Canine PMNs Research Articles

The effect of doxycycline on canine neutrophil functions

Doxycycline is a broad-spectrum tetracycline-class antibiotic that is frequently used to treat bacterial infections. Its use has also been described in immune-mediated diseases due to its immunomodulatory properties. The aim of this study was to evaluate the immunomodulatory effect of doxycycline on canine neutrophil functions. Therefore, the release of reactive oxygen species (ROS) and the formation of neutrophil extracellular traps (NETs) were determined after incubation of canine PMNs with doxycycline in three different concentrations (4 µg/mL, 20 µg/mL and 200 µg/mL) for one and three hours, respectively. Additionally, a neutrophil killing assay with a doxycycline-resistant Staphylococcus aureus was performed to determine the bactericidal effect of doxycycline treated PMNs in presence of plasma. Doxycycline significantly diminished the production of ROS. However, doxycycline concentrations of 4 µg/mL and 20 µg/mL significantly induced NETs. A synergistic bacteriostatic effect of PMNs and doxycycline on a doxycycline-resistant Staphylococcus aureus isolate was detectable. However, already PMNs and especially doxycycline alone inhibited the growth. In summary, doxycycline showed a concentration-dependent immunomodulatory property in canine PMNs with a reduced ROS production and increased NET-induction. This immunomodulatory effect resulted in a slightly increased elimination of a doxycycline-resistant Staphylococcus aureus by the doxycycline plasma concentrations achieved in dogs.

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.

Effects of Acer okamotoanum sap on the function of polymorphonuclear neutrophilic leukocytes in vitro and in vivo

Sap is a plant fluid that primarily consists of water and small amounts of mineral elements, sugars, hormones and other nutrients. Acer mono (A. mono) is an endemic Korean mono maple which was recently suggested to have health benefits due to its abundant calcium and magnesium ion content. In the present study, we examined the effects of sap from Acer okamotoanum (A. okamotoanum) on the phagocytic response of mouse neutrophils in vivo and rat and canine neutrophils in vitro. We tested the regulation of phagocytic activity, oxidative burst activity (OBA) and the levels of filamentous polymeric actin (F-actin) in the absence and presence of dexamethasone (DEX) in vitro and in vivo. Our results showed that DEX primarily reduced OBA in the mouse neutrophils, and that this was reversed in the presence of the sap. By contrast, the phagocytic activity of the mouse cells was not regulated by either DEX or the sap. Rat and canine polymorphonuclear neutrophilic leukocytes (PMNs) responded in vitro to the sap in a similar manner by increasing OBA. However, regulation of phagocytic activity by the sap was different between the species. In canine PMNs, phagocytic activity was enhanced by the sap at a high dose, while it did not significantly modulate this activity in rat PMNs. These findings suggest that the sap of A. okamotoanum stimulates neutrophil activity in the mouse, rat and canine by increasing OBA in vivo and in vitro, and thus may have a potential antimicrobial effect in the PMNs of patients with infections.

Ketamine inhibits the phagocytic responses of canine peripheral blood polymorphonuclear cells through the upregulation of prostaglandin E 2 in peripheral blood mononuclear cells in vitro

Ketamine has been reported to decrease the immune functions of phagocytes. Previously, we observed that the phagocytic capacity and oxidative burst activity (OBA) of canine peripheral blood polymorphonuclear cells (PMNs) were inhibited by the supernatant from canine peripheral blood mononuclear cells (PBMCs) cultures treated with ketamine. In the present study, we examined whether in vitro treatment with ketamine modulates prostaglandin E 2 (PGE 2) production in PBMCs. Treatment with ketamine or with ketamine-treated PBMCs culture supernatant simultaneously decreased the phagocytic capacity and OBA of PMNs. Ketamine increased PGE 2 production by PBMCs. Recombinant PGE 2 decreased the phagocytic capacity and OBA of PMNs. AH-6809, an E-prostanoid 2 (EP2) antagonist, restored the phagocytic capacity and OBA of PMNs, decreased by either the ketamine-treated PBMCs culture supernatant or recombinant PGE 2. These results suggest that ketamine inhibits the phagocytic responses of canine PMNs, and that this results from the increase in PGE 2 produced by canine PBMCs.

Effect of a Short‐Term Infusion with Soybean Oil‐Based Lipid Emulsion on Phagocytic Responses of Canine Peripheral Blood Polymorphonuclear Neutrophilic Leukocytes

The use of soybean oil-based lipid emulsion (SO-based LE) in parenteral nutrition has been reported to impair neutrophil functions in humans and rodents. As yet, little is understood about the effects of SO-based LE on canine immune responses. A short-term infusion with SO-based LE affects the phagocytic responses of canine peripheral blood polymorphonuclear neutrophilic leukocytes (PMNs). Twenty-four healthy Beagle dogs. Experimental study. Dogs were randomly assigned into groups of six and administered a 2-hour IV infusion with 0.9% NaCl solution or sufficient SO-based LE (INTRALIPOS 20%) to supply 40, 100, and 200% of the basal energy requirement (BER). PMN functions were determined after collecting blood samples before, immediately after, and 24 hours after the infusion. None of the treatments significantly affected the phagocytic capacity of PMNs or circulating leukocyte numbers. The infusion providing 200% of BERs significantly reduced PMN oxidative burst activity, filamentous actin polymerization, and Cdc42 Rho guanosine triphosphatase activity immediately after its delivery. However, these functions were restored to pre-infusion values 24 hours after the infusion. The lower calorie infusions did not have these effects. These results suggest that short-term infusions with a supraphysiological dose of SO-based LE may decrease the immune functions of canine PMNs. However, more long-term studies will be needed to extrapolate the effect of SO-based LE with clinically relevant doses in a practical situation.

In vitro evaluation of the effect of trans-10, cis-12 conjugated linoleic acid on phagocytosis by canine peripheral blood polymorphonuclear neutrophilic leukocytes exposed to methylprednisolone sodium succinate

To examine whether in vitro treatment with trans-10, cis-12 conjugated linoleic acid (t10c12-CLA) restores the phagocytic capacity and oxidative burst activity (OBA) of canine polymorphonuclear neutrophilic leukocytes (PMNs) exposed to methylprednisolone sodium succinate (MPSS). Peripheral blood PMNs obtained from 12 healthy Beagles. The experimental design involved administration of a high dose of MPSS, which is the recommended protocol for dogs with acute spinal cord injury. To evaluate PMN function, blood samples were collected from dogs before IV injections of doses of MPSS or saline (0.9% NaCl) solution (time 0) and 2, 12, and 24 hours after injections ceased. Polymorphonuclear neutrophilic leukocytes were isolated from blood samples and incubated with t10c12-CLA alone or t10c12-CLA in combination with N-acetylcysteine (an antioxidant agent). Phagocytic capacity and OBA were measured simultaneously by use of flow cytometry. The phagocytic capacity and OBA of PMNs were suppressed by IV injection of MPSS and restored 12 hours after injection ceased. In vitro treatment with t10c12-CLA enhanced the phagocytic capacity and OBA of PMNs, regardless of whether dogs had been treated with MPSS. Effects of t10c12-CLA on OBA were detected only when phagocytosis was stimulated by microspheres. Use of N-acetylcysteine attenuated the stimulatory effects of t10c12-CLA. Exposure to t10c12-CLA enhanced the phagocytic capacity and OBA of canine PMNs, and this effect may have involved t10c12-CLA-induced generation of reactive oxygen species.

In vitro effect of recombinant human tumor necrosis factor-α on canine neutrophil apoptosis

Neutrophils (polymorphonuclear leukocytes: PMNs) are essential for the host defense against various infections and are often injurious to the host, causing inflammatory diseases where tumor necrosis factor-α (TNF-α) is suggested to play an important role. Since an effect of TNF-α on canine PMN apoptosis has not been studied, canine PMNs were stimulated with recombinant human (rh)TNF-α in the present study to investigate the effect of TNF-α on canine PMN apoptosis. PMN apoptosis and function to produce ROS were assessed by flow cytometry. Delayed apoptosis was observed in the PMNs treated with rhTNF-α at 100 ng/ml, accompanied by retention of capability to produce ROS. However, PMN apoptosis was accelerated by rhTNF-α combined with cycloheximide. Therefore, it is indicated that TNF-α is able to activate anti- and pro-apoptotic pathways in PMNs and that the inhibition of PMN apoptosis by TNF-α requires protein synthesis in the PMNs.

In vitro effect of recombinant human granulocyte colony-stimulating factor on canine neutrophil apoptosis

Apoptosis is essential in eliminating neutrophils (polymorphonuclear leukocytes: PMNs) in animals. The suppression of PMN apoptosis is believed to be beneficial in eradicating pathogens and is implicated in the pathogenesis of human inflammatory diseases. In the present study, canine PMNs were stimulated with recombinant human granulocyte colony-stimulating factor (rhG-CSF) to investigate the in vitro effect on the apoptosis of canine PMNs. Apoptotic cell rates were assessed by flow cytometry in relation to the ability of PMNs to produce reactive oxygen species (ROS). Canine PMN apoptosis was markedly suppressed by rhG-CSF treatment, in association with the retention of the PMN ability to produce ROS. The addition of cycloheximide abolished this suppression by rhG-CSF. Moreover, canine PMNs, which were stimulated by rhG-CSF, expressed high levels of anti-apoptotic mcl-1 gene mRNA, as quantified by real-time polymerase chain reaction method. The results suggest that PMNs, stimulated by G-CSF, could work effectively over a longer period to eliminate pathogens, and that the prolongation of the PMN life-span might occasionally aggravate tissue injuries in dogs. In addition, the suppression of PMN apoptosis seems to be mediated by the induction of anti-apoptotic mcl-1 gene expression.

Expression and function of Toll-like receptor 2 in canine blood phagocytes

Toll-like receptors (TLRs) are a family of highly conserved pattern recognition receptors (PRR) of mammals that participate in the activation of innate immune responses against microbial infections. Among these receptors, TLR2 is essential for the recognition of conserved structural components of bacteria, protozoa and fungi. Until now, expression of TLR2 in dogs has not been investigated. In this work we describe a partial sequence of the gene coding for canine TLR2 and show that TLR2 mRNA is constitutively expressed in canine blood PMNs. We also show that stimulation of purified PMNs with lipoteichoic acid (LTA), a ligand of TLR2, leads to the release of proinflammatory chemokine IL-8. Furthermore, TLR2 protein is easily detectable by flow cytometry on the canine peripheral blood granulocyte and monocyte cell surface, and slightly on lymphocytes. These findings suggest that, also in dogs as in humans the initial antibacterial response of PMNs could be elicited through engagement of TLR2.

Acceleration of superoxide generation in polymorphonuclear leukocytes and inhibition of platelet aggregation by alk(en)yl thiosulfates derived from onion and garlic in dogs and humans

We recently identified sodium n-propyl thiosulfate (NPTS) and sodium 2-propenyl thiosulfate (2PTS) from boiled onion and garlic, respectively, as causative agents of hemolytic anemia in dogs. We present here data concerning the effects of these alk(en)yl thiosulfates on superoxide (O 2 −) generation in peripheral polymorphonuclear leukocytes (PMNs) and on adenosine 5′-diphosphate (ADP)-induced platelet aggregation in dogs and humans in vitro. Both NPTS and 2PTS increased O 2 − generation significantly ( P<0.05 at 1 mM NPTS, P<0.005 at 0.1 and 1 mM 2PTS) and reduced its reaction time significantly ( P<0.05 between 0.01 and 1 mM NPTS and at 1 mM 2PTS) in canine PMNs stimulated by phorbol 12-myristate 13-acetate, compared with the control without alk(en)yl thiosulfates. However, a tendency to return to the control level was observed at 10 mM of the alk(en)yl thiosulfates in both O 2 − generation and its reaction time. Although NPTS and 2PTS did not exert any significant effect on the O 2 − generation in human PMNs, 2PTS reduced its reaction time significantly ( P<0.05) at 1 and 10 mM compared with the control, showing that 2PTS accelerated O 2 − generation in human PMNs. The difference in effects on O 2 − generation may be due to that in susceptibility to alk(en)yl thiosulfates between canine and human PMNs. On the other hand, NPTS and 2PTS were shown to significantly inhibit ADP-induced platelet aggregation at 0.01 mM ( P<0.01) in canine platelets and at 0.001–0.1 mM ( P<0.05) in human platelets. In contrast, the maximal aggregation percentage returned to the control level at 1 mM of alk(en)yl thiosulfates in both canine and human platelets. From these results, we conclude that NPTS and 2PTS have the potential to promote immune functions and prevent cardiovascular diseases.

Chitin and chitosan stimulate canine polymorphonuclear cells to release leukotriene B4 and prostaglandin E2.

The effects of chitin and chitosan on the release of arachidonic acid products were investigated in this study. Supernatants of canine polymorphonuclear cell (PMN) suspensions incubated with chitin and chitosan contained a leukotriene B4 (LTB4) concentration high enough to induce canine PMN migration in vitro. The supernatants also contained the same concentration of prostaglandin E2 (PGE2) as that normally found in the peripheral blood of dogs. Intraperitoneal administration of chitosan to dogs induced peritoneal exudative fluid (PEF), but chitin did not. The PEF contained numerous PMNs and macrophages. The supernatant of PEF contained both heat-stable and heat-labile chemotactic factors for canine PMNs. It also contained enough LTB4 to attract the canine PMNs in vitro.

Effect of N-acetyl- d-glucosamine and d-glucosamine oligomers on canine polymorphonuclear cells in vitro

The effect of N-acetyl- d-glucosamine (GlcNAc) and d-glucosamine (GlcN) oligomers on the activation and viability of canine polymorphonuclear cells (PMNs) was evaluated. Checkerboard assays of both oligomers revealed that the GlcNAc oligomer induced chemotactic migration, while GlcN oligomer induced chemotactic and chemokinetic migration. The GlcN oligomer showed about twice the chemotactic activity of the GlcNAc oligomer, and induced slight enhancement of the chemiluminescence response (CL) of canine PMNs to zymosan, as well as shortening of the peak time of the CL response compared with the control and the GlcNAc oligomer. Neither oligomer showed cytotoxicity for canine PMNs at a concentration of 100 μg/ml. These results suggest that GlcNAc and GlcN oligomers might be activating factors for canine PMN in vivo.

Factors modifying protective effect of anti-CD18 antibodies on myocardial reperfusion injury in dogs.

Anti-CD18 monoclonal antibodies (MAb) have demonstrated variable protection against neutrophil (PMN)-mediated myocardial reperfusion injury. To identify factors contributing to this variability, open-chest dogs underwent coronary artery occlusion for 90 min followed by reperfusion for 3.5 h. Ten minutes before reperfusion the dogs received saline (n = 18) or one of three anti-CD18 MAb: MHM.23, R15.7, or PLM-2 (2, 1, and 1 mg/kg and n = 19, 8, and 4, respectively). Collateral flow was measured with radioactive microspheres, area at risk was assessed with monastral blue dye, and infarct size was measured postmortem by triphenyltetrazolium chloride. In vitro, all three MAb bound to canine PMNs, but only MHM.23 and R15.7 inhibited their adherence to keyhole limpet hemocyanin-coated plastic. In vivo, only MHM.23 and R15.7 significantly reduced infarct size after adjusting for the effect of collateral flow. MHM.23 afforded protection in dogs with moderate ischemia (epicardial collateral flow > 0.1 ml.min-1.g-1, infarct size reduced 46%) but not in dogs with more severe ischemia. Only R15.7 was effective in dogs with severe ischemia. Although MHM.23 and R15.7 produced similar inhibition of tissue PMN accumulation, as reflected by myeloperoxidase activity. R15.7 markedly inhibited H2O2 production by PMNs after exposure to platelet-activating factor, whereas MHM.23 had only a minimal effect. The effectiveness of different anti-CD18 MAb in preventing reperfusion injury appears to be 1) highly dependent on the specific anti-CD18 MAb employed, 2) predicted only partially by in vitro binding to PMNs, static in vitro tests of PMN adherence, or the extent of inhibition of PMN accumulation in vivo, 3) related more to their ability to inhibit oxidant release from activated PMNs, and 4) strongly influenced by the severity of myocardial ischemia before reperfusion.

Characterization of leukotriene B 4 synthesis in canine polymorphonuclear leukocytes

The synthesis and release of leukotriene B 4 (LTB 4) from canine polymorphonuclear leukocytes (PMNs) was characterized in terms of incubation time, temperature and effects of calcium ionophore A23187 concentrations. Maximal LTB 4 concentrations were determined when canine PMNs were incubated with 10 μM A23187. Increasing LTB 4 concentrations were determined through 10 min incubation. The maximal LTB 4 concentrations ( 310 ± 30 pg LTB 4 2.5 × 10 5 cells ) determined at 10 min did not change through a 55 min incubation period. Greater LTB 4 concentrations were synthesized by canine PMNs at 37°C ( 268 ± 12 pg LTB 4 2.5 × 10 5 cells ) than at 25°C ( 206 ± 11 pg LTB 4 2.5 × 10 5 cells ) or 5°C ( 59 ± 3 pg LTB 4 2.5 × 10 5 cells ). The synthesis of LTB 4 in canine PMNs was inhibited by incubation of the cells with either of two known lipoxygenase inhibitors, BWA4C or BW755C. BWA4C inhibited LTB 4 synthesis with an approximate IC 50 = 0.1 μM, whereas BW755C inhibited LTB 4 synthesis with an approximate IC 50 = 10 μM. These results indicate canine PMNs have the capability to synthesize large quantities of LTB 4 when stimulated with calcium ionophore A23187. Furthermore, the 5-lipoxygenase inhibitors BWA4C, an acetohydroxyamic acid, and BW755C, a phenyl pyrazoline, can readily inhibit LTB 4 synthesis in canine PMNs.

Aggregation, chemotaxis, and chemiluminescence of canine granulocytes. Studies utilizing improved cell preparation techniques.

Wishing to extrapolate in vitro observations of granulocyte function and pharmacology made with human cells to animal models of diseases in which we believe granulocyte stimulation to play a major role, we examined techniques for preparation of canine granulocytes and conducted a survey of the function and pharmacology of those cells. Isotonic density gradients of Percoll proved a simple and highly satisfactory method of preparation. Canine granulocytes in most respects paralleled human cells in function and pharmacology, except that canine cells lacked receptors for formylated oligopeptides and resisted them as stimuli; canine plasma contained a heat-labile inhibitor of canine PMN aggregation, oxidative metabolism, and myeloperoxidase release; canine PMNs were not inhibited in aggregation by protease inhibitors such as aprotinin; canine response to ibuprofen and steroids was more variable than that of human cells, and synergy between those agents was less readily demonstrated; heterologous stimulation (canine cells by human C5a or vice versa) led to a different time course and maximum response from those observed in the homologous systems. Canine granulocytes were readily marked with indium-111, and functioned normally in vitro and survived well in vivo after marking. We conclude that the dog is a suitable animal for studying the role of stimulated PMNs in disease, as long as the observed differences are taken into account in experimental design and data interpretation.

Chemiluminescence of Human and Canine Polymorphonuclear Leukocytes in the Absence of Phagocytosis

Polymorphonuclear leukocytes (PMNs) have increased oxidative metabolism during phagocytosis and emit light (chemiluminescence, CL) as a result of metabolic activation. The present study examined PMN CL in the absence of phagocytosis using sodium fluoride (NaF), a nonparticulate agent and known stimulator of cellular oxidative metabolism. Normal human and canine PMNs were assayed in a CL spectrometer which permitted continuous sample mixing and constant temperature regulation during CL measurement. PMNs treated with 20 mM NaF demonstrated maximum CL responses of 10,000-20,000 cpm above background, 13-17 min after addition of NaF at 37 degrees C. Temperature regulation of reaction mixtures was found to be a critical factor in assaying PMN CL responses to NaF, because a small decrease in temperature (i.e. 1.5 degrees C) substantially depressed and delayed the CL response. Superoxide anion production correlated closely with CL responses in NaF-treated human PMNs. CL responses were completely suppressed in the presence of the oxidative metabolic inhibitors, iodoacetamide, and N-ethylmalemide; and were partially suppressed in the presence of either superoxide dismutase or sodium azide.CL responses of NaF-treated PMNs were significantly lower than responses generated by PMNs phagocytizing opsonized yeast. When NaF was evaluated for its effect on light generation from a singlet oxygen dependent CL reaction, it was found that NaF did not quench singlet oxygen light. This study demonstrates that PMN CL can occur in the absence of phagocytosis, and it proposes that a nonphagocytic PMN CL assay may be useful in evaluating leukocyte metabolic defects.