Migration Of PMNs Research Articles

Distinct stimulus-dependent neutrophil dynamics revealed by real-time imaging of intestinal mucosa after acute injury.

Clinical symptoms in many inflammatory diseases of the intestine are directly related to neutrophil (PMN) migration across colonic mucosa and into the intestinal lumen, yet in-vivo studies detailing this process are lacking. Using real-time intravital microscopy and a new distal colon loop model, we report distinct PMN migratory dynamics in response to several models of acute colonic injury. PMNs exhibited rapid swarming responses after mechanically induced intestinal wounds. Similar numbers of PMNs infiltrated colonic mucosa after wounding in germ-free mice, suggesting microbiota-independent mechanisms. By contrast, acute mucosal injury secondary to either a treatment of mice with dextran sodium sulfate or an IL-10 receptor blockade model of colitis resulted in lamina propria infiltration with PMNs that were largely immotile. Biopsy wounding of colonic mucosa in DSS-treated mice did not result in enhanced PMN swarming however, intraluminal application of the neutrophil chemoattractant LTB4 under such conditions resulted in enhanced transepithelial migration of PMNs. Analyses of PMNs that had migrated into the colonic lumen revealed that the majority of PMNs were directly recruited from the circulation and not from the immotile pool in the mucosa. Decreased PMN motility parallels upregulation of the receptor CXCR4 and apoptosis. Similarly, increased expression of CXCR4 on human PMNs was observed in colonic biopsies from people with active ulcerative colitis. This new approach adds an important tool to investigate mechanisms regulating PMN migration across mucosa within the distal intestine and will provide new insights for developing future anti-inflammatory and pro-repair therapies.

Transepithelial channels for leukocytes in the junctional epithelium

The junctional epithelium (JE) has been recognized as a defensive organ rich in polymorphonuclear leukocytes (PMNs). However, the migration of PMNs through the JE has not been clearly documented. For mucosal defense, PMNs migrate outwards over the epithelium to defend the intestinal or respiratory tract on the epithelial surface. With this background, the present study investigated whether there is any structural evidence showing the transepithelial migration of PMNs through the JE in gingival mucosa. Three-dimensional modeling of gingiva surrounding mouse molars at varying ages was performed by array tomography. Images of the serial sections for array tomography at the 800 nm thickness were obtained using back scattered electron (BSE) detector equipped in the field-emission scanning electron microscopy (FESEM). Expressions of neutrophil marker or CD47 were immunohistochemically examined on the frozen sections. Array tomography using FESEM and 3-dimensional modeling clearly showed that a system of epithelial channels developed between keratinocytes and generally ran along the long axis of the JE. Most PMNs were found inside the channels, rather than being scattered throughout the JE. The channels could be traced from the base of the JE to the bottom of the gingival sulcus, although some channels were fragmented and interrupted with short intercellular gaps. These findings suggest that the JE may be an organ for transepithelial migration of PMNs to the bottom of the gingival sulcus through epithelial channels, as occurs in the epithelial lining of other organs such as the intestinal or respiratory tract.

Synergistic Antibacterial Effect of Zinc Oxide Nanoparticles and Polymorphonuclear Neutrophils.

Zinc oxide nanoparticles (ZnONPs) are inorganic nano-biomaterials with excellent antimicrobial properties. However, their effects on the anti-infection ability of the innate immune system remains poorly understood. The aim of the present study was to explore the potential immunomodulatory effects of ZnONPs on the innate immune system, represented by polymorphonuclear leukocytes (PMNs), and determine whether they can act synergistically to resist pathogen infections. In vitro experiment showed that ZnONPs not only exhibit obvious antibacterial activity at biocompatible concentrations but also enhance the antibacterial property of PMNs. In vivo experiments demonstrated the antibacterial effect of ZnONPs, accompanied by more infiltration of subcutaneous immune cells. Further ex vivo and in vitro experiments revealed that ZnONPs enhanced the migration of PMNs, promoted their bacterial phagocytosis efficiency, proinflammatory cytokine (TNF-α, IL-1β, and IL-6) expression, and reactive oxygen species (ROS) production. In summary, this study revealed potential synergistic effects of ZnONPs on PMNs to resist pathogen infection and the underlying mechanisms. The findings suggest that attempts should be made to fabricate and apply biomaterials in order to maximize their synergy with the innate immune system, thus promoting the host’s resistance to pathogen invasion.

Neutrophils-From Bone Marrow to First-Line Defense of the Innate Immune System.

Neutrophils (polymorphonuclear cells; PMNs) form a first line of defense against pathogens and are therefore an important component of the innate immune response. As a result of poorly controlled activation, however, PMNs can also mediate tissue damage in numerous diseases, often by increasing tissue inflammation and injury. According to current knowledge, PMNs are not only part of the pathogenesis of infectious and autoimmune diseases but also of conditions with disturbed tissue homeostasis such as trauma and shock. Scientific advances in the past two decades have changed the role of neutrophils from that of solely immune defense cells to cells that are responsible for the general integrity of the body, even in the absence of pathogens. To better understand PMN function in the human organism, our review outlines the role of PMNs within the innate immune system. This review provides an overview of the migration of PMNs from the vascular compartment to the target tissue as well as their chemotactic processes and illuminates crucial neutrophil immune properties at the site of the lesion. The review is focused on the formation of chemotactic gradients in interaction with the extracellular matrix (ECM) and the influence of the ECM on PMN function. In addition, our review summarizes current knowledge about the phenomenon of bidirectional and reverse PMN migration, neutrophil microtubules, and the microtubule organizing center in PMN migration. As a conclusive feature, we review and discuss new findings about neutrophil behavior in cancer environment and tumor tissue.

Supramolecular Toxin Complexes for Targeted Pharmacological Modulation of Polymorphonuclear Leukocyte Functions.

The targeted pharmacological modulation of polymorphonuclear leukocytes (PMNs) is of major medical interest. These innate immune cells play a central role in the defense against pathogenic microorganisms. However, their excessive chemotactic recruitment into tissues after traumatic injury is detrimental due to local and systemic inflammation. Rho-GTPases, being the master regulators of the actin cytoskeleton, regulate migration and chemotaxis of PMNs, are attractive pharmacological targets. Herein, supramolecular protein complexes are assembled in a "mix-and-match" approach containing the specific Rho-inhibiting clostridial C3 enzyme and three PMN-binding peptides using an avidin platform. Selective delivery of the C3 Rho-inhibitor with these complexes into the cytosol of human neutrophil-like NB-4 cells and primary human PMNs ex vivo is demonstrated, where they catalyze the adenosine diphosphate (ADP) ribosylation of Rho and induce a characteristic change in cell morphology. Notably, the complexes do not deliver C3 enzyme into human lung epithelial cells, A549 lung cancer cells, and immortalized human alveolar epithelial cells (hAELVi), demonstrating their cell type-selectivity. The supramolecular complexes represent attractive molecular tools to decipher the role of PMNs in infection and inflammation or for the development of novel therapeutic approaches for diseases that are associated with hyperactivity and reactivity of PMNs such as post-traumatic injury.

Abstract 2799: Targeting CXCR2-mediated neutrophil recruitment to lung cancer

Abstract With recent advances in immunotherapy, it is evident that targeting the tumor microenvironment (TME) is an effective strategy to treat lung cancer (LC), however, more than half LC patients are still resistant to therapy. Limited attention was given to the relevance of the innate immune system despite its critical role in triggering adaptive responses. Neutrophils (PMNs) are the predominant circulating leukocyte in humans. PMNs are associated with developing lesions and are the main immune component of primary non-small cell LC (NSCLC). Multiple studies support the notion that PMNs promote tumor progression, however, the exact mechanisms in which these PMNs are recruited to the primary and metastatic lung TMEs remain unclear. To this end, we examined available genomic databases of > 1,000 NSCLC primary adenocarcinoma (ADC) patients and observed that high expression of all CXCR2 ligands (CXCL1-8 and MIF) correlate with poor survival in lung ADC. Lung ADC patients display one of the highest fold increases of these ligands as compared to all other cancers. We then performed shRNA knock down (KD) of CXCL1 and MIF in A549 and tested the migration of PMNs towards treated and control cell lines using the novel microfluidic device. We observe 3-fold increase of PMN migration towards A549 compared to control. This increase was significantly inhibited in MIF and CXCL1 KDs as well as using MIF and CXCL1 neutralizing antibodies (NA) as compared to controls. PMN migration was higher to A549 then to PC9EN, and treatment of PMNs with a CXCR2 NA led to a decrease in their migration to A549 while unaffecting their migration to PC9EN. Due to the lack of similar genomic databases on LC metastasis, we profiled liver homogenates of mice intrasplenically injected with liver-metastatic Lewis lung carcinoma (LLC) and observed that Cxcl1 was the most overexpressed gene as compared to non-tumor bearing mice (non-TBM). We then KD CXCL1 from the liver metastatic LLC cell line and compared its capacity to recruit PMNs in live mice using intravital microscopy. We observe a decrease in the number of PMNs around developing CXCL1 KD LLC tumors compared to control LLC. We also observe a decrease in PMN migration toward the CXCL1 KD LLC tumors as compared the control LLC. This resulted in a significant decrease in liver metastasis of the CXCL1 KD LLC as compared to control LLC injected mice. Altogether, our data highlight the importance of CXCR2-mediated PMN migration in primary LC and the establishment of liver metastasis from LC. Thus, inhibiting CXCR2 represents a promising strategy to impede primary tumor growth and metastatic dissemination of LC. Citation Format: Roni F. Rayes, Jack G. Mouhanna, Claire Wang, Simon Milette, Carson Wong, Mariana Usatii, Betty Giannias, France Bourdeau, Rachel Mot, Arvind Chandrasekaran, Christopher Moraes, Sidong Huang, Daniela Quail, Logan Walsh, Veena Sangwan, Nicholas Bertos, Pierre-Olivier Fiset, Jonathan Cools-Lartigue, Lorenzo E. Ferri, Jonathan D. Spicer. Targeting CXCR2-mediated neutrophil recruitment to lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2799.

IL-1β-Mediated Activation of Adipose-Derived Mesenchymal Stromal Cells Results in PMN Reallocation and Enhanced Phagocytosis: A Possible Mechanism for the Reduction of Osteoarthritis Pathology

Background: Injection of adipose-derived mesenchymal stromal cells (ASCs) into murine knee joints after induction of inflammatory collagenase-induced osteoarthritis (CiOA) reduces development of joint pathology. This protection is only achieved when ASCs are applied in early CiOA, which is characterized by synovitis and high S100A8/A9 and IL-1β levels, suggesting that inflammation is a prerequisite for the protective effect of ASCs. Our objective was to gain more insight into the interplay between synovitis and ASC-mediated amelioration of CiOA pathology.Methods: CiOA was induced by intra-articular collagenase injection. Knee joint sections were stained with hematoxylin/eosin and immunolocalization of polymorphonuclear cells (PMNs) and ASCs was performed using antibodies for NIMP-R14 and CD271, respectively. Chemokine expression induced by IL-1β or S100A8/A9 was assessed with qPCR and Luminex. ASC-PMN co-cultures were analyzed microscopically and with Luminex for inflammatory mediators. Migration of PMNs through transwell membranes toward conditioned medium of non-stimulated ASCs (ASCNS-CM) or IL-1β-stimulated ASCs (ASCIL-1β-CM) was examined using flow cytometry. Phagocytic capacity of PMNs was measured with labeled zymosan particles.Results: Intra-articular saline injection on day 7 of CiOA increased synovitis after 6 h, characterized by PMNs scattered throughout the joint cavity and the synovium. ASC injection resulted in comparable numbers of PMNs which clustered around ASCs in close interaction with the synovial lining. IL-1β-stimulation of ASCs in vitro strongly increased expression of PMN-attracting chemokines CXCL5, CXCL7, and KC, whereas S100A8/A9-stimulation did not. In agreement, the number of clustered PMNs per ASC was significantly increased after 6 h of co-culturing with IL-1β-stimulated ASCs. Also migration of PMNs toward ASCIL-1β-CM was significantly enhanced (287%) when compared to ASCNS-CM. Interestingly, association of PMNs with ASCs significantly diminished KC protein release by ASCs (69% lower after 24 h), accompanied by reduced release of S100A8/A9 protein by the PMNs. Moreover, phagocytic capacity of PMNs was strongly enhanced after priming with ASCIL-1β-CM.Conclusions: Local application of ASCs in inflamed CiOA knee joints results in clustering of attracted PMNs with ASCs in the synovium, which is likely mediated by IL-1β-induced up-regulation of chemokine release by ASCs. This results in enhanced phagocytic capacity of PMNs, enabling the clearance of debris to attenuate synovitis.

The nitrated fatty acid, 10-nitrooleate inhibits the neutrophil chemotaxis via peroxisome proliferator-activated receptor gamma in CLP-induced sepsis in mice

The inhibition of polymorphonuclear neutrophils' (PMNs) migration to the source of injury is among the most prominent aspects of immunosuppression following sepsis, although the precise mechanisms involved remain unclear and multifaceted. Increasing evidence connects this immunosuppression to nitric oxide (NO), as NO production is a classic feature of inflammation probably through neutrophil activation and migration. Nitrated fatty acids (NFA) such as 10-nitrooleate (OA-NO2), nitrolinoleic acid etc. produced endogenously by the non-enzymatic reaction of NO with unsaturated fatty acids, are found to be potent activators of the transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ). Upregulation of PPARγ during immunosuppression and the subsequent inhibition of neutrophil migration in sepsis have been reported. However, the interplay of OA-NO2, NO and PPARγ in polymicrobial-induced immunosuppression has not been established. Hence to understand this, we have studied the role of OA-NO2 in blood PMNs migration, the effects of iNOS inhibitor on PMNs migration and PPARγ activity in cecal ligation and puncture (CLP)-induced sepsis in mice. We found increased expression of PPARγ and its DNA-binding activity in the lungs and blood PMNs from CLP mice. CLP or OA-NO2 treatment inhibited PMNs' migration in response to fMLP stimulation. Pharmacological inhibition of iNOS resulted in decreased PPARγ DNA-binding activity with a concomitant increase in the migration of PMNs to the site of infection. OA-NO2 treatment also inhibited the production of inflammatory cytokines (TNFα and IL-1β) secretion from PMNs stimulated with lipopolysaccharide. We have also established that, OA-NO2 mediated inhibition of PMNs migration in vivo and ex vivo are regulated through PPARγ-dependent pathway. This study further highlights the fact that the activation of PPARγ by the NFA has a pivotal role in PMNs' migration and immunosuppression.

Pseudomonas aeruginosa-induced lung epithelial production of IL-8 contributes to PMN sub-mucosa recruitment but not trans-epithelial migration in vitro.

Abstract Background Numerous lung associated diseased states such as acute pneumonia and cystic fibrosis (CF) are associated with increased concentrations of IL8 and infiltration of neutrophils (PMNs) in the airspace. IL8 production by lung epithelial cells (ECs) is stimulated by Pseudomonas aeruginosa infection, an opportunistic pathogen involved in acute pneumonia, and lung infection of CF patients. The exact role IL8 plays in migration of circulating PMNs into the infected lung and airspace is still uncertain. Objective Assess the role of IL8 in mediating PMN migration in response to P. aeruginosa infected lung epithelium. Methods CRISPR/Cas9 was used to delete the IL8 gene from human lung ECs, which were grown as polarized monolayers in a co-culture transwell model. Basolateral to apical PMN migration to PAO1 infection was assessed. A novel double transwell model was developed to observe PMN submucosa migration in response to PAO1 infection in the presence and absence of IL8 lung EC production. Results PAO1 induced PMN trans-epithelial (TE) migration was not impacted in the absence of IL8 expression. When PMNs were placed in a separate transwell suspended 5 mm above the basolateral side of the infected epithelium, the IL8 gradient produced was sufficient to cause relevant migration of PMNs to the basolateral side of the infected epithelium. The level of migrated PMNs in the double transwell system with tissues devoid of the IL8 gene was equivalent to the presence of PMNs which responded to un-infected epithelial tissues. Conclusion These findings further support that IL8 is integral in PMN recruitment, and suggests that IL8 is most important towards recruiting PMNs to the submucosa during P. aeruginosa PAO1 infection of lung epithelium.

Metabolic Abnormalities in G6PC3 Deficient Human Neutrophils Result in Severe Functional Defects

IntroductionSevere congenital neutropenia, type 4 results from a mutation in the G6PC3 gene. This gene encodes the enzyme glucose-6-phosphatase (G6Pase) which is ubiquitously expressed in endoplasmic reticulum and is responsible for the final step of gluconeogenesis and glycogenolysis. G6pc3-/- mouse models have demonstrated decreased neutrophil (PMN) function including impaired chemotaxis and superoxide anion production and increased apoptosis. Metabolically, g6pc3-/-mice have impaired intracellular glucose transport and reduced levels of ATP and lactic acid. We present the most comprehensive functional and metabolic analysis to date on a patient with a novel compound heterozygous mutation in the G6PC3 gene, c.325G>A and c.758G>A, validating mouse model findings in a human.MethodsPMNs were isolated from heparinized whole blood by dextran sedimentation and hypotonic lysis of red cells. Chemotactic assays were performed under agarose techniques with zymosan activated serum (ZAS), fMLF, and buffer control. Expression of CD18 and F-actin assembly were analyzed after incubation with buffer, PMA, and fMLF using standard flow cytometric techniques. Bactericidal activity was measured using variable ratios of S Aureus:PMNs in the presence of 10% normal human serum. Superoxide anion production was measured with SOD-inhibitable chemiluminescence with Diogenes reagent. Chemiluminescence was employed to measure caspase expression. Metabolomics and tracing experiments were performed incubating cells with U-13C-glucose prior to UHPLC/MS analysis.ResultsCell motility was significantly impaired in human G6Pase deficient (patient) PMNs. Non-directed migration was reduced by over 50% compared to controls. Directed migration of patient PMNs was only 35% and 23% of control when stimulated with ZAS and 0.1 µM fMLF respectively. Surface expression of CD18 in patient cells did not increase in response to either stimulus but more than doubled in normal PMNs. F-actin was present at significantly higher levels in unstimulated patient PMNs and did not increase in the presence of 200 ng/mL PMA or 1 µM fMLF.Bactericidal activity was normal at a bacteria:PMN ratio of 1:1 but abnormal at ratios > 5:1 with over a 20% increase in viable bacteria at 120 minutes for patient PMNs. Superoxide anion production was markedly impaired with over 70% reduction compared to control when stimulated with either 200 ng/mL PMA or 1 µM fMLF. Caspase expression was not significantly different between patient and control PMNs, but the patient was on filgrastim which has antiapoptotic effects.Tracing experiments with 13C6-glucose showed decreased flux through glycolysis (significant reductions in labeled isotopologues for G6P and lactate) and marked inhibition of the NADPH-generating pentose phosphate pathway (PPP) in patient PMNs stimulated with 1µM fMLF. Glutaminolysis appears to be increased with increased glutamine consumption in patient PMNs, decreased glucogenic amino acids, and increased TCA cycle intermediates.DiscussionMetabolic reprogramming is a recently appreciated hallmark of immune cell activation. Decreases in G6P affect multiple metabolic pathways including glycolysis, PPP and TCA cycle. Deficiency of NADPH-dependent superoxide anion production likely results from impairment in the NADPH-generating PPP. Interestingly the bactericidal defect is not as severe as would be predicted given the degree of impairment in superoxide anion production. The most striking functional defect is with PMN motility. Defects in the glycolytic/PPP likely contribute to this phenotype. ATP-dependent actin assembly is critical for cell migration. Our data demonstrate that in human patient PMNs, nearly all actin is in the filamentous form and does not increase further in response to chemoattractants. This could result in impaired hydrolysis of the F-actin filament and reassembly at the leading edge. We also see reduction in surface expression of CD11b/CD18 which is vital for PMN adhesion and migration.In conclusion, metabolic defects resulting from G6PC3 deficiency contribute to global PMN dysfunction and absolute neutropenia. Patients may be at increased risk for infections in spite of correction of neutropenia with filgrastim. These novel findings in human patient cells elucidate further the pathogenesis of this disease which has largely been dependent on data from mouse models. DisclosuresNo relevant conflicts of interest to declare.

CC chemokine ligand 2 and CXC chemokine ligand 8 as neutrophil chemoattractant factors in canine idiopathic polyarthritis

Canine idiopathic polyarthritis (IPA) is characterized by increased numbers of polymorphonuclear leukocytes (PMNs) in the synovial fluid (SF). In humans, CC chemokine ligand 2 (CCL2) and CXC chemokine ligand 8 (CXCL8) recruit monocytes and neutrophils, respectively, and are involved in various inflammatory disorders. The aim of this study was to assess the roles of these chemokines in driving PMNs infiltration into the joints of dogs with IPA. SF samples were collected from dogs with IPA (n=19) and healthy controls (n=8), and the concentrations of SF CCL2 and CXCL8 were determined by ELISA. Dogs with IPA had significantly higher concentrations of CCL2 (3316±2452pg/ml, mean±SD) and CXCL8 (3668±3879pg/ml) compared with the healthy controls (235±45pg/ml and <15.6pg/ml, respectively). Then, an in vitro chemotaxis assay was performed using a modified Boyden chamber (pore size: 3μm). SF from IPA dogs had a chemoattractant activity for PMNs that purified from the peripheral blood of a healthy dog. We subsequently found that combination treatment with MK-0812 (an antagonist of CCL2 receptor) and repertaxin (an antagonist of CXCL8 receptors) significantly inhibited the migration of PMNs to SF from IPA dogs. Thus, expression of the CCL2 receptor (chemokine (CC motif) receptor 2 (CCR2)) was examined using polymerase chain reaction and immunocytochemistry. Canine peripheral blood PMNs exhibited significantly higher CCR2 mRNA expression levels than those in monocytes. In addition, we observed strong CCR2 expression on PMNs obtained from healthy controls and IPA dogs, although mononuclear cells did not express CCR2. Taken together, the data suggest that CCL2 acts as a canine PMNs chemotactic factor as well as CXCL8 and both CCL2 and CXCL8 facilitate the infiltration of PMNs into the joints of dogs with IPA.

Targeting of Neutrophil Lewis X Blocks Transepithelial Migration and Increases Phagocytosis and Degranulation

Polymorphonuclear leukocytes (PMNs) are innate immune cells whose principal function is to migrate from the blood to sites of inflammation, where they exert crucial anti-infectious and immunomodulatory effects. However, dysregulated migration of PMNs into mucosal epithelial tissues is characteristic of chronic inflammatory disorders, including inflammatory bowel disease. Carbohydrate-mediated binding interactions between PMN Lewis glycans and endothelial glycan-binding proteins are critical for initial migration of PMN out of the vasculature. However, the role of Lewis glycans during transepithelial migration (TEM) has not been well characterized. Herein, we show that antibody blockade of Lewis X (Le(x)) displayed as terminal glycan residues on the PMN surface blocks chemotaxis and TEM while enhancing PMN-adhesive interactions with intestinal epithelia. Unexpectedly, targeting of subterminal Le(x) residues within glycan chains had no effect on PMN migration or adhesive interactions. There was increased surface expression of Le(x) on PMN after TEM, and blockade of terminal Le(x) regulated post-migratory PMN functions, increasing PMN phagocytosis and the surface mobilization of azurophilic (CD63, myeloperoxidase, and neutrophil elastase) and specific (CD66b and lactoferrin) granule markers. These findings suggest that terminal Le(x) represents a potential target for regulating PMN trafficking and function in inflamed mucosa. Furthermore, given its abundant expression on migrating PMN, Le(x) may be a rational target for modulating inflammation in diseases where dysregulated PMN influx is associated with host tissue damage.

Effect of semen extenders on frozen-thawed boar sperm characteristics and distribution in the female genital tract after deep intrauterine insemination in sows

We compared the effects of extenders of frozen-thawed semen on post-thaw sperm characteristics and the distribution of frozen-thawed spermatozoa in the female genital tract after fixed-timed deep intrauterine insemination (DIUI) in sows. Frozen semen samples were thawed and diluted in either modified Modena solution (mMS) or porcine fertilization medium (PFM) containing theophylline, adenosine and cysteine. Sperm quality, assessed in vitro based on motility using a computer-assisted sperm analyzer and the integrity of the plasma and acrosomal membranes using flow cytometry, was evaluated at 0.5, 1.5, 3 and 6h after thawing. Progressive motility and the percentage of spermatozoa with damaged acrosomal membranes in PFM were significantly better than in mMS throughout the 6h. Sows with estrus synchronized using prostaglandin F2 alpha, equine chorionic gonadotropin and human chorionic gonadotropin (hCG) were inseminated once with mMS- or PFM-diluted 5×108 frozen-thawed spermatozoa by DIUI at 34h after the hCG injection. At 4h after DIUI, reproductive tracts were recovered from 30 sows. There were significantly fewer polymorphonuclear leukocytes (PMNs) and more spermatozoa outside PMNs in the uterine horn after PFM treatment than with mMS. When 22 sows were administered DIUI with 10×108 frozen-thawed spermatozoa at 36h after hCG, the pregnancy rates did not differ significantly between the mMS- (36%) and PFM- (64%) treated groups. Thus, PFM enhanced progressive sperm motility but increased sperm membrane damage compared with mMS; it also suppressed the migration of PMNs into the uterine lumen.

Exocytosis of toxic primary granules by human neutrophils is associated with high surface TRPM2 expression and pinocytosis (INC6P.401)

Abstract Polymorphonuclear neutrophils (PMNs) play a key role in host defense against microbial infection. PMN responses culminate in the mobilization of highly toxic intracellular primary granules toward either the phagosome (during phagocytosis), nucleus (during NETosis), or plasma membrane (exocytosis). Mechanisms regulating the fate of PMN primary granules remain unclear. Here, we studied the dynamics of PMN primary granule exocytosis in two relevant sets of conditions: 1- in vivo migration of blood PMNs into chronically infected airways of patients with cystic fibrosis; 2- in vitro treatment of blood PMNs with latrunculin B and fMLF. Using flow and image cytometry, we show a striking resemblance between these two sets of conditions, with a close association between primary granule exocytosis (based on surface CD63 expression) and increased surface expression of the inflammatory calcium channel TRPM2. In addition, high primary granule exocytosis associates with major membrane rearrangements, as evidenced by the loss of surface phagocytic receptors (e.g., CD16 and CD35) and high pinocytosis (based on Lucifer Yellow uptake), a process previously linked to TRPM2 activation. All the while, exocytosing PMNs maintain their nuclear integrity. Thus, high primary granule exocytosis, surface TRPM2 upregulation and high pinocytosis are a concerted set of events indicative of a functional program in activated PMNs that is distinct from phagocytosis and NETosis.

Integrin-dependent neutrophil migration in the injured mouse cornea

As an early responder to an inflammatory stimulus, neutrophils (PMNs) must exit the vasculature and migrate through the extravascular tissue to the site of insult, which is often remote from the point of extravasation. Following a central epithelial corneal abrasion, PMNs recruited from the peripheral limbal vasculature migrate into the avascular corneal stroma. In vitro studies suggest PMN locomotion over 2-D surfaces is dependent on integrin binding while migration within 3-D matrices can be integrin-independent. Electron micrographs of injured mouse corneas show migrating PMNs make extensive surface contact not only with collagen fibrils in the extracellular matrix (ECM), but also keratocytes. Evidence supporting involvement of integrins in corneal inflammation has prompted research and development of integrin blocking agents for use as anti-inflammatory therapies. However, the role of integrin binding (cell–cell; cell–ECM) during stromal migration in the inflamed cornea has previously not been clearly defined. In this study in vivo time lapse imaging sequences provided the means to quantify cell motility while observing PMN interactions with keratocytes and other stromal components in the living eye. The relative contribution of β1, β2 and β3 integrins to PMN locomotion in the inflamed mouse cornea was investigated using blocking antibodies against the respective integrins. Of the 3 integrin families (β1, β2 and β3) investigated for their potential role in PMN migration, only β1 antibody blockade produced a significant, but partial, reduction in PMN motility. The preferential migration of PMNs along the keratocyte network was not affected by integrin blockade. Hence, the dominant mechanism for PMN motility within the corneal stroma appears to be integrin-independent as does the restriction of PMN migration paths to the keratocyte network.

In Vitro Inhibitory Effects of Moringa oleifera Leaf Extract and Its Major Components on Chemiluminescence and Chemotactic Activity of Phagocytes

The ethanol extract of Moringa oleifera Lam. leaves and its major constituents, crypto-chlorogenic acid, quercetin 3-O-glucoside and kaempferol 3-O-glucoside, were investigated on the respiratory burst of human whole blood and isolated human polymorphonuclear leukocytes (PMNs) using a luminol-based chemiluminescence assay. The chemotactic migration of PMNs was also investigated using the Boyden chamber technique. The ethanol extract demonstrated inhibitory activities on the oxidative burst and the chemotactic migration of PMNs. Quercetin 3-O-glucoside, crypto-chlorogenic acid, and kaempferol 3-O-glucoside, isolated from the extract, expressed relatively strong inhibitory activity on the oxidative burst of PMNs with IC50 values of 4.1, 6.7 and 7.0 microM, respectively, comparable with that of aspirin. They also demonstrated strong inhibition of chemotatic migration of PMNs with IC50 values of 9.5, 15.9 and 18.2 microM, respectively. The results suggest that M. oleifera leaves could modulate the immune response of human phagocytes, linking to its ethnopharmacological use as an anti-inflammatory agent. The immunomodulating activity of the plant was mainly due to its major components.

Immunomodulatory effects of 1-(6-hydroxy-2-isopropenyl-1-benzofuran-5-yl)-1-ethanone from Petasites hybridus and its synthesized benzoxazepine derivatives

1-(6-Hydroxy-2-isopropenyl-1-benzofuran-5-yl)-1-ethanone (1), isolated from the roots of Petasites hybridus L., and a series of synthetic benzoxazepine derivatives of compound 1 (2-6) were evaluated for their immunomodulatory effects. The compounds were evaluated for their effects on the respiratory burst of human whole blood and isolated human polymorphonuclear leukocytes (PMNs) using luminol- and lucigenin-based chemiluminescence (CL) assays, and their effect on chemotactic migration of PMNs was assessed using the Boyden chamber technique. Compound 1 exhibited stronger inhibition than acetylsalicylic acid (ASA) on luminol-enhanced CL of PMNs. It also inhibited PMN chemotaxis with an IC50 value comparable to that of ibuprofen. Of the compounds tested, 5 was the most effective in inhibiting luminol-enhanced CL and also strongly inhibited lucigenin-enhanced CL with IC50 values lower than that of ASA. Compound 2 was the most active in inhibiting migration of PMNs and was five times stronger than ibuprofen. The results suggest that compound 1 and its synthesized benzoxazepine derivatives, especially compounds 2 and 5, were able to modulate the innate immune response of phagocytes at different steps, emphasizing their potential as leads for the development of new immunomodulatory agents.

Synthesis and Effects of Pyrazolines and Isoxazoles on the Phagocytic Chemotaxis and Release of Reactive Oxygen Species by Zymosan Stimulated Human Neutrophils

A series of novel isoxazole and pyrazoline derivatives has been synthesized and evaluated for their effects on the chemiluminescence and chemotactic activity of human phagocytes. Their effects on the chemotactic migration of isolated polymorphonuclear leukocytes (PMNs) and on the release of reactive oxygen species (ROS) during respiratory burst of human whole blood and PMNs were carried out using the Boyden chamber technique and luminol-based chemiluminescence assay, respectively. Of the compounds tested, compounds 8, 9, 11 and 12 exhibited higher inhibitory activity on the release of ROS (with IC50 values ranging from 5.6 to 8.4 μM) than acetylsalicylic acid (IC50 = 9.5 μ M). These compounds also showed strong inhibitory activity on the migration of PMNs with compound 8 exhibiting an IC50 value lower than that of ibuprofen. The results suggest that some of these isoxazole and pyrazoline derivatives have ability to modulate the innate immune response of phagocytes at different steps, indicating their potential as a source of new immunomodulatory agents.

Effects of caffeine on sperm characteristics after thawing and inflammatory response in the uterus after artificial insemination with frozen-thawed boar semen

We previously reported that AI with frozen-thawed boar semen supplemented with caffeine increased the number of uterine sperm by inhibiting migration of polymorphonuclear leukocytes (PMNs) into the uterine lumen, and also improved fertility of gilts and sows. The objective of the present study was to determine the effects of the addition of caffeine to a thawing solution on postthaw sperm quality and uterine inflammatory response after AI with frozen-thawed boar semen. Incubation of frozen-thawed sperm in Modena solution supplemented with 10 mM caffeine for 90 minutes improved (P < 0.05) percentages of progressive motility, straightness, and linearity of sperm movement compared with no caffeine, without causing damage to plasma or acrosomal membranes. Gilts inseminated once with 2 × 109 frozen-thawed sperm suspended in Modena solution with or without caffeine, and gilts that did not receive AI, were slaughtered 4 hours later. Uteri were recovered for analysis of number of uterine PMNs and mRNA expression (quantitative reverse transcription polymerase chain reaction) of tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-8, monocyte chemoattractant protein-1, and cyclooxygenase 2 in the endometrium. Caffeine decreased (P < 0.05) both the number of total uterine PMNs and expression of IL-8 mRNA in the endometrium after AI. The amount of IL-8 and cyclooxygenase 2 mRNA after AI in the absence of caffeine were higher than samples from gilts that did not receive AI (P < 0.05), whereas there were no significant differences between treatments in expression levels of tumor necrosis factor-α, IL-1β, IL-6, or monocyte chemoattractant protein-1 mRNA. Pregnancy rate in sows inseminated with sperm supplemented with caffeine (16 of 23; 70%) tended (P < 0.1) to exceed that without caffeine (12 of 26; 46%), but litter size was not affected. In conclusion, the addition of caffeine to the thawing solution inhibited migration of uterine PMNs, probably by downregulating IL-8 mRNA expression in the endometrium.

Chemo-attractant N-acetyl proline–glycine–proline induces CD11b/CD18-dependent neutrophil adhesion

BackgroundChronic inflammation in lung diseases contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated proline–glycine–proline (N-ac-PGP). In the current study, we investigate whether N-ac-PGP influences β2-integrin activation and function in neutrophilic firm adhesion to endothelium. MethodsHuman polymorphonuclear leukocytes (PMNs) were isolated from fresh human blood. Subsequently, a transmigration assay was performed to evaluate the active migration of PMNs towards N-ac-PGP. Furthermore, the effect of the tripeptide on β2-integrin activation was assessed by performing the adhesion assay using fibrinogen as a ligand. To determine whether this effect was due to conformational change of β2-integrins, antibodies against CD11b and CD18 were used in the adhesion assay and the expression pattern of CD11b was determined. ResultsHuman neutrophils transmigrated through an endothelial cell layer in response to basolateral N-ac-PGP. N-ac-PGP induced also a neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that CD11b/CD18 (Mac-1) was responsible for the N-ac-PGP-induced firm adhesion of neutrophils to fibrinogen. Pertussis toxin decreased the Mac-1 activation indicating the involvement of G-proteins. N-ac-PGP most likely activated Mac-1 by initiating a conformational change, since the expression pattern of Mac-1 on the cell surface did not change significantly. ConclusionsChemo-attractant N-acetyl proline–glycine–proline induces CD11b/CD18-dependent neutrophil adhesion. General significanceThis is the first study to describe that the chemo-attractant N-ac-PGP also activates Mac-1 on the surface of neutrophils, which can additionally contribute to neutrophilic transmigration into the lung tissue during lung inflammation.