Dear Lucia,

The following publications demonstrate specific blocking of the neutrophil migration:

Blood. 2012 Aug 16; 120(7): 1489–1498.

Prepublished online 2012 Jun 1. doi:  10.1182/blood-2012-01-404046

PMCID: PMC3423786

Ly6G ligation blocks recruitment of neutrophils via a β2-integrin–dependent mechanism

Jun-Xia Wang,1 Angela M. Bair,2 Sandra L. King,3 Ruslan Shnayder,1 Ya-Fang Huang,4 Chi-Chang Shieh,4 Roy J. Soberman,2 Robert C. Fuhlbrigge,3,5 and Peter A. Nigrovic 1,5

Author information ► Article notes ► Copyright and License information ►

This article has been cited by other articles in PMC.

 Abstract

Ly6G is a glycosylphosphatidylinositol (GPI)–anchored protein of unknown function that is commonly targeted to induce experimental neutrophil depletion in mice. In the present study, we found that doses of anti-Ly6G Abs too low to produce sustained neutropenia remained capable of inhibiting experimental arthritis, leaving joint tissues free of infiltrating neutrophils. Thioglycollate-stimulated peritonitis was also attenuated. No alteration in neutrophil apoptosis was observed, implicating impaired recruitment. Indeed, Ly6G ligation abrogated neutrophil migration toward LTB4 and other chemoattractants in a transwell system. Exploring the basis for this blockade, we identified colocalization of Ly6G and β2-integrins by confocal microscopy and confirmed close association by both coimmunoprecipitation and fluorescence lifetime imaging microscopy. Anti-Ly6G Ab impaired surface expression of β2-integrins in LTB4-stimulated neutrophils and mimicked CD11a blockade in inhibiting both ICAM-1 binding and firm adhesion to activated endothelium under flow conditions. Correspondingly, migration of β2-integrin–deficient neutrophils was no longer inhibited by anti-Ly6G. These results demonstrate that experimental targeting of Ly6G has functional effects on the neutrophil population and identify a previously unappreciated role for Ly6G as a modulator of neutrophil migration to sites of inflammation via a β2-integrin–dependent mechanism.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3423786/

In vitro, FPR1-specific antibodies block neutrophil migration

NATURE REVIEWS IMMUNOLOGY | REVIEW

Neutrophil migration in infection and wound repair: going forward in reverse

Sofia de Oliveira,

Emily E. Rosowski & Anna Huttenlocher

Nature Reviews Immunology 16, 378–391 (2016) doi:10.1038/nri.2016.49

http://www.nature.com/nri/journal/v16/n6/full/nri.2016.49.html?WT.feed_name=subjects_cell-biology

Peptide-Mediated Inhibition of Neutrophil Transmigration by

Blocking CD47 Interactions with Signal Regulatory Protein

Yuan Liu,2 * Miriam B. O’Connor,2 * Kenneth J. Mandell,* Ke Zen,* Axel Ullrich,‡

Hans-Jo¨rg Bu¨hring,† and Charles A. Parkos3

CD47, a cell surface transmembrane Ig superfamily member, is an extracellular ligand for signal regulatory protein (SIRP). Interactions between CD47 and SIRP regulate many important immune cell functions including neutrophil (PMN) transmigration. Here we report identification of a novel function-blocking peptide, CERVIGTGWVRC, that structurally mimics an epitope on CD47 and binds to SIRP. The CERVIGTGWVRC sequence was identified by panning phage display libraries on the inhibitory CD47 mAb, C5D5. In vitro PMN migration assays demonstrated that peptide CERVIGTGWVRC specifically inhibited PMN migration across intestinal epithelial monolayers and matrix in a dose-dependent fashion. Further studies using recombinant proteins indicated that the peptide specifically blocks CD47 and SIRP binding in a dose-dependent fashion. Protein binding assays using SIRP domain-specific recombinant proteins demonstrated that this peptide directly bound to the distal-most Ig loop of SIRP, the same loop where CD47 binds. In summary, these findings support the relevance of CD47-SIRP interactions in

regulation of PMN transmigration and provide structural data predicting the key residues involved on the surface of CD47. Such peptide reagents may be useful for studies on experimental models of inflammation and provide a template for the design of anti-inflammatory agents. The Journal of Immunology, 2004, 172: 2578–2585.

https://www.biochem.mpg.de/292456/018-04.pdf

The Endogenous Opioid Spinorphin Blocks fMet-Leu-Phe-Induced Neutrophil Chemotaxis by Acting as a Specific Antagonist at the N-Formylpeptide Receptor Subtype FPR

Thomas S. Liang, Ji-Liang Gao, Omid Fatemi, Mark Lavigne, Thomas L. Leto and

Philip M. Murphy1

Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892

Abstract

Spinorphin is an endogenous heptapeptide (leucylvalylvalyltyrosylprolyltryptophylthreonine), first isolated from bovine spinal cord, whose sequence matches a conserved region of β-hemoglobin. Also referred to as LVV-hemorphin-4 and a member of the nonclassical opioid hemorphin family, spinorphin inhibits enkephalin-degrading enzymes and is analgesic. Recently, spinorphin was reported to block neutrophil activation induced by the chemotacticN-formylpeptide N-formylmethionylleucylphenylalanine (fMLF), suggesting a potential role as an endogenous negative regulator of inflammation. Here we use both gain- and loss-of-function genetic tests to identify the specific mechanism of spinorphin action on neutrophils. Spinorphin induced calcium flux in normal mouse neutrophils, but was inactive in neutrophils from mice genetically deficient in the fMLF receptor subtype FPR (N-formylpeptide receptor). Consistent with this, spinorphin induced calcium flux in human embryonic kidney 293 cells transfected with mouse FPR, but had no effect on cells expressing the closely related fMLF receptor subtype FPR2. Despite acting as a calcium-mobilizing agonist at FPR, spinorphin was a weak chemotactic agonist and effectively blocked neutrophil chemotaxis induced by fMLF at concentrations selective for FPR. Spinorphin did not affect mouse neutrophil chemotaxis induced by concentrations of fMLF that selectively activate FPR2. Thus, spinorphin blocks fMLF-induced neutrophil chemotaxis by acting as a specific antagonist at the fMLF receptor subtype FPR.

http://www.jimmunol.org/content/167/11/6609.abstract

Therapeutic Effect of Blocking CXCR2 on Neutrophil Recruitment and Dextran Sodium Sulfate-Induced Colitis

Shukkur Muhammed Farooq, RoseMarie Stillie, Majlis Svensson, Catharina Svanborg1, Robert M. Strieter and Andrew W. Stadnyk

http://jpet.aspetjournals.org/content/329/1/123.full

Structural Insights into Neutrophilic Migration Revealed by the Crystal Structure of the Chemokine Receptor CXCR2 in Complex with the First PDZ Domain of NHERF

Guorong Lu , Yanning Wu , Yuanyuan Jiang , Shuo Wang, Yuning Hou, Xiaoqing Guan, Joseph Brunzelle, Nualpun Sirinupong, Shijie Sheng, Chunying Li , Zhe Yang

Published: October 2, 2013http://dx.doi.org/10.1371/journal.pone.0076219

Abstract

Neutrophil plays an essential role in host defense against infection, but uncontrolled neutrophilic infiltration can cause inflammation and severe epithelial damage. We recently showed that CXCR2 formed a signaling complex with NHERF1 and PLC-2, and that the formation of this complex was required for intracellular calcium mobilization and neutrophilic transepithelial migration. To uncover the structural basis of the complex formation, we report here the crystal structure of the NHERF1 PDZ1 domain in complex with the C-terminal sequence of CXCR2 at 1.16 Å resolution. The structure reveals that the CXCR2 peptide binds to PDZ1 in an extended conformation with the last four residues making specific side chain interactions. Remarkably, comparison of the structure to previously studied PDZ1 domains has allowed the identification of PDZ1 ligand-specific interactions and the mechanisms that govern PDZ1 target selection diversities. In addition, we show that CXCR2 can bind both NHERF1 PDZ1 and PDZ2 in pulldown experiments, consistent with the observation that the peptide binding pockets of these two PDZ domains are highly structurally conserved. The results of this study therefore provide structural basis for the CXCR2-mediated neutrophilic migration and could have important clinical applications in the prevention and treatment of numerous neutrophil-dependent inflammatory disorders.

http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0076219

Hoping this will be helpful,

Rafik

 Dear Rafik , Thanks so much for your input. I'll read these ASAP.  Best Wishes!

Sometimes it, somehow, depends on the pathological condition you are looking for. You may want to decide what kind of disease/pathological condition you want to study. Then you can pick the positive and negative controls properly.  

Dear Lucia

Neutrophil recruitment is an integral part of the immune response to infection as well as of inflammatory disorders. The process of neutrophil extravasation comprises a complex multistep cascade that is orchestrated by a tightly coordinated sequence of adhesive interactions with vessel wall endothelial cells. Adhesion receptors as well as signaling molecules in both neutrophils and endothelial cells regulate the recruitment of neutrophils into the site of inflammation or infection.

One of method demonstrated the neutrophil blocking and you can read details in this title as:Platelet endothelial cell adhesion molecule-1 (PECAM-1) and CD99.

PECAM-1 is a member of the immunoglobulin superfamily consisting of six Ig domains and is expressed at the intercellular borders of endothelial cells as well on platelets, neutrophils, monocytes and some T cells (91;92). The two amino terminal Ig domains of PECAM-1 are involved in a homophilic interaction which is considered to operate during neutrophil transendothelial migration. Blocking the homophilic interaction of PECAM-1 inhibits transendothelial migration in vitro and in vivo (93-97), and leukocytes blocked at the PECAM-1-dependent step remain adherent on the apical surface of the endothelial cells. Endothelial PECAM-1 recycles between the junctions and the subjunctional plasmalemma, and is targeted to the zone of active leukocyte transmigration (97). PECAM-1 knockout mice in the mouse strain C57Bl/6 did not show a reduction in neutrophil recruitment (98), however, breeding these mice into different mouse strains did result in a decrease in the recruitment of neutrophils into inflamed peritoneum (99). Interestingly, PECAM-1 deficiency was found to affect preferentially interleukin (IL)-1β but not if TNF-α induced inflammation (100).

Besides the cation-independent Ig domain 1 and 2-dependent homophilic binding of PECAM-1, a cation-dependent PECAM-1 heterophilic interaction mediated by Ig domain 6 of the molecule has been reported to participate at a late step of diapedesis, namely the migration through the basement membrane (94). In this context, we recently identified a member of Ly-6 family, CD177, as a novel heterophilic binding partner of PECAM-1. CD177 is a 58- to 64-kDa glycosyl-phosphatidyl-inositol (GPI)-anchored glycoprotein, which is expressed exclusively on neutrophils but not on other blood cells. In contrast to the homophilic PECAM-1 interaction, the heterophilic interaction between CD177 and PECAM-1 could be blocked by antibodies to Ig domain 6 of PECAM-1. In addition, we demonstrated that this heterophilic interaction functions in neutrophil transendothelial migration (101).

Another interesting observation is that PECAM-1 homophilic ligation results in an upregulation of the laminin receptor α6β1-integrin on transmigrating neutrophils thereby enhancing the subsequent penetration of the basement membrane by neutrophils. Consequently, neutrophils treated with antibodies to α6β1-integrin are trapped between endothelium and the basal lamina (102). These findings also point to the fact that PECAM-1 acts a signaling receptor. PECAM-1 contains two immunoreceptor tyrosine-based inhibitory motifs, which are involved in signals mediated by Src-homology-2 containing phosphatases, such as SHP-1 and SHP-2 (103-105). In addition, antibody crosslinking of PECAM-1 has been shown to upregulate the activity of β1- and β2-integrins on neutrophils and other leukocytes (106;107).

CD99 is a highly O-glycosylated molecule expressed on both neutrophils and other leukocytes and at the interendothelial junctions that acts in a homophilic manner to mediate transmigration. It controls a step in diapedesis that is distinct from and distal to the step mediated by PECAM-1, as cells blocked at the CD99-dependent step are arrested halfway across the endothelial junction. Mouse CD99 was recently cloned and antibodies to mouse CD99 inhibited the recruitment of antigen-specific T cells into inflamed areas of the skin and edema formation. In addition, endothelial CD99 was found to participate in neutrophil diapedesis in vivo. CD99L2 is a recently identified molecule expressed on both leukocytes and endothelial cells that shares 32% amino acid identity with CD99. CD99L2 was shown to specifically participate in neutrophil but not lymphocyte diapedesis in vivo and blocking CD99L2 inhibited neutrophil transmigration through the vessel wall at the level of the perivascular basement membrane.

Find the full publication:Eun Young Choi, Sentot Santoso, and Triantafyllos Chavakis. Front Biosci. 2009 Jan 1; 14: 1596–1605. 

Good luck

Marius