease. Traffic (Copenhagen, Denmark) 4, 57-64 (2003).

12.       Jentsch, T.J. Chloride and the endosomal-lysosomal pathway: emerging roles of CLC chloride transporters. The Journal of physiology 578, 633-640 (2007).