PMN Infiltration Research Articles

PP-032 Predictors of mortality in sepsis intensive care unit patients

Background: Glycogen synthase kinase-3 (GSK-3) modulates a wide array of cellular processes including embryonic development, cell differentiation survival and apoptosis. Recently, it was reported that GSK-3 inhibitor functions as an attenuator of LPS-induced septic shock and regulates mortality of endotoxemic mice. However, detailed mechanism of improved mortality via GSK-3 inhibitor was less defined yet. Method: LPS-induced endotoxemia model, Generation and culture of DC, Measurement of Cytokines, Measurement of PMN infiltration. Result: Here, we show that the inhibition of GSK-3 induces ERK1/2 activation in LPS-stressed condition via PKCd activation. These PKCd-induced ERK1/2 activation by the inhibition of GSK-3 provokes IL-10 expression, which has a crucial role as a crucial regulator in endotoxemia condition. We confirmed that GSK-3 inhibition-induced PKCd, ERK1/2 activation provokes IL-10 expression in LPStreated condition using MEK1 and PKCd inhibitor. Next, we observed that septic shock is caused by LPS is attenuated on GSK-3 inhibition using a GSK-3 inhibitor. These relieved endotoxemia induced by GSK-3 inhibition restored in ERK1/2-dependent manner. Conclusion: Taken together, IL-10 expression produced by GSK-3 inhibition-induced ERK activation via PKCd relieves LPS-mediated endotoxemia. This finding suggests that IL-10 hyper-expression-mediated by GSK-3 inhibition-induced ERK activation could be a new therapeutic pathway in the endotoxemial state.

IGF-1 Can Either Protect Against or Increase LPS-Induced Damage in the Developing Rat Brain

Periventricular leukomalacia (PVL) is a major form of brain damage in premature infants. This study was to test whether IGF-1 can prevent PVL-like brain damage induced by lipopolysaccharide (LPS) in the neonatal rat. Intraventricular delivery of LPS resulted in an acute brain inflammatory response, i.e., rapid recruitment of polymorphonuclear leukocytes (PMNs), activation of microglia and astrocytes, and induction of IL-1beta (IL1beta) expression. Brain inflammation was associated with the loss of O4+ preoligodendrocytes (preOLs), a decrease of myelin basic protein (MBP) in the white matter and an increase of pyknotic cells in the cortex. IGF-1 at a low dose significantly prevented LPS-induced deleterious effects without alteration of IL-1beta expression and microglia/astrocytes activation. On the other hand, the low dose of IGF-1 enhanced LPS-induced PMNs recruitment and blood-brain barrier (BBB) permeability, and caused intracerebral hemorrhage. At higher doses, co-application of IGF-1 with LPS resulted in a high mortality rate. Brains from the surviving rats showed massive PMN infiltration and intracerebral hemorrhage. However, these adverse effects were not found in rats treated with IGF-1 alone. This study provides the alarming evidence that in an acute inflammatory condition, IGF-1 may have severe, harmful effects on the developing brain.

S1776 Chronic Lung and Liver Inflammation in a Mouse Model of Crohn's Colitis

Recent population-based studies demonstrate that in addition to the more common joint and hepatobiliary extraintestinal manifestations observed in patients with one of the inflammatory bowel diseases (IBD), pulmonary inflammation is a very common comorbidity increased in Crohn's disease. Because few studies have reported the presence of extraintestinal inflammation in mouse models of IBD, we undertook a systematic histopathological study to document the presence (or absence) of lung and liver inflammation in a mouse model of CD. Chronic colitis was induced in RAG-1-deficient (RAG-/-) mice by the adoptive transfer of naive (CD4+CD45RBhigh) T-cells (5x105 cells per mouse) obtained from the spleens of healthy donor mice. PBS-injected RAG-/mice were used as controls. Colons, lungs and liver as well as heart, kidneys, and brain were excised, fixed in formalin, embedded, sectioned and stained with H&E. Blinded histopathological analysis was performed on each organ by a pathologist not familiar with the experimental groups. We found that at 6-8 weeks post Tcell transfer, the majority of T-cell injected mice (80%) became moribund and were euthanized whereas PBS-injected animals appeared healthy throughout the entire 8week observation period. Blinded histopathological examination of the colons obtained from RAG-/mice injected with naive T-cells revealed moderate-to-severe colitis characterized by dramatic transmural T-cell and PMN infiltration, bowel wall thickening, Goblet cell dropout and epithelial cell erosions. Intracellular cytokine analysis of T-cells obtained from the colon and MLNs of colitic mice showed enhanced production of both IFNγ and IL-17. Surprisingly, we found that virtually all mice injected with naive T-cells presented with chronic bronchitis/ pulmonary inflammation. In many cases (>60%) confluent inflammation merged together between small and medium sized bronchi. Varying degrees of perivascular lymphocytic cuffing were also observed in virtually all of the mice however few PMNs were observed and vasculitis was absent. Lung parenchyma involvement was mild and patchy. In addition to gut and lung inflammation, we also observed significant hepatocellular necrosis with periportal and lobular lymphocytic inflammation. There was no evidence of bile ductal damage. No significant inflammation was observed in the heart, kidneys or brain of the colitic animals and no inflammation was observed in any organ system of the PBS controls. Taken together, these data demonstrate, for the first time, that pulmonary and liver inflammation is a occurrence in the T-cell transfer model of chronic colitis (Supported by NIH PO1 43785).

Anti-inflammatory and pro-resolving properties of benzo-lipoxin A 4 analogs

Lipoxins (LXs) are potent endogenous counter-regulatory lipid mediators that dampen acute inflammation and promote its resolution. Here, we present our investigation of a new class of thermally and metabolically stable benzo-LXA 4 analogs that are potently anti-inflammatory and easier to synthesize. Replacement of the tetraene unit of native LXA 4 with a benzo-fused ring system not only increases the thermal stability but also enables highly convergent and efficient syntheses of these analogs. In addition, they resist rapid catalysis and inactivation by eicosanoid oxidoreductase. Like native LXs, o-[9, 12]-benzo-ω6-epi-LXA 4, o-[9, 12]-benzo-deoxy-LXA 4, m-[9, 12]-benzo-ω6-epi-LXA 4 and [9, 14]-benzo-ω6-(R/S)-LXA 4 demonstrated potent time-dependent reduction, at nanogram dosages, of PMN infiltration and pro-inflammatory cytokine generation in vivo in murine peritonitis and were organ protective in hind limb ischemia-reperfusion injury of the lung. The o-[9, 12]-benzo-ω6-epi-LXA 4 and m-[9, 12]-benzo-ω6-epi-LXA 4 were most potent in nanogram doses; both decreased PMN infiltration by ∼32%, while o-[9, 12]-benzo-deoxy-LXA 4 and [9, 15]-ω6-(R/S)-LXA 4 were less potent. The [9,12]-benzo-ω6-epi-LXA 4 also activated a lipoxin A 4 GPCR and increased macrophage phagocytic activity. Taken together, these findings demonstrate a new generation of LXA 4 stable analogs that are easy to synthesize and anti-inflammatory. These benzo-LXA 4 analogs are promising tools for new therapeutic approaches as well as assessing endogenous mechanisms in anti-inflammation and resolution.

Toll‐like receptor ligands induce polymorphonuclear leukocyte migration: key roles for leukotriene B4and platelet‐activating factor

Activation of toll-like receptors (TLRs) and polymorphonuclear leukocyte (PMN) accumulation at infection sites are critical events of host defense. The involvement of leukotriene (LT) B(4) and platelet-activating factor (PAF) in TLR ligand-induced activation of inflammatory cell functions is essentially unknown. Using an in vitro model of human PMN migration through human endothelial cell monolayers, we demonstrate that prototypic ligands of TLR1/2, 2/6, 3, 4, 5, and 7/8 promote PMN migration, an effect markedly inhibited by 3 LTB(4) receptor antagonists (70-80% inhibition at 100 nM compared to vehicle-treated cells), 3 PAF receptor antagonists (20-50% inhibition at 10 nM), 3 LT biosynthesis inhibitors (75-85% inhibition at 100 nM), and 1 cytosolic phospholipase A(2)alpha (cPLA(2)alpha) inhibitor (90% inhibition at 1 microM). Accordingly, selected TLR ligands caused Ser-505-phosphorylation of cPLA(2)alpha and measurable LTB(4) and PAF biosynthesis in the transmigration assay. As negative controls, interleukin-8- and formyl-methionyl-leucyl-phenylalanine-elicited migration in vitro was not inhibited either by an LTB(4) receptor antagonist or by the cPLA(2)alpha inhibitor. Finally, LTB(4) and PAF receptor antagonists inhibited (up to approximately 65% at optimal doses) TLR ligand-induced PMN infiltration in the mouse air-pouch model. These studies unravel the critical involvement of de novo LTB(4) and PAF biosynthesis in PMN migration elicited by TLR ligands.

Lipopolysaccharide-Induced Epithelial Monoamine Oxidase Mediates Alveolar Bone Loss in a Rat Chronic Wound Model

Reactive oxygen species (ROS) production is an antimicrobial response to pathogenic challenge that may, in the case of persistent infection, have deleterious effects on the tissue of origin. A rat periodontal disease model was used to study ROS-induced chronic epithelial inflammation and bone loss. Lipopolysaccharide (LPS) was applied for 8 weeks into the gingival sulcus, and histological analysis confirmed the onset of chronic disease. Junctional epithelium was collected from healthy and diseased animals using laser-capture microdissection, and expression microarray analysis was performed. Of 19,730 genes changed in disease, 42 were up-regulated >/=4-fold. Three of the top 10 LPS-induced genes, monoamine oxidase B (MAO/B) and flavin-containing monooxygenase 1 and 2, are implicated in ROS signaling. LPS-associated induction of the ROS mediator H(2)O(2), as well as MAO/B and tumor necrosis factor (TNF)-alpha levels were validated in the rat histological sections and a porcine junctional epithelial cell culture model. Topical MAO inhibitors significantly counteracted LPS-associated elevation of H(2)O(2) production and TNF-alpha expression in vivo and in vitro, inhibited disease-associated apical migration and proliferation of junctional epithelium and inhibited induced systemic H(2)O(2) levels and alveolar bone loss in vivo. These results suggest that LPS induces chronic wounds via elevated MAO/B-mediated increases in H(2)O(2) and TNF-alpha activity by epithelial cells and is further associated with more distant effects on systemic oxidative stress and alveolar bone loss.

Ginsenoside Re downregulates ICAM-1 expression, inhibits polymorphonuclear infiltration, and ameliorates ischemia-reperfusion injury

In this study, we investigate the effects of ginsenoside Re on the expression of intercellular adhesion molecule-1 (ICAM-1), neutrophil infiltration, and myeloperoxidase (MPO) activity during reperfusion of ischemic tissue. The model of acute ischemia reperfusion was established by ligating the left anterior descending branch of the coronary artery in Wistar rats. After ginsenoside Re administration, expression of ICAM-1 was measured by Western blotting; hematoxylin and eosin was used to analyze myocardial damage and polymorphonuclear leukocyte (PMN) infiltration. The number of PMNs per high-power field was calculated. The infarcted area and the level of MPO were also measured. This showed that ginsenoside Re administration markedly decreased the infarct size compared with that in the saline-treated I/R group (P < 0.05), significantly decreased the expression of ICAM-1 (P < 0.05) and the level of MPO (P < 0.05), and inhibited PMN infiltration (P < 0.05). In addition, a significant correlation was found between the number of PMNs infiltrating cardiac tissue and the MPO level (r = 0.981, P < 0.01).

Phosphoinositide 3-kinase required for lipopolysaccharide-induced transepithelial neutrophil trafficking in the lung

Phosphoinositide 3-kinase gamma(PI3Kgamma) is a critical mediator of directional cell movement. Here, we sought to characterise the role of PI3Kgamma in mediating the different steps of polymorphonuclear leukocyte (PMN) trafficking in the lung. In a murine model of lipopolysaccharide (LPS)-induced lung injury, PMN migration into the different lung compartments was determined in PI3Kgamma gene-deficient (PI3Kgamma(-/-)) and wild-type mice. Bone marrow chimeras were created to characterise the role of PI3Kgamma on haematopoietic versus nonhaematopoietic cells. A small-molecule PI3Kgamma inhibitor was tested in vitro and in vivo. PMN adhesion to the pulmonary endothelium and transendothelial migration into the lung interstitium was enhanced in PI3Kgamma(-/-) mice. However, transepithelial migration into the alveolar space was reduced in these mice. When irradiated PI3Kgamma(-/-) mice were reconstituted with bone marrow from wild-type mice, migratory activity into the alveolar space was restored partially. A small-molecule PI3Kgamma inhibitor reduced chemokine-induced PMN migration in vitro when PMNs or epithelial cells, but not when endothelial cells, were treated. The inhibitor also reduced LPS-induced PMN migration in vivo. We conclude that PI3Kgamma is required for transepithelial but not for transendothelial migration in LPS-induced lung injury. Inhibition of PI3Kgamma activity may be effective at curbing excessive PMN infiltration in lung injury.

Protective effects of interleukin-6 in lipopolysaccharide (LPS)-induced experimental endotoxemia are linked to alteration in hepatic anti-oxidant enzymes and endogenous cytokines

Endogenous interleukin-6 has been considered as an important anti-inflammatory cytokine controlling both local and systemic acute inflammatory responses; the usefulness of IL-6 in endotoxemia aiming to block the production of reactive oxygen species and the release of inflammatory cytokines is under discussion The aim of the study was to evaluate the protective effects of IL-6 in experimentally induced endotoxemia in mice correlating the changes in tissue anti-oxidant enzymes and circulating cytokines. Liver injury in low-dose bacterial lipopolysaccharide (5 μg/mouse)-induced endotoxemic mice receiving IL-6 (300 ng/mouse) treatment either before or after LPS injection was detected by the estimation of serum oxaloacetate transaminase and serum glutamate pyruvate transaminase. This finding supports that liver injury during experimental endotoxemia could be lowered by IL-6. Current data also demonstrate the critical role of IL-6 in inducing SOD in liver, whereas IL-6 prior and after LPS challenge group showed reduced PMN infiltration in the liver as evident by decreased hepatic MPO content in those mice. IL-6 treatment also showed higher IL-10 production in serum than endotoxic group as IL-10 is a potent and pleiotropic anti-inflammatory cytokine that inhibits the synthesis of pro-inflammatory cytokines and also has a suppressive effect on pro-inflammatory cytokine like TNF-α, IFN-γ and IL-12. It is also directly involved in the modulation of other aspects of inflammation, particularly cytokine responses and inflammatory cell infiltration.

Ischemic Preconditioning (IP) of the Liver as a Safe and Protective Technique against Ischemia/Reperfusion Injury (IRI)

The aim of the study was to evaluate safety and efficacy of IP in LT, particularly in marginal grafts. From 2007 to 2008, 75 LT donors were randomized to receive IP (IP+) or not (IP-). Considering the graft quality, we divided the main groups in two subgroups (marg+/marg-). IP was performed by 10-min inflow occlusion (Pringle maneuver utilizing a toruniquet). Donor variables considered were gender, age, AST/ALT, ischemia time and steatosis. Recipient variables were gender, age, indication to LT and MELD/CHILD/UNOS score. AST/ALT levels, INR, bilirubin, lactic acid, bile output on postoperative days 1, 3 and 7 were evaluated. Histological analysis was performed evaluating necrosis/steatosis, hepatocyte swelling, PMN infiltration and councilman bodies. Thirty patients received IP+ liver. No differences were seen between groups considering recipient and donor variables. Liver function and AST/ALT levels showed no significant differences between the main two groups. Marginal IP+ showed lower AST levels on day1 compared with untreated marginal livers (936.35 vs. 1268.23; p = 0.026). IP+ livers showed a significant reduction of moderate-severe hepatocyte swelling (33.3% vs. 65.9%; p = 0.043). IP+ patients had a significant reduction of positive early microbiological investigations (36.7% vs. 57.1%; p = 0.042). In our experience IP was safe also in marginal donors, showing a protective role against IRI.

Effect of soluble JAM‐C on leukocyte transmigration in models of ischemia/reperfusion injury

Numerous in vitro studies have implicated the adhesion molecule JAM‐C in leukocyte transendothelial cell migration but the role of JAM‐C in leukocyte migration in vivo requires further investigations. Here we used soluble JAM‐C (sJAM‐C) as a blocker of JAM‐C‐mediated reactions to investigate the role of this molecule in leukocyte transmigration in vivo as induced by ischemia/reperfusion (I/R). The study employed a murine model of kidney I/R injury and investigated I/R‐induced leukocyte migration by intravital microscopy in mouse cremasteric venules. In the kidney model, mice were subjected to an acute bi‐lateral renal ischemia (30 min) followed by a 24hr reperfusion period after which kidneys were removed and PMN infiltration was quantified in tissue sections by immunohistochemistry. Kidneys subjected to I/R injury exhibited a significant PMN infiltration response as compared to sham‐operated kidneys (~22 fold increase) and mice pre‐treated with sJAM‐C (3mg/kg, i.v.) showed a significant reduction in PMN infiltration (49.6% inhibition, p&lt;0.05). In cremasteric venules, sJAM‐C suppressed I/R‐induced (30min ischemia/120 min reperfusion) leukocyte adhesion and transmigration. The results demonstrate a role for JAM‐C in PMN migration as induced by I/R injury.Funded by The Wellcome Trust and The British Heart Foundation.

FPR1 does not mediate neutrophil infiltration in experimental colitis

One hallmark of the inflammatory bowel diseases is the infiltration of neutrophils (PMN) into the mucosa. The mechanism behind this infiltration is unclear but bacterial N‐formyl peptides are candidates since these are potent chemoattractants and activating factors for PMN. We investigated this hypothesis by inducing colitis in mice deficient for the formyl peptide receptor, FPR1. FPR1‐/‐ and wild type (WT) mice were administered dextran sodium sulphate (DSS) through their drinking water for 5 days followed by one day of pure water (acute phase) or 4 days of water (recovery phase), or for 2 DSS cycles (chronic phase) with the second cycle commencing on day 22. During acute colitis FPR1‐/‐ mice lost up to 5% of their starting body weight whereas WT mice lost up to 20%. However, the histopathology was similar between the two groups at both the acute and recovery phase points. This included ulcers, PMN infiltration, and loss of crypt architecture. In contrast, during the chronic phase FPR1‐/‐ mice experienced weight loss of up to 20%, greater clinical illness, and 15% shorter colons than WT mice, all indications of a more severe disease. Whether this is reflected in differences in the histopathology remains to be evaluated. We conclude that FPR1 is not necessary for PMN infiltration into the acutely inflamed colon; however, it may play a role in chronic colitis. Funded by the CCFC and an IWK Health Centre Fellowship to SMF.

Modulatory Role of Mammalian Target of Rapamycin in Regulating Endothelial ICAM‐1 Expression and Lung PMN Recruitment

PKC‐δ, PI 3‐kinase, and the downstream target Akt are important signaling intermediates in mediating NF‐κB activation and ICAM‐1 expression in endothelial cells. Here, we show that these pathways also activate mTOR to ensure a tight regulation of NF‐κB activation and ICAM‐1 expression these cells. Exposure of human vascular endothelial cells to thrombin resulted in phosphorylation of mTOR and its downstream target p70S6 kinase in a PKC‐δ‐ and PI 3‐kinase/Akt‐dependent manner. Consistent with this, expression of constitutively active mutant of PKC‐δ or Akt was sufficient to induce NF‐κB activation and ICAM‐1 expression, and co‐expression of mTOR suppressed whereas inhibition/depletion of mTOR augmented these responses. Increasing or impairing mTOR signaling produced similar effects on NF‐κB activation and ICAM‐1 expression induced by thrombin. These data reveal an important role of mTOR as an endogenous modulator that may be engaged in PKC‐δ‐ and PI3K/Akt‐dependent manner to dampen thrombin‐induced NF‐κB signaling of ICAM‐1 expression in endothelial cells. We also determined the in vivo consequence of mTOR overexpression in a mouse model of sepsis. Introduction of the construct encoding mTOR via cationic liposomes caused a marked reduction in lung PMN infiltration in mice challenged i.p. with LPS. Together, these results indicate that mTOR may be a general modifier of lung inflammatory responses.

Polymorphonuclear Neutrophil Infiltration Intensity as Consequence ofEntamoeba histolyticaDensity in Amebic Colitis

It has been suggested that the damage observed in invasive amebiasis is related to interactions between polymorphonuclear leukocytes (PMN) and Entamoeba histolytica trophozoites. We analyzed the relation between infiltrating inflammatory cell populations and E. histolytica density in intestinal amebic lesions. Biopsies obtained endoscopically from patients with amebic colitis were analyzed to describe their morphologic abnormalities. Cellular populations and E. histolytica trophozoites were measured quantitatively in order to assess the correlation between infiltrating inflammatory cell populations and parasite density. Amebic lesions were most often located in the colon (55%). The histopathologic diagnoses were colitis in 31%, erosive colitis in 26%, and ulcerated colitis in 24%. The predominant cellular populations found in the lesions were lymphocytes (59.8%) (3,672 +/- 2,413/mm(2)) followed by PMN (17%) (1,038 +/- 1,171 PMN/mm(2)) (p < 0.01). A higher density of PMN infiltration was observed in severe cases. Cellular populations predictive of the presence of E. histolytica trophozoites (p = 0.047) were PMN (p = 0.019) and lymphocytes (p = 0.033). The highest association was found between E. histolytica trophozoites and PMN (p = 0.0221). Neutrophils and lymphocytes, particularly the former, are associated significantly with the density of parasites. Our findings support the theory that PMN interaction with E. histolytica contributes to the pathogenesis of amebic intestinal lesions.

Mice that overexpress human heat shock protein 27 have increased renal injury following ischemia reperfusion

We previously showed that activation of the A1 adenosine receptor protected the kidney against ischemia-reperfusion injury by induction and phosphorylation of heat shock protein 27 (HSP27). Here, we used mice that overexpress human HSP27 (huHSP27) to determine if kidneys from these mice were protected against injury. Proximal tubule cells cultured from the transgenic mice had increased resistance to peroxide-induced necrosis compared to cells from wild-type mice. However, after renal ischemic injury, HSP27 transgenic mice had decreased renal function compared to wild-type mice, along with increased renal expression of mRNAs of pro-inflammatory cytokines (TNF-alpha, ICAM-1, MCP-1) and increased plasma and kidney keratinocyte-derived cytokine. Following ischemic injury, neutrophils infiltrated the kidneys earlier in the transgenic mice. Flow cytometric analysis of lymphocyte subsets showed that those isolated from the kidneys of transgenic mice had increased CD3(+), CD4(+), CD8(+), and NK1.1(+) cells 3 h after injury. When splenocytes or NK1.1(+) cells were isolated from transgenic mice and adoptively transferred into wild-type mice there was increased renal injury. Further, depletion of lymphocytes by splenectomy or neutralization of NK1.1(+) cells resulted in improved renal function in the transgenic mice following reperfusion. Our study shows that induction of HSP27 in renal tubular cells protects against necrosis in vitro, but its systemic increase counteracts this protection by exacerbating renal and systemic inflammation in vivo.

Effects of mitomycin C on infiltration of polymorphonuclear leukocytes after epithelial scrape injury in the mouse cornea

To determine whether mitomycin C (MMC) alters appearance and disappearance of polymorphonuclear leucocytes (PMN) in the cornea stroma, using an epithelial scrape injury in eye mouse model. Twenty-mice underwent mechanical epithelium debridement in the central cornea using 20% ethanol. After the scrape, the right eye received 0.02% MMC for one minute, while the left eye received physiological saline. The animals were sacrificed on days 1, 2, 5, and 14 after surgery, and corneal whole mounts were prepared for histology. PMN distribution was analyzed in digitized microscope images. Cell division in the cornea was determined by immunohistochemical detection of bromodeoxyuridine (BrdU), which was injected intraperitoneally before the mice were sacrificed. Epithelial scrape injury triggered infiltration of PMNs into the corneal stroma. An analysis of PMN distribution revealed that there was no difference between eyes treated with and without MMC at all time points. BrdU labeling showed that 0.02% MMC for one minute blocked keratocyte proliferation completely. MMC treatment regimen, which is common in clinical practice, inhibits keratocyte proliferation during wound healing, but when used at 0.02% for one minute, it does not affect PMN infiltration into the corneal stroma, and subsequent movement toward the injury site, or the disappearance of PMNs from the stroma, in the mouse epithelial injury model.

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

Anti-inflammatory actions of aprotinin provide dose-dependent cardioprotection from reperfusion injury.

Myocardial injury following ischaemia and reperfusion has been attributed to activation and transmigration of polymorphonuclear leukocytes (PMNs) with release of mediators including oxygen-derived radicals and proteases causing damage. We studied the serine protease inhibitor aprotinin in an in vivo rabbit model of 1 h of myocardial ischaemia followed by 3 h of reperfusion (MI+R). Aprotinin (10,000 Ukg(-1)) or its vehicle were injected 5 min prior to the start of reperfusion. Myocardial injury was significantly reduced with aprotinin treatment as indicated by a reduced necrotic area (11+/-2.7% necrosis as percentage of area at risk after aprotinin; 24+/-3.1% after vehicle; P<0.05) and plasma creatine kinase activity (12.2+/-1.5 and 17.3+/-2.3 IU g(-1) protein in aprotinin and vehicle groups, respectively, P<0.05). PMN infiltration (assessed by myeloperoxidase activity) was significantly decreased in aprotinin-treated animals compared to vehicle (P<0.01). Histological analysis also revealed a substantial increase in PMN infiltration following MI+R and this was significantly reduced by aprotinin therapy (44+/-15 vs 102+/-2 PMN mm2 in aprotinin vs vehicle-treated animals, P<0.05). In parallel in vitro experiments, aprotinin inhibited neutrophil-endothelium interaction by reducing PMN adhesion on isolated, activated aortic endothelium. Finally, immunohistochemical analysis illustrated aprotinin significantly reduced myocardial apoptosis following MI+R. Inhibition of serine proteases by aprotinin inhibits an inflammatory cascade initiated by MI+R. The cardioprotective effect appears to be at least partly due to reduced PMN adhesion and infiltration with subsequently reduced myocardial necrosis and apoptosis.

Chlamydiae and polymorphonuclear leukocytes: unlikely allies in the spread of chlamydial infection

While much is known about the attachment of the chlamydiae to the host cell and intracellular events during the developmental cycle, little is known about the mechanism(s) by which elementary bodies exit the cell. In this report, we use the guinea-pig conjunctival model of Chlamydia caviae infection to present in vivo ultrastructural evidence supporting two mechanisms for release of chlamydiae from the mucosal epithelia. Four days after infection, histopathologic observation shows an intense infiltration of polymorphonuclear leukocytes (PMN) in the conjunctival epithelium. Using transmission electron microscopy, a gradient-directed PMN response to chlamydiae-infected epithelial cells was observed. As PMN infiltration intensifies, epithelial hemidesmosome/integrin/focal adhesion adherence with the basal lamina is disconnected and PMNs literally lift off and release infected superficial epithelia from the mucosa. Many of these infected cells appear to be healthy with intact microvilli, nuclei, and mitochondria. While lysis of some infected cells occurs with release of chlamydiae into the extracellular surface milieu, the majority of infected cells are pushed off the epithelium. We propose that PMNs play an active role in detaching infected cells from the epithelium and that these infected cells eventually die releasing organisms but, in the process, move to new tissue sites via fluid dynamics.

Neutrophils Invade Atrophied Skeletal Muscles Without Causing Muscle Dysfunction Unless They Are Stimulated With LPS.

Important muscle atrophy and loss of force are observed following unloading and early reloading periods, with subsequent muscle recovery within 14 to 28 d. The reloading period is associated with neutrophil (PMN) and macrophage infiltration. PMNs phagocyte necrotic debris and release various cytokines and oxidative factors. Although PMNs can induce secondary damage in different pathologies, their roles in muscle dysfunction and regrowth following unloading and reloading periods have never been fully investigated. PURPOSE: To determine the contribution of PMNs to muscle injury, dysfunction and regrowth in an unloading and reloading model. METHODS: Mice were submitted to 10 d of hindlimb unloading followed by reloading periods of 1, 3 or 14 d. They were intraperitoneally injected with 50 μL (0.025 mg) of rat anti-PMN (anti-Ly6G/Ly6C) or placebo 1 d before and after reloading. This dose was sufficient to deplete 95 % of circulating PMNs. Ambulatory mice were used as control. Soleus muscles were incubated in a physiological solution for measurements of contractile properties. In a second set of experiments, solei were incubated in vitro with lipopolysaccharide (LPS, 100 μg/mL) to activate PMNs. Contractile properties were obtained every hour for 6 h and muscles were subsequently incubated in 1 % procion orange at 4 °C to assess muscle damage. Comparisons between groups were performed with a one-way ANOVA followed by a Fisher's a posteriori test. RESULTS: Our findings show that PMN depletion neither affects the loss of muscle force nor the time of recovery. Interestingly, reloaded soleus muscles that contain a high PMN density showed a 20 % greater loss of force when compared to reloaded solei depleted in PMNs following LPS incubation. These significant differences appear after 4 h of incubation in LPS (p<0.05). Procion orange staining also confirms that presence of PMNs in atrophied muscles induces a 3-fold increase in muscle membrane damage following LPS stimulation (26 % vs 9 %, p<0.05). CONCLUSIONS: Together, these results show that secondary damage following PMN infiltration is strongly limited during muscle atrophy and regrowth. However, when activated by other stimuli, such as LPS, PMNs can potentially damage skeletal muscle. Supported by the Canadian Space Agency and Canadian Institutes of Health Research.