PMN Infiltration Research Articles

Injection of phosphatidylcholine and deoxycholic acid regulates gene expression of lipolysis-related factors, pro-inflammatory cytokines, and hormones on mouse fat tissue

Injection of phosphatidylcholine (PC) and deoxycholic acid (DA) preparation is widely used as an alternative to liposuction for the reduction of subcutaneous fat. Nevertheless, its physiological effects and mechanism of action are not yet fully understood. In this report, PC and deoxycholic acid (DA) were respectively injected into adipose tissue. PC decreased tissue mass on day 7, but DA did not. On the other hand, a decrement of DNA mass was observed only in DA-injected tissue on day 7. Both PC and DA reduced the mRNA expression of adipose tissue hormones, such as adiponectin, leptin, and resistin. In lipolysis-related gene expression profiles, PC increased hormone-sensitive lipase (HSL) transcription and decreased the expression other lipases, perilipin, and the lipogenic marker peroxisome proliferator-activated receptor-γ (PPARγ); DA treatment diminished them all, including HSL. Meanwhile, the gene expression of pro-inflammatory cytokines and a chemokine was greatly elevated in both PC-injected and DA-injected adipose tissue. Microscopic observation showed that PC induced lipolysis with mild PMN infiltration on day 7. However, DA treatment did not induce lipolysis but induced much amount of PMN infiltration. In conclusion, PC alone might induce lipolysis in adipose tissue, whereas DC alone might induce tissue damage.

Oxytocin Ameliorates the Immediate Myocardial Injury in Rat Heart Transplant Through Downregulation of Neutrophil-Dependent Myocardial Apoptosis

BackgroundCardiac oxytocin (OT) is structurally identical to that found in the hypothalamus, indicating that this active form of OT is derived from the same gene. The abundance of OT and its receptors in atrial myocytes suggests that, directly and/or via the release of the cardiac hormone atrial natriuretic peptide, this hormone regulates the force of cardiac contractions. Previous studies have demonstrated a role of OT in myocardial inflammatory responses. We sought to study protective myocardial and anti-inflammatory effects of OT in the immediate post-transplant period. MethodsWe grouped adult male Albino rats into sham, control, and OT-treated groups. Control and treated groups underwent heterotopic cervical heart transplantation. Myocardial injury was assessed by measuring plasma cardiac troponin I, and myocardial proinflammatory cytokines as well as by performing histopathologic assessments injury score, and of apoptotic degree. Myocardial myeloperoxidase, neutrophil infiltration, neutrophil chemotactic mediators as well as formation of reactive oxygen and reactive nitrogen species were measured in the myocardium at 3 hours after reperfusion to assess neutrophil-dependent myocardial injury. ResultsOT downregulates neutrophil chemotactic molecules — KC/CXCL1 and MIP-2/CXCL2. The decrement in myocardial PMN infiltration was associated with reduced reactive oxygen and reactive nitrogen species formation in the myocardium at 3 hours after reperfusion following global ischemia. OT reduced postmyocardial ischemia/reperfusion apoptotic processed. ConclusionOT ameliorated immediate myocardial injury in heart grafts, through downregulation of the inflammatory response, of reactive oxygen species, and of neutrophil dependent apoptosis.

Inhaled Carbon Monoxide Accelerates Resolution of Inflammation via Unique Proresolving Mediator–Heme Oxygenase-1 Circuits

Resolution of acute inflammation is an active event accompanied by biosynthesis of specialized proresolving mediators (SPM). We employed a systems approach to determine the impact of CO in resolution active programs during self-limited inflammation in mice. Compared with ambient air, inhaled CO gas (250 ppm) significantly limited PMN infiltration (∼44%, 6 h) into peritoneum and shortened resolution interval from 4 to 2 h. We profiled exudate lipid mediators (LM) via metabololipidomics, CO reduced leukotriene B4 (21 ± 11 versus 59 ± 24 pg/mouse, 6 h), and elevated SPM including resolvin (Rv) D1 (27 ± 4 versus 16 ± 5 pg/mouse) and maresin 1 (26 ± 9 versus 15 ± 3 pg/mouse). With human macrophages, SPM (10 pM-10 nM) elevated heme oxygenase (HO)-1 (∼50%, 8 h). CO also enhanced HO-1 expression and accumulation of RvD1 and RvD5, an action reversed by blockage of a key SPM biosynthesis enzyme 15-lipoxygenase type 1. Compared with normoxia, CO increased ∼30% phagocytosis of opsonized zymosan with human macrophage, which was further enhanced by SPM (∼100%). This CO increased phagocytosis was blocked by 15-lipoxygenase inhibition, and SPM stimulated phagocytosis was diminished by HO-1 inhibition. In murine peritonitis, both pre- and posttreatment with CO inhalation significantly increased macrophages carrying ingested apoptotic PMN in exudates and enhanced PMN apoptosis. Taken together, these results indicate that CO accelerates resolution of acute inflammation, shortens resolution intervals, enhances macrophage efferocytosis, and temporally regulates local levels of lipid mediator/SPM. Moreover, they provide proresolving mechanisms for HO-1/CO, which is part of the SPM-initiated resolution circuit.

G protein-coupled receptor kinase 6 deficiency promotes angiogenesis, tumor progression, and metastasis.

G protein-coupled receptor kinases (GRKs) phosphorylate the activated form of G protein-coupled receptors leading to receptor desensitization and downregulation. We have recently shown that the chemokine receptor, CXCR2, couples to GRK6 to regulate cellular responses including chemotaxis, angiogenesis, and wound healing. In this study, we investigate the role of GRK6 in tumorigenesis using murine models of human lung cancer. Mice deficient in GRK6 (GRK6(-/-)) exhibited a significant increase in Lewis lung cancer growth and metastasis relative to control littermates (GRK6(+/+)). GRK6 deletion had no effect on the expression of proangiogenic chemokine or vascular endothelial growth factor, but upregulated matrix metalloproteinase (MMP)-2 and MMP-9 release, tumor-infiltrating PMNs, and microvessel density. Because β-arrestin-2-deficient (βarr2(-/-)) mice exhibited increased Lewis lung cancer growth and metastasis similar to that of GRK6(-/-), we developed a double GRK6(-/-)/βarr2(-/-) mouse model. Surprisingly, GRK6(-/-)/βarr2(-/-) mice exhibited faster tumor growth relative to GRK6(-/-) or βarr2(-/-) mice. Treatment of the mice with anti-CXCR2 Ab inhibited tumor growth in both GRK6(-/-) and GRK6(-/-)/βarr2(-/-) animals. Altogether, the results indicate that CXCR2 couples to GRK6 to regulate angiogenesis, tumor progression, and metastasis. Deletion of GRK6 increases the activity of the host CXCR2, resulting in greater PMN infiltration and MMP release in the tumor microenvironment, thereby promoting angiogenesis and metastasis. Because GRK6(-/-)/βarr2(-/-) showed greater tumor growth relative to GRK6(-/-) or βarr2(-/-) mice, the data further suggest that CXCR2 couples to different mechanisms to mediate tumor progression and metastasis.

Abstract 2821: GRK6 deficiency promotes tumor aggressiveness and metastasis in a murine model of human lung cancer .

Abstract G protein coupled receptor kinases (GRKs) phosphorylate the activated form of G protein coupled receptors (GPCRs) followed by the association of β-arrestin (βarr) with the phosphorylated receptor, desensitization and down-regulation. We have recently shown that the chemokine receptor, CXCR2, couples to GRK6 to regulate cellular responses including chemotaxis, angiogenesis and wound healing. In this study, we sought to investigate the role of GRK6 in CXCR2-mediated tumorigenesis using a murine model of human lung cancer. Mice deficient in GRK6 (GRK6−/−) exhibited a significant increase in Lewis lung cancer (LLC) growth and metastasis relative to control littermates (GRK6+/+). GRK6 deletion had no effect on the expression of pro-angiogenic chemokine or vascular endothelial growth factor (VEGF), but up-regulated matrix metalloproteinase (MMP)-2 and MMP-9 release, tumor-infiltrating PMNs and microvessel density. Treatment of the mice with a CXCR2 inhibitor (SB225002) or a murine anti-CXCR2 antibody blocked the effect of GRK6 deletion. Surprisingly, a GRK6−/−/βarr2−/− double knockout animal model exhibited faster tumor growth relative to GRK6−/− or βarr2−/− animals. Exposure of GRK6−/−/βarr2−/− mice to the anti-CXCR2 antibody totally inhibited tumor growth. This suggest that the effects of βarr2 and GRK6 knockdown in CXCR2-mediated tumor development are additive. Altogether, the results indicate that CXCR2 couples to GRK6 to regulate angiogenesis, tumor progression and metastasis. Deletion of GRK6 increases the activity of the host CXCR2, resulting in greater PMN infiltration and MMP release in the tumor microenvironment thereby promoting angiogenesis and metastasis. Since GRK6−/−/βarr2−/− mice showed greater tumor growth relative to GRK6−/− or βarr2−/− mice, the data also suggest that CXCR2 couples to a different mechanism to mediate tumor progression and metastasis. Citation Format: Natalie Sutton, Nikia Smith, Ariel J. Thomas, Sandeep K. Raghuwanshi, Ricardo M. Richardson. GRK6 deficiency promotes tumor aggressiveness and metastasis in a murine model of human lung cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2821. doi:10.1158/1538-7445.AM2013-2821

Inhaled Carbon Monoxide Accelerates Resolution of Inflammation via Novel Pro‐resolving Mediators and Heme Oxygenase‐1

Resolution of acute inflammation is an active event accompanied by biosynthesis of specialized pro‐resolving mediators (SPM). We employed a systems approach to determine the impact of carbon monoxide (CO) in resolution active programs. In zymosan induced murine peritonitis model, inhaled CO gas (250 ppm) significantly limited PMN infiltration (~44% reduction at 6h) into peritoneum and shortened resolution interval. We profiled exudate lipid mediators (LM), CO reduced leukotriene (LT) B4 (21±11 vs. 59±24 pg/mouse at 6h) and elevated SPM including resolvin (Rv) E2 (1,201±197 vs. 864±125 pg/mouse), RvD1 (27±4 vs. 16±5 pg/mouse) and maresin 1 (MaR1; 26±9 vs. 15±3 pg/mouse). With human macrophages, SPM (10 pM–10 nM) elevated heme oxygenase (HO)‐1 (~50% at 8h). CO also enhanced HO‐1 expression (~25%), an action reversed by blockage of a key SPM biosynthesis enzyme 15‐lipoxygenase (LOX) type 1. CO increased phagocytosis with human macrophage, that was further enhanced by SPM. This CO increased phagocytosis was blocked by 15‐LOX inhibition and SPM stimulated phagocytosis was diminished by HO‐1 inhibition. In murine peritonitis, CO inhalation significantly increased numbers of macrophages carrying ingested apoptotic PMN at 12h in both exudates (1.9 vs. 3.4%) and inguinal lymph nodes (2.0 vs. 4.7%).These results indicate that CO accelerates resolution of acute inflammation, shortens resolution intervals, enhances macrophage efferocytosis and temporally regulates local levels of LM/SPM. Moreover, they provide pro‐resolving mechanisms for HO‐1/CO that is part of the SPM‐initiated resolution circuit.

Neutrophil‐epithelial interactions modulate the inflammatory microenvironment during colitis

Neutrophil (PMN) accumulation in crypt abscesses is a pathological hallmark of ulcerative colitis. We hypothesized that transmigrating neutrophils influence the transcriptional profile of intestinal epithelia. Microarray studies revealed a cohort of hypoxia‐responsive genes regulated by PMN‐epithelial crosstalk. Transmigrating PMNs rapidly depleted microenvironmental O2 sufficiently to stabilize epithelial hypoxia‐inducible factor (HIF). Utilizing HIF reporter mice in a TNBS colitis model, we investigated the contribution of PMNs and the oxidative burst to “inflammatory hypoxia”. Gp91phox−/− mice, lacking functional NADPH oxidase, developed exacerbated colitis with enhanced PMN infiltration and diminished inflammatory hypoxia. Pharmacological intervention with a HIF‐stabilizing compound resulted in restoration of mucosal HIF‐signaling concomitant with abrogation of colitis‐severity in the gp91phox−/− mice. In conclusion, transcriptional imprinting of host tissue by infiltrating PMNs modulates host responses to inflammation. Likewise, the oxidative burst contributes fundamentally to localized O2 depletion, resultant microenvironmental hypoxia and effective inflammatory resolution. Funding: CCFA and NIH.

Critical Role of Non-Muscle Myosin Light Chain Kinase in Thrombin-Induced Endothelial Cell Inflammation and Lung PMN Infiltration

The pathogenesis of acute lung injury (ALI) involves bidirectional cooperation and close interaction between inflammatory and coagulation pathways. A key molecule linking coagulation and inflammation is the procoagulant thrombin, a serine protease whose concentration is elevated in plasma and lavage fluids of patients with ALI and acute respiratory distress syndrome (ARDS). However, little is known about the mechanism by which thrombin contributes to lung inflammatory response. In this study, we developed a new mouse model that permits investigation of lung inflammation associated with intravascular coagulation. Using this mouse model and in vitro approaches, we addressed the role of non-muscle myosin light chain kinase (nmMLCK) in thrombin-induced endothelial cell (EC) inflammation and lung neutrophil (PMN) infiltration. Our in vitro experiments revealed a key role of nmMLCK in ICAM-1 expression by its ability to control nuclear translocation and transcriptional capacity of RelA/p65 in EC. When subjected to intraperitoneal thrombin challenge, wild type mice showed a marked increase in lung PMN infiltration via expression of ICAM-1. However, these responses were markedly attenuated in mice deficient in nmMLCK. These results provide mechanistic insight into lung inflammatory response associated with intravascular coagulation and identify nmMLCK as a critical target for modulation of lung inflammation.

Further studies on anti-inflammatory activity of maprotiline in carrageenan-induced paw edema in rat

Antidepressant drugs are commonly used for treatment of different medical disorders besides of psychiatric diseases. Accumulating evidence suggests that antidepressants exhibit anti-inflammatory activity in vivo and in vitro conditions, but the mechanisms of this property are not clear very well. In our earlier work, we demonstrated that i.c.v. and i.p. injection of maprotiline, as an antidepressant, decreased paw edema at the fourth hour after subplantar injection of carrageenan. Therefore, this work was undertaken to investigate anti-inflammatory effects of maprotiline in more details. Our results verified that i.p. (25 and 50mg/kg) and i.c.v. (100μg/rat) application of maprotiline significantly reduced paw edema at 1, 2, 3 and 4h intervals after carrageenan challenge. Pathological examinations and MPO activity also showed that both i.p. and i.c.v. maprotiline considerably inhibited infiltration of PMN leucocytes into the inflamed paws. Additionally, i.p. and i.c.v. maprotiline at all applied doses noticeably declined levels of IL-1β into the site of inflammation, while only i.p. maprotiline at a dose of 50mg/kg significantly decreased TNF-α levels in the carrageenan-injected paws.These results confirmed anti-edematogenic activity of i.p. and i.c.v. maprotiline in the carrageenan induced paw edema model and showed that these properties of maprotiline might be mediated through inhibition of PMN infiltration and release of IL-1β and TNF-α.

Tobacco smoke modulates ozone-induced toxicity in rat lungs and central nervous system

Adult Sprague-Dawley (SD) male rats were exposed for a single 3 h period to air, ozone (O3) or O3 followed by tobacco smoke (O3/TS). For pulmonary effects, bronchoalveolar lavage (BAL) cells and fluid were analyzed. Data revealed a significant increase in polymorphonuclear leukocytes (PMN), total protein and albumin concentrations in the O3 group, reflecting inflammatory and toxic responses. A subsequent exposure to TS attenuated PMN infiltration into the airspaces and their recovery in the BAL. A similar reduction was observed for BAL protein and albumin in the O3/TS group, but it was not statistically significant. We also observed a significant increase in BAL total antioxidant capacity following O3 exposure, suggesting development of protective mechanisms for oxidative stress damage from O3. Exposure to TS attenuated the levels of total antioxidant capacity. Lung tissue protein analysis showed a significant reduction of extracellular superoxide dismutase (EC-SOD) in the O3 or O3/TS group and catalase in the O3/TS group. TS further altered O3-induced EC-SOD and catalase protein expression, but the reductions were not significant. For effects in the central nervous system (CNS), we measured striatal dopamine levels by HPLC with electrochemical detection. O3 exposure produced a nonsignificant decrease in the striatal dopamine content. The effect was partially reversed in the O3/TS group. Overall, the results show that the toxicity of O3 in the lung is modulated by TS exposure, and the attenuating trend, though nonsignificant in many cases, is contrary to the synergistic toxicity predicted for TS and O3, suggesting limited cross-tolerance following such exposures.

Transmigrating Neutrophils Shape the Mucosal Microenvironment During Colitis

Neutrophil (PMN) accumulation in crypt abscesses is a pathological hallmark of ulcerative colitis. PMN transepithelial migration is orchestrated through a complex series of cell-cell interactions involving bi-directional signaling molecules. Thus we hypothesized that migrating PMN influence the transcriptional profile of epithelia as they transmigrate, priming them for either resolution or chronic inflammation - a concept we term “transcriptional imprinting”. Employing a novel approach to a PMN-transepithelial migration model, we attempted to ascertain the influence of transmigration on epithelial gene expression by microarray analysis. DNA Microarray was used to identify transcriptional changes in intestinal epithelial cells post-PMN transmigration. Real-time oxygen sensing was performed with an SDR-OxoDish (PreSens). Wild-type C57/B6 mice, ODD-Luciferase (HIF reporter) mice and gp91phox-null (NADPH oxidase deficient) mice were subjected to chemically-induced colitis (TNBS). Microarray studies revealed a cohort of hypoxia-responsive genes regulated by PMN-epithelial crosstalk. Real-time oxygen measurements effectively demonstrated that activated PMN rapidly depleted microenvironmental oxygen. Subsequent studies indicated that activated PMNs are sufficient to induce hypoxia-inducible factor (HIF) stabilization and activity in epithelial cells. Intestinal epithelial hypoxia induced by activated PMNs during transmigration was found to be dependent on the PMN respiratory burst. HIF is known to influence the expression of barrier-protective genes in the epithelium, initiate glycolytic metabolism and attenuates the clinical course/disease parameters in murine colitis. To ascertain the clinical relevance of transmigrating PMN and the induction of epithelial hypoxia, we stained biopsies from UC patients for evidence of Glut1 expression. PMN transmigration increased crypt epithelial Glut1 expression (see Figure). We investigated the relative contribution of PMN to “inflammatory hypoxia” in vivo, utilizing a colitis model. Antibody-mediated depletion of PMNs worsened the course of colitis, exhibited reduced tissue hypoxia visualized with Hypoxyprobe-1 staining and attenuated the induction of hypoxia-dependent genes. Patients with chronic granulomatous disease (CGD) lack functional NADPH oxidase and develop IBD-like symptoms. We demonstrated that murine gp91phox−/− mice mirror human CGD, develop a severe colitis, with exaggerated PMN infiltration. However, these infiltrating PMN are incapable of inducing a hypoxic microenvironment. Finally, pharmacological intervention with a HIF-stabilizing compound (PHD inhibitor), resulted in recapitulation of mucosal HIF-signaling concomitant with abrogation of colitis-severity in the gp91phox−/− mice. In conclusion, transcriptional imprinting of host tissue by infiltrating neutrophils significantly modulates the host response to inflammation. Moreover, the respiratory burst contributes fundamentally to localized oxygen depletion and resultant microenvironmental hypoxia. We propose that this microenvironment established by infiltrating PMN is protective during colitis.

The Effect of Co-Stimulation Blockade on Memory T Cell Responses in a Rat-To-Mouse Islet Xenotransplantation Model

Background: Co-stimulatory signals have been reported to play an important role in islet xenograft rejection. Co-stimulation blockade cannot inhibit graft rejection in sensitized recipients. We investigated whether pre-existing donor-specific antibodies influenced islet xenograft survival in recipients treated with co-stimulation blockade. Methods: MaleC57BL/6 mice with alloxan-induced diabetes were used as recipients and male Lewis rats as donors. Xenoimmune serum was collected from mice transplanted with 200 Lewis rat islets and used as source of immune antibodies. Diabetic naïve C57BL/6 mice were transplanted under the left kidney capsule with 100 Lewis rat islets and the recipients were divided into three groups receiving: i) isotype control antibodies; ii) anti-CD154 and CTLA4Ig; and iii) anti-CD 154, CTLA4Ig, and anti-LFA-1 every second day from day 0 to day 8. Half of the animals in each group received naïve mouse serum (as source of control antibody) and half xenoimmune serum at the time of transplantation. Graft survival was followed by daily recordings of non-fasting blood glucose levels and body weight. Cured mice were examined by the IPGT test at 4 and 6-8 weeks after transplantation. Results: Anti-donor antibody was detected in the immune antibody injected recipient serum at 48 hours after transplantation but disappeared after 96 hour. There was no significant difference in graft survival between immune antibody and naive antibody injected groups. Mice receiving isotype control antibodies rejected their grafts within 13 days, and histology showed severe infiltration of both PMN and lymphocytes with few or no remaining grafts. The graft function in mice receiving double and triple treatment survived longer (>70 days), however, approximately half of them lost graft function after 100 days. In cured mice, islet grafts were verified by histological examination. Most of them showed mononuclear cell infiltration around the grafts. In mice rejecting the grafts, no or very little graft mass was seen with severe infiltration of mononuclear cells and some PMNs. In the IPGTT at 6-8 weeks after transplantation, return of normoglycemia was significantly delayed compared to the response in the IPGTT done at 4 weeks, indicating a gradual graft loss during the observation period. Mixed lymphocyte cultures in vitro showed no significant difference in T-cell proliferation between islet rejecting and accepting hosts. Anti-donor antibodies developed even in mice maintaining graft function. Conclusion: Xenoimmune antibody alone did not affect rat islet xenograft survival in naïve mice when their T cell immunity was impaired through co-stimulation blockade. Co-stimulation blockade could not induce permanent graft survival in the rat-to-mouse islet xenotransplantation model.

Severe multiple organ injury in HSF1 knockout mice induced by lipopolysaccharide is associated with an increase in neutrophil infiltration and surface expression of adhesion molecules

We have reported previously that HSF1 is essential in protection against the lethal systemic inflammation induced by LPS. However, the mechanism by which HSF1 protects against LPS-induced systemic inflammation remains unknown. In this study, HSF1(-/-) mice were subjected to endotoxemia by a bolus injection of LPS (10 mg/kg, i.p.). The serum levels of LDH, BUN, and transaminase (ALT and AST) were measured. PMN infiltration in lung, liver, and kidney tissues after endotoxemia was observed with immunohistochemistry. Comparing with the WT control, LPS administration induced more severe multiple organ dysfunction and lower survival rates in the HSF1(-/-) mice. Moreover, PMN infiltration into lungs, liver, and kidneys in HSF1(-/-) mice was more than that in the WT mice. The augmented tissue PMN infiltration in HSF1(-/-) mice was associated with their enhanced adhesive properties to endothelium in vivo. In addition, HSF1(-/-) caused greater surface expression of PSGL-1 and CD11b on the PMN surface after LPS treatment. These findings suggested that HSF1 alleviated LPS-induced multiple organ injury in mice by suppressing the surface expression of adhesion molecules on PMNs and subsequent infiltration of PMNs in tissues.

ICAM-1–activated Src and eNOS signaling increase endothelial cell surface PECAM-1 adhesivity and neutrophil transmigration

AbstractPolymorphonuclear neutrophil (PMN) extravasation requires selectin-mediated tethering, intercellular adhesion molecule-1 (ICAM-1)–dependent firm adhesion, and platelet/endothelial cell adhesion molecule 1 (PECAM-1)–mediated transendothelial migration. An important unanswered question is whether ICAM-1–activated signaling contributes to PMN transmigration mediated by PECAM-1. We tested this concept and the roles of endothelial nitric oxide synthase (eNOS) and Src activated by PMN ligation of ICAM-1 in mediating PECAM-1–dependent PMN transmigration. We observed that lung PMN infiltration in vivo induced in carrageenan-injected WT mice was significantly reduced in ICAM-1−/− and eNOS−/− mice. Crosslinking WT mouse ICAM-1 expressed in human endothelial cells (ECs), but not the phospho-defective Tyr518Phe ICAM-1 mutant, induced SHP-2–dependent Src Tyr530 dephosphorylation that resulted in Src activation. ICAM-1 activation also stimulated phosphorylation of Akt (p-Ser473) and eNOS (p-Ser1177), thereby increasing NO production. PMN migration across EC monolayers was abolished in cells expressing the Tyr518Phe ICAM-1 mutant or by pretreatment with either the Src inhibitor PP2 or eNOS inhibitor L-NAME. Importantly, phospho–ICAM-1 induction of Src signaling induced PECAM-1 Tyr686 phosphorylation and increased EC surface anti–PECAM-1 mAb-binding activity. These results collectively show that ICAM-1–activated Src and eNOS signaling sequentially induce PECAM-1–mediated PMN transendothelial migration. Both Src and eNOS inhibition may be important therapeutic targets to prevent or limit vascular inflammation.

Staphylococcus Aureus Infection of the Rabbit Cornea Following Topical Administration

Purpose: To determine the ability of diverse S. aureus strains to infect the rabbit cornea following topical inoculation, with special emphasis on a strain of unusual virulence.Materials and Methods: S. aureus strains (5 × 105 colony forming units; CFU) were topically applied onto scarified rabbit corneas or 100 CFU were intrastromally injected into rabbit corneas. Eyes were scored by slit lamp examination (SLE) and corneas were cultured to determine the log CFU. Polymorphonuclear leukocytes (PMN) were quantified by myeloperoxidase assays and corneas underwent histopathological analysis. Hemolysin titers of S. aureus strains were determined and S. aureus interactions with rabbit tears or human corneal epithelial cells were investigated.Results: All strains injected into the cornea produced high SLE scores and multi-log increases in CFU. Following topical inoculation, four strains produced low SLE scores with no bacterial replication. One strain (UMCR1) topically infected the cornea, causing high SLE scores, extensive PMN infiltration, and multi-log increases in CFU. Histopathologic analysis demonstrated a PMN influx into the UMCR1-infected cornea, destruction of the corneal epithelium, and severe edema. Strain UMCR1 did not demonstrate a high hemolysin titer or resistance to the bactericidal activity of rabbit tears, but did invade human corneal epithelial cells with relatively high efficiency.Conclusions: One S. aureus strain demonstrated the ability to topically infect the rabbit cornea. This strain was previously found to be unique in its ability to infect the anterior chamber and conjunctiva, suggesting that a key mechanism may be employed to overcome the host defenses of these three ocular sites.

The Anti-inflammatory Effect of Z-Ligustilide in Experimental Ovariectomized Osteopenic Rats

The purpose of the present study was to investigate the anti-inflammatory activity of Z-Ligustilide (LIG) in experimental ovariectomized (OVX) osteopenic rats. The anti-inflammatory potential of LIG in the regulation of nuclear factor kappa B (NF-κB), maleic dialdehyde (MDA), polymorphonuclear cells (PMN), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α), adhesion molecule (ICAM-1) and cyclooxygenase-2 (COX-2) was determined by ELISA. LIG significantly inhibited OVX-induced up-regulation of NF-κB activation and the production of IL-1β, TNF-α, iNOS, ICAM-1 and COX-2. Moreover, LIG suppressed MDA and infiltration of PMN. The results of the present study clearly demonstrate that there may be an inflammatory component in the etiology of osteoporosis. It revealed a significant anti-inflammatory effect of Z-Ligustilide in experimental OVX osteopenic rats.

Role of Mouse Peptidoglycan Recognition Protein PGLYRP2 in the Innate Immune Response to Salmonella enterica Serovar Typhimurium Infection In Vivo

Peptidoglycan recognition proteins (PGRPs) are a family of innate pattern recognition molecules that bind bacterial peptidoglycan. While the role of PGRPs in Drosophila innate immunity has been extensively studied, how the four mammalian PGRP proteins (PGLYRP1 to PGLYRP4) contribute to host defense against bacterial pathogens in vivo remains poorly understood. PGLYRP1, PGLYRP3, and PGLYRP4 are directly bactericidal in vitro, whereas PGLYRP2 is an N-acetylmuramyl-L-alanine amidase that cleaves peptidoglycan between the sugar backbone and the peptide stem. Because PGLYRP2 cleaves muramyl peptides detected by host peptidoglycan sensors Nod1 and Nod2, we speculated that PGLYRP2 may act as a modifier of Nod1/Nod2-dependent innate immune responses. We investigated the role of PGLYRP2 in Salmonella enterica serovar Typhimurium-induced colitis, which is regulated by Nod1/Nod2 through the induction of an early Th17 response. PGLYRP2 did not contribute to expression of Th17-associated cytokines, interleukin-22 (IL-22)-dependent antimicrobial proteins, or inflammatory cytokines. However, we found that Pglyrp2-deficient mice displayed significantly enhanced inflammation in the cecum at 72 h postinfection, reflected by increased polymorphonuclear leukocyte (PMN) infiltration and goblet cell depletion. Pglyrp2 expression was also induced in the cecum of Salmonella-infected mice, and expression of green fluorescent protein under control of the Pglyrp2 promoter was increased in discrete populations of intraepithelial lymphocytes. Lastly, Nod2(-/-) Pglyrp2(-/-) mice displayed increased susceptibility to infection at 24 h postinfection compared to Pglyrp2(-/-) mice, which correlated with increased PMN infiltration and submucosal edema. Thus, PGLYRP2 plays a protective role in vivo in the control of S. Typhimurium infection through a Nod1/Nod2-independent mechanism.

Regulation of the inflammatory response: enhancing neutrophil infiltration under chronic inflammatory conditions (118.5)

Abstract Neutrophil (PMN) infiltration plays a central role in inflammation and is a major cause of tissue damage. Thus, PMN infiltration must be tightly controlled. Using zymosan-induced peritonitis as an in vivo PMN infiltration model, we show in this study that PMN response and infiltration were significantly enhanced in mice experiencing various systemic inflammation including colitis and diabetes. Adoptive transfer of leukocytes from mice with inflammation into healthy recipients or from healthy into inflammatory recipients followed by inducing peritonitis demonstrated that both circulating PMN and tissue macrophages were altered under inflammation and that they collectively contributed to enhanced PMN infiltration. Detailed analyses of dextran sulfate sodium (DSS)-elicited colitis revealed that enhancement of PMN infiltration and macrophage function occurred only at the post-acute/chronic phase of inflammation and was associated with markedly increased IL-17A in serum. In vitro and ex vivo treatment of isolated PMN and macrophages confirmed that IL-17A directly modulates these cells and significantly enhances their inflammatory responses. Neutralization of IL-17A eliminated the enhancement of PMN infiltration and IL-6 production and also prevented severe tissue damage in DSS-treated mice. Thus, IL-17A produced at the chronic stage of colitis serves as an essential feedback signal that enhances PMN infiltration and promotes inflammation.

Neutrophil infiltration related to the methotrexate‐induced intestinal barrier dysfunction

ObjectiveMethotrexate (MTX) is used for the treatment of acute leukemia, rheumatoid arthritis and some intraosseous sarcomas. It causes severe side effects like mucosal inflammation, nausea and diarrhea. We found that MTX‐induced ROS resulted in neutrophil (PMN) accumulation and migration in rat intestine. MTX‐induced secretion of inflammatory cytokines seemed to be a triggering factor for the PMN migration. Thus, we built the in vitro assay system for PMN migration and studied the molecular mechanism.MethodsT84 cells derived from human colon carcinoma were seeded on to the lower side of inverted transwell and cultured for 3 weeks. PMNs isolated from human venous blood were applied to the upper chamber of transwell. PMNs in the lower chamber were evaluated by myeloperoxidase (MPO) activity.ResultsPMN number in the lower side was increased by adding MTX treated conditioned medium, but not changed by MTX exposure alone. IL‐8 mRNA expression was increased in a time‐dependent manner. IL‐8 secretion into MTX treated conditioned medium was increased. PMN migration was suppressed by IL‐8 neutralizing antibody, but not by control IgG. Treatment with SB203580, a p38 MAPK inhibitor regulated the MTX‐induced PMN transmigration.ConclusionsMTX caused PMN infiltration across T84 monolayer, which was caused by secreted IL‐8. It was likely triggered by IL‐1β production and p38 MAPK signal pathway.

Resolvin D1 limits PMN recruitment to inflammatory loci: receptor dependent bioactions

Resolvins are a newly discovered family of lipid mediators biosynthesized from omega‐3 PUFAs (DHA & EPA) that actively resolve inflammation. Bioactions of resolvins are mediated via specific G‐protein coupled receptors (GPCRs). Recent studies identified two GPCRs that RvD1 specifically binds on human leukocytes, the lipoxin A4/annexin A1 receptor (ALX/FPR2) and GPR32 (that has no known murine orthologue). Using zymosan stimulated peritonitis as an acute model of inflammation; RvD1 reduced peritoneal PMN infiltration at 4 and 24h with doses as low as 1ng/mouse. Importantly, this effect was not observed in fpr2 null mice, indicating the crucial role of this GPCR in RvD1 mediated PMN responses in the mouse. Moreover, the pro‐resolving properties of RvD1 were ALX/FPR2 specific, as RvD1 enhanced zymosan phagocytosis by WT but not fpr2 null murine macrophages. RvD1 also displayed potent actions on human PMN; reducing recruitment to endothelial cells under shear conditions as quantified using a flow chamber system. Interestingly, receptor specific antibodies blocked these anti‐inflammatory actions of RvD1, with low 1nM concentrations sensitive to GPR32 blockade, whilst 10nM RvD1 appeared ALX/FPR2 specific. These results highlight GPCR dependent actions of RvD1 in limiting overzealous PMN recruitment.Supported by Arthritis Research UK Foundation Fellowship 18445 to LVN & National Institutes of Health USA GM38765 and DE019938 to CNS