Functional competence and monoclonal antibody reactivity of neutrophils from cows injected with Escherichia coli endotoxin

Neutrophils of cows injected with endotoxin were evaluated for their functional competence and monoclonal antibody binding in relation to morphological maturity. Six clinically healthy lactating Holstein cows were each injected intravenously with 100 g Escherichia coli endotoxin. Blood was collected at 0, 6, 30, 54, 78 and 150 hours postinjection for total and differential leucocyte counts and to isolate neutrophils for functional assays. Marked leucopenia, neutropenia with left shift to band neutrophils, lymphopenia and monocytopenia were evident at 6 h postinjection. Neutrophil and lymphocyte counts normalised by 30 hours, while band neutrophils remained elevated until 54 hours postinjection.Neutrophils isolated at 6, 30 and 54 hours postinjection revealed significantly increased relative phagocytic index, reduced chemotactic activity, and increased binding of a bovine neutrophil monoclonal antibody (36H10). Ingested bacteria per cell were increased at 6 hours. Phorbol myristate acetate-induced oxidative product formation was significantly reduced at 30 hours, but chemiluminescence activity did not change significantly. In vivo binding of autologous IgG₂ and IgA to neutrophils significantly decreased at 30 and 54 hours and IgM binding was reduced at 78 hours postinjection.Band neutrophils were chemotactically and phagocytically less active than segmented neutrophils. The number of band neutrophils in blood correlated directly with the relative phagocytic index and the percentage of cells binding monoclonal antibody 361110 and inversely with the chemotactic activity of neutrophils. These observations indicate that functional heterogeneity of bovine neutrophils can be attributed, at least in part, to morphological maturity of cells and to differences in expression of surface antigens.     

Dectin-1 promotes fungicidal activity of human neutrophils

Human polymorphonuclear leukocytes (PMN) are a first line of defense against fungal infections. PMN express numerous pattern recognition receptors (PRR) that facilitate identification of invading microorganisms and ultimately promote resolution of disease. Dectin-1 (beta-glucan receptor) is a PRR expressed on several cell types and has been studied on monocytes and macrophages. However, the role played by dectin-1 in the recognition and killing of fungi by PMN is unknown. We investigated the ability of dectin-1 to mediate human PMN phagocytosis and fungicidal activity. Dectin-1 was expressed on the surface of PMN from all subjects tested (n=29) and in an intracellular compartment that co-sedimented with azurophilic granules in Percoll density gradients. Soluble beta-glucan and mAb GE2 (anti-dectin-1) inhibited binding and phagocytosis of zymosan by human PMN (e.g., ingestion was inhibited 40.1% by 30 min, p<0.001), and blocked reactive oxygen species production. Notably, soluble beta-glucan and GE2 inhibited phagocytosis and killing of Candida albicans by PMN (inhibition of killing was 54.8% for beta-glucan and 36.2% for GE2, p<0.01). Our results reveal a mechanism whereby PMN dectin-1 plays a key role in the recognition and killing of fungal pathogens by the innate immune system.     

Blockade of TNF does not alter oxygen burst and phagocytosis of human neutrophils in patients with rheumatoid arthritis

Clinical trials evaluating tumor necrosis factor alpha (TNF-alpha) binding agents in patients with rheumatoid arthritis (RA) have demonstrated significant efficacy in reducing symptoms of disease and slowing radiographic progression. However, infectious complications are the most severe and common adverse effects of anti-TNF therapy. The functional capacities of neutrophils (PMNs) as the first line of defense in bacterial and fungal infections are enhanced by soluble TNF as a potent neutrophil primer. The aim of this study was to assess the influence of in vivo TNF blockade on oxygen burst (OB) and phagocytosis of human neutrophils. PMNs were derived from 20 patients with RA on anti-TNF-alpha therapy and 13 patients using conventional DMARDs. By flow cytometry we measured OB upon stimulation with Escherichia coli and N-formyl-1-methionyl-1-leucyl-phenylalanine (FMLP) with and without priming with granulocyte-colony stimulating factor (G-CSF) and/or TNF-alpha using dihydrorhodamine (DHR) 123. Phagocytosis of fluorescein isothiocyanate (FITC)-labeled E. coli was also assessed by flow cytometry. Thirty-three healthy volunteers served as controls. Upon stimulation with E. coli and FMLP, there was no significant difference in OB between the two patient groups and healthy controls. Priming was effective in all groups. Phagocytosis of E. coli by PMNs was equally effective in controls and patients independent from the treatment regimen. These data show that OB, phagocytosis and responsiveness to priming with TNF and G-CSF of PMNs are not impaired in patients with RA treated with anti-TNF agents in comparison with patients on conventional DMARDs or healthy controls. Thus, the infectious complications observed in patients with TNF blockade cannot be explained by functional impairment of PMNs; however, the neutralization of TNF as a potent primer of neutrophil response may increase the susceptibility for infections in these patients.     

Study of the phagocytic process in neutrophils from elite sportswomen

Summary All the stages of the phagocytic process of blood neutrophils were compared in sedentary young women (no formal exercise during the previous 24 months) and elite sportswomen (basketball players from the Siglo XXI Spain Selection, in the middle of their competitive season) at rest. The sportswomen had performed no exercise since the day before taking the blood samples. Adherence of neutrophils to nylon fibre, which is similar to endothelium adherence, was not different between the two groups [62 (SD 14) and 58 (SD 18) in control and sport groups respectively]. Chemotaxis (studied in a Boyden chamber using a filter with 3 m pore diameter) was found to be stimulated (P<0.001) in the sportswomen [105 (SD 30)] with respect to the controls [39 (SD 9)]. Attachment, ingestion and killing by neutrophils was measured by incubation of the neutrophils with serum and a suspension ofCandida albicans. While no statistical differences were found in attachment ofC. albicans after 15 min incubation [71 (SD 8) in the control group, and 74 (SD 20) in the sport group], the sportswomen showed a higher (P<0.001) ingestion capacity forC. albicans at both 15 min [53 (SD 13) and 111 (SD 32) in control and sportswomen respectively] and 60 min [control 90 (SD 10), and sport group 224 (SD 21)] incubation. The greater phagocytic capacity in sportswomen was correlated with a higher plasma cortisol concentration (P <0.05) and a lower plasma ACTH concentration (P <0.001) in this group. It is concluded that elite women basketball players have a greater phagocytic capacity than sedentary women, possibly mediated by the higher plasma cortisol concentration in the sportswomen.     

Human neutrophils in auto-immunity

Human neutrophils have great capacity to cause tissue damage in inflammatory diseases via their inappropriate activation to release reactive oxygen species (ROS), proteases and other tissue-damaging molecules. Furthermore, activated neutrophils can release a wide variety of cytokines and chemokines that can regulate almost every element of the immune system. In addition to these important immuno-regulatory processes, activated neutrophils can also release, expose or generate neoepitopes that have the potential to break immune tolerance and result in the generation of autoantibodies, that characterise a number of human auto-immune diseases. For example, in vasculitis, anti-neutrophil cytoplasmic antibodies (ANCA) that are directed against proteinase 3 or myeloperoxidase are neutrophil-derived autoantigens and activated neutrophils are the main effector cells of vascular damage. In other auto-immune diseases, these neutrophil-derived neoepitopes may arise from a number of processes that include release of granule enzymes and ROS, changes in the properties of components of their plasma membrane as a result of activation or apoptosis, and via the release of Neutrophil Extracellular Traps (NETs). NETs are extracellular structures that contain chromatin that is decorated with granule enzymes (including citrullinated proteins) that can act as neo-epitopes to generate auto-immunity. This review therefore describes the processes that can result in neutrophil-mediated auto-immunity, and the role of neutrophils in the molecular pathologies of auto-immune diseases such as vasculitis, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We discuss the potential role of NETs in these processes and some of the debate in the literature regarding the role of this phenomenon in microbial killing, cell death and auto-immunity.     

A technique for isolating heterophils from blood of orange-winged Amazon parrots (Amazona amazonica amazonica)

Blood samples from adult orange-winged Amazon parrots (Amazona amazonica amazonica) were collected to develop a rapid and efficient technique for isolating pure populations of morphologically intact and functional heterophils. In addition, normal haematological parameters for the orange-winged Amazon parrots (n=20) were established and found to be within the reported range of other Amazon species. Heterophil isolation (n=16) was maximally achieved by concentrating the white blood cells with 3% hetastarch prior to running the sample through a polysucrose and disodium diatrizoate centrifugation. The isolation procedure yielded an average heterophil recovery of 61.9%, the purity exceeded 92.6% and viability was 98.9%. Cell integrity was evaluated utilising flow cytometry, cytochemistries, and light microscopy. In this study, isolated heterophils exhibited morphological and cytochemical characteristics similar to whole blood heterophils and were phagocytically active. The results of this study demonstrate that an intact, viable and functionally active heterophil population can successfully be isolated from relatively small volumes of blood and that these cell populations can be further studied employing in vitro bioassaying techniques.     

Phagocytosis of opsonised fluorescent platelets by neutrophils to detect antiplatelet antibody

A platelet immunofluorophagocytosis method was developed to detect the presence of antiplatelet antibody. The method combines the advantages of immunofluorescence of antibody-coated platelets, the ability of neutrophils to phagocytose opsonised platelets, and the property of methylene blue to quench FITC-generated fluorescence of extracellular particles. Paraformaldehyde-fixed equine platelets in suspension were treated with rabbit antiequine platelet serum and FITC-conjugated goat antirabbit IgG. Viable equine neutrophils were incubated with these platelets at 37°C for 15–30 minutes. Phagocytosis was evaluated by fluorescence microscopy immediately after addition of 1% methylene blue to neutrophil platelet suspensions. Extracellular and free platelets were rendered nonfluorescent by methylene blue, whereas intracellular platelets remained unaffected and appeared highly fluorescent depending on the antiserum dilution. Phagocytosis was significantly (P <0.05) increased in the presence of complement. Addition of divalent cations (Ca²⁺ and Mg²⁺) did not increase phagocytosis, whereas EDTA significantly (P <0.05) reduced phagocytosis irrespective of the presence of complement. Sensitivity of the method can be increased by quantifying phagocytosis using flow cytometry.     

Functional expression of CD134 by neutrophils

CD134 (OX40) is a member of the tumor necrosis factor (TNF) receptor superfamily expressed on activated T cells. Here, we show that human peripheral blood neutrophils express CD134. Activation of CD134 by soluble CD134 ligand (OX40 ligand/gp34) resulted in delayed caspase-3 activation and consequently in delayed neutrophil apoptosis in vitro. Moreover, CD134 ligand, like G-CSF, maintained anti-apoptotic Mcl-1 levels and inhibited cleavage of the pro-apoptotic Bcl-2 family members Bid and Bax in these cells, suggesting that CD134-mediated signals block apoptosis pathways proximal to mitochondria activation. In conclusion, CD134 regulates neutrophil survival, suggesting that this molecule does not only contribute to adaptive but also to innate immune responses.     

Signal transduction pathways involved in phagocytic and oxidative burst activities of cytokine-treated bovine neutrophils

In vitro studies were conducted to determine the relative importance of various signal transduction factors involved in the phagocytic and oxidative burst activities of cytokine-primed bovine neutrophils. These neutrophil functions were assayed in the presence of known signal transduction pathways inhibitors which included nicotinamide, staurosporine, genistein, pertussis toxin, RO 20-1724 and U-73122.Neutrophils were isolated (purity >91%, viability >97%) from EDTA-anticoagulated jugular blood from five healthy Holstein-Friesian heifers. Freshly isolated neutrophils (6 ml, 10 x 106 cells/ml) were incubated separately for 1 h at 37 °C with equal volumes of recombinant human cytokines such as tumour necrosis factor-alpha (500 ng/ml), interleukin-l-alpha (1 ng/ml), granulocyte colony-stimulating factor (25 ng/ml), granulocyte-macrophage colony-stimulating factor (10 ng/ml) and interferon-gamma (10 ng/ml). Aliquots (1.8 ml) of various cytokine-treated neutrophils were exposed to each signal transduction pathways inhibitor for 20 min at 37 °C in the dark. Then, percentage phagocytosis and average number of intracellular bacteria per cell were evaluated microscopically using FITC-labelled opsonised bacteria (Escheria coli 0111:134). Unlabelled opsonised bacteria and dichlorofluorescin diacetate were used to evaluate H₂O₂ production, a measure of oxidative burst, by flow cytometry.Phagocytic activity and H₂O₂ production by bovine neutrophils treated with various cytokines were increased by 52.4–86.1% and 31.3–58.2%, respectively.These functional activities were significantly (p <0.05) reduced after exposure to different inhibitors of signal transduction pathways. The reduction in phagocytic activity of cytokine-primed neutrophils varied greatly depending on the site of action of various inhibitors, with pertussis toxin and U-73122 being the most inhibitory. In comparison, H₂O₂ production decreased moderately, with pertussis toxin and U-73122 being the most inhibitory and other inhibitors inducing minimal variations. It was concluded that G-inhibitory proteins (pertussis toxin-sensitive) and phospholipase C play a major role, whereas tyrosine kinase plays a minor role in the phagocytic activity and H₂O₂ production by cytokine-primed bovine neutrophils.     

FcgammaRIIa is a target for modulation by TNFalpha in human neutrophils

Activation of neutrophils by the interaction of immune complexes with Fc gamma receptors (FcgammaR) is amplified in tumor necrosis factor-alpha (TNFalpha)-primed cells, whereas interleukin-10 (IL-10) has been reported to suppress cytokine-mediated neutrophil activation. We examined whether the expression and function of FcgammaR in human neutrophils is modulated by TNFalpha and IL-10 in vitro, and whether FcgammaRIIa expression is altered following treatment with the TNFalpha inhibitor infliximab in rheumatoid arthritis (RA) patients in vivo. TNFalpha treatment induced upregulation of expression and function of the major activating Fc receptor, FcgammaRIIa, in neutrophils from healthy donors. Unexpectedly, treatment with IL-10 led to gain of FcgammaRIIa function in TNFalpha-primed neutrophils. In neutrophils from RA patients initiating infliximab therapy and followed longitudinally through consecutive treatments, FcgammaRIIa protein decreased during the course of TNFalpha blockade, indicating that FcgammaRIIa is a target of TNFalpha modulation in human neutrophils in vivo.     

Cholesterol-rich domains are involved in Bordetella pertussis phagocytosis and intracellular survival in neutrophils

Bordetella pertussis-specific antibodies protect against whooping cough by facilitating host defense mechanisms such as phagocytosis. However, the mechanism involved in the phagocytosis of the bacteria under non-opsonic conditions is still poorly characterized. We report here that B. pertussis binding and internalization is cholesterol dependent. Furthermore, we found cholesterol to be implicated in B. pertussis survival upon interaction with human neutrophils. Pre-treatment of PMN with cholesterol sequestering drugs like nystatin or methyl-β-cyclodextrin (MβCD) resulted in a drastic decrease of uptake of non-opsonized B. pertussis. Conversely, phagocytosis of opsonized bacteria was not affected by these drugs, showing that cholesterol depletion affects neither the viability of PMN nor the route of entry of opsonized B. pertussis. Additionally, intracellular survival rate of non-opsonized bacteria was significantly decreased in cholesterol-depleted PMN. Accordingly, confocal laser microscopy studies showed that non-opsonized B. pertussis co-localized with lysosomal markers only in cholesterol-depleted PMN but not in normal PMN. Our results indicate that B. pertussis docks to molecules that eventually prevent cellular bactericidal activity.     

Ultrastructural localization of lactoferrin and iron-binding protein in human neutrophils and rabbit heterophils.

Lactoferrin in marrow and blood granulocytes from rabbits and humans was stained with an immunoferritin method. Iron-binding protein(s) was localized by the staining of granulocytes with acid ferrocyanide after saturation of the iron-binding protein with iron. The latter was most readily accomplished by treatment of the glutaraldehyde-fixed cell suspension with 1% saponin, followed by treatment with an iron-nitrilotriacetate (Fe-NTA 3mM:4mM) solution, adjusted to pH 7.0 with NaHCO3. The affinity of purified lactoferrin and transferrin for radioiron after such treatment was minimally diminished. Both immunoferritin and iron-binding methods heavily stained osmiophiliuc (phospholipid-containing) mature primary granules in late promyelocytes, myelocytes, and polymorphonuclear cells. Early promyelocytes containing abundant immature primary granules lacked immunoferritin or iron staining. Trypsin digestion of rabbit marrow cells considerably diminished the cytochemically demonstrable iron-binding capability of the mature primary granules. Specimens sequentially stained for peroxidase and immunostained for lactoferrin or cytochemically stained for iron-binding protein confirmed that lactoferrin and iron-binding protein were in peroxidase-positive primary granules. Some peroxidase positive granules appeared to lack staining for lactoferrin and iron-binding proteining protein, and all secondary granules uniformly lacked staining. Treatment of human neutrophils with phorbol myristate acetate demonstrated early release of granules containing iron-binding protein with subsequent agglutination of neutrophils and attachment of iron-binding protein to the cell surface. In summary, this study distinguishes at least two subpopulations of primary granules and identifies lactoferrin and an iron-binding protein(s) in a subpopulation of peroxidase-positive primary granules in rabbit heterophils and human neutrophils.     

The Effect of Mare's Milk Consumption on Functional Elements of Phagocytosis of Human Neutrophil Granulocytes From Healthy Volunteers

We investigated functional parameters of phagocytosis in 18 healthy volunteers drinking 250 mL of mare's milk, deep-frozen (FMM) or lyophilized (LMM), or cow's milk (CM) daily for three weeks. Blood was taken before, weekly during, and one week after intervention. Chemotaxis of isolated polymorphonuclear leukocytes (PMN) was investigated by a micropore filter assay, migration was determined fluorimetrically. The activity of phagocytosis and respiratory burst of PMN in whole blood was analysed with Phago- and Bursttest ® by flow cytometry. Contrary to phagocytosis activity, chemotactic index (CI) and burst activity diminished significantly in the group FMM. One week after intervention, CI rose tendentiously, burst activity significantly. In conclusion, immunostimulating effects ascribed to mare's milk consumption could not be observed in healthy volunteers, at least concerning phagocytosis. Our results suggest that drinking FMM modulates inflammation processes by decreasing chemotaxis and respiratory burst, which might be favourable for giving relief to diseases with recurrent inflammation.     

Expression and alternative processing of IL-18 in human neutrophils

Interleukin-18 (IL-18), a member of the IL-1 cytokine superfamily, is an important regulator of both innate and acquired immune responses. We demonstrate here constitutive expression of IL-18 by human neutrophils. Unexpectedly, we observed that neutrophils from peripheral blood or rheumatoid synovial compartments contained not only pro and mature IL-18, but also several novel smaller-molecular-weight IL-18-derived species. Using specific protease inhibitors, and serine protease gene-targeted mice, we demonstrate that these IL-18-derived products arose through caspase-independent cleavage events mediated by the serine proteases, elastase and cathepsin G. Moreover, we report that the net effect of elastase treatment of mature recombinant IL-18 was to reduce its IFN-gamma-inducing activity. Thus, human neutrophils contain IL-18 and IL-18-derived molecular species that can arise through novel enzymatic processing pathways. Through cytosolic, membrane or secretory expression of such processing enzymes, together with generation of IL-18 itself, neutrophils likely play a critical role in regulating IL-18 activities during early innate immune responses.     

Development of an in vitro model system to study the interactions between Mycobacterium marinum and teleost neutrophils

The lack of a reliable mammalian neutrophil in vitro culture system has restricted our ability to examine their precise roles in mycobacterial infections. Previously, we developed the procedures for the isolation and culture of primary kidney-derived neutrophil-like cells from goldfish that are functionally and morphologically similar to mammalian neutrophils. The cultured primary goldfish neutrophils exhibited prolonged viability and functional effector responses. In this study, we demonstrate that when exposed to live or heat-killed Mycobacterium marinum, goldfish neutrophils increased their mRNA levels for several pro-inflammatory cytokines (il-1β1, il-1β2, tnfα-1, tnfα-2) and the cytokine receptors (ifngr1-1, tnfr1, tnfr2). These neutrophils also exhibited chemotaxis toward live mycobacteria, internalized the bacilli, and produced reactive oxygen intermediates (ROI) in response to pathogen exposure. The survival of intracellular mycobacteria was significantly reduced in activated neutrophils, indicating a robust killing response by these teleost granulocytes. We suggest that this goldfish primary neutrophil in vitro model system will provide important information regarding neutrophil-mediated host defense mechanisms against mycobacteria in teleosts as well as in higher vertebrates.     

Influence of catecholamines on cytokine production and expression of adhesion molecules of human neutrophils in vitro

The impact of catecholamines on cytokine production and expression of adhesion molecules by human neutrophils was evaluated in vitro. Neutrophils were separated from venous blood of healthy subjects. The generation of intracellular cyclic adenosine monophosphate (cAMP) and Ca2+ was determined after incubation with catecholamines. Resting and lipopolysaccharide (LPS)-activated neutrophils were tested for synthesis of interleukins (IL-6, IL-8) and tumor necrosis factor alpha (TNF-alpha). In addition, the expression of the adhesion molecules CD15, CD44, and CD54 was evaluated in resting and activated neutrophils. Increasing concentrations (1 nM-1 mM) of epinephrine (EPI) were used to study the influence of activation of beta2-adrenergic receptors (beta2R) on cytokine production and adhesion molecule expression. Incubation with catecholamines induced an increase in cAMP but not Ca2+ in neutrophils. Only IL-8 was detected following stimulation with LPS and was unchanged upon co-incubation with EPI. The expression of CD15 and CD44 decreased spontaneously in vitro. The density of CD44 increased in the presence of very high doses of EPI (1 mM). Expression of CD54 on resting neutrophils increased upon activation. The density of CD54 on activated neutrophils was reduced upon co-incubation with 1 mM EPI for 6 h. However, 1 mM EPI for 12 and 18 h decreased the spontaneous loss of CD54 on resting neutrophils. Beta2R are functionally coupled to signalling cascades in human neutrophils. Nevertheless, the impact of catecholamines on IL-8 synthesis and expression of CD15, CD44, and CD54 is limited.     

丹参酮对人中性粒细胞氧自由基释放的抑制效应

本文作者用化学发光测定法研究了丹参酮对趋化寡肽介导的人中性粒细胞活性氧产生的抑制效应。当分别用５、１０、２５、５０ｎｇ／ｍｌ丹参酮与中性粒细胞孵育后，再用ｆｍＬＰ刺激，发现各浓度的丹参酮均对其化学发光反应有显著抑制作用，抑制率分别为４５．２９％，６４．２３％，７５．２１％，８７．６４％。当用ＰＭＡ作为刺激剂时，亦发现丹参酮强烈抑制其介导的中性粒细胞化学发光反应。本实验结果提示丹参酮抑制中性粒细胞在炎症反应中产生氧自由基，可能是其抗炎症损伤的重要机制之一。     

The influence of human neutrophils on N-nitrosodimethylamine (NDMA) synthesis

N-nitrozodimethyloamine (NDMA) is a carcinogenic compound that can be formed in vivo. NDMA is synthesized from precursors-amines and nitrosating agents. Nitrosating agents are formed through the reaction of oxide, reactive oxygen species and nitric oxide (NO). Human neutrophils (PMN) are an important source of the most reactive oxygen species as well as of the nitric oxide. The increase in oxygen metabolism of PMN can lead to the increase nitrosating agent and nitroso-forms. Inflammatory process is associated with locally decreased pH that may favor nitrosation reaction.

In the present study, we estimated the NDMA synthesis by LPS-stimulated PMN in the presence of the iNOS inhibitor - N-nitro-L-arginine methyl ester (L-NAME). In the nitrosation reaction dimethylamine (DMA) was used as substrat. The viability of the cells was measured by cytometric method. NDMA concentrations the culture media was measured by GCMS method. NO production was estimated by Griess's method. Expression of iNOS was determined by western blotting.

Results obtained showed that DMA nitrosation is most effective in pH between 3-4.5. Nonstimulated PMN produced lower concentrations of NO than LPS-stimulated cells (1.27 μg/cm³ and 1.57 μg/cm³, respectively). In the culture of nonstimulated PMN supplemented with DMA, there was NDMA (mean - 0.99 ng/cm³). In the culture of LPS-stimulated PMN in the presence of DMA, the concentration of NDMA was higher than in the culture of nonstimulated PMN (median - 1.45 ng/cm³). In the supernatants of cells incubated without DMA and with DMA, LPS and L-NAME, no NDMA was detected. These results indicate that PMN can be one of sources of nitrosating agents and can play a role in endogenous NDMA synthesis. Stimulation of PMN can lead to the increase of NDMA concentration following the increase of NO production. Different pathological conditions associated with PMN activation as well as the decreased pH may favor endogenous NDMA synthesis.     

Expression and function of triggering receptor expressed on myeloid cells-1 (TREM-1) on canine neutrophils

The dog is both a valued veterinary species and a widely used translational model for sepsis research. However, relatively little work has been performed evaluating potential biomarkers present during canine infection. Triggering receptor expressed on myeloid cells-1 (TREM-1) has shown promise as a biomarker for infection and pneumonia in humans. Here we describe, for the first time, the expression and function of the canine orthologue of TREM-1. Expression of TREM-1 on canine neutrophils is significantly up-regulated by stimulation with microbial agonists of TLR2/6, TLR1/2, and TLR4/MD2. Kinetics of TREM-1 protein up-regulation are rapid, with significant increases observed within 2 hr of neutrophil activation. Functionally, canine TREM-1 synergistically enhances LPS-induced production of IL-8, TNF-α and a canine orthologue of CXCL1. Collectively, these data suggest that TREM-1 expression in dogs, as it is in humans, is an amplifier of pro-inflammatory responses to microbial products. These results have direct application to veterinary diagnostics as well as the potential to enhance the utility of canine disease models in the assessment of potential therapeutics in the treatment of human sepsis.