PMN Transmigration Research Articles

Cleavage of the CD11b extracellular domain by the leukocyte serprocidins is critical for neutrophil detachment during chemotaxis (173.7)

Abstract The β(2)-integrin CD11b/CD18 mediates the firm adhesion of neutrophils (PMNs) to epithelial monolayers, a key step in PMN transepithelial migration. To complete the transmigration process, adherent PMNs must detach from epithelial surfaces to move forward. The mechanism that governs the detachment of adherent PMNs, however, is not clear. Here, we present evidence that cleavage of the CD11b extracellular domain containing the ligand-binding I-domain by 3 structural and functional related serine proteases (elastase, proteinase-3 and cathepsin G) serves as a novel mechanism for PMN detachment after the initial cell adhesion. Kinetic studies showed that the cleavage of CD11b is positively correlated with PMN detachment and subsequent transmigration. Moreover, the results demonstrated that elastase, proteinase-3 and cathepsin G all cleaved the purified, functionally active form of CD11b in a pattern similar to the CD11b shedding that occurs during PMN transmigration. Their cleavage sites on purified CD11b were located at (761)Thr-Ala(762) (elastase/proteinase-3) and (760)Phe-Thr(761) (cathepsin G), respectively. CD11b cleavage and PMN detachment and chemotaxis, were impaired in elastase/cathepsin G-deficient Beige mice; this defect could be restored by the addition of extracellular elastase. By illustrating CD11b shedding by elastase, proteinase-3 and cathepsin G as a novel mechanism for PMN detachment, our study provides novel therapeutic targets for controlling inflammation.

Phospholipase A2 plays a key role in PMN transepithelial migration during S. pneumoniae infection (55.18)

Abstract Streptococcus pneumoniae lung infection is characterized by a massive alveolar infiltration of neutrophils (PMNs). Using an in vitro model of lung epithelium and human PMNs, we have shown that S. pneumoniae-induced PMN transepithelial migration is mediated by hepoxilin A3 (HXA3), an arachidonic acid (AA)-derived eicosanoid. Generation of AA is thought to be the rate-limiting step in generation of eicosanoids. Therefore, we studied the role of phospholipase A2 (PLA2), which generates AA from membrane phospholipids, on S. pneumoniae-elicited PMN transpeithelial migration. We found that pneumococcal infection of in vitro lung epithelium resulted in release of AA. This event was partially inhibited by pan-PLA2 inhibitors ONO-RS-082 and ACA, and also by diacylglycerol (DAG) lipase inhibitor RHC 80267. However, while the pan-PLA2 inhibitors significantly reduced S. pneumoniae-induced PMN transmigration, RHC 80267 had no effect on this event. We found that, cytosolic PLA2 (cPLA2) and calcium-independent PLA2 (iPLA2) are important for generation of free AA and inhibition of these enzymes lead to dramatic reduction in PMN transepithelial migration. On the other hand, secretory PLA2 (sPLA2) did not seem to pay a key role in this event. We observed that S. pneumoniae infection caused an increase in the phosphorylation of cPLA2, suggesting a mechanism whereby S. pneumoniae activates PLA2 to generate free AA that may ultimately be converted to the PMN chemoattractant HXA3.

Abstract 220: Interleukin 17 Promotes Endothelial Activation and Neutrophil Recruitment Through Synergistic Action with Tumor Necrosis Factor α

Introduction: Interleukin 17 (IL-17) is the signature cytokine produced by TH17 cells, a novel subset of pro-inflammatory CD4+ T cells. Prior studies have noted elevated serum IL-17 in patients presenting with acute coronary syndrome but little is known regarding the effect of IL-17 on endothelial (EC) activation, a key step in thrombosis and the process of leukocyte and platelet recruitment to sites of inflammation. Methods and results: We tested the hypothesis that IL-17 would induce a distinct pattern of EC activation and leukocyte recruitment when compared to the TH1 cytokine IFNγ. We found that activation of mouse heart EC with the combination of TNFα and IL-17 but not IFNγ had a synergistic effect on mRNA expression of P-selectin (2.6 fold over TNFα alone, p<0.01) and E-selectin (4.0 fold, p<0.01); as well as the CXCR2-ligands CXCL1 (29.0 fold, p<0.01), CXCL2 (15.2 fold, p<0.01), and CXCL5 (53.4 fold, p<0.05), which are highly chemotactic for neutrophils (PMN). Furthermore, EC activation with TNFα and IL-17 but not IFNγ had a synergistic effect on the level of PMN transmigration (46.9% versus 9.1% for TNFα alone, p<0.01) in a parallel-plate flow chamber model, which was prevented upon perfusion of PMN from CXCR2 (-/-) mice. In addition, TNFα and IL-17 had a synergistic effect on microvascular inflammation in vivo as determined by intravital microscopy of cremasteric leukocyte rolling velocity (8.9 μm/s versus 23.9 μm/s for TNFα alone, p<0.01) and perivascular infiltration (22.4 cells/field versus 13.9 cells/field, p<0.01) at 16hr after cytokine injection. By contrast, treatment with TNFα and IFNγ but not IL-17 induced the expression of ICAM1 (2.0 fold over TNFα alone, p<0.05) and VCAM1 (1.6 fold, p<0.01), as well as the T-cell chemokines, CXCL9 (187.4 fold, p<0.01), CXCL10 (2.9 fold, p<0.01), and CCL5 (2.6 fold, p<0.01). Accordingly, treatment with TNFα and IFNγ but not IL-17 preferentially enhanced the transmigration of effector CD4+ T cells under shear flow (41.9% versus 29% for TNFα alone, p<0.01). Conclusion: IL-17 and TNFα act in a synergistic manner to induce a pattern of EC activation that favors selective PMN recruitment to sites of inflammation, providing a potential link between IL-17 and risk of atherosclerotic plaque rupture and thrombosis.

Phospholipase A2 is involved in S. pneumoniae‐elicited PMN transepithelial migration

Alveolar infiltration of neutrophils (PMNs) characterizes Streptococcus pneumoniae lung infection. Using in vitro lung epithelium and human PMNs, we have shown that S. pneumoniae‐induced PMN transepithelial migration is mediated by the PMN chemoattractant hepoxilin A3 (HXA3), an eicosanoid metabolized from arachidonic acid (AA). Release of AA from membrane phospholipids is the rate‐limiting step in eicosanoid generation. We studied the role of phospholipase A2 (PLA2), which generates AA, on S. pneumoniae‐elicited PMN transpeithelial migration. We found that pneumococcal infection resulted in release of AA from lung epithelial cells. This event was partially inhibited by pan‐ PLA2 inhibitors ONO‐RS‐082 and ACA, and also by diacylglycerol (DAG) lipase inhibitor. Pan‐PLA2 inhibitors significantly reduced S. pneumoniae‐induced PMN transmigration, but the DAG lipase inhibitor had no effect. We found that, both cytosolic PLA2 (cPLA2) and secretory PLA2 (sPLA2) are important for generation of free AA and inhibition of these enzymes lead to dramatic reduction in PMN migration. We observed that S. pneumoniae infection caused increase in the phosphorylation of cPLA2, suggesting a mechanism whereby S. pneumoniae activates cPLA2 and sPLA2 to generate free AA that may be converted to HXA3 which ultimately mediate PMN transmigration. This research is supported by institutional funds of Professor Leong.

Spreading of human neutrophils on an ICAM-1-immobilized substrate under shear flow

Neutrophil (PMN) spreading on endothelium, mediated by the interactions between surface-bound β2 integrin and intercellular adhesion molecule-1 (ICAM-1) in the inflammatory cascade, is crucial for PMN post-adhesion and trans-migration in blood flow. The underlying mechanisms by which shear flow regulates PMN spreading dynamics are not well understood. Here, a parallel-plate flow chamber assay was applied to quantify the time course of PMN adhesion and spreading on an ICAM-1-immobilized substrate. Two types of shear flow, steady flows at shear stresses of 0.2, 0.5, and 1 dyne/cm2 and stepwise flows at 0, 1, and 10 dyne/cm2, were used to elucidate the impact of shear flow on cell adhesion and spreading. The number of adhered PMNs, the fraction of spreading PMNs and the projected area of spread PMNs were determined and were found to correlate with the distribution of surface-bound β2 integrin subunit (CD11a, CD11b, or CD18). The results indicate that PMN spreading on an ICAM-1 substrate is bi-directionally regulated under shear flow. CD11a, CD11b and CD18 subunits of β2 integrin contribute distinctly to PMN spreading on ICAM-1 substrates. This work provides new insights into understanding PMN spreading on the endothelium, mediated by β2 integrin and ICAM-1 under shear flow.

Cleavage of the CD11b extracellular domain by the leukocyte serprocidins is critical for neutrophil detachment during chemotaxis

AbstractThe β2-integrin CD11b/CD18 mediates the firm adhesion of neutrophils (PMNs) to epithelial monolayers, a key step in PMN transepithelial migration. To complete the transmigration process, adherent PMNs must detach from epithelial monolayer surfaces to move forward. The mechanism that governs the detachment of adherent PMNs, however, is not clear. Here, we present evidence that cleavage of the CD11b extracellular domain containing the ligand-binding I-domain by 3 structural and functional related serine proteases (elastase, proteinase-3 and cathepsin G) serves as a novel mechanism for PMN detachment after the initial cell adhesion. Kinetic studies showed that the cleavage of CD11b is positively correlated with PMN detachment and subsequent transmigration. Moreover, the results demonstrated that elastase, proteinase-3 and cathepsin G all cleaved the purified, functionally active form of CD11b in a pattern similar to the CD11b shedding that occurs during PMN transmigration. Their cleavage sites on purified CD11b were located at 761Thr-Ala762 (elastase/proteinase-3) and 760Phe-Thr761 (cathepsin G), respectively. CD11b cleavage and PMN detachment and chemotaxis, were impaired in elastase/cathepsin G–deficient Beige mice; this defect could be restored by the addition of extracellular elastase. By illustrating CD11b shedding by elastase, proteinase-3 and cathepsin G as a novel mechanism for PMN detachment, our study provides novel therapeutic targets for controlling inflammation.

Endothelial damage induced by Shiga toxins delivered by neutrophils during transmigration

The endothelial damage induced by Stx represents the main pathogenic event in the HUS associated with STEC infections in humans. Stx, released in the gut by bacteria, enter the bloodstream and are targeted to renal endothelia. The role of PMN as a toxin carrier has been the object of controversy. In this paper, we confirm the binding of Stx1 to PMN, also showing its degranulating effects on full-loaded leukocytes, and support the carrier role of PMN by using a two-chamber transmigration device, in which PMN, loaded in vitro with different amounts of Stx1, transmigrated through confluent monolayers of endothelial cells, mimicking the toxin-induced renal endothelial injury. Stx1 was transferred during PMN transmigration, impairing protein synthesis and triggering production of proinflammatory cytokines in endothelial cells. PMN, carrying low toxin amounts, induced the release of high levels of cytokines in viable endothelial cells, whereas cytokine production was blocked in cells challenged with PMN fully loaded with Stx as a result of an almost total impairment of translation and of the activation of the apoptotic program. In agreement with previous unexplained observations in animal models, the results obtained with our experimental setting suggest that a self-amplifying circle triggered by low doses of toxin may lead to the production of proinflammatory mediators of renal damage in HUS.

Inflammation-induced Expression of Immunoreceptor Tyrosine-based Inhibition Motif (ITIM)-less SIRPα Enhances Neutrophil Transmigration (89.39)

Abstract Previous studies by us and others have demonstrated that SIRPα, an immunoreceptor tyrosine-based inhibition motif (ITIMs)-containing cell surface receptor, regulates neutrophil (PMN) and monocyte transmigration in response to inflammatory stimuli. In this study, we further characterized leukocyte SIRPα and found that PMN express multiple forms of SIRPα including the typical SIRPα with a long cytoplasmic domain containing the full ITIMs (ITIM+SIRPα) and shorter, ITIM-depleted SIRPα (ITIM-SIRPα). Importantly, our studies in both human and inflammatory animals revealed that, while ITIM+SIRPα is generally expressed in PMN of healthy donors, active inflammatory conditions induce diminishing of ITIM+SIRPα but a dominant expression of ITIM-SIRPα. In vitro and in vivo functional assays indicate that, compared to PMN from healthy donors bearing ITIM+SIRPα, PMN with chiefly ITIM-SIRPα display a higher tendency of activation and exhibit enhanced transmigration under inflammation. However, despite the differences in the intracellular domains, purified ITIM+SIRPα and ITIM-SIRPα from healthy and inflammatory donors had similar extracellular binding with the ligand CD47. In addition, our results suggest that conversion of SIRPα into ITIM-less receptor is through inflammation-induced proteolytic cleavage in PMN and such process serves as an important mechanism to enhance inflammatory response and dynamically modulate PMN transmigration.

Critical role of Ral GTPase as a gatekeeper in controlling neutrophil selective degranulation of secondary granules (140.11)

Abstract Neutrophil (PMN) inflammatory responses, including cell adhesion, diapedesis and phagocytosis, are dependent on the mobilization and release of various intracellular granules and vesicles. In this study, we observed that inhibition of the Ras family GTPases by damnacanthal and manumycin A resulted in specifically release of PMN secondary granules, but not primary or tertiary granules, in the absence of stimulation. By screening Ras family members for activity changes during PMN activation and degranulation, we identified Ral GTPase to be a critical regulator that selectively controls secondary granule release. In particular, we found that Ral exists as an active GTPase in resting PMN, while quick inactivation correlates with chemoattractant-induced degranulation. Indeed, we demonstrated that over-expression of a constitutively active Ral mutant (Ral23V) in PMN completely blocked secondary granule release. Subcellular fractionation and immunofluorescence labeling further found that Ral is associated with the plasma membrane and redistributed to secondary granules following chemoattractant stimulation. Blocking clathrin-mediated plasma membrane endocytosis, however, inhibited Ral intracellular translocation and PMN degranulation. Thus, these results provide strong evidence demonstrating Ral to be an essential gatekeeper for secondary granules in PMN. Dysregulation of Ral results in impairment of PMN transmigration in response to inflammatory stimuli.

Differential expression of two pathways of chemokine induction protect CD14- and TLR4-deficient mice from localized E. coli infections (94.16)

Abstract Sepsis, characterized by a systemic bacterial infection, is a life-threatening condition that can lead to multi-organ failure and death from septic shock. We have observed that CD14- and TLR4-deficient mice are resistant to death by sepsis from several different clinical isolates of E. coli injected intra-peritoneally. This resistance has been attributed to the 10-fold lower levels of the proinflammatory cytokines TNF and IL-6 induced in these mice. Surprisingly, however, neither TLR4- nor CD14-deficient mice were resistant to two closely related organisms that differed only in that they expressed a K1 polysaccharide capsule, even though these mice continued to express low levels of the proinflammatory cytokines. These and other observations suggest that the ability to limit bacterial dissemination may be equally or even more important than reducing the levels of these proinflammatory cytokines in preventing death from sepsis. We present evidence here, that the pathogenic role for the CD14/TLR4 pathway in severe infection, whereby it mediates the induction of proinflammatory cytokines, may be secondary to a more important insidious role whereby differential activation of local and systemic pathways of chemokine induction via CD14/TLR4-dependent and independent mechanisms generate chemokine gradients that prevent PMN transmigration from the blood to infected tissue and facilitates the dissemination of bacteria from a localized site of infection.

Uncoupling Protein-2 Negatively Regulates Polymorphonuclear Leukocytes Chemotaxis via Modulating [Ca 2+ ] Influx

Previous studies demonstrated that uncoupling protein 2 (UCP2) plays a negative role in modulating leukocyte inflammatory responses. The mechanism underneath the role of UCP2 in modulating leukocyte inflammatory responses, however, is incompletely understood. Here, we investigated the effect of UCP2 in polymorphonuclear leukocyte (PMN) chemotaxis. First, we assessed PMN chemotaxis in zymosan-induced murine peritonitis and found that UCP2(-/-) mice had significantly more migrated PMN in peritoneal lavage compared to their wild-type littermates. In vitro transmigration assays using isolated PMN also showed that PMN from UCP2(-/-) mice migrated faster than those from wild-type mice in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP). Second, in supporting an inhibitory role of UCP2 in PMN transmigration, migrated PMN had a decreased UCP2 expression compared to nonmigrated PMN. In contrast, in streptozotocin-induced diabetic mice in which UCP2 expression was enhanced, PMN chemotaxis was reduced. Third, comparing to UCP2(+/+) PMN, UCP2(-/-) PMN had a stronger upregulation of fMLP-induced surface CD11b/CD18 and CD11a/CD18. Finally, UCP2(-/-) PMN showed a quicker and larger fMLP-triggered intracellular calcium mobilization compared to UCP2(+/+) PMN. Our study demonstrates that UCP2 serves as a brake in controlling PMN chemotaxis and that the effect of UCP2 on PMN chemotaxis may be through modulating calcium influx.

CD47 and TLR-2 Cross-Talk Regulates Neutrophil Transmigration

Neutrophil (PMN) infiltration into tissues is a hallmark of acute inflammation and is crucial for the rapid removal of microbial pathogens. Previous studies have shown that PMN transmigration is regulated by the cell surface protein CD47. However this phenomenon in the context of microbial invasion and subsequent TLR signaling is poorly understood. In this study, we assessed the role of TLR2 and CD47 costimulation in regulating PMN transmigration. Human PMN transmigration across acellular collagen-coated filters toward the bacterial chemoattractant fMLP was more significantly inhibited by MALP-2 (TLR2/6 agonist) than Pam(3)CSK(4) (TLR2/1 agonist). Subsequent experiments demonstrated that treatment with MALP-2 or anti-human CD47 mAbs delayed human PMN transfilter migration, while combined treatment led to further delayed inhibition. Interestingly, stimulation of PMNs with MALP-2 resulted in an increase in surface expression of CD11b, but not CD47. In experiments addressing the role of TLR agonists in regulating CD47-mediated PMN transmigration, incubation with MALP-2 or with anti-mouse CD47 mAbs did not inhibit transfilter migration of TLR2(-/-) or MyD88(-/-)-deficient murine bone marrow-derived PMNs. Similarly, inhibition of MyD88 homodimerization reversed the attenuation of human PMN transmigration induced by MALP-2 or anti-human CD47 mAbs. Separate experiments demonstrated that CD47(-/-) murine bone marrow-derived PMNs exhibited 4-fold decreased sensitivity toward MALP-2. Collectively, these findings suggest that activation of CD47 signaling enhances PMN sensitivity toward TLR2 activation which, in turn, signals their arrival at a site of invasion and may facilitate antimicrobial function.

Ca2+ signaling in airway epithelial cells facilitates leukocyte recruitment and transepithelial migration

In airway cells, TLR2 stimulation by bacterial products activates Ca2+ fluxes that signal leukocyte recruitment to the lung and facilitates transepithelial migration into the airway lumen. TLR2 is apically displayed on airway cells, where it senses bacterial stimuli. Biochemical and genetic approaches demonstrate that TLR2 ligands stimulate release of Ca2+ from intracellular stores by activating TLR2 phosphorylation by c-Src and recruiting PI3K and PLCgamma to affect Ca2+ release through IP3Rs. This Ca2+ release plays a pivotal role in signaling TLR2-dependent NF-kappaB activation and chemokine expression to recruit PMNs to the lung. In addition, TLR2-initiated Ca2+ release activates Ca2+-dependent proteases, calpains, which cleave the transmembrane proteins occludin and E-cadherin to promote PMN transmigration. This review highlights recent findings that demonstrate a central role for Ca2+ signaling in airway epithelial cells to induce proinflammatory gene transcription and to initiate junctional changes that accommodate transmigration of recruited PMNs.

Resolvins E1 and D1 in Choroid-Retinal Endothelial Cells and Leukocytes: Biosynthesis and Mechanisms of Anti-inflammatory Actions

To investigate the biosynthesis of resolvins E1 and D1 (RvE1 and RvD1) in choroid-retinal endothelial cells (CRECs) and leukocytes under inflammatory conditions and to define the mechanisms of anti-inflammatory actions of RvE1 and RvD1 in CRECs and leukocytes, cells crucial to posterior ocular inflammation. RvE1, RvD1, and markers of their biosynthesis were determined by lipidomic analysis. After CRECs or cocultures of CRECs and leukocytes were treated with RvE1 or RvD1 and inflammatory stimuli, inflammatory signaling molecules were quantified by Western blot analysis, ELISA, or protein array. Transmigration of polymorphonuclear leukocytes (PMNs) across CRECs monolayers was quantified. Inflammatory stimulation increased the biosynthesis of RvE1 and RvD1 from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), respectively, in coculture of CRECs and leukocytes. CRECs alone did not produce RvE1 and RvD1. RvE1 or RvD1 inhibited the expressions of vascular cell adhesion molecule-1, IL-8, macrophage inflammatory protein-1beta, regulated on activation normal T cell expressed and secreted, and tumor necrosis factor-alpha from CRECs or cocultures of CRECs and leukocytes. RvD1 reduced prostaglandin E(2) generation from CRECs. However, neither resolvin affected cyclooxygenase-2 formation. Treating CRECs or PMNs with RvE1 or RvD1 inhibited PMN transmigration across CREC barriers. The interplay of inflammatory stimuli-activated CRECs and leukocytes biosynthesizes RvE1 and RvD1 from EPA and DHA. These resolvins inhibit inflammatory signaling from CRECs and leukocytes and inflammatory activity as PMN transmigration across CRECs barriers. Thus, these resolvins and their biosynthesis pathways are potential targets for novel treatment of inflammatory ocular diseases.

Influence of Inseminate Components on Porcine Leucocyte Migration In Vitro and In Vivo after Pre‐ and Post‐Ovulatory Insemination

A post-breeding migration of leucocytes (PMN) into the uterus is considered to be an important reason for sperm losses. Minimizing such effects may be necessary for successful insemination with low sperm numbers, as required with sex-sorted spermatozoa. We examined the magnitude of PMN influx 3 h after pre- or post-ovulatory insemination with various combinations of seminal plasma (SP), semen extender Androhep (AH; Minitüb, Tiefenbach, Germany) and sperm preparations (S). Pre-ovulatory inseminations with preparations containing 98% AH caused a massive influx of PMN, independent of whether spermatozoa were present (628 +/- 189 x 10(6) leucocytes/uterine horn) or not (580 +/- 153 x 10(6)). Post-ovulatory, 98% AH caused a comparable immigration only in the absence of sperm cells (AH: 569 +/- 198 x 10(6), AH+S: 162 +/- 102 x 10(6)). The presence of SP significantly dampened the numbers of recruited uterine leucocytes. The reaction to all inseminates containing 98% SP both with and without spermatozoa, used before ovulation (SP: 14 +/- 6 x 10(6), SP+S: 73 +/- 27 x 10(6)) and after ovulation (SP: 60 +/- 32 x 10(6), SP+S: 51 +/- 33 x 10(6)) did not differ significantly from controls using phosphate buffered saline (PBS) (pre-ovulatory: 1 +/- 1 x 10(6), post-ovulatory: 11 +/- 9 x 10(6)). Quantitative in vitro transmigration assays with blood-derived PMN proved that AH-induced leucocyte migration into the uterus to be not as a result of direct chemotaxis, because, on account of the chelator citrate, AH significantly inhibited the transmigration towards recombinant human Interleukin-8 (rhCXCL8) (AH: 14 +/- 5% migration rate vs controls: 37 +/- 6%, p < 0.05). Supernatants of spermatozoa incubated in PBS for 1, 12 or 24 h showed neither chemoattractive nor chemotaxis-inhibiting properties. SP at > or =0.1% [v/v] significantly inhibited the in vitro transmigration of PMN. With respect to in vivo migration of neutrophils, the striking difference in the results between semen extender and seminal plasma suggests that adaptation of extender composition is needed to reflect more closely the in vivo regulatory potential of natural seminal plasma.

Abstract P124: CGEN-855A, a Small Peptide Agonist of the Formyl Peptide Receptor-Like 1 Reduces Myocardial Reperfusion Injury

Background: Neutrophils (PMNs) mediate reperfusion injury in part. Reperfusion (R) accelerates the accumulation of PMNs into ischemic myocardium. Postconditioning (PC) inhibits PMN activation and accumulation in area at risk (AAR) myocardium. Formyl-peptide receptor-like1 (FPRL1) agonists directly inhibit PMN transmigration. Accordingly, this study tests the hypotheses that the FPRL1 agonist CGEN-855A applied at R reduces infarct size by inhibition of PMN infiltration; the infarct reduction by CGEN-855A is comparable to that of the mechanical intervention Postconditioning; and since both CGEN-855A and PC share a common property of PMN inhibition, the combination of CGEN-855A and Postconditioning (PC) will not have an additive effect on infarct size reduction. Methods: In anesthetized open-chest rats, the left coronary artery (LCA) was occluded for 30 min and reperfused for 3 hours. Rats (n=10) were randomly assigned to: Control- equal volume vehicle 5 min before R; PC: three cycles of 10-s R followed by 10-s re-occlusion during the first minute of R; 2 mg/kg CGEN-855A administered 5 min before R; 2 mg/kg CGEN-855A + PC at R. Separate experiments were performed to assess PMN accumulation in AAR tissue by immunohistochemistry in control, CGEN-855A and PC groups. Results: The AAR (% left ventricular mass, AAR/LV) was comparable among groups. Infarct size (necrosis as % AAR, AN/AAR), was significantly less in the PC (41.2 ± 2.7*) and CGEN-855A (43.6 ± 2.9*) groups vs Control (57.0 ± 2.3). There was no additive effect of the combination of CGEN-855A and PC at R (44.6 ± 1.3). Both CGEN-855A (30.1 ± 0.6/HPF) and PC (34.8 ± 1.5/HPF) significantly reduced PMN accumulation in AAR tissue vs control (43.2 ± 0.7/HPF). Conclusions: stimulation of the FPRL1 with agonist CGEN-855A exerts cardioprotection at R by reducing infarct size; CGEN-855A peptide inhibits PMN accumulation; the extent of cardioprotection and PMN inhibition by CGEN-855A is similar to that of PC; there is no additive effect of their combination, consistent with a similar physiological pathway of PMN inhibition. *P&lt;0.05 vs Control

PMNs facilitate translocation of platelets across human and mouse epithelium and together alter fluid homeostasis via epithelial cell–expressed ecto-NTPDases

Mucosal diseases are often characterized by an inflammatory infiltrate that includes polymorphonuclear leukocytes (PMNs), monocytes, lymphocytes, and platelets. A number of studies have suggested that the interaction of platelets with leukocytes has an essential proinflammatory role. Here, we examined whether platelets migrate across mucosal epithelium, as PMNs are known to do, and whether platelets influence epithelial cell function. Initial studies revealed that human platelets did not efficiently transmigrate across human epithelial cell monolayers. However, in the presence of human PMNs, platelet movement across the epithelium was proportional to the extent of PMN transmigration, and strategies that blocked PMN transmigration diminished platelet movement. Furthermore, platelet-PMN comigration was observed in intestinal tissue derived from human patients with inflammatory bowel disease (IBD). The translocated platelets were found to release large quantities of ATP, which was metabolized to adenosine via a 2-step enzymatic reaction mediated by ecto-nucleotidases, including CD73 and ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTPDases), expressed on the apical membrane of the intestinal epithelial cells. In vitro studies and a mouse model of intestinal inflammation were employed to define a mechanism involving adenosine-mediated induction of electrogenic chloride secretion, with concomitant water movement into the intestinal lumen. These studies demonstrate that ecto-NTPDases are expressed on the apical membrane of epithelial cells and are involved in what we believe to be a previously unappreciated function for platelets in the inflamed intestine, which might promote bacterial clearance under inflammatory conditions.

Toll‐like receptor‐2 and CD47 co‐stimulatory signaling regulates neutrophil transmigration

Tissue infiltration by neutrophils (PMN) is a hallmark of acute inflammation and is crucial for the rapid removal of microbial pathogens. Previous studies have shown that PMN transmigration is regulated by the cell surface protein CD47, however this phenomenon in the context of microbial invasion and subsequent Toll‐like receptor (TLR) signaling is poorly understood. In this study we assessed the role of TLR2 and CD47 co‐stimulation in regulating PMN transmigration. PMN transepithelial migration (TEM; T84 monolayers) towards the bacterial chemoattractant fMLP was significantly delayed for 30min by 1μM Pam3CSK4 (TLR2/1 agonist), 100nM MALP‐2 (TLR2/6 agonist) or 20μg/ml anti‐human CD47 mAbs (C5/D5). In addition, Pam3CSK4 or MALP‐2 and C5/D5 added together further delayed PMN TEM by 2h. C57BL/6 murine bone marrow‐derived PMN transfilter migration (TFM) towards fNLP was inhibited (50%) by 200nM MALP‐2 or 20μg/ml anti‐mouse CD47 mAbs (MIAP), while, in combination, blocked PMN TFM by 75%. Conversely TFM using TLR2−/− or MyD88−/− bone marrow PMN was not inhibited by MALP‐2 or MIAP. TLR2 agonists did not increase human or murine PMN surface CD47 expression (flow cytometry). Collectively, these findings suggest that TLR activation may serve as “stop” signals, and facilitate antimicrobial functions including CD47‐mediated PMN transmigration.

Neutrophil adhesion under flow conditions induces zyxin nuclear translocation in endothelial cells

High shear stress causes zyxin translocation from focal adhesions (FA) to stress fibers. We previously showed that leukocyte adhesion to stimulated endothelial cells (EC) activated mechanosensitive signaling pathways in ECs at low shear stress, which in turn modulated leukocyte transmigration. In this study, we examined the remodeling of the FA protein zyxin following neutrophil (PMN) adhesion under shear stress and determined if downregulating this protein altered PMN transmigration. Zyxin localized to FAs and stress fibers under static conditions. Buffer perfusion across TNFα‐stimulated ECs did not change zyxin localization at lower shear stresses, but resulted in modest nuclear translocation of zyxin at a shear stress of 4 dyne/cm2. In contrast, PMN recruitment to TNFα‐stimulated ECs resulted in a loss of zyxin from FAs and translocation of zyxin to the nucleus at shear stresses as low as 1 dyne/cm2. Translocation specifically occured in ECs with adherent PMNs and did not occur in ECs distant from regions containing adherent PMNs. Perfusion of fixed PMNs, which roll but to do not adhere or transmigrate, did not induce changes in zyxin localization. Finally, zyxin downregulation in ECs attenuated PMN transmigration. These data suggest that PMN trafficking across stimulated ECs leads to focal adhesion remodeling and that EC zyxin nuclear translocation during this process may play a potential role in gene transcription.Funding: Canadian Institutes of Health Research, Alberta Heritage Foundation for Medical Research.

Subendothelial infiltration of neutrophil granulocytes and liberation of matrix-destabilizing enzymes in an experimental model of human neo-intima

It was the objective of this study to examine the role of human neutrophil granulocytes (PMN) in an in-vitro model of human neo-intima developed for the study of atherosclerosis. Human granulocytes were subjected to a co-culture model of human endothelial and smooth muscle cells. Subendothelial lipid accumulation was achieved by addition of native LDL to the culture medium. Tissue samples were analyzed by immunohistochemistry and scanning/transmission electron microscopy, and culture supernatants were examined for the presence of interleukin-8 (IL-8), MCP-1, GRO-alpha, elastase and matrixmetalloproteinase-8 (MMP-8). Following addition of 2 mg/ml LDL, adherence, transmigration and infiltration depth of PMN was increased significantly when compared to controls. LDL challenging was paralleled by a time- and dose-dependent secretion of IL-8 from intimal smooth muscle cells. PMN infiltration was mediated by the IL-8-signalling pathway and accompanied by release of elastase and MMP-8 into the supernatant and induction of endothelial cell apoptosis. In conclusion, LDL-induced secretion of IL-8 by intimal smooth muscle cells provides a potential mechanism of PMN-recruitment into culprit lesions. The concomitant release of potent matrix-degrading enzymes and the induction of EC apoptosis may have implications for plaque destabilization and cardiovascular events.