Polymorphonuclear Neutrophils Depletion Research Articles

NO-producing Polymorphonuclear Neutrophils Confer Protection against Chlamydia psittaci in Mouse Lung Infection.

Whether polymorphonuclear neutrophils (PMN) exert a protective role upon chlamydial infection by expressing inducible nitric oxide (NO) synthase (iNOS) and producing NO remains unclear. This issue was addressed using BALB/c mice infected with C. psittaci (Cps) 6BC strain. Methods mainly included flow cytometry, immunofluorescence, qRT-PCR, and Western blot. The number of PMN was significantly increased during Cps infection, which was accompanied by the increased iNOS expression and NO production in the mouse lungs. PMN were the major source of NO during pulmonary Cps infection and inhibited Cps multiplication in an iNOS/NO-dependent manner. Depletion of PMN aggravated Cps-induced disease and increased Cps burden. Mechanically, NF-κB and STAT1 signaling pathways, but not MAPK signaling pathways, were required for the induction of iNOS expression and NO production in PMN by Cps infection. Thus, our findings highlight the protective role of NO-producing PMN in Cps infection. NO-producing PMN confer a protective role during pulmonary Cps infection in mice, and thus our study sheds new light on PMN function during Chlamydia infection.

Effect of neutrophil extracellular traps on tumor lymph nodes.

4033 Background: In most human cancers, regional lymph nodes (LNs) are the first sites of metastasis. In addition to being an important part of the tumor staging system, with the advent of novel therapies, lymph node metastasis has become a crucial clinical intervention point before distant metastasis, the leading cause of cancer-associated deaths. To initiate metastasis, the conditions of LNs need to be optimized for tumor cell deposition and growth. This process is believed to be mediated by the activation of immune cells including polymorphonuclear neutrophils (PMNs). However, the cellular mechanism is not well defined. Our early observations suggest that PMNs and neutrophil extracellular traps (NETs), DNA comprising structures that are extruded in response to inflammatory cues, are associated with adverse oncologic outcomes. Thus, one potential mechanism of increased LN metastasis is that tEVs recruit PMNs and propend NETs formation. Methods: Human tissue micro-arrays (TMAs) of gastroesophageal (GEA) cancer patients were stained with PMN and NETs markers and quantified by HALO software. C57BL/6 or pad4-/- mice were injected with B16F10 or H59 cells alone or treated with neutrophil elastase inhibitor (NEi) or PMN depletion antibody. LN sections were stained with NETs markers and quantified by ImageJ (NIH). Results: In the study of 175 GEA cancer patients, lymphatic NET deposition was observed in both tumor infiltrated lymph nodes and tumor negative lymph nodes. We also demonstrated high LN NETs deposition was associated with reduced survival, even in the absence of overt metastasis ( p=0.03). Next, we sought to investigate the dynamic and the consequence of LN NETs deposition using animal models. We found that LN Neutrophil Recruitment and NET deposition happens in a pre-metastatic manner. Moreover, LN metastasis was abrogated through different kinds of NETs inhibition (neutrophil depletion, pad4 knockout and NEi treatment, n=10, p<0.001), demonstrating the consequences of LN NETs deposition and its potential as a treatment target. Finally, we showed that the LN PMN recruitment and NETs formation was mediated by increased production of IL-8 by Lymphatic Endothelial Cells (LEC). Conclusions: Together, we demonstrated that NETs can contribute to LN metastasis, and can serve as a potential therapeutic targets. By further investigating the detailed mechanism, this project will lead to major advances in the management of cancer patients.

Stamp2 Protects From Maladaptive Structural Remodeling and Systolic Dysfunction in Post-Ischemic Hearts by Attenuating Neutrophil Activation.

The six-transmembrane protein of prostate 2 (Stamp2) acts as an anti-inflammatory protein in macrophages by protecting from overt inflammatory signaling and Stamp2 deficiency accelerates atherosclerosis in mice. Herein, we describe an unexpected role of Stamp2 in polymorphonuclear neutrophils (PMN) and characterize Stamp2’s protective effects in myocardial ischemic injury. In a murine model of ischemia and reperfusion (I/R), echocardiography and histological analyses revealed a pronounced impairment of cardiac function in hearts of Stamp2-deficient- (Stamp2-/- ) mice as compared to wild-type (WT) animals. This difference was driven by aggravated cardiac fibrosis, as augmented fibroblast-to-myofibroblast transdifferentiation was observed which was mediated by activation of the redox-sensitive p38 mitogen-activated protein kinase (p38 MAPK). Furthermore, we observed increased production of reactive oxygen species (ROS) in Stamp2-/- hearts after I/R, which is the likely cause for p38 MAPK activation. Although myocardial macrophage numbers were not affected by Stamp2 deficiency after I/R, augmented myocardial infiltration by polymorphonuclear neutrophils (PMN) was observed, which coincided with enhanced myeloperoxidase (MPO) plasma levels. Primary PMN isolated from Stamp2-/- animals exhibited a proinflammatory phenotype characterized by enhanced nuclear factor (NF)-κB activity and MPO secretion. To prove the critical role of PMN for the observed phenotype after I/R, antibody-mediated PMN depletion was performed in Stamp2 -/- mice which reduced deterioration of LV function and adverse structural remodeling to WT levels. These data indicate a novel role of Stamp2 as an anti-inflammatory regulator of PMN and fibroblast-to-myofibroblast transdifferentiation in myocardial I/R injury.

Homozygous Smpd1 deficiency aggravates brain ischemia/ reperfusion injury by mechanisms involving polymorphonuclear neutrophils, whereas heterozygous Smpd1 deficiency protects against mild focal cerebral ischemia

By cleaving sphingomyelin into ceramide, which is an essential component of plasma membrane microdomains, acid sphingomyelinase (Asm) pivotally controls cell signaling. To define how the activation of the Asm/ceramide pathway, which occurs within seconds to minutes upon stress stimuli, influences brain ischemia/reperfusion (I/R) injury, we exposed male and female wildtype mice carrying both alleles of Asm’s gene sphingomyelinase phosphodiesterase-1 (Smpd1+/+), heterozygously Asm-deficient mice (Smpd1+/−) and homozygously Asm-deficient mice (Smpd1−/−) of different age (8, 12 or 16 weeks) to 30, 60 or 90 min intraluminal middle cerebral artery occlusion (MCAO). For studying the contribution of brain-invading polymorphonuclear neutrophils (PMN) to I/R injury, PMNs were depleted by delivery of a PMN-specific Ly6G antibody. In male and female mice exposed to 30 min, but not 60 or 90 min MCAO, homozygous Smpd1−/− consistently increased I/R injury, blood–brain barrier permeability and brain leukocyte and PMN infiltration, whereas heterozygous Smpd1+/− reduced I/R injury. Increased abundance of the intercellular leukocyte adhesion molecule ICAM-1 was noted on cerebral microvessels of Smpd1−/− mice. PMN depletion by anti-Ly6G delivery prevented the exacerbation of I/R injury in Smpd1−/− compared with wildtype mice and reduced brain leukocyte infiltrates. Our results show that Asm tempers leukocyte entry into the reperfused ischemic brain, thereby attenuating I/R injury.

Mesenchymal Stromal Cell-Derived Small Extracellular Vesicles Induce Ischemic Neuroprotection by Modulating Leukocytes and Specifically Neutrophils.

Background and Purpose- Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) were shown to induce neurological recovery after focal cerebral ischemia in rodents and to reverse postischemic lymphopenia in peripheral blood. Since peripheral blood cells, especially polymorphonuclear neutrophils (PMNs), contribute to ischemic brain injury, we analyzed brain leukocyte responses to sEVs and investigated the role of PMNs in sEV-induced neuroprotection. Methods- Male C57Bl6/j mice were exposed to transient intraluminal middle cerebral artery occlusion. After reperfusion, vehicle or sEVs prepared from conditioned media of MSCs raised from bone marrow samples of 3 randomly selected healthy human donors were intravenously administered. sEVs obtained from normoxic and hypoxic MSCs were applied. PMNs were depleted in vehicle and MSC-sEV-treated mice. Neurological deficits, ischemic injury, blood-brain barrier integrity, peripheral blood leukocyte responses, and brain leukocyte infiltration were evaluated over 72 hours. Results- sEV preparations of all 3 donors collected from normoxic MSCs significantly reduced neurological deficits. Preparations of 2 of these donors significantly decreased infarct volume and neuronal injury. sEV-induced neuroprotection was consistently associated with a decreased brain infiltration of leukocytes, namely of PMNs, monocytes/macrophages, and lymphocytes. sEVs obtained from hypoxic MSCs (1% O2) had similar effects on neurological deficits and ischemic injury as MSC-sEVs obtained under regular conditions (21% O2) but also reduced serum IgG extravasation-a marker of blood-brain barrier permeability. PMN depletion mimicked the effects of MSC-sEVs on neurological recovery, ischemic injury, and brain PMN, monocyte, and lymphocyte counts. Combined MSC-sEV administration and PMN depletion did not have any effects superior to PMN depletion in any of the readouts examined. Conclusions- Leukocytes and specifically PMNs contribute to MSC-sEV-induced ischemic neuroprotection. Individual MSC-sEV preparations may differ in their neuroprotective activities. Potency assays are urgently needed to identify their therapeutic efficacy before clinical application. Visual Overview- An online visual overview is available for this article.

Beneficial Role of Neutrophils Through Function of Lactoferrin After Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) is a devastating disease with a 30-day mortality of ~50%. There are no effective therapies for ICH. ICH results in brain damage in 2 major ways: through the mechanical forces of extravasated blood and then through toxicity of the intraparenchymal blood components including hemoglobin/iron. LTF (lactoferrin) is an iron-binding protein, uniquely abundant in polymorphonuclear neutrophils (PMNs). After ICH, circulating blood PMNs enter the ICH-afflicted brain where they release LTF. By virtue of sequestrating iron, LTF may contribute to hematoma detoxification. ICH in mice was produced using intrastriatal autologous blood injection. PMNs were depleted with intraperitoneal administration of anti-Ly-6G antibody. Treatment of mouse brain cell cultures with lysed RBC or iron was used as in vitro model of ICH. LTF mRNA was undetectable in the mouse brain, even after ICH. Unlike mRNA, LTF protein increased in ICH-affected hemispheres by 6 hours, peaked at 24 to 72 hours, and remained elevated for at least a week after ICH. At the single cell level, LTF was detected in PMNs in the hematoma-affected brain at all time points after ICH. We also found elevated LTF in the plasma after ICH, with a temporal profile similar to LTF changes in the brain. Importantly, mrLTF (recombinant mouse LTF) reduced the cytotoxicity of lysed RBC and FeCl3 to brain cells in culture. Ultimately, in an ICH model, systemic administration of mrLTF (at 3, 24, and 48 hours after ICH) reduced brain edema and ameliorated neurological deficits caused by ICH. mrLTF retained the benefit in reducing behavioral deficit even with 24-hour treatment delay. Interestingly, systemic depletion of PMNs at 24 hours after ICH worsened neurological deficits, suggesting that PMN infiltration into the brain at later stages after ICH could be a beneficial response. LTF delivered to the ICH-affected brain by infiltrating PMNs may assist in hematoma detoxification and represent a powerful potential target for the treatment of ICH.

Vaginal Heparan Sulfate Linked to Neutrophil Dysfunction in the Acute Inflammatory Response Associated with Experimental Vulvovaginal Candidiasis.

ABSTRACTDespite acute inflammation by polymorphonuclear neutrophils (PMNs) during vulvovaginal candidiasis (VVC), clearance of Candida fails to occur. The purpose of this study was to uncover the mechanism of vaginal PMN dysfunction. Designs included assessing PMN migration, proinflammatory mediators, and tissue damage (by analysis of the activity of lactate dehydrogenase [LDH]) in mice susceptible (C3H/HeN-C57BL/6) or resistant (CD-1) to chronic VVC (CVVC-S or CVVC-R) and testing morphology-specific Candida albicans strains under conditions of preinduced PMN migration (CVVC-S mice) or PMN depletion (CVVC-R mice). In vitro designs included evaluation of C. albicans killing by elicited vaginal or peritoneal PMNs in standard or vaginal conditioned medium (VCM). Results showed that despite significant migration of PMNs and high levels of vaginal beta interleukin-1 (IL-1β) and alarmin S100A8, CVVC-S mice failed to reduce vaginal fungal burden irrespective of morphology or whether PMNs were present pre- or postinoculation, and had high LDH levels. In contrast, CVVC-R mice had reduced fungal burden and low LDH levels following PMN recruitment and IL-1β/S100A8 production, but maintained colonization in the absence of PMNs. Elicited vaginal and peritoneal PMNs showed substantial killing activity in standard media or VCM from CVVC-R mice but not in VCM from CVVC-S mice. The inhibitory effect of VCM from CVVC-S mice was unaffected by endogenous or exogenous estrogen and was ablated following depletion/neutralization of Mac-1 ligands using Mac-1+/+ PMNs or recombinant Mac-1. Heparan sulfate (HS) was identified as the putative inhibitor as evidenced by the rescue of PMN killing following heparanase treatment of VCM, as well as by inhibition of killing by purified HS. These results suggest that vaginal HS is linked to PMN dysfunction in CVVC-S mice as a competitive ligand for Mac-1.

Antibodies to major histocompatibility complex class II antigens directly prime neutrophils and cause acute lung injury in a two-event in vivo rat model.

Transfusion-related acute lung injury (TRALI) is a significant cause of mortality, especially after transfusions containing antibodies to major histocompatibility complex (MHC) class II antigens. We hypothesize that a first event induces both 1) polymorphonuclear neutrophils (PMNs) to express MHC class II antigens, and 2) activation of the pulmonary endothelium, leading to PMN sequestration, so that the infusion of specific MHC class II antibodies to these antigens causes PMN-mediated acute lung injury (ALI). Rats were treated with saline (NS), endotoxin (lipopolysaccharide [LPS]), or cytokines (interferon-γ [IFNγ], macrophage colony-stimulating factor [MCSF], tumor necrosis factor-α [TNFα]); the PMNs were isolated; and the surface expression of the MHC class II antigen OX6 and priming by OX6 antibodies were measured by flow cytometry or priming assays. A two-event model of ALI was completed with NS, LPS, or IFNγ/MCSF/TNFα (first events) and the infusion of OX6 (second event). Compared with NS incubation, rats treated with either LPS or IFNγ/MCSF/TNFα exhibited OX6 PMN surface expression, OX6 antibodies primed the formyl-methionyl-leucyl phenylalanine (fMLF)-activated respiratory burst, and PMN sequestration was increased. OX6 antibody infusion into LPS-incubated or IFNγ/MCSF/TNFα-incubated rats elicited ALI, the OX6 antibody was present on the PMNs, and PMN depletion abrogated ALI. Proinflammatory first events induce PMN MHC class II surface expression, activation of the pulmonary endothelium, and PMN sequestration such that the infusion of cognate antibodies precipitates TRALI.

Female-Specific Downregulation of Tissue Polymorphonuclear Neutrophils Drives Impaired Regulatory T Cell and Amplified Effector T Cell Responses in Autoimmune Dry Eye Disease.

Immune-driven dry eye disease primarily affects women; the cause for this sex-specific prevalence is unknown. Polymorphonuclear neutrophils (PMN) have distinct phenotypes that drive inflammation but also regulate lymphocytes and are the rate-limiting cell for generating anti-inflammatory lipoxin A4 (LXA4). Estrogen regulates the LXA4 circuit to induce delayed female-specific wound healing in the cornea. However, the role of PMNs in dry eye disease remains unexplored. We discovered an LXA4-producing tissue PMN population in the corneal limbus, lacrimal glands, and cervical lymph nodes of healthy male and female mice. These tissue PMNs, unlike inflammatory PMNs, expressed a highly amplified LXA4 circuit and were sex-specifically regulated during immune-driven dry eye disease. Desiccating stress in females, unlike in males, triggered a remarkable decrease in lymph node PMN and LXA4 formation that remained depressed during dry eye disease. Depressed lymph node PMN and LXA4 in females correlated with an increase in effector T cells (Th1 and Th17), a decrease in regulatory T cells (Treg), and increased dry eye pathogenesis. Ab depletion of tissue PMN abrogated LXA4 formation in lymph nodes, as well as caused a marked increase in Th1 and Th17 cells and a decrease in Tregs. To establish an immune-regulatory role for PMN-derived LXA4 in dry eye, females were treated with LXA4. LXA4 treatment markedly inhibited Th1 and Th17 and amplified Treg in draining lymph nodes, while reducing dry eye pathogenesis. These results identify female-specific regulation of LXA4-producing tissue PMN as a potential key factor in aberrant effector T cell activation and initiation of immune-driven dry eye disease.

ADAMTS13 Regulates Neutrophil Recruitment in a Mouse Model of Invasive Pulmonary Aspergillosis

Von Willebrand factor (VWF) is secreted as an acute phase protein during inflammation. The main mechanism regulating the size and prothrombotic activity of VWF is the specific proteolytic activity of ADAMTS-13. To determine the relevance of this regulatory pathway for the innate inflammatory response by polymorphonuclear neutrophils (PMN), we employed a mouse model of invasive pulmonary aspergillosis (IPA) where PMN functionality is crucial for fungal clearance and survival. IPA was induced by intratracheal application of Aspergillus fumigatus conidia in wild-type (129/Sv/Pas) or Adamts13 deficient (Adamts13-/-) mice. After PMN depletion using a anti-Gr-1 specific antibody, all mice infected with Aspergillus fumigatus conidia developed neutropenia and succumbed due to lethal IPA. In contrast, all undepleted wild-type mice survived the infection. Interestingly, Aspergillus fumigatus infection in Adamts13-/- mice was lethal in 20% of the animals displaying a more severe course of IPA, as indicated by an increased fungal burden in lung homogenates along with increased levels of albumin and the inflammatory mediators IL-1β, IL-6, TNF-α, KC and MCP-1 in the bronchio-alveolar lavage fluid (BALF) compared to wild-type controls. Beyond this, we observed a decreased number of PMN in BALF of infected Adamts13-/- mice compared to wild-type mice. Lung histology sections demonstrated a more pronounced perivascular leukocyte infiltration in further support of a dysregulated inflammatory response in Adamts13-/- mice. Importantly, we observed no general defect in the activation of neutrophil effector functions as demonstrated by the normal induction of the oxidative burst, phagocytosis, degranulation, L-selectin shedding and apoptosis in response to formyl-peptide receptor agonists or exposure to Aspergillus fumigatus conidia or hyphae in vitro. Therefore, we conclude that the proteolytic regulation of VWF by ADAMTS-13 in an important mechanism to control PMN recruitment in the regulation of the innate inflammatory response in invasive fungal infections. DisclosuresRadsak:Celgene: Research Funding.

Roles of the Hemostatic System and Neutrophils in Liver Injury From Co-exposure to Amiodarone and Lipopolysaccharide

It has been demonstrated that co-treatment of rats with amiodarone (AMD) and bacterial lipopolysaccharide (LPS) produces idiosyncrasy-like liver injury. In this study, the hypothesis that the hemostatic system and neutrophils contribute to AMD/LPS-induced liver injury was explored. Rats were treated with AMD (400 mg/kg, ip) or vehicle and 16 h later with LPS (1.6×10⁶ endotoxin units/kg, iv) or saline (Sal). AMD did not affect the hemostatic system by itself but significantly potentiated LPS-induced coagulation activation and fibrinolysis impairment. Increased hepatic fibrin deposition and subsequent hypoxia were observed only in AMD/LPS-treated animals, starting before the onset of liver injury. Administration of anticoagulant heparin abolished AMD/LPS-induced hepatic fibrin deposition and reduced AMD/LPS-induced liver damage. Polymorphonuclear neutrophils (PMNs) accumulated in liver after treatment with LPS or AMD/LPS, but PMN activation was only observed in AMD/LPS-treated rats. Rabbit anti-rat PMN serum, which reduced accumulation of PMNs in liver, prevented PMN activation and attenuated AMD/LPS-induced liver injury in rats. PMN depletion did not affect hepatic fibrin deposition. Anticoagulation prevented PMN activation without affecting PMN accumulation. In summary, both the hemostatic system alteration and PMN activation contributed to AMD/LPS-induced liver injury in rats, in which fibrin deposition was critical for the activation of PMNs.

Neutrophil–cytokine interactions in a rat model of sulindac-induced idiosyncratic liver injury

Previous studies indicated that lipopolysaccharide (LPS) interacts with the nonsteroidal anti-inflammatory drug sulindac (SLD) to produce liver injury in rats. In the present study, the mechanism of SLD/LPS-induced liver injury was further investigated. Accumulation of polymorphonuclear neutrophils (PMNs) in the liver was greater in SLD/LPS-cotreated rats compared to those treated with SLD or LPS alone. In addition, PMN activation occurred specifically in livers of rats cotreated with SLD/LPS. The hypothesis that PMNs and proteases released from them play critical roles in the hepatotoxicity was tested. SLD/LPS-induced liver injury was attenuated by prior depletion of PMNs or by treatment with the PMN protease inhibitor, eglin C. Previous studies suggested that tumor necrosis factor-α (TNF) and the hemostatic system play critical roles in the pathogenesis of liver injury induced by SLD/LPS. TNF and plasminogen activator inhibitor-1 (PAI-1) can contribute to hepatotoxicity by affecting PMN activation and fibrin deposition. Therefore, the role of TNF and PAI-1 in PMN activation and fibrin deposition in the SLD/LPS-induced liver injury model was tested. Neutralization of TNF or inhibition of PAI-1 attenuated PMN activation. TNF had no effect on PAI-1 production or fibrin deposition. In contrast, PAI-1 contributed to fibrin deposition in livers of rats treated with SLD/LPS. In summary, PMNs, TNF and PAI-1 contribute to the liver injury induced by SLD/LPS cotreatment. TNF and PAI-1 independently contributed to PMN activation, which is critical to the pathogenesis of liver injury. Moreover, PAI-1 contributed to liver injury by promoting fibrin deposition.

Neutrophils, Not Monocyte/Macrophages, are the Major Splenic Source of Postburn IL-10

Burn induces myeloid-derived suppressor cells (MDSCs), a heterogeneous population of immature polymorphonuclear neutrophils (PMNs) and monocytes, which protect against infection. Previous work from our laboratory demonstrated that inflammatory monocytes (iMos) were the major MDSC source of TNF-α in the postburn spleen, and we hypothesized that they were also the major source of postburn IL-10. To test this hypothesis, we examined cytokine production by postburn CCR2 knockout (KO) mice, which have fewer iMos than burn wild-type (WT) splenocytes, but equal numbers of PMNs and F4/80 macrophages. Using cell sorting and/or intracellular cytokine techniques, we examined IL-10 production by postburn PMNs and iMos. Finally, we compared IL-10 production by postburn PMNs and iMos with culture-derived MDSCs. Splenocytes from postburn CCR2 KO mice produced less IL-6 and TNF-α than WT burn splenocytes in response to LPS, but KO and WT burn splenocytes produced equal amounts of IL-10 in response to peptidoglycan. Depletion of PMNs from postburn splenocytes led to reductions in IL-10 and increases in IL-6 and TNF-α in response to peptidoglycan, but not in response to LPS. Sorting or intracellular cytokine techniques gave consistent results: Burn PMNs made more IL-10 than sham PMNs and also more IL-10 than burn or sham iMos. Polymorphonuclear neutrophil and iMos subpopulations from culture-derived MDSCs produced the same cytokine profiles in response to LPS and peptidoglycan as did the PMNs and iMos from postburn spleens: PMNs made IL-10, whereas iMos made IL-6. Finally, LPS-induced mortality of burn mice was made worse by anti-Gr-1 depletion of all PMNs and 66% of iMos from burn mice. This suggests that PMNs play a primarily anti-inflammatory role in vitro and in vivo.

Role of Neutrophils, MyD88-Mediated Neutrophil Recruitment, and Complement in Antibody-Mediated Defense againstPseudomonas aeruginosaKeratitis

Purpose. Ulcerative keratitis due to Pseudomonas aeruginosa is a sight-threatening disease leading to loss of vision due to corneal inflammation. A human IgG1 monoclonal antibody (MAb F429) to the alginate capsule significantly reduces pathology and bacterial burdens in the cornea when applied topically starting 8 hours post-infection. The purpose of this study was to determine whether local polymorphonuclear neutrophils (PMN) recruitment and complement were important lipopolysaccharide co-factors in MAb F429-mediated reductions in P. aeruginosa tissue levels and corneal pathology. Methods. MyD88 knock-out mice unable to recruit PMN to tissues, mice depleted of PMNs, or mice depleted of complement component C3 were topically treated with MAb F429 starting 8 hours post-infection and evaluated for bacterial levels and corneal pathology 48 hours after infection with two P. aeruginosa isolates. Results. An inability to recruit PMN or systemic PMN depletion plus topical application of MAb F429 resulted in less pathology in the eye, but bacterial burdens were markedly increased in the cornea, brains, and spleens of these mice, indicative of systemic spread. Intraperitoneal injection of cobra venom factor (CVF) reduced C3 levels in the cornea approximately 40%, which did not change the beneficial effects of MAb F429. Both systemic injection and topical application of CVF reduced local C3 levels >60%, which eliminated MAb-mediated reductions in corneal pathology and bacterial levels. Conclusions. PMN recruitment and complement are both needed for maximal in vivo efficacy of MAb F429 in therapeutically treating P. aeruginosa keratitis, and attempts to reduce pathology by limiting PMN influx could have consequences leading to more extensive local and systemic infection.

Neutrophils contribute to intracerebral haemorrhages after treatment with recombinant tissue plasminogen activator following cerebral ischaemia

Polymorphonuclear neutrophils (PMNs) contribute to the vascular damage caused by transient cerebral ischaemia. Here we have evaluated the role of PMNs in intracerebral haemorrhage (ICH) induced in a model of thrombolysis with recombinant tissue plasminogen activator (t-PA) during the acute phase of cerebral ischaemia. The middle cerebral artery (MCA) of male spontaneously hypertensive rats was occluded for 1 h followed by reperfusion and, 5 h later, infusion of thrombolytic products (generated in vitro by t-PA on autologous clots). Effects of pretreatment (before the MCA occlusion) with vinblastine (4 days before; 0.5 mg.kg(-1)), monoclonal anti-neutrophil antibody (mAbRP3; 12 h, 0.3 mg.kg(-1)) or saline on ICH, neutrophil infiltration, MCA vascular reactivity and brain infarct volume were assessed, 24 h after the beginning of reperfusion. Depletion of circulating neutrophils significantly reduced t-PA-induced ICH (vinblastine, 4.6 +/- 1.0; mAbRP3, 5.2 +/- 1.0 vs. saline, 10.8 +/- 2.7 haemorrhages; P < 0.05). This depletion was associated with a decrease in cerebral infiltration by neutrophils and a decrease of endothelium-dependent, vascular dysfunction in isolated MCA, induced by the ischaemia/reperfusion and t-PA treatment. Brain infarct volume was significantly decreased after vinblastine treatment (159 +/- 13 mm(3) vs. 243 +/- 16 mm(3) with saline; P < 0.01) but not after depletion with mAbRP3 (221 +/- 22 mm(3)). Our results showed that pharmacological depletion of PMNs prevented t-PA-induced ICH, in parallel with a decrease in cerebral infiltration by PMNs and a decreased endothelial dysfunction in cerebral blood vessels.

Dynamics of neutrophil extravasation and vascular permeability are uncoupled during aseptic cutaneous wounding

Transport of macromolecules and transmigration of leukocytes across vascular endothelium are regulated by a tight molecular junction, but the mechanisms by which these two inflammatory events are differentially controlled in time and magnitude during aseptic cutaneous wounding remain elusive. A real-time fluorescence imaging technique was developed to simultaneously track influx of Alexa 680-labeled albumin and genetically tagged enhanced green fluorescent protein-neutrophils [polymorphonuclear neutrophils (PMN)] within the wound bed. Vascular permeability increased approximately threefold more rapidly than the rate of PMN influx, reaching a maximum at 12 h, on the order of approximately 0.15% per minute versus approximately 0.05% per minute for PMN influx, which peaked at 18 h. Systemic depletion of PMN with antibody blocked their extravasation to the wound but did not alter the increase in vascular permeability. In contrast, pretreatment with antiplatelet GPIb decreased permeability by 25% and PMN influx by 50%. Hyperpermeability stimulated by the endothelium-specific agonists VEGF or thrombin at 24 h postwounding was completely inhibited by blocking Rho-kinase-dependent signaling, whereas less inhibition was observed at 1 h and neutrophil influx was not perturbed. These data suggest that in aseptic wounds, the endothelium maintains a tight junctional barrier to protein leakage that is independent of neutrophil transmigration, partially dependent on circulating platelets, and associated with Rho-kinase-dependent signaling.

Substance P Delays Apoptosis, Enhancing Keratitis afterPseudomonas aeruginosaInfection

Apoptosis was examined after Pseudomonas aeruginosa corneal infection in C57BL/6 (B6, susceptible) and BALB/c (resistant) mice. TUNEL staining, real-time RT-PCR, polymorphonuclear neutrophils (PMNs) and macrophage (Mphi) depletion, and immunostaining were used. Intense TUNEL staining was seen in BALB/c versus B6 cornea at 1 versus 3 days after infection (PI) and correlated with mRNA levels for caspase-3. TUNEL staining (with or without PMN depletion) and PMN immunostaining revealed the PMN as the major apoptotic cell for both groups. Next, B6 mice with high corneal levels of the antiapoptosis neuropeptide, substance P (SP), were treated with the SP antagonist, Spantide I (with/without Mphi depletion), resulting in earlier apoptosis and diminished disease only when M(phi)s were present. SP interactions with M(phi)s were explored further by eliciting cells from both groups and stimulating them with lipopolysaccharide (LPS), with or without SP. LPS with SP treatment decreased the number of apoptotic M(phi)s in B6 but not BALB/c mice and correlated with reduced mRNA expression of NK-1R (major SP receptor) on BALB/c cells. In addition, mRNA expression for IL-12 was upregulated in LPS-stimulated B6 M(phi)s, although cells from BALB/c mice expressed more IL-10. These studies provide evidence that PMN apoptosis is delayed in the cornea of B6 versus BALB/c mice after bacterial infection; that in B6 mice, blocking SP interaction with the NK-1R promotes earlier apoptosis and improves disease outcome; that M(phi)s regulate PMN apoptosis; and that M(phi)s from B6 versus BALB/c mice differ in expression of the NK-1R and cytokines produced after LPS challenge.

Neutrophil Interaction with the Hemostatic System Contributes to Liver Injury in Rats Cotreated with Lipopolysaccharide and Ranitidine

Cotreatment of rats with nontoxic doses of ranitidine (RAN) and lipopolysaccharide (LPS) causes liver injury, and this drug-inflammation interaction might be a model for idiosyncratic adverse drug responses in humans. Both polymorphonuclear neutrophils (PMNs) and the hemostatic system have been shown to be important in the injury. We tested the hypothesis that PMNs cause liver injury by interacting with the hemostatic system and producing subsequent hypoxia. In rats cotreated with LPS/RAN, PMN depletion by anti-PMN serum reduced fibrin deposition and hypoxia in the liver. PMN depletion also reduced the plasma concentration of active plasminogen activator inhibitor-1 (PAI-1), a major down-regulator of the fibrinolytic system. This suggests that PMNs promote fibrin deposition by increasing PAI-1 concentration. PMNs were activated in the livers of LPS/RAN-cotreated rats as evidenced by increased staining for hypochlorous acid-modified proteins generated by the myeloperoxidase-hydrogen peroxide-chloride system of activated phagocytes. Antiserum against the PMN adhesion molecule CD18 protected against LPS/RAN-induced liver injury. Because CD18 is important for PMN transmigration and activation, these results suggest that PMN activation is required for the liver injury. Furthermore, anti-CD18 serum reduced biomarkers of hemostasis and hypoxia, suggesting the necessity for PMN activation in the interaction between PMNs and the hemostatic system/hypoxia. Liver injury, liver fibrin, and plasma PAI-1 concentration were also reduced by eglin C, an inhibitor of proteases released by activated PMNs. In summary, PMNs are activated in LPS/RAN-cotreated rats and participate in the liver injury in part by contributing to hemostasis and hypoxia.

Role of polymorphonuclear neutrophils (PMNs) and NK cells in the protection conferred by different vaccines against Chlamydophila abortus infection

Ovine enzootic abortion (OEA) is caused by Chlamydophila abortus, an intracellular bacterium which acts by infecting the placenta, causing abortion in the last term of gestation. The main prevention strategy against OEA is the vaccination of flocks. An effective vaccine against C. abortus must induce a Th1-like specific immune response, which is characterized by the early production of IFN-γ and the activation of CD8 +T cells. Moreover, vaccine effectiveness could be modulated by the functioning of the innate immunity. The purpose of this study was to ascertain how polymorphonuclear neutrophils (PMNs) and NK cells might influence vaccine-induced protection. The live attenuated 1B vaccine and two inactivated experimental vaccines, adjuvated with aluminium hydroxide (AH) or QS-21 (QS), were used in PMN-depleted or NK cell-depleted mice. For PMN depletion, RB6-8C5 monoclonal antibody, which recognizes GR1 +receptors (Robben, P.M., LaRegina, M., Kuziel, W.A., Sibley, L.D. 2005. Recruitment of Gr-1 + monocytes is essential for control of acute toxoplasmosis. The Journal of Experimental Medicine 201, 1761–1769.) was used, while for NK cell-depletion the anti-asialo GM1 polyclonal antibody was used. The depletion of PMNs caused 100% mortality in non-vaccinated mice (NV) and 60% mortality in the AH-vaccinated mice by day 10 p.i., while both groups showed a significant increase in their bacterial burden in the liver by day 4 p.i. The depletion of NK cells caused mortality only in the NV group (50% by day 10 p.i.), although this group and the 1B vaccinated mice showed an increased bacterial burden in the liver at day 4 p.i. Our results suggest that the importance of PMNs in inactivated vaccines depends on the adjuvant chosen. The results also demonstrated that the importance of NK cells is greater in live vaccines than in inactivated vaccines.

Bacterial Lipoprotein Induces Resistance to Gram-Negative Sepsis in TLR4-Deficient Mice via Enhanced Bacterial Clearance

TLRs are highly conserved pathogen recognition receptors. As a result, TLR4-deficient C3H/HeJ mice are highly susceptible to Gram-negative sepsis. We have previously demonstrated that tolerance induced by bacterial lipoprotein (BLP) protects wild-type mice against polymicrobial sepsis-induced lethality. In this study, we assessed whether pretreatment of C3H/HeJ mice with BLP could induce resistance to a subsequent Gram-negative Salmonella typhimurium infection. Pretreatment with BLP resulted in a significant survival benefit in TLR4-deficient C3H/HeJ mice (p < 0.0002 vs control C3H/HeJ) after challenge with live S. typhimurium (0.25 x 10(6) CFU/mouse). This survival benefit was associated with enhanced bacterial clearance from the circulation and in the visceral organs (p < 0.05 vs control C3H/HeJ). Furthermore, pretreatment with BLP resulted in significant increases in complement receptor type 3 (CR3) and FcgammaIII/IIR expression on polymorphonuclear neutrophils (PMNs) and macrophages (p < 0.05 vs control C3H/HeJ). There was impaired bacterial recognition and phagocytosis in TLR4-deficient mice compared with wild-type mice. However, a significant augmented uptake, ingestion, and intracellular killing of S. typhimurium by PMNs and peritoneal macrophages was evident in BLP-pretreated C3H/HeJ mice (p < 0.05 vs control C3H/HeJ). An up-regulation of inducible NO synthase and increased production of intracellular NO were observed in peritoneal macrophages from BLP-pretreated C3H/HeJ mice (p < 0.05 vs control C3H/HeJ). Depletion of PMNs did not diminish the beneficial effects of BLP with regard to both animal survival and bacterial clearance. These results indicate that BLP, a TLR2 ligand, protects highly susceptible TLR4-deficient mice from Gram-negative sepsis via enhanced bacterial clearance.