Leukocyte recruitment/activation

The inflammatory response is the cornerstone of the body’s defense mechanism against bacterial and viral pathogens, as well as physical-, chemical- and environmental-mediated tissue and organ damage. Leucocyte recruitment at the site of pathogen evasion or sterile tissue injury is a critical adaptation for the preservation of tissue integrity. Neutrophils are the cell population that acutely responds to the alterations of inflammatory micro-environment. Neutrophil infiltration takes place within 6-8 hours from the initiation of the inflammatory process and is followed by the recruitment of other cell populations, like monocytes, lymphocytes, and eosinophils, which either promote or drive the resolution of inflammation. Leukocyte infiltration into sites of infection or sterile inflammation is a tightly regulated process that follows a sequence of adhesive events, termed as leukocyte adhesion cascade. One can broadly generalize that most leukocytes follow a similar multi-step cascade in the peripheral (non-lymphoid) vasculature with some exceptions. Accordingly, an updated adhesion cascade in postcapillary venules involves free-flowing leukocytes initial attachment or tethering and slow velocity rolling (step 1),stable adhesion (arrest) on endothelial cells (step 2), leukocyte flattening (step 3), and subsequent crawling on the vascular endothelium, transendothelial cell migration (TEM) between (paracellular route) or through (transcellular) the vascular endothelium (step 4), and uropod elongation to complete transmigration of postcapillary venules (step 5). The initial attachment and rolling steps are initiated by interactions of endothelial E- and P-selectins and their counterreceptors on leukocytes L-selectin (Leick et al., 2014).

Each of these steps is necessary for effective leukocyte recruitment; these steps are not phases of inflammation, but represent the sequence of events from the perspective of each leukocyte. At any given moment they all happen in parallel, involving different leukocytes in the same microvessels.

From the initial selectin-dependent leukocyte tethering to endothelial cells to the final migration of leukocytes into the sub-endothelium, this process depends on the interplay between leukocyte receptors and endothelial cell counter-receptors, as well as on the presence of endogenous inhibitors of leukocyte adhesion enabling the targeted recruitment of leukocytes to inflamed tissues.

To enable the infiltration of leukocytes at the site of inflammation, a series of alterations in endothelial cells and leukocytes takes place:

• regulation of the expression of adhesion molecules in leukocytes
• increased secretion of chemokines by endothelial cells
• increased expression of adhesion molecules in the luminal surface of endothelial cells

(Kourtzelis and Mitroulis, 2015) (Robbins and Cotran: Pathologic Basis of Disease 2010).

After recruitment, activation includes phenotype modification with morphologic alterations, changes in marker proteins (MHC, adhesion molecules, co-stimulatory signal), expression of mediators, enzymes, and pro-inflammatory proteins/lipids. Recruited monocytes recruited mature into macrophages with phagocytic activity and elaboration of a myriad of mediators of inflammation. The macrophage can replicate within tissues or die, including by apoptosis.

in vivo imaging:

• Flow cytometry (FC/FACS),
• immunhistochemistry
• two photon-intravital microscopy (TP-IVM) (van Grinsven et al., 2017)
• Spinning Disk Confocal Microscopy-IVM (Jenne et al., 2011)
• Histology, increased cell numbers and altered composition

In vitro

• transwell Migration Assay (Justus et al., 2014)
• T-Lymphocyte & Innate Immune Cell Activation Assays
• Leukocyte Surface Markers (Monoclonal Antibodies to Leukocyte Surface Markers)
• Markers of leukocyte activation – protease release, ROS/RNS, NADPH oxidase (NOX), defense response - expression of anti-oxidants.
• organs-on-a-chip (Bnam et al., 2016; Ribas et al., 2017; Wufuer et al. 2016)

REFERENCES:

Benam KH, Villenave R, Lucchesi C, Varone 1, Hubeau C, Lee HH, Alves SE, Salmon M, Ferrante TC, Weaver JC, Bahinski A, Hamilton GA, Ingber DE., Small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro, Nat Methods. 2016 Feb;13(2):151-7.

Ribas, J., Zhang, Y. S., Pitrez, P. R., Leijten, J., Miscuglio, M., Rouwkema, J., Dokmeci, M. R., Nissan, X., Ferreira, L. and Khademhosseini, A. (2017), Organ-On-A-Chip: Biomechanical Strain Exacerbates Inflammation on a Progeria-on-a-Chip Model doi:10.1002/smll.201770087

Wufuer M, Lee G, Hur W, Jeon B, Kim BJ, Choi  TH, Lee SH, Skin-on-a-chip model simulating inflammation, edema and drug-based treatment,  Nature Scientific Reports 6, Article number: 37471 (2016) doi:10.1038/srep37471

Kourtzelis I and Mitroulis I, Encyclopedia of Inflammatory Diseases, Leukocyte Recruitment, pp 1-9, Compendium of Inflammatory Diseases, Editors: Michael J. Parnham , Springer Basel, 2015, DOI 10.1007/978-3-0348-0620-6_177-1

Kumar, V.; Abbas, AK.; Fausto, N.; Aster, J. Robbins and Cotran: Pathologic Basis of Disease. 8. Elsevier; Philadelphia: 2010.

Leick M, Azcutia V, Newton G, Luscinskas FW., Leukocyte recruitment in inflammation: basic concepts and new mechanistic insights based on new models and microscopic imaging technologies, Cell Tissue Res. 2014 Mar;355(3):647-56

Nourshargh S, Alon R., Leukocyte migration into inflamed tissues., Immunity. 2014 Nov 20;41(5):694-707