Number Of Polymorphonuclear Neutrophils Research Articles

Characterization of oral polymorphonuclear neutrophils in periodontitis patients: a case-control study

BackgroundMaintaining oral health is a continuous and dynamic process that also involves the immune system. Polymorphonuclear neutrophils (PMNs) migrate from blood circulation and become apparent in the oral fluid. Controversies exist regarding the specific role of the oral PMNs (oPMNs) in the presence of chronic oral inflammation, such as periodontitis. In this study we characterized cell counts, activation status, apoptosis, and reactive oxygen species (ROS) generation by oPMNs and circulatory (cPMNs), and the salivary protease activity, in subjects with and without periodontitis.MethodsVenous blood and oral rinse samples were obtained from 19 patients with untreated periodontitis and 16 control subjects for PMN isolation. Apoptosis and expression of cell activation markers CD11b, CD63, and CD66b were analyzed using flow cytometry. Constitutive ROS generation was detected using dihydrorhodamine123. Additionally, ROS production in response to stimulation was evaluated in samples incubated with 10 μM phorbol myristate acetate (PMA) or Fusobacterium nucleatum. Total protease activity was measured using substrate PEK-054.ResultsPeriodontitis patients presented with over 4 times higher oPMN counts compared to controls (p = 0.007), which was a predictor for the total protease activity (r2 = 0.399, P = 0.007). More oPMNs were apoptotic in periodontitis patients compared to the controls (P = 0.004). All three activation markers were more expressed on the oPMNs compared to the cPMNs (p < 0.05), and a higher expression of CD11b on the oPMNs from periodontitis patients was observed compared to the control subjects (P = 0.024). Constitutive ROS production per oPMN was higher compared to the cPMN (P < 0.001). Additional analysis showed that the oPMNs retained their ability to respond to stimulation, with no apparent differences between the periodontitis and control subjects.ConclusionsHigher numbers of oral PMNs, being more apoptotic and having increased levels of degranulation markers were found in periodontitis compared to periodontal health. However, since the oPMNs in periodontitis were responsive to ex vivo stimulation, we conclude that the oPMNs are active in the oral ecosystem. It is currently unknown whether the oPMN counts, which correlated with the detected protease levels, are detrimental in the long term for the oral mucosa integrity.Trial registrationThis study was retrospectively registered at the ISRCTN registry (trial ID ISRCTN15252886). Registration date August 11, 2017.

Neutrophils cause obstruction of eyelid sebaceous glands in inflammatory eye disease in mice.

Meibomian glands (MGs) are sebaceous glands of the eyelid margin that secrete lipids needed to avert tear evaporation and to help maintain ocular surface homeostasis. Obstruction of MGs or other forms of MG dysfunction can promote chronic diseases of the ocular surface. Although chronic eyelid inflammation, such as allergic eye disease, is an associated risk factor for obstructive MG dysfunction, it is not clear whether inflammatory processes contribute to the pathophysiology of MG obstruction. We show that polymorphonuclear neutrophils (PMNs) promoted MG obstruction in a chronic inflammatory model of allergic eye disease in mice. Analysis of leukocytes in tears of patients with MG dysfunction showed an increase in PMN numbers compared to healthy subjects. Moreover, PMN numbers in tears positively correlated with clinical severity of MG dysfunction. Our findings point to a role for PMNs in the pathogenesis and progression of MG dysfunction.

Experimental Study of the Protective Effect of Simvastatin on Lung Injury in Rats with Sepsis

Simvastatin, which is primarily prescribed to lower cholesterol, may also mitigate lung injury caused by sepsis, although the mechanisms remain elusive. This study aimed to evaluate the protective effect of simvastatin on acute lung injury in rats with sepsis and to investigate possible mechanisms. Male Wistar rats were pretreated with simvastatin (0.2μg/g) for 1week before cecal ligation and puncture. Treatment with simvastatin demonstrated significant decreases in the concentration of protein, TNF-α, IL-1β, IL-6, and lipocalin 2, and the number of polymorphonuclear neutrophils in bronchoalveolar lavage fluid in septic rats. In addition, simvastatin also reduced levels of Evans blue, malondialdehyde, 8-hydroxy-2'-deoxyguanosine, and wet/dry lung weight ratios, and increased the activity of superoxide dismutase in lung tissue. Furthermore, expression levels of TLR4, NF-κB p65, and active caspase-3 proteins and Bax mRNA were also decreased by simvastatin. H&E staining showed that severe lung injury occurred in the sepsis group and that lung injury was reduced by treatment with simvastatin. In conclusion, simvastatin improved endothelial permeability and mitigated the inflammatory response of lung tissue, the oxidative stress response, and cell apoptosis by inhibiting the TLR4/NF-κB signaling pathway, thereby alleviating sepsis-induced acute lung injury in rats.

α-Toxin Regulates Local Granulocyte Expansion from Hematopoietic Stem and Progenitor Cells in Staphylococcus aureus-Infected Wounds.

The immune response to Staphylococcus aureus infection in skin involves the recruitment of polymorphonuclear neutrophils (PMNs) from the bone marrow via the circulation and local granulopoiesis from hematopoietic stem and progenitor cells (HSPCs) that also traffic to infected skin wounds. We focus on regulation of PMN number and function and the role of pore-forming α-toxin (AT), a virulence factor that causes host cell lysis and elicits inflammasome-mediated IL-1β secretion in wounds. Infection with wild-type S. aureus enriched in AT reduced PMN recruitment and resulted in sustained bacterial burden and delayed wound healing. In contrast, PMN recruitment to wounds infected with an isogenic AT-deficient S. aureus strain was unimpeded, exhibiting efficient bacterial clearance and hastened wound resolution. HSPCs recruited to infected wounds were unaffected by AT production and were activated to expand PMN numbers in proportion to S. aureus abundance in a manner regulated by TLR2 and IL-1R signaling. Immunodeficient MyD88-knockout mice infected with S. aureus experienced lethal sepsis that was reversed by PMN expansion mediated by injection of wild-type HSPCs directly into wounds. We conclude that AT-induced IL-1β promotes local granulopoiesis and effective resolution of S. aureus-infected wounds, revealing a potential antibiotic-free strategy for tuning the innate immune response to treat methicillin-resistant S. aureus infection in immunodeficient patients.

Role of carbonyl sulfide in acute lung injury following limb ischemia/reperfusion in rats

ObjectiveTo investigate the effect of carbonyl sulfide (COS) on limb ischemia/reperfusion (I/R)-induced acute lung injury (ALI) and the associated mechanism in rats.MethodsALI was induced by bilateral hind limb I/R in Sprague–Dawley (SD) rats. Sixty-four SD rats were randomly divided into the control group, I/R group, I/R + COS group, and I/R + AIR group. We observed the survival rate of the rats and the morphological changes of lung tissues, and we measured the change in the lung coefficient, the expression levels of the intercellular adhesion factor-1 (ICAM-1) protein in lung tissue, the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-lβ, and interleukin (IL)-6 in both lung tissue and serum, and cell apoptosis.ResultsLimb I/R caused significant lung tissue damage. The number of polymorphonuclear neutrophil in alveolar septa, the expression level of the ICAM-1 protein in lung tissue, the expression levels of TNF-α, IL-1, and IL-6 in lung tissue and serum, the lung coefficient, and cell apoptosis all increased. When a low dose of COS gas was administered prior to limb I/R, the variation of the above indicators was significantly reduced, while an increase in the dose of COS did not reduce the lung injury but rather increased the mortality rate.ConclusionCarbonyl sulfide is another new gaseous signaling molecule, and a low dose of exogenous COS may play a protective role in I/R-induced ALI by acting as an anti-inflammatory agent by promoting the production of antioxidants and by inhibiting the expression of adhesion molecule proteins.

Apicidin mediated attenuation of MRSA virulence corresponds with quorum sensing inhibition and enhanced immune effector responses.

Abstract As the leading cause of infectious mortality in the United States, S. aureus-induced disease represents a major healthcare problem. The alarming rise of infections caused by virulent, antibiotic resistant strains, such as emerging methicillin-resistant S. aureus (MRSA) isolates, highlight the need for new interventions that inhibit MRSA pathogenicity and potentiate host defense responses. The expression of MRSA virulence factors is a function of agr-driven quorum sensing, making this system a promising target for anti-MRSA therapies. Through agr-reporter-based screens, a class of compounds, called apicidins, were found to inhibit quorum-sensing activity across MRSA isolates. To test the efficacy of apicidin in vivo, mice were challenged intradermally with 2×107 MRSA (+/−) 5 mg apicidin. The abatement MRSA virulence in the apicidin-treated group was demonstrated by reduced: weight loss, dermonecrosis and cutaneous bacterial burden. By challenging mice with an agr-reporter strain, we also found that the apicidin-mediated attenuation of MRSA pathogenesis corresponded with reduced quorum sensing activity in vivo. To evaluate apicidin’s impact upon anti-MRSA effector responses, we assessed polymorphonuclear neutrophil (PMN) accumulation and function at cutaneous sites of infection. Flow cytometric analysis revealed that apicidin increased the density of PMNs within infected wounds 24 hours post infection. In addition, we found that the number of PMNs that phagocytosed MRSA organisms in vivo was increased in lesional skin preparations from apicidin treated mice. Together, these results indicate that apicidin-mediated quorum quenching represents a novel strategy to limit MRSA virulence and promote host defense.

Inactivation of the miR-183/96/182 Cluster Decreases the Severity of Pseudomonas aeruginosa-Induced Keratitis.

PurposeThe microRNA-183/96/182 cluster (miR-183/96/182) plays important roles in sensory organs. Because the cornea is replete with sensory innervation, we hypothesized that miR-183/96/182 modulates the corneal response to bacterial infection through regulation of neuroimmune interactions.MethodsEight-week-old miR-183/96/182 knockout (ko) mice and their wild-type littermates (wt) were used. The central cornea of anesthetized mice was scarred and infected with Pseudomonas aeruginosa (PA), strain 19660. Corneal disease was graded at 1, 3, and 5 days postinfection (dpi). Corneal RNA was harvested for quantitative RT-PCR. Polymorphonuclear neutrophils (PMN) were enumerated by myeloperoxidase assays; the number of viable bacteria was determined by plate counts, and ELISA assays were performed to determine cytokine protein levels. A macrophage (Mϕ) cell line and elicited peritoneal PMN were used for in vitro functional assays.ResultsMicroRNA-183/96/182 is expressed in the cornea, and in Mϕ and PMN of both mice and humans. Inactivation of miR-183/96/182 resulted in decreased corneal nerve density compared with wt mice. Overexpression of miR-183/96/182 in Mϕ decreased, whereas knockdown or inactivation of miR-183/96/182 in Mϕ and PMN increased their capacity for phagocytosis and intracellular killing of PA. In PA-infected corneas, ko mice showed decreased proinflammatory neuropeptides such as substance P and chemoattractant molecules, MIP-2, MCP1, and ICAM1; decreased number of PMN at 1 and 5 dpi; increased viable bacterial load at 1 dpi, but decreased at 5 dpi; and markedly decreased corneal disease.ConclusionsMicroRNA-183/96/182 modulates the corneal response to bacterial infection through its regulation of corneal innervation and innate immunity.

Abstract 171: The Effect of Ischemia and Reperfusion and Tissue Plasminogen Activator on Cerebrovascular Function, Oxidative Stress and Polymorphonuclear Neutrophils in Ovariectomized Female Rats

Introduction: Stroke is a growing health problem in women. We previously showed that ischemia/reperfusion (I/R) in male rats reduced tone in middle cerebral arteries (MCA) but enhanced tone in parenchymal arterioles (PA). Tissue plasminogen activator (tPA) may contribute to vascular dysfunction, oxidative stress and inflammation. However, vascular dysfunction and the effect of tPA have not been studied in females. Hypothesis: I/R in ovariectomized (OVX) female rats impairs MCA and PA function differentially, increases vascular oxidative stress and intravascular polymorphonuclear neutrophils (PMN). tPA has an additive effect on the vascular dysfunction after I/R. Methods: OVX female rats underwent 2 h MCA filament occlusion (n=16) or sham surgery (n=17) with 30 min reperfusion. tPA (1 mg/kg, i.v.) or vehicle (Vh) was given 10 min prior to reperfusion. Myogenic tone and reactivity to nitric oxide synthase (L-NNA, 10-4M) and Rho kinase (Y27632, 10-8 to 10-5M) inhibition were measured in pressurized MCA and PA. MCA were stained for F-actin and 3-nitrotyrosine (NT), a marker of oxidative stress. Brain intravascular PMN were identified with myeloperoxidase and collagen IV using immunohistochemistry, and counted blinded to group. Results: In MCA, I/R decreased tone at 75 mmHg (Sham Vh: 26±3%, Sham tPA: 27±3% vs. MCAO Vh: 21±2%, MCAO tPA: 21±3%, P&lt;0.05) and reduced constriction to L-NNA (Sham Vh: 35±3%, Sham tPA: 34±2% vs. MCAO Vh: 20±3%, MCAO tPA: 25±3%, P&lt;0.01) with no effect on dilation to Y27632. In PA, myogenic tone (at 40 mmHg) and reactivity to L-NNA and Y27632 was not affected by I/R. PA were less sensitive to Y27632 than MCA after I/R with tPA (EC50: 2.0±0.5 vs. 0.8±0.1 μM, P&lt;0.05). F-actin and NT in the MCA were not affected by I/R or tPA. The number of PMN was small (&lt;0.8±0.3/mm2) in all groups but there was a trend to increased ipsilateral intravascular PMN after I/R with tPA. Conclusions: The differential effect of I/R on MCA vs. PA in OVX females on tone suggest parenchymal blood flow may be restricted during reperfusion that could increase perfusion deficits. tPA had no effect on tone in MCA and PA but might enhance intravascular PMN, implicated in no-reflow after I/R. Therapies to alleviate parenchymal vasoconstriction may improve stroke outcome in females.

Inhibition of bone marrow mesenchymal stem cell autologous transplantation on inflammation following rabbit corneal alkali burn

Background Ocular alkali burns leads to corneal ulcer and angiogenesis and even corneal opacity. There is still no ideal treatment method. Studies showed that mesenchymal stem cells (MSCs) can repair corneal wound in vivo, but the specific mechanism is still not clear. Objective This study aimed to observe the histopathological change after the early transplatation of bone marrow MSCs (BMSCs) for corneal alkali burn model in rabbits and explore the anti-inflammatory effects of MSCs after corneal alkali burn. Methods Bone marrow of 4 ml was collected from 2-3 month-old Japanese rabbit. BMSCs were isolated and cultured from the bone marrow of rabbits, and the third generation of cells were used in this study. Cultured cells were identified by morphology and the expressions of surface markers. Corneal alkali burn models were extablished in the right eyes of 24 rabbits by attaching the filter paper with 0. 1% NaOH at the central cornea for 30 seconds, and then the models were randomized into 2 groups. BMSCs suspension of 300 μl (concentration 5×106/μl) was subconjunctivally injected 1 hour after modeling in the BMSCs group, and equal volume of PBS was used in the same way in the PBS group. Corneal opacification was scored under the slim lamp microscope in 3, 14 and 28 days after injection. The polymorphonuclear neutrophils (PMNs) were counted by histopathological examination, and the expression of matrix metalloproteinase-2 (MMP-2) in the corneal tissue was evaluated by immunochemistry in various time points. The use and care of the rabbits followed the statement of ARVO. Results The rabbit BMSCs were plastic-adherent cells that exhibited a fibroblast-like shape. Cultrued cells highly expressed surface adhesion molecular markers CD29 and CD90 (99. 18% and 97. 94%) and lowly expressed hematopoietic cell markers CD34 and CD31 (0. 74% and 0. 15%). Opacification of cornea, defect of corneal epithelium, stromal edema and neovascularization appeared after modeling. In 14 days and 28 days after modeling, the opacification scores in the BMSCs group were 2. 37±0. 52 and 2. 25±0. 50, which were significantly lower than 3. 00±0. 53 and 3. 25±0. 50 in the PBS group (t=2. 376, 2. 828, both at P<0. 05). After subconjunctival injection, the number of PMNs was (34. 17±1. 85) /12 fields and (25. 64±3. 86)/12 fields in the BMSCs group, showing significant decrease in comparison with (42. 70±1. 54) /12 fields and (32. 67±1. 42)/12 fields in the PBS group (t=10. 021, 4. 832, both at P=0. 000). The expression levels of MMP-2 (A value) in cornea were 0. 388±0. 016 and 0. 384±0. 006 in the BMSCs group, with considerable decreases in comparison with 0. 438±0. 006 and 0. 412±0. 005 in the PBS group (t=10. 205, 13. 514, both at P=0. 000). Conclusions Early transplantation of BMSCs can arrest the occurrance of corneal ulcer by suppressing the infiltration of PMNs, alleviateing the inflammation reaction, downregulating the expression of MMP-2 in cornea and inhibiting the degradation of stromal collagen fibers. Key words: Bone marrow mesenchymal stem cell transplantation/methods; Biological markers; Burns, chemical/therapy; Corneal diseases/therapy; Neovascularization, pathologic/chemically induced; Inflammation/prevention & control; Matrix metalloproteinase; Disease models, animal

Brucella abortus Induces the Premature Death of Human Neutrophils through the Action of Its Lipopolysaccharide.

Most bacterial infections induce the activation of polymorphonuclear neutrophils (PMNs), enhance their microbicidal function, and promote the survival of these leukocytes for protracted periods of time. Brucella abortus is a stealthy pathogen that evades innate immunity, barely activates PMNs, and resists the killing mechanisms of these phagocytes. Intriguing clinical signs observed during brucellosis are the low numbers of Brucella infected PMNs in the target organs and neutropenia in a proportion of the patients; features that deserve further attention. Here we demonstrate that B. abortus prematurely kills human PMNs in a dose-dependent and cell-specific manner. Death of PMNs is concomitant with the intracellular Brucella lipopolysaccharide (Br-LPS) release within vacuoles. This molecule and its lipid A reproduce the premature cell death of PMNs, a phenomenon associated to the low production of proinflammatory cytokines. Blocking of CD14 but not TLR4 prevents the Br-LPS-induced cell death. The PMNs cell death departs from necrosis, NETosis and classical apoptosis. The mechanism of PMN cell death is linked to the activation of NADPH-oxidase and a modest but steadily increase of ROS mediators. These effectors generate DNA damage, recruitments of check point kinase 1, caspases 5 and to minor extent of caspase 4, RIP1 and Ca++ release. The production of IL-1β by PMNs was barely stimulated by B. abortus infection or Br-LPS treatment. Likewise, inhibition of caspase 1 did not hamper the Br-LPS induced PMN cell death, suggesting that the inflammasome pathway was not involved. Although activation of caspases 8 and 9 was observed, they did not seem to participate in the initial triggering mechanisms, since inhibition of these caspases scarcely blocked PMN cell death. These findings suggest a mechanism for neutropenia in chronic brucellosis and reveal a novel Brucella-host cross-talk through which B. abortus is able to hinder the innate function of PMN.

Morphofunctional state of neutrophils and cytokine status in patients with ovarian cancer

The purpose of the study was to evaluate the morphofunctional state of polymorphonuclear neutrophils and cytokine levels in the lysate of blood neutrophils of ovarian cancer patients. The absolute and the relative number of peripheral blood polymorphonuclear neutrophils in 120 primary ovarian cancer patients (III-IV clinical stage FIGO) and 80 healthy women were determined. The expression of CD11a, 11b, 15 and CD95 in polymorphonuclear neutrophils was assessed by fluorescent microscopy. Cytochemically the myeloperoxidase activity, the content of cationic proteins, the activity in the spontaneous NBT-test and phagocytic activity in polymorphonuclear neutrophils was determined. The amounts of cytokines TNF-α, IL-1β, 1Ra, 6, 18, and G-CSF in serum were measured by the enzyme immunoassay method. Rigidity and topology of native polymorphonuclear neutrophils were assessed by scanning probe microscopy. To detect the differences between the data obtained in the evaluation of ovarian cancer patients and healthy people, a non-parametric U-Mann-Whitney test was used. Reliable difference was considered at p ≤ 0.05. These studies found an increase of the total number of polymorphonuclear neutrophils in peripheral blood, while reducing their cytotoxicity parameters. We observed reduced myeloperoxidase activity and level of cationic proteins. Reducing in the levels of CD15 and CD95 expression on the background of increasing polymorphonuclear neutrophils activity in the spontaneous NBT-test may indicate a decrease in ability to complete phagocytosis. The dynamics of CD11a and CD11b expression in polymorphonuclear neutrophils was multidirectional, and the changes were not statistically significant. Polymorphonuclear neutrophils membrane stiffness decreased in stage IV ovarian cancer. We also observed the levels of IL-1β reduced up to 248.5 ± 9.00 pg / ml in stage III ovarian cancer, up to 230.9 ± 15.04 pg / ml in stage IV ovarian cancer vs 36.8 ± 10.74 pg / ml in control, reduced levels of 1Ra, and IL-18. We found that the level of IL-6 increased at stage III ovarian cancer up to 34.38 ± 9.74 pg / ml vs 7.66 ± 2.28 pg / ml in control, and the levels of TNF-α and G-CSF decreased. Thus, these results suggest a state of secondary immunodeficiency in patients with ovarian cancer at III-IV clinical stages of the 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.

Effect of recombinant bovine granulocyte colony-stimulating factor covalently bound to polyethylene glycol injection on neutrophil number and function in periparturient dairy cows

Dairy cows often experience decreased immune function around the time of calving, typified by impaired polymorphonuclear neutrophil (PMN) function and a transient neutropenia. This is associated with increased disease incidence, including mastitis, retained placenta, and metritis. In an attempt to improve PMN functional capacity during the periparturient period, we injected cows with recombinant bovine granulocyte colony-stimulating factor covalently bound to polyethylene glycol (PEG rbG-CSF) twice subcutaneously, about 6d before calving and within 24h after calving. Twenty-one cows in their second pregnancy were enrolled in this study and divided into 2 groups: PEG rbG-CSF treated (n=11) and saline-treated controls (n=10). The PMN numbers quickly and dramatically increased after PEG rbG-CSF administration and remained elevated through the end of the experiment (13d after calving). Exocytosis of myeloperoxidase by stimulated PMN, which is generally decreased in periparturient cows, was markedly increased by PEG rbG-CSF after injection. Higher myeloperoxidase exocytosis persisted for at least 10d after calving. The PMN superoxide anion release and phagocytosis activity did not differ between groups. Injection of PEG rbG-CSF was safe for cows, with no significant negative effects observed. The greatest single effect of PEG rbG-CSF administration was a dramatic increase in circulating numbers of PMN. The increased numbers of PMN ready to move to a site of infection early in the course of an infection may improve the ability of the cow to ward off clinical disease in the periparturient period.

The effect of intraperitoneally injection of S100A9 protein on the early immune function in mice with sepsis

Objective To investigate the effect of S100A9 protein when injected intraperitoneally on early immune function in mice with sepsis.Methods Experimental sepsis was induced by cecal ligation and puncture (CLP).36 C57BL/6 mice were randomly divided into 3 groups (n=12):a Sham group,a CLP group and a S100A9 group.1 h after the CLP operation,S100A9 protein was injected intraperitoneally into mice in the S100A9 group,while the same volume of normal saline was given to the mice of Sham and CLP group.24 h after surgery,the peripheral blood and peritoneal lavage fluid(PLF) were collected for detection of bacterial loads,while the livers and lungs were harvested from the living mice from each group.The levels of inflammatory factors tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) in blood were measured using enzyme linked immunosorbent assay.Counts of polymorphonuclear neutrophils (PMN),CD4+ and CD8+T cells in PLF were assayed using flow cytometry.The sections of liver and lung tisssues were stained by hematoxylin and eosin for histopathological examination.Results Bacterial loads in the blood and PLF after CLP were (179-±52)×103 and (247±50)×107 clonal formation unit(CFU) respectively,and they were significantly higher compared to Sham group (P＜0.01).Bacterial loads in blood and PLF from the S100A9 group were (20±8)×103 and (48±19)×107 CFU respectively,and they were significantly lower when compared to the CLP group (P＜0.01).The level of anti-inflammatory cytokine IL-10 in CLP group was (296±7) ng/L,and (907±93) ng/L in S100A9 group(P＜0.01),while the level of TNF-α was almost similar in these two groups.In CLP group only the number of PMN in PLF,as (767±142) cells/μl,increased significantly when compared with sham group(P＜0.01).Number of PMN in PLF increased even more in S100A9 group as (1 569±195) cells/μl(P＜0.01).Moreover,CD4+ and CD8+T cells in the S100A9 group also increased significantly when compared with CLP group,as (702±167) cells/μl and(287± 115) cells/μl (P＜0.05 and ＜0.01) respectively.Histopathology results also showed less severe injuries in liver and lung tissues in S100A9 group.Conclusions Intraperitoneally injection of S100A9 could enhance the ability of clearing pathogens in peritoneal cavity,inhibit the over-aggressive inflammatory response,and attenuate liver and lung injury in mice with sepsis. Key words: S100A9; Sepsis; Cecal ligation and puncture; Polymorphonuclear neutrophil; Inflammation

CELL BIOLOGY SYMPOSIUM: Perspectives: Possible roles of polymorphonuclear neutrophils in angiogenesis and lymphangiogenesis in the corpus luteum during development and early pregnancy in ruminants1

The establishment of pregnancy requires well-balanced regulation of the endocrine and immune systems and involves interactions among the conceptus, oviduct-uterus, and corpus luteum (CL). In particular, a rapid increase in plasma progesterone during the first week after ovulation is critical for the growth of the conceptus and successful pregnancy in cattle. Events involved in maternal recognition of pregnancy (MRP) may commence within 1 wk from AI, when interferon-stimulated gene expression in circulating polymorphonuclear neutrophils (PMN) increases in pregnant cows. To regulate optimal endocrine conditions within this time, the CL must develop rapidly, with active angiogenesis and lymphangiogenesis. The major angiogenic factors, vascular endothelial growth factor and fibroblast growth factor 2, contribute to the development of the CL but may also act as chemoattractants for PMN. Indeed, the number of PMN is greatest in the new CL, where PMN together with IL-8 induce active angiogenesis and lymphangiogenesis. During MRP, the conceptus secretes interferon tau (IFNT), which prevents CL regression by inhibiting luteolytic release of PGF2α from uterine endometrium. In addition, IFNT and PGE2 reach the CL and may contribute to desensitizing the CL to the luteolytic effects of PGF2α. In the bovine CL, lymphangiogenesis, stimulated by IFNT, may occur during MRP, and thus a shift of local immunity might occur at this timing. The aforementioned evidence supports the possible involvement of PMN in the establishment of pregnancy via CL regulation. Further investigation could expand our understanding of the communication between zygotes, PMN, and reproductive organs during early pregnancy. This should provide new insight into the contribution of neutrophils to CL function and immune tolerance during early pregnancy in ruminants.

Influence of seminal plasma on leucocyte migration and amount of COX-2 protein in the jenny endometrium after insemination with frozen–thawed semen

After mating, seminal plasma has an immuno-modulatory effect on the endometrium in some mammals. In jennies, achieving conception via artificial insemination (AI) with frozen–thawed semen is generally much more difficult than in mares. The endometrial inflammatory response is hypothesized to be a contributing factor to the lesser fertility. Following a cross-over experimental design, the uterine inflammatory response of six jennies was evaluated at 6h after AI with frozen–thawed semen (deposited in the uterine body) in the presence or absence of autologous seminal plasma (+SP or −SP). The endometrial cytology and histology of the animals were examined by uterine lavage, uterine swabbing and biopsy. The amount of cyclooxygenase-2 (COX-2) protein in endometrial cells was also evaluated. As a control (C), the same examinations were made before any AI procedure (i.e., when the jennies were in oestrus). Large numbers of polymorphonuclear neutrophils (PMN) were observed in the −SP and +SP cytology and biopsy samples; more than in the C samples. The −SP samples also had intense COX-2 labelling; less labelling was detected in the +SP and C samples (no significant difference between these latter two types). Thus, while the presence of SP does not change the post-AI number of PMNs with regard to that detected in its absence, it does reduce COX-2 protein. Further research into the complex mix of molecules in SP and its effects during AI might help increase the pregnancy rates achieved in jennies.

Secretion of prostaglandins and leukotrienes by endometrial cells in cows with subclinical and clinical endometritis

The aims of this study were (1) to measure the secretion of prostaglandin F2α (PGF2α), prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and leukotriene C4 (LTC4) by endometrial cells collected by a cytobrush from healthy cows and cows with subclinical and clinical endometritis in the fourth week postpartum, and (2) to evaluate the relationship between the mediators' levels of secretion and the number of polymorphonuclear neutrophils (PMNs) in the uterine smears of cows with subclinical endometritis. The study included cows without any signs of clinical endometritis (n = 63) and cows with clinical endometritis as a positive control (ENDOM, n = 12). Two different threshold ratios (>5% and >18% of PMNs) were used to categorize the cows without clinical signs as with or without cytologic endometritis (CE). Considering the first or second threshold, the animals with CE were included in group CE POS I or CE POS II, whereas the healthy cows were assigned to group CE NEG I or CE NEG II, respectively.The prevalence of CE was 68.25% (42/63) and 57.14% (36/63) according to the first and second thresholds, respectively. The highest level of secretion of all of the measured mediators occurred in the ENDOM group and differed significantly (P < 0.05) from the CE POS and CE NEG groups, regardless of the threshold. PGF2α secretion in the CE POS II group (1629 pg/mL) was significantly lower (P < 0.05) when compared with the CE NEG II group (2797 pg/mL), whereas there was no significant difference between the CE POS I and CE NEG I groups. PGE2 secretion differed between both groups with CE; higher concentrations were measured in the CE POS II group (6.68 ng/mL) when compared with the CE POS I (2.4 ng/mL) and CE NEG II (2.37 ng/mL) groups (P < 0.05). No significant differences were observed in the LTB4 and LTC4 secretion between the CE POS and CE NEG groups, considering both thresholds. It seems that CE does not fully mimic the inflammatory cascade associated with clinical signs. The response in the subclinical cases was limited to enhanced production of PGE2, which was particularly well-pronounced in cows with high numbers of PMNs (>18%) in the endometrial scrapings.

Comparison of cytological and histological examinations in different locations of the equine uterus—an in vitro study

Endometritis constitutes a major problem in managing broodmares. The histological occurrence of polymorphonuclear neutrophils (PMNs) in the stratum compactum of the endometrium is accepted as the reference standard to diagnose endometritis in mares. The objective of this study was to determine the distribution of PMNs within different sampling locations of the uterus by cytological examinations and to compare it with PMN numbers in endometrial biopsies of the corresponding location. Cytological and endometrial samples were obtained from 37 uteri within 2 ± 1 hours after slaughter through small incisions from five different, predefined locations of each uterus. The cytological samples were smeared on microscopic slides, stained, and classified as negative (<2% PMNs) or positive (≥2% PMNs) for endometritis. Histologically, the numbers of PMNs were counted in three high power fields by an experienced pathologist and classified as positive for this type of endometritis if ≥5 PMNs occurred in three high power fields (×40 magnification). The biopsies were also evaluated for lymphoplasmacellular endometritis, periglandular fibrosis (endometrosis), and angiosclerosis. The prevalence of positive cytological and histological samples was 14.6% and 17.8%, respectively. A fair agreement between the two diagnostic methods could be detected (k = 0.29; P < 0.01). The numbers of PMNs differed between the sampling locations, resulting in positive and negative locations within a positive scored uterus, in both cytologically positive scored uteri (8/10) and histologically positive scored uteri (13/14). No significant differences were found in PMN numbers in the different locations, either the cytological (P = 0.78) or histological (P = 0.79) examination. Additionally, no significant differences were observed in the assessment of endometrosis (P = 0.96) and angiosclerosis (P = 0.67) within the locations.In conclusion, PMN numbers of a cytological examination of the endometrium showed fair agreement to the occurrence of PMN in the stratum compactum of the histological examination at the same sampling location. Although variations were found in the number of PMN using both methods (cytology and histology), statistically significant differences were not detected within the different locations (P = 0.78; P = 0.79), implyingies that the decision to take more than one sample should be critically considered. Additional research is warranted to determine the number of sampling locations necessary for reliable examination results.

Effect of ketoprofen treatment on the uterine inflammatory response after AI of jennies with frozen semen

Artificial insemination (AI) involving the placing of frozen-thawed semen directly into the jenny uterine body is associated with very low pregnancy rates. This might be because of an exacerbation of the acute response of the endometrium to sperm, as seen in mares with persistent induced mating endometritis. Pregnancy rates can be increased in such mares, however, by including anti-inflammatory treatments in the insemination protocol (Bucca S, Carli A, Buckley T, Dolci G, Fogarty U. The use of dexamethasone administered to mares at breeding time in the modulation of persistent mating induced endometritis. Theriogenology 2008;70:1093–100; Rojer H, Aurich C. Treatment of persistent mating-induced endometritis in mares with the non-steroid anti-inflammatory drug vedaprofen. Reprod Domest Anim 2010;45:e458–60). To investigate the endometritis caused by the use of frozen-thawed semen in jennies, and to assess the response to ketoprofen treatment, endometrial cytological samples and biopsies from six healthy jennies were examined in a crossover design experiment. Samples were taken from jennies in estrus (E; control) and at 6 hours after AI with or without ketoprofen (+K and −K, respectively). Ketoprofen was administered iv 24 hours before and for 4 days after insemination (total = 2.2 mg/kg/24 hours for 5 days). All animals showed a severe inflammatory response to semen deposition. Polymorphonuclear neutrophil numbers in the cytological smears and biopsies differed significantly between the +K and E animals. No significant differences were recorded, however, between the +K and −K treatments. Eosinophils were observed in all sample types from all groups; these cells appear to be a feature of the normal jenny endometrium. Slight fibrosis was observed in some biopsies, but no significant relationship with inflammation was found. Intense cyclooxygenase-2 (COX-2) immunohistochemical labeling was detected in the −K biopsies. Less intense labeling was seen in those of the +K animals, and mainly localized in the stratum compactum. No differences in COX-2 labeling were observed between the +K and E animals. Plasma concentrations of ketoprofen remained detectable until 2 hours after administration, after which the compound was rapidly eliminated. In summary, jennies are susceptible to endometritis after insemination with frozen-thawed semen. Ketoprofen reduces this inflammation by inhibiting COX-2; no reduction in the number of polymorphonuclear neutrophils occurs. The physiological and pharmacological characteristics of jennies should be taken into account when designing treatments for acute endometritis aimed at enhancing pregnancy rates after insemination with frozen-thawed sperm.

Investigations on the Endometrial Response to Intrauterine Administration of N‐Acetylcysteine in Oestrous Mares

In mares, mating-induced persistent endometritis contributes to low fertility. The condition is in part related to delayed clearance of mucus accumulated within the uterine lumen. The objective of this study was to investigate the endometrial response of healthy mares to intrauterine (i.u.) treatment with N-acetylcysteine (NAC). Oestrous mares (n=12) were randomly assigned to a treatment (TM) or control (C) group and received an i.u. infusion of 5% NAC and saline (total volume 140ml), respectively. Endometrial biopsies were collected in five of the mares 24h after treatment, in the remaining seven mares 72h after treatment. Endometrial biopsies were evaluated for integrity of the luminal epithelium, number of polymorphonuclear neutrophils (PMN), staining for cyclooxygenase 2 (COX2), staining with Kiel 67 antigen (Ki-67), lectins and periodic acid-Schiff (PAS). The integrity of endometrial epithelial cells was not affected by treatment (no statistical differences between groups or times). At 24h after treatment, the mean number of PMN in endometrial biopsies from NAC- and C-mares did not differ, but at 72h after treatment, number of PMN was significantly higher (p<0.05) in C (3.9±0.6 PMN/field) compared with NAC-treated mares (2.3±0.2 PMN/field). At 72h after treatment, the intensity of staining for COX2 was significantly higher after saline than after NAC treatment (p<0.05). In the epithelium, no differences in staining for the proliferation marker Ki-67 were seen with respect to time and treatment. Score for the lectin wheat germ agglutinin (WGA) was slightly higher in NAC-treated mares than in C-mares 72h after treatment (p<0.05). Score for PAS staining of mucus in deep uterine glands differed significantly between groups at 24h after treatment (p<0.05). The present study demonstrates that NAC does not adversely affect the endometrial function. Moreover, an anti-inflammatory effect on the equine endometrium was observed.