Protein Kinase C-θ Is Required for Murine Neutrophil Recruitment and Adhesion Strengthening under Flow This information is current as Anna Bertram, Hong Zhang, Sibylle von Vietinghoff, of September 26, 2021. Carmen de Pablo, Hermann Haller, Nelli Shushakova and Klaus Ley J Immunol 2012; 188:4043-4051; Prepublished online 7 March 2012; doi: 10.4049/jimmunol.1101651 Downloaded from http://www.jimmunol.org/content/188/8/4043 References This article cites 51 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/188/8/4043.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Protein Kinase C-u Is Required for Murine Neutrophil Recruitment and Adhesion Strengthening under Flow Anna Bertram,*,† Hong Zhang,† Sibylle von Vietinghoff,*,† Carmen de Pablo,† Hermann Haller,* Nelli Shushakova,* and Klaus Ley† Protein kinase C (PKC)-u is involved in T cell activation via regulating the avidity of the b2 integrin LFA-1 in the immunological synapse. LFA-1 also mediates leukocyte adhesion. To investigate the role of PKC-u in neutrophil adhesion, we performed intravital microscopy in cremaster venules of mice reconstituted with bone marrow from LysM-GFP+ (wild-type [WT]) and PKC-u gene- deficient (Prkcq2/2) mice. Following stimulation with CXCL1, both WT and Prkcq2/2 cells became adherent. Although most WT neutrophils remained adherent for at least 180 s, 50% of Prkcq2/2 neutrophils were detached after 105 s and most by 180 s. Upon CXCL1 injection, rolling of all WT neutrophils stopped for 90 s, but rolling of Prkcq2/2 neutrophils started 30 s after CXCL1 2 2 2 2 stimulation. A similar neutrophil adhesion defect was seen in vitro, and spreading of Prkcq / neutrophils was delayed. Prkcq / Downloaded from neutrophil recruitment was impaired in fMLP-induced transmigration into the cremaster muscle, thioglycollate-induced perito- nitis, and LPS-induced lung injury. We conclude that PKC-u mediates integrin-dependent neutrophil functions and is required to sustain neutrophil adhesion in postcapillary venules in vivo. These findings suggest that the role of PKC-u in outside–in signaling following engagement of neutrophil integrins is relevant for inflammation in vivo. The Journal of Immunology, 2012, 188: 4043– 4051. http://www.jimmunol.org/ nflammation is the body’s natural defense mechanism against stimuli, the b2 integrin macrophage-1 Ag (Mac-1) can be rapidly different forms of injury, such as invading microorganisms or mobilized to the cell surface after exposure to inflammatory I physical trauma. Although inflammation is critical for sur- stimuli (4). Neutrophil arrest is LFA-1–dependent and Mac-1– vival, massive inflammation can also be deleterious, leading to independent, whereas Mac-1 is required for many b2 integrin- systemic inflammatory response syndrome or other disorders. A dependent neutrophil functions such as phagocytosis and respi- critical step in the inflammatory process is the attraction and ac- ratory burst (5, 6). tivation of leukocytes that transmigrate through the vascular en- Recently, several intracellular kinases have been shown to dothelium and then migrate to the site of inflammation. Neutrophil participate in neutrophil adhesion. E-selectin engagement induces by guest on September 26, 2021 granulocytes are the first line of defense against bacteria and fungi. b2 integrin activation via a signaling cascade involving the Src They migrate to the site of injury in acute inflammation, where they family tyrosine kinase FGR (7), spleen tyrosine kinase (SYK) (8), kill and phagocytose the attacking bacteria and where they release and Bruton’s tyrosine kinase (9). Once adherent, the ligand-bound oxygen radicals, bactericidal peptides, and inflammatory cytokines. integrins initiate outside–in signaling events that involve SYK To get to the site of inflammation, neutrophils follow a sequence of (10) and Src kinases (11, 12). Using pseudosubstrate inhibition, well-orchestrated events. This adhesion cascade involves tethering, the atypical isoform protein kinase C (PKC)-z has been implicated rolling and slow rolling, arrest, adhesion strengthening and intra- in outside–in signaling downstream of the b2 integrin Mac-1 (13), vascular crawling, and transmigration through the endothelium, but Mac-1 has no known role in neutrophil adhesion under flow. followed by migration through the tissue (1, 2). The functional role of PKC has not yet been investigated in the Inflammation induces surface expression of selectins on endo- context of neutrophil adhesion to the vascular endothelium (14). thelial cells, increasing the portion of rolling cells. Activation of PKC isoforms are involved in cell differentation, migration, rolling leukocytes by chemokines leads to a conformational change proliferation, and activation (15). In other cell types, PKC isoforms of the b2 integrin LFA-1, enabling it to bind to ICAM-1 and other have been shown to be involved in the activation of different ligands (3), which leads to neutrophil arrest. Whereas the level of integrins (16–19). PKCs have been grouped into the conventional, LFA-1 expression on neutrophils is unaltered by inflammatory novel, and atypical isoforms. PKCs are known to be activated by G protein-coupled receptors (GPCRs), tyrosine kinase receptors, *Department of Nephrology and Hypertensiology, Hannover Medical School, 30625 and growth factor receptors (15). Using knockout strategies, PKC Hannover, Germany; and †Division of Inflammation Biology, La Jolla Institute for isoforms have been implicated in a multitude of diseases and or- Allergy and Immunology, La Jolla, CA 92037 gan dysfunctions, including inflammatory processes (20). In this Received for publication June 6, 2011. Accepted for publication February 7, 2012. study, we investigate the role of PKC-u, a novel PKC isoform (14), This work was supported by Dr. Werner Jackstaedt Foundation (Germany) Grant which is calcium-insensitive, but activated by 1,2-diacylglycerol S134-10.056 (to A.B.). or phorbol esters, in neutrophil adhesion. The main PKC isoforms Address correspondence and reprint requests to Dr. Anna Bertram, Department of Nephrology and Hypertensiology, Hannover Medical School, Carl-Neuberg-Strasse expressed in neutrophils are thought to be PKC-a, PKC-b, PKC-d, 1, 30625 Hannover, Germany. E-mail address: [email protected] and PKC-z (21, 22). Expression of PKC-u and PKC-i/l has also Abbreviations used in this article: GPCR, G protein-coupled receptor; KO, knockout; been demonstrated (23). MPO, myeloperoxidase; PKC, protein kinase C; RGD, Arg-Gly-Asp; SYK, spleen PKC-u has a well-documented role in T cell activation. Upon tyrosine kinase; WT, wild-type. TCR stimulation, proliferation, IL-2 production, and anti- Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 apoptotic mechanisms were markedly reduced in PKC-u knock- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101651 4044 PKC-u IN NEUTROPHIL ADHESION out T cells (24–26). Recently, PKC-u has been shown to regulate For the degranulation assay, isolated neutrophils were primed with 5 the affinity of the b integrin LFA-1 downstream of the TCR by mg/ml cytochalasin B (Sigma-Aldrich, Munich, Germany) for 15 min on 2 ice and 15 min at 37˚C, and 2 3 106 cells in 400 ml were incubated at 37˚ phosphorylating the guanine nucleotide exchange factor Rap- C on a 24-well plate coated with 5 mg/cm2 poly-RGD (Sigma-Aldrich, GEF2, an activator of the small GTPase Rap1. This leads to Munich, Germany), with 100 ng/ml PMA (Sigma-Aldrich, Munich, a stable immunological synapse (27). The signaling cascade Germany) as positive control or the vehicle PBS as negative control. Cell- leading to LFA-1 activation after TCR stimulation shares some free supernatants were collected after 15, 30, and 60 min and were an- similarities with the inside–out signaling pathway following the alyzed for myeloperoxidase (MPO) activity as described (30). Briefly, 20 ml supernatant was incubated with 230 ml 0.167 mg/ml o-dianisidine activation of neutrophil GPCRs by chemokines. Binding of the dihydrochloride solution (Sigma-Aldrich, Munich, Germany) with 160 chemokine CXCL1 to its GPCR CXCR2 is followed by a rapid mM hydrogen peroxide (Sigma-Aldrich, Munich, Germany) for 15 min at signaling cascade involving PLC-b, the guanine exchange factor room temperature. The reaction was stopped by adding 50 ml1Msulfuric CALDAG-GEF1, and the small GTPase Rap1 (3, 28). This inside– acid (Sigma-Aldrich, Munich, Germany). MPO activity was assessed via changes in OD at 405 nm on a Multiskan Ascent plate reader (Thermo out signaling results in the activation of LFA-1, its conformational Scientific, Schwerte, Germany). Total cellular content was determined in change to the high-affinity state, and subsequent arrest of the cell lysates in 0.5% Triton X-100 (Sigma-Aldrich, Munich, Germany) neutrophil on the endothelium (2). The role of PKC-u in neu- and 0.5% hexadecyltrimethylammonium bromide (Sigma-Aldrich, trophils has not been studied. Munich, Germany) in 50 mM potassium phosphate buffer (pH 6.0), and The objective of this study was to elucidate the role of PKC-u MPO degranulation was determined by dividing MPO activity of the supernatants by MPO activity of whole-cell lysates.