Inflammation

Leukocyte Trafficking

The key feature of any effective immune response is the movement of immune cells (leukocytes) out of the blood stream to provide aid in tissue repair and host defense against invading pathogens. However besides these important effector functions leukocytes could also cause in some cases damage like in inflammatory diseases as rheumatoid arthritis, multiple sclerosis or inflammatory bowel disease. Therefore, elucidating the molecular mechanisms underlying the migration of leukocytes from the blood to tissues is critical to our understanding of immune function.

Our goal is to identify the mechanisms underlying the recruitment of leukocytes out of the blood stream to sites of tissue damage and inflammation

Using intravital microscopy techniques (brightfield, spinning disc confocal fluorescence microscopy, 2-Photon laser scanning fluorescence microscopy) in different organ systems of the mouse the research in the lab spans the sequence of events from rolling and adhesion in blood vessels to how leukocytes know where to go along the vessel to emigrate. (Fig 1. Phillipson, M. Nat Med. 2011)

Fig 1. Phillipson, M. Nat Med. 2011: Fig 1. The neutrophil recruitment cascade. Intravital confocal microscopy image of a cremasteric postcapillary venule with added cartoon cells illustrating the consecutive steps of the recruitment of circulating neutrophils to localized inflammation (infection or necrosis). The white boxes show the adhesion molecules involved in each step. Endothelial cell junctions are stained with monoclonal antibodies to CD31 (red). Endothelial upregulation of adhesion molecules results in interactions between selectins and their ligands on neutrophils, leading to neutrophil tethering and rolling. Chemokines sequestered on the luminal endothelium induce conformation changes of neutrophil β2 integrins, which results in neutrophil adhesion and crawling. Mechanotactic and chemotactic guidance signals direct crawling neutrophils to junctional transmigration sites closer to the source of chemotactic agent, examples of which are given in the green box. (Nature Medicine 2011).

The type of leukocyte, the vascular bed, the type of stimulus and the organ specific environment dictates how leukocytes are recruited (Fig 2. Ref. Petri et al. JI 2008).

One research focus is the liver (Fig. 2B) where neutrophil recruitment within the hepatic microvasculature differs from most tissues in 3 fundamental ways: 1) Neutrophils adhere to the endothelium of capillaries called sinusoids, 2) neutrophils do not appear to roll for a significant distance, but rather tether and immediately adhere within the sinusoidal capillaries and 3) neutrophil adhesion within the sinusoids is selectin-independent. In a recent study, our group clearly demonstrates that adhesion molecules CD44 and hyaluronan are responsible for neutrophil sequestration within the sinusoids of the liver. (Fig.3; McDonald et al., 2008, J. Exp. Med).

Our lab is furthermore interested in the crawling step identified as part of the leukocyte recruitment cascade and demonstrated to play an important role in vivo (Ref. Phillipson et al., JEM 2006) and which signaling pathways as well as adhesion molecules are involved.

Another part of the research regarding leukocyte migration focuses on how leukocytes are able to migrate either at junctions (paracellular) or through the endothelial cell body (transcellular). Recently endothelial domes have been identified in our lab (Fig.4: Phillipson et al. PLoS ONE 2008).


Fig 2. Ref. Petri et al. JI 2008: Fig.2: Organ-specific recruitment of leukocytes in inflamed vessels of three different organs (A-C). (1): mononuclear and polymorphonuclear cells; (2): neutrophils; *: as demonstrated for neutrophils; #: as demonstrated for monocytes. §: only shown for PMN. (J Immunol 2008)

Fig.3; McDonald et al., 2008, J. Exp. Med: Fig.3: Adherent neutrophils (red, visualized with PE-labeled anti GR-1) in liver sinusoids after LPS treatment of the liver (green, auto fluorescence). (J Exp Med 2008)

Fig.4 Phillipson et al. PLoS ONE 2008: Fig.4: (A) Crawling neutrophil with podosome like pertusions (arrows) invaginating the endothelium. e: endothelial cell: n: neutrophil; L: lumen. (B) Novel “dome” like structures (arrows) in vivo may be formed from docking structures or transmigratory cups. e: endothelial cell: n: neutrophil; L: lumen. (PLoS One 2008)

Endothelial Domes

We have further demonstrated that endothelial LSP1 is recruited to the cytoskeleton in inflammation and plays an important role in forming endothelial domes thereby regulating neutrophil transendothelial migration.(Petri et al Blood 2011) (Fig. 5).

Fig.5 Petri et al Blood 2011: Fig. 5  In vivo endothelial dome formation in WT mice visualized by whole-mount staining of the cremaster muscle and 2-photon microscopy. (A) Overview merge of a postcapillary venule showing a migrating neutrophil covered by a dome. (B-D) Magnifications of the area in panel A showing the single channels for PECAM-1 (B), MRP-14 (C), and the merge (D). The dome is highlighted by arrows. (F) PECAM-1 positive stained endothelium demonstrating the formation of a dome (arrow). (G) Neutrophils (green) migrate and are encapsulated by endothelial domes (red) as highlighted by arrows. Insets represent magnifications of the dome and the encapsulated neutrophil from panels G and F. The dome reaches into the vessel lumen (L) and is highlighted by the arrow. (Blood 2011).

Platelets in Inflammation

A new area of interest has been to demonstrate that platelets are central players in inflammation and are an important component of the innate immune response. The ability to visualize platelets within the live host is essential to understanding their role in these processes. Following LPS-induced inflammation, we were able to measure a significant increase in both the number and size of platelet aggregates observed within the vasculature of a number of different tissues. Real-time observation of these platelet aggregates reveals them to be large, dynamic structures that are continually expanding and sloughing-off into circulation.

Fig.6 Jenne, et al. PLoS One. 2011: Fig 6. Platelet recruitment to livers of mice treated with intravenous LPS using CD41-YFPki/+ mice.  Representative fields of view of livers from untreated (Ai, Aii) and LPS treated (Bi, Bii) CD41-YFPki/+ mice. Neutrophils are labelled with Alexa Fluor 647-conjugated anti-Gr-1 (blue); YFP+ platelets and liver autofluorescence (green). (PLoS One 2012)  

*VIDEOS

References:

  • Phillipson M, Kubes P. The neutrophil in vascular inflammation. Nat Med. 2011 Nov 7;17(11):1381-90.
  • Petri B, Kaur J, Long EM, Li H, Parsons SA, Butz S, Phillipson M, Vestweber D, Patel KD, Robbins SM, Kubes P. Endothelial LSP1 is involved in endothelial dome formation, minimizing vascular permeability changes during neutrophil transmigration in vivo. Blood. 2011 Jan 20;117(3):942-52.
  • Petri B, Phillipson M, Kubes P: The Physiology of Leukocyte Recruitment: An In Vivo Perspective. The Journal of Immunology, 2008, 2008 May 15;180(10):6439-46.
  • McDonald B, McAvoy EF, Lam F, Gill V, de la Motte C, Savani RC, Kubes P: Interaction of CD44 and hyaluron is the dominant mechanism for neutrophil sequestration in inflamed liver sinusoids. J Exp Med, 2008, Apr 14;205(4):915-27.
  • Phillipson M, Heit B, Colarusso P, Liu L, Ballantyne BM, Kubes P: Intraluminal crawling of neutrophils to emigration sites: a molecularly distinct process from adhesion in the recruitment cascade. J Exp Med, 2006, 203(12):2569-75.
  • Phillipson M, Kaur J, Colarusso P, Ballantyne BM, Kubes P: Endothelial domes encapsulate adherent neutrophils and minimize increases in vascular permeability in paracellular and transcellular emigration. PLoS ONE, 2008, 3(2):e1649.
  • Jenne CN, Wong CH, Petri B, Kubes P. The use of spinning-disk confocal microscopy for the intravital analysis of platelet dynamics in response to systemic and local inflammation. PLoS One. 2011;6(9):e25109.