Nitric oxide- and oxygen-derived free radical generation from control and lipopolysaccharide-treated rat polymorphonuclear leukocyte.

Previous studies from this lab have shown NO-mediated modulation of free radical generation from polymorphonuclear leukocytes (PMNs), following hypoxic-reoxygenation as well as in the normoxic cells. The present study is an attempt to investigate further the regulation of NO and free radical generation in the lipopolysaccharide (LPS)-treated PMNs. PMNs were isolated from the rat blood and peritoneal cavity, 4 h after LPS (1 mg/kg, i.p.) treatment. Nitric oxide synthase (NOS) activity and nitrite content were increased in the peripheral and peritoneal PMNs following LPS treatment. An increase in the apparent V(max) for l-arginine uptake was also observed in the LPS-treated peripheral PMNs, while peritoneal PMNs exhibited increase in both apparent V(max) and affinity for l-arginine. Synthesis of nitrite did not augment after increasing the availability of substrate to control PMNs, however, peripheral and peritoneal PMNs from LPS-treated rats utilized l-arginine more efficiently for nitrite synthesis. NOS activity, l-arginine uptake, and its utilization were maximal in the peritoneal PMNs. Arachidonic acid (AA, 1 x 10(-6) M)-induced free radical generation from PMNs was also enhanced significantly after LPS treatment. Preincubation of PMNs with nitrite elevated the free radical generation and myeloperoxidase (MPO) release. MPO and antioxidant enzyme activity in the PMNs was significantly augmented after LPS treatment. NOS inhibitors, aminoguanidine and 7-nitroindazole, inhibited arachidonic acid-induced free radical generation from LPS treated PMNs. The results obtained thus indicate that augmentation of free radical generation from rat PMNs following LPS treatment appears to be regulated by NO and MPO.     

Role of Vegetation-Associated Protease Activity in Valve Destruction in Human Infective Endocarditis

Aims

Infective endocarditis (IE) is characterized by septic thrombi (vegetations) attached on heart valves, consisting of microbial colonization of the valvular endocardium, that may eventually lead to congestive heart failure or stroke subsequent to systemic embolism. We hypothesized that host defense activation may be directly involved in tissue proteolytic aggression, in addition to pathogenic effects of bacterial colonization.

Methods and Results

IE valve samples collected during surgery (n = 39) were dissected macroscopically by separating vegetations (VG) and the surrounding damaged part of the valve from the adjacent, apparently normal (N) valvular tissue. Corresponding conditioned media were prepared separately by incubation in culture medium. Histological analysis showed an accumulation of platelets and polymorphonuclear neutrophils (PMNs) at the interface between the VG and the underlying tissue. Apoptotic cells (PMNs and valvular cells) were abundantly detected in this area. Plasminogen activators (PA), including urokinase (uPA) and tissue (tPA) types were also associated with the VG. Secreted matrix metalloproteinase (MMP) 9 was also increased in VG, as was leukocyte elastase and myeloperoxidase (MPO). The presence of neutrophil extracellular traps (NETs) associating MPO and externalized nucleosomes, was shown by immunostaining in the VG. Both MPO and cell-free DNA were released in larger amounts by VG than N samples, suggesting bacterial activation of PMNs within the vegetation. Finally, evidence of proteolytic tissue damage was obtained by the release of fragments of extracellular matrix components such as fibrinogen and fibronectin, as well as protease-sensitive receptors such as the uPA receptor.

Conclusion

Our data obtained using human IE valves suggest that septic vegetations represent an important source of proteases originating from massive leukocyte recruitment and activation of the host plasminergic system. The latter forms a potential therapeutic target to minimize valvular tissue degradation independently from that induced by bacterial proteases.     

Cytokines Induced Neutrophil Extracellular Traps Formation: Implication for the Inflammatory Disease Condition

Neutrophils (PMNs) and cytokines have a critical role to play in host defense and systemic inflammatory response syndrome (SIRS). Neutrophil extracellular traps (NETs) have been shown to extracellularly kill pathogens, and inflammatory potential of NETs has been shown. Microbial killing inside the phagosomes or by NETs is mediated by reactive oxygen and nitrogen species (ROS/RNS). The present study was undertaken to assess circulating NETs contents and frequency of NETs generation by isolated PMNs from SIRS patients. These patients displayed significant augmentation in the circulating myeloperoxidase (MPO) activity and DNA content, while PMA stimulated PMNs from these patients, generated more free radicals and NETs. Plasma obtained from SIRS patients, if added to the PMNs isolated from healthy subjects, enhanced NETs release and free radical formation. Expressions of inflammatory cytokines (IL-1β, TNFα and IL-8) in the PMNs as well as their circulating levels were significantly augmented in SIRS subjects. Treatment of neutrophils from healthy subjects with TNFα, IL-1β, or IL-8 enhanced free radicals generation and NETs formation, which was mediated through the activation of NADPH oxidase and MPO. Pre-incubation of plasma from SIRS with TNFα, IL-1β, or IL-8 antibodies reduced the NETs release. Role of IL-1β, TNFα and IL-8 thus seems to be involved in the enhanced release of NETs in SIRS subjects.     

Metabolic activation of chlorpromazine by stimulated human polymorphonuclear leukocytes. Induction of covalent binding of chlorpromazine to nucleic acids and proteins.

Human polymorphonuclear leukocytes (PMNs) have been stimulated with either phorbol 12-myristate 13-acetate (PMA), calcium ionophore A23187 or a combination of both to induce the respiratory burst and myeloperoxidase (MPO) release. Chlorpromazine (CPZ) but not chlorpromazine sulfoxide (CPZSO) inhibited the respiratory burst as measured with lucigenin chemiluminescence. The inhibition was due to interference with processes in the cell leading to the respiratory burst and not to scavenging of produced oxygen radicals that provoke the luminescence. CPZ was metabolized by stimulated PMNs. HPLC analysis revealed formation of CPZSO and an unidentified product. Both products result from decay of chlorpromazine radical cation (CPZ+.), indicating formation of this radical intermediate in CPZSO oxidation by stimulated PMNs. CPZ conversion correlated with H2O2 production and MPO release. The largest CPZ conversion was observed with phorbol ester plus A23187 stimulation. The conversion was reduced by catalase and sodium azide, an inhibitor of MPO, with 70% and 40%, respectively. This indicates only partial involvement of extracellularly released MPO in CPZ metabolism by PMNs. Considerable covalent binding of [3H]CPZ to nucleic acids and proteins of intact stimulated PMNs was observed. This binding was larger upon co-stimulation with phorbol ester and A23187. Azide did not reduce covalent binding. This indicates that covalent binding is not mediated by extracellularly released MPO and that CPZ is probably activated intracellularly. Activation of PMNs and production of H2O2 is a prerequisite for both CPZ conversion and covalent binding. This study demonstrates that phagocytic cells might contribute to drug metabolism and drug-induced toxicity.     

Calcium activity in response to nAChR ligands in murine bone marrow granulocytes with different Gr-1 expression

Polymorphonuclear neutrophilic granulocytes (PMNs) are the largest proportion of leukocytes in adult human blood that perform numerous functions, including phagocytosis, degranulation, generation of reactive oxygen species, and NETosis. Excessive neutrophil activity associates with hyperinflammation and tissue damage during pathologies such as inflammatory bowel disease, diabetes mellitus, tuberculosis, and coronavirus disease 2019. Nicotinic acetylcholine receptors (nAChRs) can modulate immune cells, including neutrophils, functions, therefore, nAChR ligands are considered as the potent agents for therapy of inflammation. Earlier it was shown, that about 30% of PMNs from the acute inflammatory site responded to nicotine by calcium spikes. In this study, we studied the generation of calcium spikes in murine granulocytes with different maturity level (evaluated by Gr-1 expression) isolated from bone marrow in response to ligands of nAChRs in control and under chronic nicotine consumption. It was found that nearly 20%–25% cells in the granulocyte population responded to nicotine or selective antagonists of different type of nAChRs (α-cobratoxin, GIC, and Vc1.1). We demonstrated that in the control group Ca²⁺-mobilizing activity was regulated through α7 and α9α10 nAChRs in immature granulocytes (Gr-1^int), whereas in mature granulocytes (Gr-1^hi) it was regulated through α7, α3β2, and α9-contained nAChRs. Sensitivity of PMNs to nicotine depended on their maturity level after chronic nicotine consumption. Gr-1^int cells responded to nicotine through α7 and α9-contained nAChRs, while Gr-1^hi did not respond to nicotine. Thus, calcium response to nAChR ligands in bone marrow PMNs depends on their maturity level.     

Inducible nitric oxide synthase expression is inhibited by myeloperoxidase.

Nitric oxide (NO) plays key roles in vasodilation and host defense, yet the overproduction of NO by inducible nitric oxide synthase (iNOS) at inflammatory sites can also be pathogenic. Here, we investigate the role of MPO in modulating the induction of iNOS by IFNgamma/LPS (IL). In monocyte-macrophages (Mvarphi) treated with IL, MPO gene expression was found to be downregulated as iNOS was upregulated. In Mvarphi from MPO-knockout (KO) mice, the induction of iNOS by IL was earlier and higher than in MPO-positive cells, suggesting MPO is inhibitory. Consistent with that interpretation, the addition of purified MPO enzyme to cultured macrophages inhibited iNOS induction by IL. In addition, an inhibitor of MPO enzyme, 4-aminobenzohydrazide, enhanced iNOS induction in MPO-positive cells, but not in MPO-KO cells. Similarly, taurine, a scavenger of MPO-generated HOCl, enhanced iNOS induction in MPO-positive cells, but not in MPO-KO cells. MPO affects an early event, suppressing iNOS induction when added within 2h of IL, but not when added several hours after IL. The suppression by MPO was alleviated by NO donor, sodium nitroprusside, suggesting the suppression results from scavenging of NO by MPO. This interpretation is consistent with earlier reports that MPO consumes NO, and that low levels of NO donor augment induction of iNOS by IFNgamma/LPS. The implication of these findings is that MPO acts as gatekeeper, suppressing the deleterious induction of iNOS at inflammatory sites by illegitimate signals. The combined signaling of IFNgamma/LPS overrides the gatekeeper function by suppressing MPO gene expression.     

Enhanced killing of penicillin-treated gram-positive cocci by human granulocytes: role of bacterial autolysins, catalase, and granulocyte oxidative pathways

Staphylococci pretreated with subminimal inhibitory concentrations (subMIC) of cell-wall active antibiotics exhibit increased susceptibility to killing by human polymorphonuclear leukocytes (PMNs), even when phagosome information is impaired by the mold metabolite, cytochalasin B. To investigate the role of specific bacterial factors in the process, studies were carried out with organisms lacking catalase (streptococci) or cell-wall autolytic enzymes and compared to findings with Staphylococcus aureus 502A. Neutrophil factors were studied using inhibitors, oxygen radical scavengers, myeloperoxidase (MPO)-deficient PMNs, or PMNs from a patient with chronic granulomatous disease (CGD). Documentation of the enhanced susceptibility of the streptococcal strains to killing by PMNs following subMIC penicillin pretreatment required the use of cytochalasin B. Enhancement of killing occurred independent of the presence or absence of bacterial autolysins or catalase. SubMIC penicillin pretreatment of S. pneumoniae R36A specifically promoted the susceptibility of these organisms to killing by myeloperoxidase (MPO)-mediated mechanisms (enhancement lost using MPO-deficient or azide-treated cells). Factors other than MPO or toxic oxygen products generated by the PMN respiratory burst are responsible for enhanced killing of penicillin-pretreated S. aureus 502A (enhancement preserved using MPO-deficient, azide-treated, or chronic granulomatous disease patient cells). These studies define methods to study the interaction of antimicrobial agents and PMNs in the killing of microorganisms. They also demonstrate that penicillin treatment can change the susceptibility of gram-positive cocci to the action of specific PMN microbicidal mechanisms. The mechanism of the enhancement appears to be bacterial strain-dependent and not predictable by bacterial autolysin or catalase activity.     

Biosynthesis, processing, and sorting of human myeloperoxidase

Exclusively synthesized by normal neutrophil and monocyte precursor cells, myeloperoxidase (MPO) functions not only in host defense by mediating efficient microbial killing but also can contribute to progressive tissue damage in chronic inflammatory states such as atherosclerosis. The biosynthetic precursor, apoproMPO, is processed slowly in the ER, undergoing cotranslational N-glycosylation, transient interactions with the molecular chaperones calreticulin and calnexin, and heme incorporation to generate enzymatically active proMPO that is competent for export into the Golgi. After exiting the Golgi the propeptide is removed prior to final proteolytic processing in azurophil granules, resulting in formation of a symmetric MPO homodimer linked by a disulfide bond. Some proMPO escapes granule targeting and becomes constitutively secreted to the extracellular environment. Although the precise mechanism is unknown, the pro-segment is required for normal processing and targeting, as propeptide-deleted MPO precursor is either degraded or constitutively secreted. Characterizing the molecular consequences of naturally occurring mutations that cause inherited MPO deficiency provides unique insight into the structural determinants of MPO involved in biosynthesis, processing and targeting.     

Measuring Myeloperoxidase Activity in Biological Samples

Background

Enzymatic activity measurements of the highly oxidative enzyme myeloperoxidase (MPO), which is implicated in many diseases, are widely used in the literature, but often suffer from nonspecificity and lack of uniformity. Thus, validation and standardization are needed to establish a robust method that is highly specific, sensitive, and reproducible for assaying MPO activity in biological samples.

Principal findings

We found conflicting results between in vivo molecular MR imaging of MPO, which measures extracellular activity, and commonly used in vitro MPO activity assays. Thus, we established and validated a protocol to obtain extra- and intracellular MPO from murine organs. To validate the MPO activity assays, three different classes of MPO activity assays were used in spike and recovery experiments. However, these assay methods yielded inconsistent results, likely because of interfering substances and other peroxidases present in tissue extracts. To circumvent this, we first captured MPO with an antibody. The MPO activity of the resultant samples was assessed by ADHP and validated against samples from MPO-knockout mice in murine disease models of multiple sclerosis, steatohepatitis, and myocardial infarction. We found the measurements performed using this protocol to be highly specific and reproducible, and when performed using ADHP, to be highly sensitive over a broad range. In addition, we found that intracellular MPO activity correlated well with tissue neutrophil content, and can be used as a marker to assess neutrophil infiltration in the tissue.

Conclusion

We validated a highly specific and sensitive assay protocol that should be used as the standard method for all MPO activity assays in biological samples. We also established a method to obtain extra- and intracellular MPO from murine organs. Extracellular MPO activity gives an estimate of the oxidative stress in inflammatory diseases, while intracellular MPO activity correlates well with tissue neutrophil content. A detailed step-by-step protocol is provided.     

Nitric oxide modulates the catalytic activity of myeloperoxidase

Myeloperoxidase (MPO), an abundant protein in neutrophils, monocytes, and subpopulations of tissue macrophages, is believed to play a critical role in host defenses and inflammatory tissue injury. To perform these functions, an array of diffusible radicals and reactive oxidant species may be formed through oxidation reactions catalyzed at the heme center of the enzyme. Myeloperoxidase and inducible nitric-oxide synthase are both stored in and secreted from the primary granules of activated leukocytes, and nitric oxide (nitrogen monoxide; NO) reacts with the iron center of hemeproteins at near diffusion-controlled rates. We now demonstrate that NO modulates the catalytic activity of MPO through distinct mechanisms. NO binds to both ferric (Fe(III), the catalytically active species) and ferrous (Fe(II)) forms of MPO, generating stable low-spin six-coordinate complexes, MPO-Fe(III).NO and MPO-Fe(II).NO, respectively. These nitrosyl complexes were spectrally distinguishable by their Soret absorbance peak and visible spectra. Stopped-flow kinetic analyses indicated that NO binds reversibly to both Fe(III) and Fe(II) forms of MPO through simple one-step mechanisms. The association rate constant for NO binding to MPO-Fe(III) was comparable to that observed with other hemoproteins whose activities are thought to be modulated by NO in vivo. In stark contrast, the association rate constant for NO binding to the reduced form of MPO, MPO-Fe(II), was over an order of magnitude slower. Similarly, a 2-fold decrease was observed in the NO dissociation rate constant of the reduced versus native form of MPO. The lower NO association and dissociation rates observed suggest a remarkable conformational change that alters the affinity and accessibility of NO to the distal heme pocket of the enzyme following heme reduction. Incubation of NO with the active species of MPO (Fe(III) form) influenced peroxidase catalytic activity by dual mechanisms. Low levels of NO enhanced peroxidase activity through an effect on the rate-limiting step in catalysis, reduction of Compound II to the ground-state Fe(III) form. In contrast, higher levels of NO inhibited MPO catalysis through formation of the nitrosyl complex MPO-Fe(III)-NO. NO interaction with MPO may thus serve as a novel mechanism for modulating peroxidase catalytic activity, influencing the regulation of local inflammatory and infectious events in vivo.     

Spatial mapping of pulmonary and vascular nitrotyrosine reveals the pivotal role of myeloperoxidase as a catalyst for tyrosine nitration in inflammatory diseases

Nitrotyrosine (NO(2)Tyr) formation is a hallmark of acute and chronic inflammation and has been detected in a wide variety of human pathologies. However, the mechanisms responsible for this posttranslational protein modification remain elusive. While NO(2)Tyr has been considered a marker of peroxynitrite (ONOO(-)) formation previously, there is growing evidence that heme-protein peroxidase activity, in particular neutrophil-derived myeloperoxidase (MPO), significantly contributes to NO(2)Tyr formation in vivo via the oxidation of nitrite (NO(2)(-)) to nitrogen dioxide (.NO(2)). Coronary arteries from a patient with coronary artery disease, liver and lung tissues from a sickle cell disease patient, and an open lung biopsy from a lung transplant patient undergoing rejection were analyzed immunohistochemically to map relative tissue distributions of MPO and NO(2)Tyr. MPO immunodistribution was concentrated along the subendothelium in coronary tissue and hepatic veins as well as in the alveolar epithelial compartment of lung tissue from patients with sickle cell disease or acute rejection. MPO immunoreactivity strongly colocalized with NO(2)Tyr formation, which was similarly distributed in the subendothelial and epithelial regions of these tissues. The extracellular matrix protein fibronectin (FN), previously identified as a primary site of MPO association in vascular inflammatory reactions, proved to be a major target protein for tyrosine nitration, with a strong colocalization of MPO, NO(2)Tyr, and tissue FN occurring. Finally, lung tissue from MPO(-/-) mice, having tissue inflammatory responses stimulated by intraperitoneal zymosan administration, revealed less subendothelial NO(2)Tyr immunoreactivity than tissue from wild-type mice, confirming the significant role that MPO plays in catalyzing tissue nitration reactions. These observations reveal that (i) sequestration of neutrophil-derived MPO in vascular endothelial and alveolar epithelial compartments is an important aspect of MPO distribution and action in vivo, (ii) MPO-catalyzed NO(2)Tyr formation occurs in diverse vascular and pulmonary inflammatory pathologies, and (iii) extracellular matrix FN is an important target of tyrosine nitration in these inflammatory processes.     

Evaluation of the changes of immune cells during lipopolysaccharide-induced mastitis in rats

The aim of this study was to evaluate in rats, changes in peripheral blood immune cells and mammary tissue after an intramammary infusion of lipopolysaccharide (LPS). The results of the study showed that infusion of LPS induced a rapid migration of neutrophils (PMNs) from the blood to mammary alveoli, increased the activity of myeloperoxidase (MPO) and the concentration of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6) in mammary tissues, decreased the activity of myeloperoxidase in serum and reduced the CD4+/CD8+ ratio. This is the first report of changes in peripheral blood immune cells and mammary tissue in rat mastitis.     

Leukocytic cell sources of airway tissue kallikrein

Lung tissue kallikrein (TK) is a serine proteinase that putatively plays a role in the pathophysiology of asthma by generating kallidin and bradykinin, mediators that contribute to airway hyperresponsiveness. In previous studies we observed biphasic increases in TK activity in bronchoalveolar lavage fluid following airway allergen challenge in allergic sheep. Although glandular TK is likely a major source of the initial increase in TK, the sources of the late increases in TK that are associated with the development of airway hyperresponsiveness may be dependent on activated resident and recruited inflammatory cells including alveolar macrophages (AMs) and neutrophils (PMNs). These cells increase concomitantly with the late increases in TK activity. To test this hypothesis, we obtained AMs from bronchoalveolar lavage fluid and PMNs and monocytes (precursors of AMs) from sheep blood and determined whether these cells contained TK and whether these same cells could release TK upon activation. Using confocal microscopy, immunocytochemical techniques, and enzyme activity assays, we found that all three cell types contained and secreted TK. All three cell types demonstrated basal release of TK, which could be increased after stimulation with zymosan. In addition, PMNs also released TK in the presence of phorbol ester, suggesting multiple secretory pathways in these cells. Furthermore, we showed that human monocytes also contain and secrete TK. We conclude that in the airways, monocytes, PMNs, and AMs may contribute to increased TK activity. Knowing the sources of TK in the airways could be important in understanding the mechanisms of inflammation that contribute to the pathophysiology of asthma and may help in the development of new therapies to control the disease.     

Inactivation of DAP12 in PMN Inhibits TREM1-Mediated Activation in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is an autoimmune disease characterized by dysregulated and chronic systemic inflammatory responses that affect the synovium, bone, and cartilage causing damage to extra-articular tissue. Innate immunity is the first line of defense against invading pathogens and assists in the initiation of adaptive immune responses. Polymorphonuclear cells (PMNs), which include neutrophils, are the largest population of white blood cells in peripheral blood and functionally produce their inflammatory effect through phagocytosis, cytokine production and natural killer-like cytotoxic activity. TREM1 (triggering receptor expressed by myeloid cells) is an inflammatory receptor in PMNs that signals through the use of the intracellular activating adaptor DAP12 to induce downstream signaling. After TREM crosslinking, DAP12’s tyrosines in its ITAM motif get phosphorylated inducing the recruitment of Syk tyrosine kinases and eventual activation of PI3 kinases and ERK signaling pathways. While both TREM1 and DAP12 have been shown to be important activators of RA pathogenesis, their activity in PMNs or the importance of DAP12 as a possible therapeutic target have not been shown. Here we corroborate, using primary RA specimens, that isolated PMNs have an increased proportion of both TREM1 and DAP12 compared to normal healthy control isolated PMNs both at the protein and gene expression levels. This increased expression is highly functional with increased activation of ERK and MAPKs, secretion of IL-8 and RANTES and cytotoxicity of target cells. Importantly, based on our hypothesis of an imbalance of activating and inhibitory signaling in the pathogenesis of RA we demonstrate that inhibition of the DAP12 signaling pathway inactivates these important inflammatory cells.     

Involvement of endogenous nitric oxide in myeloperoxidase mediated benzo(a)pyrene induced polymorphonuclear leukocytes injury

The present study was undertaken to investigate the involvement of nitric oxide in the augmentation of benzo(a)pyrene induced cellular injury in polymorphonuclear leukocytes (PMNs). Polymorphs were isolated from the blood collected from Wistar rats treated with and without benzo(a)pyrene (50mg/kg, i.p.) through cardiac puncture. Catalase, superoxide dismutase (SOD), glutathione-s-transferase (GST), myeloperoxidase (MPO) and nitrite content were estimated in PMNs using standard procedures. Inducible nitric oxide synthase (iNOS) and cytochrome P-4501A1 (CYP1A1) expression in PMNs were also analyzed in presence or absence of nitric oxide synthase (NOS) inhibitors, aminoguanidine (AG, 5mM) and L-N^G nitro L-arginine methyl ester (L-NAME, 1mM). A significant augmentation was observed in the nitrite content, activities of superoxide dismutase, MPO and GST and the expressions of iNOS and CYP1A1, however, catalase activity was attenuated in PMNs of benzo(a)pyrene treated rats as compared with their respective controls. AG and L-NAME resulted in a significant attenuation in nitrite content, MPO activity and iNOS expression; however, no significant alteration was observed in CYP1A1 expression. CYP1A1 inhibitor alpha-naphthoflavone inhibited the expression of iNOS in PMNs of benzo(a)pyrene treated animals significantly. The results obtained thus suggest that CYP1A1 induces iNOS expression leading to the generation of endogenous nitric oxide (NO) that could be responsible for the augmentation of myeloperoxidase-mediated benzo(a)pyrene-induced injury in PMNs.     

A novel effect of polymorphonuclear leukocytes in the facilitation of angiogenesis

The purpose of this study was to examine whether the adhesion of polymorphonuclear leukocytes (PMNs) to endothelial cells and/or reactive oxygen species (ROS) released from PMNs are responsible for inducing angiogenesis. Angiogenesis was assessed by tube formation using endothelial cells obtained from bovine thoracic aorta (BAECs) grown on a layer of collagen type I. Addition of PMNs to BAECs weakly induced angiogenesis. The angiogenesis induced by PMNs alone was further enhanced by treatment of the PMNs with N-formyl-methionyl-leucyl-phenylalanine (FMLP), a selective activator of PMN. The involvement of PMN adhesion to BAECs via adhesion molecules in angiogenesis was investigated by using monoclonal antibodies against E-selectin and intercellular adhesion molecule-1 (ICAM-1). These antibodies blocked both the PMN adhesion to BAECs and the enhancement of angiogenesis induced by FMLP-treated PMNs. Furthermore, the enhancement of angiogenesis by FMLP-treated PMNs was blocked by catalase, a scavenging enzyme of H2O2, but not by superoxide dismutase (SOD). These results suggest that PMNs induce angiogenesis in vitro, and that the mechanism of stimulation of angiogenesis by PMNs may involve the adherence of PMNs to endothelial cells via E-selectin and ICAM-1, and H2O2, but not superoxide. Thus, activated PMNs in pathological states may not only induce tissue injury, but may also function as regulators of angiogenesis.     

Immunolocalization of CXC chemokine and recruitment of polymorphonuclear leukocytes in the rat molar periodontal tissue after topical application of lipopolysaccharide

This study investigated the recruitment of polymorphonuclear leukocytes (PMNs) and the immunolocalization of CXC chemokines, including macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant-2 (CINC-2) in rat periodontal tissue after topical application of lipopolysaccharide (LPS; 5 mg/ml) from Escherichia coli into the rat molar gingival sulcus. In normal periodontal tissues, a small number of MIP-2- and CINC-2-positive cells were seen in junctional epithelium (JE), especially in its coronal half. After topical application of LPS, a prominent increase of MIP-2- and CINC-2-positive JE cells was observed. Almost all JE cells strongly expressed them at day 1 and day 2, and then the number of chemokine-positive cells returned to normal at day 7. Corresponding to these chemokine expressions, LPS application induced a significant increase in the number of PMNs in the sub-JE area from 1 h to 2 days and a significant increase in JE area from 3 h to 5 days, indicating a dynamic flow of PMNs from the sub-JE area into JE. These findings indicated that JE cells produced MIP-2 and CINC-2 in response to LPS stimulation and suggested that MIP-2 and CINC-2 may be responsible for PMN migration toward the periodontal pathogen and may play an important role in the initiation of inflammation and subsequent periodontal tissue destruction.     

Effects of an immunomodulatory feed additive on the development of mastitis in a mouse infection model using four bovine-origin isolates

The goal of this study was to examine the ability of a commercially available feed additive (OmniGen-AF) to reduce mammary infections caused by a single strain of mastitic pathogens (Streptococcus uberis, Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae) and to examine the effects of the additive on markers of mammary immunity. Four experiments were completed using a murine model of bovine mastitis. Infection progression was examined using Sybr-green- and TaqMan-based quantitative PCR assays of 16S ribosomal DNA. Infection of the mammary gland with all pathogens caused rapid (24 to 48 h) appearance of pathogen DNA in mammary tissue. Provision of the feed additive for 2 weeks before infection significantly (P < 0.05) reduced the extent of pathogen DNA accumulation in models of S. uberis, E. coli and S. aureus infection. The additive was ineffective in reducing mammary infections caused by K. pneumoniae. We examined mechanisms of action of the additive through assessment of mammary concentrations of mammary myeloperoxidase (MPO), major histocompatibility complex 2 class II (MHC) and macrophage inflammatory protein-1α (MIP) messenger RNA (mRNA) concentrations and by examining serum complement C3 concentration. Infection of the mammary gland increased concentrations of MPO and MHC mRNAs (P < 0.05). Ability of the pathogen to elicit changes in mammary MPO and MHC gene expression was enhanced by the provision of the additive for 2 weeks before infection. These data imply that the additive increased the mammary inflammatory response and increased antigen presentation during a mammary infection. Value of the additive in preventing mastitis in cattle awaits additional studies using a bovine model and further evaluation of additional strains of the pathogens used in this study.     

Cigarette Smoke-Induced Collagen Destruction; Key to Chronic Neutrophilic Airway Inflammation?

Background

Cigarette smoking induces inflammatory responses in all smokers and is the major risk factor for lung disease such as chronic obstructive pulmonary disease (COPD). In this progressive disease, chronic inflammation in the lung contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated Proline-Glycine-Proline (N-ac-PGP). The generation of this tripeptide is mediated by a multistep pathway involving matrix metalloproteases (MMPs) 8 and 9 and prolyl endopeptidase (PE). Here we investigated whether cigarette smoke extract (CSE) stimulates human PMNs to breakdown whole matrix collagen leading to the generation of the chemotactic collagen fragment N-ac-PGP.

Methodology/Principal Findings

Incubating PMNs with CSE led to the release of chemo-attractant CXCL8 and proteases MMP8 and MMP9. PMNs constitutively expressed PE activity as well as PE protein. Incubating CSE-primed PMNs with collagen resulted in collagen breakdown and in N-ac-PGP generation. Incubation of PMNs with the tripeptide N-ac-PGP resulted in the release of CXCL8, MMP8 and MMP9. Moreover, we tested whether PMNs from COPD patients are different from PMNs from healthy donors. Here we show that the intracellular basal PE activity of PMNs from COPD patients increased 25-fold compared to PMNs from healthy donors. Immunohistological staining of human lung tissue for PE showed that besides neutrophils, macrophages and epithelial cells express PE.

Conclusions

This study indicates that neutrophils activated by cigarette smoke extract can breakdown collagen into N-ac-PGP and that this collagen fragment itself can activate neutrophils, which may lead in vivo to a self-propagating cycle of neutrophil infiltration, chronic inflammation and lung emphysema. MMP-, PE- or PGP-inhibitors can serve as an attractive therapeutic target and may open new avenues towards effective treatment of COPD.     

Effects of Euterpe oleracea Mart. (A?Aí) extract in acute lung inflammation induced by cigarette smoke in the mouse

Short term inhalation of cigarette smoke (CS) induces significant lung inflammation due to an imbalance of oxidant/antioxidant mechanisms. A?ai fruit (Euterpe oleracea) has significant antioxidant and anti-inflammatory actions. The present study aimed to determine whether oral administration of an a?ai stone extract (ASE) could reduce lung inflammation induced by CS. Thirty C57BL/6 mice were assigned to three groups (n=10 each): the Control+A group was exposed to ambient air and treated orally with ASE 300 mg/kg/day; the CS group was exposed to smoke from 6 cigarettes per day for 5 days; and the CS+A group was exposed to smoke from 6 cigarettes per day for 5 days and treated orally with ASE (300 mg/kg/day). On day 6, all mice were sacrificed. After bronchoalveolar lavage, the lungs were removed for histological and biochemical analyses. The CS group exhibited increases in alveolar macrophage (AMs) and neutrophil numbers (PMNs), myeloperoxidase (MPO), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase activities (GPx), TNF-α expression, and nitrites levels in lung tissue when compared with the control ones (p<0.001 for all parameters). The AMs, PMNs, MPO, SOD, CAT, GPx and nitrite were significantly reduced by oral administration of ASE when compared with CS group (p<0.001 for all parameters, with exception of AMs p<0.01). The present results suggested that systemic administration of an ASE extract could reduce the inflammatory and oxidant actions of CS. Thus, the results of this study in mice should stimulate future studies on ASE as a potential agent to protect against CS-induced inflammation in humans.