In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation

Immune-mediated pulmonary diseases are a significant public health concern. Analysis of leukocyte behavior in the lung is essential for understanding cellular mechanisms that contribute to normal and diseased states. Here, we used two-photon imaging to study neutrophil extravasation from pulmonary vessels and subsequent interstitial migration. We found that the lungs contained a significant pool of tissue-resident neutrophils in the steady state. In response to inflammation produced by bacterial challenge or transplant-mediated, ischemia-reperfusion injury, neutrophils were rapidly recruited from the circulation and patrolled the interstitium and airspaces of the lung. Motile neutrophils often aggregated in dynamic clusters that formed and dispersed over tens of minutes. These clusters were associated with CD115+ F4/80+ Ly6C+ cells that had recently entered the lung. The depletion of blood monocytes with clodronate liposomes reduced neutrophil clustering in the lung, but acted by inhibiting neutrophil transendothelial migration upstream of interstitial migration. Our results suggest that a subset of monocytes serve as key regulators of neutrophil extravasation in the lung and may be an attractive target for the treatment of inflammatory pulmonary diseases.

[1]  M. Fay,et al.  In Vivo Imaging Reveals an Essential Role for Neutrophils in Leishmaniasis Transmitted by Sand Flies , 2008, Science.

[2]  Y. Suezer,et al.  Induced bronchus-associated lymphoid tissue serves as a general priming site for T cells and is maintained by dendritic cells , 2009, The Journal of experimental medicine.

[3]  C. Haslett,et al.  Physiological neutrophil sequestration in the lung: visual evidence for localization in capillaries. , 1987, Journal of applied physiology.

[4]  Ming You,et al.  Detection of primary lung tumors in rodents by magnetic resonance imaging. , 2004, Cancer research.

[5]  M. Burdick,et al.  CXCR2/CXCR2 Ligand Biology during Lung Transplant Ischemia-Reperfusion Injury1 , 2005, The Journal of Immunology.

[6]  Mark J. Miller,et al.  Two-Photon Imaging of Lymphocyte Motility and Antigen Response in Intact Lymph Node , 2002, Science.

[7]  D. Kreisel,et al.  A Mouse Model of Orthotopic Vascularized Aerated Lung Transplantation , 2007, American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons.

[8]  M. V. D. van den Brink,et al.  CCR2 is required for CD8-induced graft-versus-host disease. , 2005, Blood.

[9]  N. Van Rooijen,et al.  Subpopulations of Mouse Blood Monocytes Differ in Maturation Stage and Inflammatory Response1 , 2004, The Journal of Immunology.

[10]  G. Winkler Review of the significance of pulmonary intravascular macrophages with respect to animal species and age. , 1989, Experimental cell biology.

[11]  N. Van Rooijen,et al.  Dendritic Cell-Induced Memory T Cell Activation in Nonlymphoid Tissues , 2008, Science.

[12]  N. Van Rooijen,et al.  Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. , 1994, Journal of immunological methods.

[13]  G. V. van Dooren,et al.  Dynamics of neutrophil migration in lymph nodes during infection. , 2008, Immunity.

[14]  M. Miller,et al.  CCR2 Regulates Monocyte Recruitment As Well As CD4+ Th1 Allorecognition After Lung Transplantation , 2010, American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons.

[15]  Marie-Claire Schanne-Klein,et al.  Three‐dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy , 2007, Microscopy research and technique.

[16]  Michael D. Cahalan,et al.  Two-photon tissue imaging: seeing the immune system in a fresh light , 2002, Nature Reviews Immunology.

[17]  T. Graf,et al.  Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages. , 2000, Blood.

[18]  Michael L. Dustin,et al.  MYELOMONOCYTIC CELL RECRUITMENT CAUSES FATAL CNS VASCULAR INJURY DURING ACUTE VIRAL MENINGITIS , 2008, Nature.

[19]  E. Parzen On Estimation of a Probability Density Function and Mode , 1962 .

[20]  F. Tacke,et al.  Modulation of Dendritic Cell Trafficking to and from the Airways , 2006, The Journal of Immunology.

[21]  Mark J. Miller,et al.  ITAM Signaling by Vav Family Rho Guanine Nucleotide Exchange Factors Regulates Interstitial Transit Rates of Neutrophils In Vivo , 2009, PloS one.

[22]  M. Grayson,et al.  Pulmonary defense mechanisms against pneumonia and sepsis , 2008, Current opinion in pulmonary medicine.

[23]  K. Ley,et al.  Therapeutic inhibition of CXCR2 by Reparixin attenuates acute lung injury in mice , 2008, British journal of pharmacology.

[24]  W. Seeger,et al.  The role of CC chemokine receptor 2 in alveolar monocyte and neutrophil immigration in intact mice. , 2002, American journal of respiratory and critical care medicine.

[25]  H. Anders,et al.  Chemokine receptors Ccr1, Ccr2, and Ccr5 mediate neutrophil migration to postischemic tissue , 2006, Journal of leukocyte biology.

[26]  Ronald N Germain,et al.  Macrophage and T cell dynamics during the development and disintegration of mycobacterial granulomas. , 2008, Immunity.

[27]  D. Kreisel,et al.  Orthotopic mouse lung transplantation as experimental methodology to study transplant and tumor biology , 2008, Nature Protocols.

[28]  F. Ginhoux,et al.  Immature monocytes acquire antigens from other cells in the bone marrow and present them to T cells after maturing in the periphery , 2006, The Journal of experimental medicine.

[29]  V. Ambrosini,et al.  Assessment of a chemically induced model of lung squamous cell carcinoma in mice by 18F-FDG small-animal PET , 2007, Nuclear medicine communications.

[30]  Ian Parker,et al.  Real-time imaging of lymphocytes in vivo. , 2003, Current opinion in immunology.

[31]  M. Tate,et al.  Neutrophils Ameliorate Lung Injury and the Development of Severe Disease during Influenza Infection1 , 2009, The Journal of Immunology.

[32]  Mark J. Miller,et al.  Cutting Edge: Acute Lung Allograft Rejection Is Independent of Secondary Lymphoid Organs1 , 2009, The Journal of Immunology.

[33]  J. Brain,et al.  Pulmonary intravascular macrophages: their contribution to the mononuclear phagocyte system in 13 species. , 1999, American journal of physiology. Lung cellular and molecular physiology.

[34]  Simon C Watkins,et al.  Intravital fluorescence microscopy in pulmonary research. , 2006, Advanced drug delivery reviews.

[35]  W. Seeger,et al.  Monocytes Are Potent Facilitators of Alveolar Neutrophil Emigration During Lung Inflammation: Role of the CCL2-CCR2 Axis1 , 2003, The Journal of Immunology.

[36]  M. Jutila,et al.  Elimination of mouse splenic macrophages correlates with increased susceptibility to experimental disseminated candidiasis. , 1994, Journal of immunology.

[37]  J. Christie,et al.  Risk factors for early primary graft dysfunction after lung transplantation: a registry study , 2009, Clinical transplantation.