Canine Granulocytes Research Articles

Characterization of Lactobacillus reuteri BCLR-42 and Lactobacillus plantarum BCLP-51 as novel dog probiotics with innate immune enhancing properties

Probiotics that are able to provide beneficial effects on animal health have become important ingredients of dog foods. This study was conducted to characterize the probiotic potentials of two strains, Lactobacillus reuteri BCLR-42 and Lactobacillus plantarum BCLP-51, that were derived from feces of healthy dogs and evaluated based on tolerance to low pH and bile acid, antimicrobial activities, enzyme profiles, sensitivity to antibiotics, and innate immune enhancing potentials. Both strains showed survival of more than 90% at pH 3 and 0.2% bile acid and exhibited broad antimicrobial activities against indicator bacteria. Moreover, both strains showed high sensitivity to antibiotics, except vancomycin, metronidazole, and gentamicin. The alkaline phosphatase was negligible (score 0), whereas they showed strong beta galactosidase activity (score range 5 or 3, respectively). The phagocytosis and oxidative burst activities of canine granulocytes were significantly enhanced in response to both strains. These results show that both strains have the capability to act as probiotics and the potential for application as ingredients in dog foods.

Up-Regulation of Cluster of Differentiation (CD) 11b Expression on the Surface of Canine Granulocytes with Human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF)

ABSTRACTGranulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine, sharing a common beta subunit (CDw131) with interleukins 3 and 5. GM-CSF is important for its direct and indirect involvement in host defense. In veterinary medicine, human (h) GM-CSF has been used as a substitute for canine GM-CSF to stimulate canine granulocytes and macrophages. In this study, we compared the effects of three distinct hGM-CSFs produced by bacteria, yeasts and Chinese hamster ovary (CHO) cells with those of Escherichia (E) coli-produced canine GM-CSF on the cluster of differentiation 11b (CD11b) expression in canine granulocytes. The median effective dose (ED50) of hGM-CSFs from bacteria, yeasts and CHO cells was 3.09, 4.09 and 4.27 ng/ml, respectively, with no significant difference among three. In contrast, a significant difference was observed between ED50 of canine GM-CSF (0.56 ng/ml) and three hGM-CSFs according to the paired t-test (P<0.05). We conclude that hGM-CSF can activate canine granulocytes, but the average activity of the three rhGM-CSFs was approximately 15% of that of canine GM-CSF.

The anti-inflammatory effects of methane*

Gastrointestinal methane generation has been demonstrated in various stress conditions, but it is not known whether nonasphyxiating amounts have any impact on the mammalian pathophysiology. We set out to characterize the effects of exogenous methane administration on the process of inflammatory events arising after reoxygenation in a large animal model of ischemia-reperfusion. A randomized, controlled in vivo animal study. A university research laboratory. Inbred beagle dogs (12.7 6 2 kg). Sodium pentobarbital-anesthetized animals were randomly assigned to sham-operated or ischemia-reperfusion groups, where superior mesenteric artery occlusion was maintained for 1 hr and the subsequent reperfusion was monitored for 3 hrs. For 5 mins before reperfusion, the animals were mechanically ventilated with normoxic artificial air with or without 2.5% methane. Biological responses to methane-oxygen respirations were defined in pilot rat studies and assay systems were used with xanthine oxidase and activated canine granulocytes to test the in vitro bioactivity potential of different gas concentrations. The macrohemodynamics and small intestinal pCO(2) gap changes were recorded and peripheral blood samples were taken for plasma nitrite/nitrate and myeloperoxidase analyses. Tissue superoxide and nitrotyrosine levels and myeloperoxidase activity changes were determined in intestinal biopsy samples; structural mucosal damage was measured by hematoxylin and eosin staining. Methane inhalation did not influence the macrohemodynamics but significantly reduced the magnitude of the tissue damage and the intestinal pCO(2) gap changes after reperfusion. Furthermore, the plasma and mucosal myeloperoxidase activity and the intestinal superoxide and nitrotyrosine levels were reduced, whereas the plasma nitrite/nitrate concentrations were increased. Additionally, methane effectively and specifically inhibited leukocyte activation in vitro. These data demonstrate the anti-inflammatory profile of methane. The study provides evidence that exogenous methane modulates leukocyte activation and affects key events of ischemia-reperfusion-induced oxidative and nitrosative stress and is therefore of potential therapeutic interest in inflammatory pathologies.

Immunohistochemical Investigation of PNL2 Reactivity of Canine Melanocytic Neoplasms and Comparison with Melan A

PNL2 is a recently generated monoclonal antibody (mAb) that recognizes normal and neoplastic melanocytes. Although the antigen recognized by PNL2 remains unknown, recent studies of human and mouse melanomas have confirmed its usefulness as a diagnostic marker. In the current study, the immunoreactivity of PNL2 in canine melanomas was tested and compared with Melan A (A103). Validation of PNL2 was performed by Western blot analysis. PNL2 and Melan A immunoreactivity were tested on frozen samples of canine melanomas and on 69 formalin-fixed, paraffin-embedded melanocytic neoplasms. Normal canine tissues and nonmelanocytic neoplasms were included as negative controls. Western blot confirmed the presence of a protein recognized by the PNL2 antibody in canine melanomas. Immunohistochemically, PNL2 stained the melanocytic neoplastic cells with an intracytoplasmic, granular pattern. Among the melanocytic neoplasms tested, 62% stained positively with PNL2 and 59% with Melan A; 50.7% stained positively with both mAbs. The overall percentage of neoplasms that stained positively with at least 1 of these 2 antibodies was 68%. The extent of staining (i.e., the percentage of cells stained per specimen) was greater with PNL2 than with Melan A. With both mAbs, staining was most intense and diffuse in the epithelioid cell phenotype. Neither nonspecific staining nor staining in cells other than melanocytes was detected with either mAb. In contrast to human granulocytes, canine granulocytes were negative by both Western blot and immunohistochemical analyses. PNL2 mAb proved to be highly specific for the identification of formalin-fixed canine melanocytic neoplasms and should be a valuable diagnostic reagent.

Priming Effects of Lipopolysaccharide and Inflammatory Cytokines on Canine Granulocytes

Granulocytes play a pivotal role in natural immunity. Under inflammatory conditions, granulocytes are universally primed by several agents, such as endotoxins and inflammatory cytokines. Primed granulocytes exert potent adhesiveness, chemotaxis, phagocytosis and reactive oxygen species (ROS) production, effectively eliminating invading agents. Reactivity against priming agents is known to vary with species; however, there have been few reports on the effects of priming agents on canine granulocytes. In the present study, we assayed the priming effects of lipopolysaccharide (LPS), recombinant canine tumor necrosis factor-alpha (rcTNF-alpha) and recombinant canine granulocyte macrophage colony-stimulating factor (rcGM-CSF) on canine granulocyte function in vitro. Isolated recombinant canine were primed with various concentrations of LPS, rcTNF-alpha and rcGM-CSF, and CD11b expression was assayed. Furthermore, actin polymerization, phagocytosis and ROS production were then assayed at primer concentrations where enhancement of CD11b expression was observed. LPS did not enhance canine granulocyte function. Phagocytosis and actin polymerization were not enhanced by priming agents; however, rcTNF-alpha and rcGM-CSF enhanced CD11b expression and ROS production in canine granulocytes. These results suggest that priming effects are mainly reflected in CD11b expression and ROS production, with rcGM-CSF and rcTNF-alpha having a priming effect similar to that observed in humans.

Antibodies specific for human or murine Toll-like receptors detect canine leukocytes by flow cytometry

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns on microbes, and ligand recognition results in production of pro-inflammatory cytokines, reactive oxygen and nitrogen intermediates, and upregulation of costimmulatory molecules. The purpose of this study was to test antibodies specific for human or murine TLR2, 3, 4, 5 and 9 for their use in dogs. Twenty-two antibodies were assessed for recognition of canine monocytes (Mo), granulocytes (Gr) or lymphocytes (Ly) by flow cytometry, and results were compared with isotype-matched controls and other antibodies against the same TLR. Nine monoclonal antibodies detected canine leukocyte subpopulations. Antibodies TL2.1 and TL2.3 (specific for human TLR2), HTA125 (specific for human TLR4), as well as 85B152.5 and 19D759.2 (specific for human TLR5) recognized Mo and Gr but not Ly without permeabilization, and putatively cross-react with canine TLR2, 4 and 5, respectively. Antibodies 40C1285.6 and TLR3.7 (specific for human TLR3) as well as 26C593.2 and 5G5 (specific for human and murine TLR9) recognized Mo, Gr and Ly after their permeabilization and putatively cross-react with canine TLR3 and 9, respectively, inside the cell. None of these nine antibodies recognized paraformaldehyde-treated (4%) canine leukocytes but all except 40C1285.6, TLR3.7 and 5G5 recognized methanol-fixed cells, suggesting that they might be useful also in immunohistochemistry.

Optimization of conditions for in vitro production of radical oxygen species and expression of tissue factor by canine mononuclear cells and granulocytes for use in high-throughput assays

The purpose of this study was to optimize conditions for high throughput measurement of radical oxygen species (ROS) production and expression of tissue factor, also termed procoagulant activity, by canine leukocytes. Granulocytes and mononuclear cells were separated by density gradient centrifugation from peripheral blood collected on several occasions from three healthy large breed dogs. To determine optimal conditions for ROS production, granulocytes were incubated for 1 or 3 h in PBG (PBS containing 0.5% BSA and 5 mM glucose) or RPMI containing 10% fetal bovine serum (FBS); lipopolysaccharide (LPS), zymosan, peptidoglycan (PGN) and phorbol myristate acetate (PMA) were used as stimuli. ROS was assessed by conversion of the nonfluorescent dye dihydrorhodamine 123 to fluorescent rhodamine 123 by radical species released into the media. To identify optimal conditions for expression of tissue factor, mononuclear cells were incubated for 5 h in RPMI containing different concentrations of heat-inactivated FBS (HI-FBS), and LPS, zymosan, PGN or PMA as stimuli. Expression of tissue factor was determined using a one-stage recalcification assay performed in an automated nephelometric coagulation analyzer. Neither LPS nor zymosan increased ROS production by granulocytes incubated in PBG media. In contrast, granulocytes incubated in RPMI had dose-dependent increases in ROS production in response to zymosan and PGN. ROS production was significantly increased by incubation with concentrations of LPS of 0.01 μg/ml or greater, and by zymosan concentrations of 0.1 μg/ml or greater. ROS production in response to incubation with PMA was signficantly increased starting at 10 −7 M, and was significantly greater for cells incubated in RPMI than cells incubated in PBG. LPS-, zymosan- and PGN-stimulated procoagulant activity increased in a dose-dependent manner, whereas PMA-stimulated procoagulant activity peaked at 10 −7 M. Increasing concentrations of HI-FBS significantly increased LPS-, zymosan- and PGN-induced procoagulant activity of mononuclear cells. Results obtained in this study indicate production of ROS by canine granulocytes is optimal when these cells are incubated for 3 h in RPMI with LPS (0.1 μg/ml), zymosan (10 μg/ml), PGN (10 μg/ml), and PMA (10 −7 M). Furthermore, canine mononuclear cells express procoagulant activity in response to LPS, zymosan, PGN, and PMA, and responses to LPS, zymosan and PGN are enhanced by the addition of HI-FBS. These findings suggest that HI-FBS retains important serum proteins that facilitate interactions between each of these bacterial or yeast derived products and the mononuclear cells. Consequently, future studies regarding the regulation of procoagulant activity by canine mononuclear cells should be performed in the presence of HI-FBS. Both assays utilized in this study allow high throughput of samples, and therefore are appropriate choices for rapid screening of conditions and/or therapeutic interventions affecting the canine inflammatory system.

Characterization of the priming effect by pituitary canine growth hormone on canine polymorphonuclear neutrophil granulocyte function.

In this report, we demonstrate that canine growth hormone (cGH) is capable of priming canine polymorphonuclear neutrophil granulocytes (PMN) in a manner resembling that of human PMN. The cGH influences important functions that are involved in the process of recruitment of PMN, i.e., shape change, chemotaxis, CD11b/CD18 expression, adhesion, and subsequent transendothelial migration. Also, intracellular O(2)(-) production was evaluated. We investigated the priming effect by incubating PMN with purified pituitary cGH at various concentrations (10 to 800 microg/liter). The capacity for shape change was significantly (P < 0.05) enhanced, whereas the chemotactic response under agarose was significantly (P < 0.05) reduced. The chemotactic migration in Boyden chambers (10-microm-thick polycarbonate filter; lower surface count technique) was significantly (P < 0.05) enhanced, presumably due to cGH-induced hyperadhesiveness to the lower surface of the filters. The adhesion in albumin-coated microtiter plates and adherence to canine pulmonary fibroblasts were significantly (P < 0.05) increased, and the increased adhesion resulted in a significant (P < 0.01) increase in transendothelial migration using canine jugular vein endothelial cells. The increase in adhesion was associated with a significant increase in CD11b/CD18 expression. Furthermore, intracellular O(2)(-) production was significantly enhanced in response to both phorbol myristate acetate (P < 0.01) and opsonized zymosan (P < 0.05). In the absence of a PMN-stimulating agent, cGH did not influence the effector functions investigated except for an increased expression of CD11b/CD18.

Determination of the adhesive capability of canine polymorphonuclear neutrophil granulocytes using a fluorometric microtiter plate cellular adhesion assay

A rapid fluorometric cellular adhesion assay in microtiter plates was evaluated for canine polymorphonuclear neutrophil granulocytes (PMN) using the fluorescent indicator calcein acetoxymethyl ester (Calcein AM). Optimum adhesive responsiveness occurred in bovine serum albumin (BSA)-coated plates after 30 min incubation at 37°C with 5 × 10 5 PMN in 100 μl per well using phorbol myristate acetate (PMA, 100 ng/ml) as stimulant. The adhesive responsiveness for 15 laboratory beagle dogs was determined and the mean percentage of adherence for unstimulated (2.9%) and stimulated (74.3%) PMN and corresponding 95% confidence intervals was determined. The mean intra-assay coefficient of variation (CV intra) for unstimulated and stimulated adhesion in the assay was: CV intra = 12.9% and CV intra = 4.1%, respectively. The mean inter-assay coefficient of variation (CV inter) for unstimulated and stimulated adhesion, respectively, in the assay was: CV inter = 22.9% and CV inter = 5.6%. The assay was highly reproducible and labelling with Calcein AM neither reduced PMN viability nor activated the PMN.

Chemotaxis of canine polymorphonuclear neutrophil granulocytes using the under-agarose method applied to glass microscope slides

The under-agarose chemotaxis assay method applied to glass microscope slides was optimised for canine polymorphonuclear granulocytes (PMN) with gelatin as protein supplement. Optimum chemotactic response occurred after approximately 135 min of incubation at 37 °C with 5 × 105 cells/well and zymosan-activated serum (ZAS) as chemoattractant. Chemotactic (C) and random migration (R) were stated separately for each dog. Storage of blood samples for 90 min before isolation of PMN did not influence the C or R values (p > 0.05). The preanalyfcal variation was investigated and the proportion of variance, expressed as a percentage, due to each source for the C and R values, respectively, was determined: dog, 92%, 96%; blood sample, 0.4%, 0%; interslide, 3%, 2% and intraslide, 3%, 3%. The mean chemotactic responsiveness against ZAS and ZAS diluted 1:1 with RPMI 1640 (dZAS) was determined using 15 laboratory beagle dogs: CZAS = 0.67 mm (range: 0.25–2.33 mm), coefficient of variation (CV) = 24%, CdZAS = 0.47 mm (range: 0.18–2.12 mm), CV = 22%. Also, the mean random migration was determined: 0.11 mm (range: 0.05–0.34 mm), CV = 25%. Neither age nor sex had any significant influence on chemotactic or random migration (p>0.05). The effect of heat-inactivated pooled canine serum (HI-serum) incorporated into agarose was examined and it was found that this had a significant stimulatory effect on both chemotactic and random migratory responses (p<0.05). Therefore, agarose with HI-serum is not useful when investigating the chemotactic function.

Expression of adhesion and Fc γ-receptors on canine blood eosinophils and neutrophils studied by anti-human monoclonal antibodies

Commercially available anti-human monoclonal antibodies were tested as markers of adhesion receptors and Fc γ-receptors on canine neutrophils and eosinophils. Purified populations of eosinophils and neutrophils were incubated with selected monoclonal antibodies and binding was measured by flow cytometry. Most of the anti-human monoclonal antibodies used in this study crossreacted with canine granulocytes and many showed expression similar to that of human granulocytes. The results suggest that the adhesion and Fc γ-receptors are well conserved among species. Canine eosinophils and neutrophils were simultaneously purified by a two-layer Percoll density gradient centrifugation. The purity of the neutrophil fraction was ≥97% after lysis of the erythrocytes. In the eosinophil fraction, 27–92% were eosinophils (mean 60%). After purification, the neutrophils and eosinophils were incubated with selected monoclonal antibodies and analysed by flow cytometry. Human anti-CD11b and CD18 antibodies bound intensely to both canine eosinophils and neutrophils. Canine neutrophils did label with anti-CD29, but eosinophils did not. Anti-CD49d bound weakly to both eosinophils and neutrophils. Both anti-CD16 and anti-CD32 antibodies labelled neutrophils, but not eosinophils. There was no binding of anti-CD9 to canine neutrophils and the binding of anti-CD9 to canine eosinophils varied between dogs.

Reactivity of antibodies directed against human antigens with surface markers on canine leukocytes.

A panel of anti-human antibodies was used in order to investigate their cross-reactivity with canine leukocytes. The labeling was carried out at the microscopic level by immunocytochemical staining. Of 50 antibodies 22 cross-reacted with canine leukocytes from afferent lymph and peripheral blood. All leukocytes reacted with MHM23 (CD18). Two anti-HLA DR antibodies, DK22 and L243, reacted mostly with veiled cells and with PHA-stimulated lymphocytes, whereas TAL1B5 was cross-reactive with all canine lymphocytes. The activation markers CD25, Ki-67, PCNA were identified on PHA-stimulated lymphocytes with ACT1, Ki-67 and PC10 clone produced antibodies. Canine eosinophils reacted with MHM6 (CD23) antibody. A large number of antibodies reacted with canine lymph veiled cells. Canine granulocytes and a subset of lymphocytes were stained with the anti-CD15 antibody only after treatment of cytospins with neuraminidase.

Characterization of monoclonal antibodies directed to canine T lymphocyte markers expressed during development

Two monoclonal antibodies (mAb) which recognize cell surface antigens of canine T lymphocytes are described. mAb RQ1 (of IgG 1 subclass) binds a 34 kDa peptide under non-reducing conditions which is expressed, on average, by 27±9% ( n = 11 dogs) of thymocytes, with preferential binding for medullary, but not cortical thymocytes. RQ1 adheres to splenic periarteriolar lymphoid sheath cells. In adult dogs ( n = 7) RQ1 binds on average 86 ± 7% of unstimulated canine peripheral blood mononuclear cells (CPBMC), 69 ± 13% ( n = 11) of lymph node cells, and ≤ 19% ( n = 2) of bone marrow cells. RQ1 does not bind canine granulocytes, erythrocytes, fibroblasts or kidney epithelial cells. The relative density of RQ1-binding antigen (expressed as fluorescence intensity per cell) was up to 83% greater in lymph node cells than in thymocytes. Thus, RQ1 appears to preferentially bind mature T cells. In contrast, mAb LQ1 (of IgG 3 subclass, which binds a 32 kDa sialic acid containing glycoprotein) recognizes both cortical and medullary canine thymocytes as well as 75± 6% CPBMC. This profile is characteristic of a pan-T cell marker. The simultaneous utilization of these two markers allows for the characterization of canine T lymphocyte development: early lymphocyte (e.g. LQ1 +, RQ1 − thymocytes), common lymphocyte (e.g. LQ1 +, RQ1 + [dim fluorescence] thymocytes), and mature lymphocyte (e.g. LQ1 +, RQ1 + [bright fluorescence]).

Inhibition of Granulocyte cAMP-Phosphodiesterase by Rolipram In Vivo Is Not Sufficient to Protect the Canine Myocardium from Reperfusion Injury

The purpose of this study was to determine whether the selective type IV cAMP-phosphodiesterase inhibitor rolipram could reduce the reperfusion injury that occurs during myocardial infarction in the anesthetized dog. This question was tested in pentobarbital-anesthetized dogs subject to 90 min of regional myocardial ischemia and 5 h of reperfusion. Dogs were treated with 1 mg/kg of rolipram (i.v., 15 min before reperfusion) followed by a 1 mg/kg/h infusion over the duration of the 5 h of reperfusion. Rolipram was tested in vitro for efficacy in inhibition of isolated human neutrophil superoxide generation. Rolipram produced significant inhibition of superoxide production over the concentration range of 0.1-100 microM rolipram when neutrophils were stimulated with a 10(-7) M concentration of the chemotactic peptide f-Met-Leu-Phe. Rolipram significantly inhibited superoxide generation from human and canine granulocytes in whole blood stimulated by zymosan. Therapeutic concentrations of rolipram in the blood of dogs were achieved during the course of the experiments with a plasma concentration of 0.761 +/- 0.095 micrograms/ml (2.76 +/- 0.34 microM) at the time of reperfusion, and 0.574 +/- 0.098 micrograms/ml (2.08 +/- 0.36 microM) at the end of the reperfusion period. The relative severity of myocardial ischemia between the two treatment groups was similar as assessed with radiolabeled microsphere measurement of myocardial blood flow. Transmural myocardial blood flows were not significantly different between the two groups after coronary occlusion (control, 0.05 +/- 0.01 ml/min/g, n = 6, vs. rolipram, 0.18 +/- 0.07 ml/min/g, n = 6; p = 0.48).(ABSTRACT TRUNCATED AT 250 WORDS)

Analyses of Ehrlichia canis and a canine granulocytic Ehrlichia infection

Ehrlichia canis and canine granulocytic Ehrlichia sp. (CGE) infect canine monocytes and granulocytes, respectively. E. canis has been cultured in vitro and used to develop an immunofluorescence assay. CGE has not been cultured, and a serologic assay is not available. The sera of dogs infected with CGE were reported to react with E. canis by immunofluorescence. In this study, the temporal response of immunoglobulin G (IgG) was determined by an enzyme-linked immunosorbent assay (ELISA) with purified E. canis antigen in four dogs experimentally infected with E. canis, in two dogs experimentally infected with CGE, and in one dog infected with E. canis and subsequently infected with CGE. E. canis-infected dogs developed an IgG ELISA result of 1.5 or greater for the optical density signal/noise ratio by 2 months postinfection. CGE challenge of a dog with a previous E. canis infection induced an anamnestic increase in the IgG ELISA result; however, CGE infection alone did not induce a significant IgG ELISA response. Western immunoblot analysis showed that dogs infected with E. canis developed antibodies initially that reacted with low-molecular-mass proteins (30, 24, and 21 kDa) and subsequently with higher-molecular-mass proteins (160, 100, 78, 64, 47, and 40 kDa). In contrast, CGE-infected dogs showed reactions with the same higher-molecular-mass proteins of E. canis but, unlike E. canis-infected dogs, not with the low-molecular-mass proteins of E. canis. Of 10 serum samples collected in the field of Indonesia from dogs with tropical canine pancytopenia, all had an optical density signal minus noise value of 2.54 or greater in the IgG ELISA and reacted with E. canis antigen in a pattern similar to that of serum samples from dogs experimentally infected with E. canis in Western immunoblotting. This study suggests that the IgG ELISA and Western immunoblotting with purified E. canis as the antigen are useful in distinguishing between E. canis and CGE infections in dogs.

Chemiluminescence and Chemotaxis Assay of Canine Granulocytes: A Methodological Study

Chemiluminescence (CL) of isolated granulocytes and of whole blood from dogs was evaluated. Chemiluminescence of whole blood samples created an undesired quenching effect by the red blood cells which makes the assay difficult to apply in pathological cases with low formation of oxygen metabolites. This problem was avoided when chemiluminescence was determined, using isolated granulocytes. A cell concentration of 5 x 10(9)/l was needed to create optimal conditions. The Boyden chamber technique was used for study of random migration and chemotaxis. Casein (0.1%), zymosan activated serum with and without epsilon-amino-n-caproic-acid and homologous serum were effective chemoattractants for canine granulocytes, while FMLP (formyl-methionyl-leucyl-phenylalanin) did not attract canine granulocytes.

Phagocytosis Induced by Yeast Cells in Canine Granulocytes: A Methodological Study

A phagocytic function assay of canine granulocytes was established. This method allows the proportion of active granulocytes to be estimated as well as the number of adhered and ingested yeast cells. The influence of different factors on phagocytosis was studied. Temperature variation within the interval 36-41 degrees C did not affect phagocytosis. The incubation time for optimal phagocytosis of yeast cells was 35 min. The opsonization procedure giving the optimal phagocytosis was purified IgG and serum together.

Luminol-dependent chemiluminescence analysis of the variables of the phagocytic response by canine granulocytes

Variables which influence the oxygen-dependent chemiluminescence (CL) response of canine polymorphonuclear leukocytes (PMN) to zymosan were examined in a luminol-dependent CL assay system. Maximal CL responses were obtained when 5 × 10 6 canine PMN, isolated from heparinized blood, were assayed at 37° C in a Luminometer. The response was enhanced by the addition of 0.05 mM Luminol and inhibited by the addition of 0.06 mM sodium azide and 60 ug superoxide dismutase. Repeatability on a given day was very good; however, day to day variations in CL activity prevented direct comparison of phagocytic activity between days. Opsonization of zymosan in equine serum significantly reduced the CL response by canine PMN as compared to opsonization of zymosan in autologous or homologous canine serum and bovine serum. The present results show that luminol-dependent CL analysis can be used to measure phagocytosis by canine granulocytes in a luminometer and has potential use in clinical situations.

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.

An indirect assay for canine leukocyte migration inhibitory factor (LIF): Demonstration of its species specificity

An indirect assay in agarose-gel was developed to measure secretion of leukocyte migration inhibitory factor (LIF) by canine lymphocytes. Supernatants from cultures of mitogen- or alloantigen-stimulated canine peripheral blood lymphocytes were assayed for LIF activity using purified granulocytes as indicator cells. Canine LIF activity could be detected provided either autologous or allogeneic canine granulocytes were used as indicator cells. In contrast, canine LIF uniformly failed to inhibit the migration of human granulocytes. Likewise, human LIF inhibited the migration of human but not canine granulocytes in this assay. The data indicated that this species specificity could be due to differences in the structures of LIF receptors on canine and human granulocytes.