Fms@andjns Leukotrie~~essad Essential Fatty Acids (1932’145,283-288 (iF Loneman Gmw UK Ltd 19%

Characterization of Leukotriene B4 Synthesis in Canine ~oly~orp~ouu~l~~r Leukocytes


The synthesis and release of leukotriene B4 (LTBJ from canine polymorpiuumciearleukocytes (PMNs) was characterizedin terms of htcubation time, temperatureand effects of calcium ionophore A23187 ventral. ~~~ LTB4 ~on~en~~~ns were deters when canine PMNs were ~~~ with 10 PM A23187. Increasing LTB4 concentrations were determined throagh 10 min incubation. The maximal LTB4 concentrations (310 -t 30 pg LTBd2.5 X 10’ ceils) determined at 10 mitt did not change through a 55 min i~u~ation period. Greater LTB4 con~entrat~ns were synthesized by canine PMNs at 37°C (268 t 12 pg LTBd2.5 X 10’ C&S) than at 25°C (206 2 11 pg LTBdi2.5 x X0 ce&s) or 5°C (59 t- 3 pg LTBd2.5 x 10s ceils), The synthesis of LTB., in canine PMNs was inhibited by incu~t~~ of the ceils witk either of two known iipoxygenase inhibitors, BWA4C or BW755C. BWA4C inhibited LTB4synthesis with an a~x~ate XC%= 0.1 PM, whereas BW755C ~hibit~ LTB4 synthesis with au ~pp~~rnate R& = 10 PM. These results indicate canine PMNs have the capability to synthesize targe quantities of LTB4 when stimu tated with calcium ionophore A23187. .Furthermore, the Slipoxygenase inhibitors BWA4C, an ~e~hydroxyami~ acid, and BW755C, a phenyl pyrazoiine, can readily inhibit LTR, synthesis in canine PMNs.

INTRODUCTION Nolecular mechanisms associated with inflammatory diseases are of interest to veterinary researchers and others who may use dogs as models for various human pathological disorders. Treatment of spo~t~eously occurring in~ammatory conditions in small animal veterinary medicine is a common occurrence, however, the molecular events associated with the inflammato~ response in dogs or cats has not been thoroughly investigated. Information derived primarily from the use of human, rabbit, and rodent experiments have indicated the significant role that arachidonic acid metabolites have in in~ammatio~ (l-6). Leukotriene B4 (LTB4) is an arachidonic acid metabolite, synthesized through the iipoxygenase pathway, that is involved in the initiation and perpetuation of various

Date received 12 August 1991 Date accepted 2 October 1991

pathological conditions. Enhanced synthesis of LTB4 has been associated with pathologic conditions such as excess fluid ac~~rnu~at~on in the lungs (7, 8), inflammatory bowel disease, (9, lo), uveitis (ll), inflammatory skin disease (12), and neuronal trauma (13). Leukotriene 94 (5s,l2R>-5,12_dihydroxy-(Z,E,E,Z)6,8,10,14-~jcosatetraenojc acid) is synthesized from arachidonic acid by the activities of two enzymes, S-lipoxygenase and leukotriene A4 hydrotase (14). While leukotriene B4 synthesis has been shown to occur in several cell types, it’s synthesis can readily be studied in isolated polymo~honuclear Ieukocytes (PMNs). The purpose of the current study was to characterize the synthesis of LTBQ induced by the calcium ionophore A23817 in canine PMNs under a variety of experimental conditions. The fact that there are no drugs used in veterinary medicine that selectively inhibit leukotriene synthesis led us to also examine the ability of two known lipoxygenase inhibitors, BWA4C and BW755C, to inhibit LTB4 synthesis in canine PMNs (15).

2&l Prostagiandins Leukotrienes and Essential Fatty Acids

MATERIALS AND METHODS Animals 20 adult Greyhound dogs, 14 males and 6 females, were used in these experiments. Dogs were kenneled in individual indoor/outdoor runs and provided commercial dog chow and tap water ad libitum. Blood was drawn between 07:OO and 08:30 before the dogs were fed in the morning. Reagents The calcium ionophore (A23187), Tris-HCI, Hanks Balanced Salt Solution (HBSS), CaQ, NH&l, Histopaque No. 1119 and No. 1077 were obtained from Sigma (St. Louis, MO, USA). Reagent grade methyl alcohol, ethyl alcohol, deionized optima water, dimethylsulfoxide (DMSO) and Scintiverse II scintillation fluid were obtained from Fisher Scientific (Fairlawn, NJ, USA). Leukotriene B4 radioimmunoassay kits were obtained from Du Pont New England Nuclear Co (Boston, MA, USA). BWA4C and BW755C were kindly supplied by G. A. Higgs, The Wellcome Research Laboratories (Beckenham, Kent, UK). Polymorphonuclear leukocyte isolation and purification techniques The dogs’ neck area was shaved with clippers and an ethanol wipe was used to clean the neck region. Blood was collected from the jugular veins with a 21 g needle connected to a 10 ml Vacutainer tube that contained 15 mg EDTA in 0.1 ml water (Sherwood Medical, St. Louis, MO, USA). Approximately 25-30 ml whole blood/dog was collected. Whole blood, 25-30 ml, was added to an equal volume of HBSS that contained 0.8 mM CaClz. The mixture was gently rotated by hand to mix. To each of eight 15 ml clear polystyrene test tubes 3 ml Histopaque No. 1119 was added. Three ml Histopaque No. 1077 was carefully layered on top of the Histopaque No. 1119. Six ml of the whole blood/HBSS mixture were layered on top of the gradient. To separate the PMNs from the rest of the cells in the blood the tubes were centrifuged at 700 x g, 23°C for 30 min. The PMN band from each tube was removed with a pipette and transferred to a 15 ml tube. NH&l was used to lysis the contaminating red blood cells. The PMNs from the eight gradient tubes were transferred to eight separate tubes and 3 ml 17 mM Tris-HCI buffer that contained 0.83% NH&l pH 7.65 (buffer A) were added to each tube. The mixtures were vortexed gently and allowed to stand for 5 min at 23°C. The mixtures were centrifuged at 260 X g, 23°C for 5 min. The supernatants were discarded and the pellets resuspended in 2.0 ml buffer A. The eight samples were combined into four tubes for a total

of 4 ml each. The 4 ml homogenates were centrifuged at 260 x g, 23°C for 5 min. The pellets were resuspended in 1 ml buffer A. The contents of two tubes were combined as above, for a total of two tubes. The wash/lysis process was repeated until the red blood cells were not visible (usually two more times for a total of 4-5 lysis steps). After the final lysis centrifugation, the pellets of two tubes were resuspended in 3 ml each HBSS minus calcium (pH 7.65). The cells were centrifuged at 260 x g, 23°C for 15 min. The pellets were resuspended in 3 ml HBSS minus calcium and then washed again. The pellets were resuspended in 3 ml/tube HBSS that contained 0.8 mM CaC12. The two resuspensions were combined for a final volume of 6 ml. One ml was used to determine both cell number with a hemocytometer and cellular purity with a Coulter Counter (Model S-550, Coulter Electronics Inc, Heleah, FL, USA). Cell viability was assessed by trypan-blue exclusion. The remaining PMNs were kept on ice until they were diluted to 5% 000 PMNs/ml in HBSS which contained 0.8 mM CaCl2 pH 7.65. Generation of leukotriene B4 using calciumLeukotriene B4 was generated stimulated synthesis of LTB4 from the endogenous arachidonic acid in the isolated PMNs. The standard 0.5 ml incubation mixture contained 0.474 ml cell preparation in HBSS with 0.8 mM CaCl* (pH 7.65), 0.013 ml A23817 (10 PM final concentration in 0.5 ml), and 0.013 ml lipoxygenase inhibitor or deionized water. Nonstimulated or basal LTB4 release was determined by replacing the A23187 with the solvent 0.2% DMSO in HBSS with no CaC12. Immediately prior to the assay, the cells were preincubated for 10 min at 37°C in a shaking water bath. For inhibition experiments the cell suspensions were mixed with a lipoxygknase inhibitor and allowed to incubate for an additional 10 min at 37°C. To initiate LTB4 synthesis and release, calcium ionophore A23817 was added. The mixtures were vortexed and incubated for 10 min at 37°C. The reactions were stopped by cold dilution with 2 ml ice-cold methanol, vortexed and then placed on ice. The homogenates were centrifuged at 750 x g for 5 min at 4°C to pellet the cellular debris. The supernatants were transferred to 12 mm X 75 mm plastic tubes, put into a rotor evaporator and the methanol removed under cold vacuum. The residues were resuspended in 0.2 ml methanol and stored overnight at -80°C. LTB4 radioimmunoassay method Leukotriene B4 concentration in each sample was determined by radioimmunoassay (RIA) and liquid scintillation spectroscopy. Immediately prior to the

LTB, and Canine Polymorphonuclear Leukocytes

RIA, the methanol was removed by rotary evaporation and the residue resuspended in 0.2 ml buffer supplied with the RIA kits which contained 0.9% NaCl, 0.1% gelatin, 0.1% sodium azide in 10 mM Tris HCI buffer, pH 8.6. The 0.2 ml samples were incubated with 0.1 ml [3H]-LTB4 and antibody each (supplied with the RIA kits) and 0.1 ml RIA buffer for 18 h in a shaking waterbath at 4°C. The LTB4 antibody obtained from the Du Pont New England Nuclear Co had a known cross-reactivity with the following compounds: LTB4 = 100%) 5,lZdiHete = 3.6%, 20-OH-LTB4 = 1.3%, 6-trans LTB4 = 1.0%) and 12-epi-LTB4 = 0.6%. All other eicosanoids tested were reported to have less than 0.08% cross reactivity with the antibody. To stop the reaction 0.5 ml dextran-coated charcoal suspension was added, the mixture was vortexed and allowed to stand for 15 min on ice. The tubes were centrifuged at 1500 x g for 15 min at 4°C. 600 ul were transferred into a 7 ml plastic scintillation vial, and then 5 ml scintillation fluid were added. The samples were counted in a computerized LKB 1214 liquid scintillation counter (Gaithersburg, MD, USA) for 10 min each. The amount of bound radioactivity in the test samples were calculated and plotted against the amount bound in the presence of unlabelled LTB4 in the standard curve solutions. The LTB4 concentrations in the standard curves were 12.5, 50, 25, 50, 100, 250 and 500 pg LTB4. The LTB4 concentrations in the test samples were determined by reference to this standard curve. Final LTB4 concentrations were expressed as pg LTBdO.25 million cells. A23187 dose-response experiments To determine a concentration of calcium ionophore that stimulated the synthesis and release of a sufficient LTB4 concentration from canine PMNs to readily detect with the available RIA methods and to perform the inhibition assays, canine PMN cell suspensions were incubated at 37°C for 10 min with either 0, 0.01, 0.1, 1, or 10 PM A23187.


suspensions were incubated with 10 PM A23187 for 10 min at 4”C, 25”C, or 37°C. 5-Lipoxygenase inhibition experiments To evaluate the ability of two known lipoxygenase inhibitors to alter the calcium-stimulated synthesis and release of LTB4 from canine PMNs, BWA4C (N-[3-phenyoxycinnamyll-acetohydroxamic acid) and BW755C (3-amino-I-[3-triflouromethylphenyl]-2-pyrazoline hydrochloride) were incubated with the PMN cell suspensions for 10 min at 37°C prior to the addition of 10 PM A23187. Following the addition of the calcium ionophore the cell suspensions were further incubated for 10 min at 37°C. Both lipoxygenase inhibitors were incubated with the cells at concentrations from 1 PM to 1 mM.

RESULTS Calcium ionophore stimulation: dose, time and temperature response studies Canine PMNs stimulated with A23187 synthesized and released LTB4 in a dose-dependent manner. Maximal LTB4 release was observed with 10 PM A23187 (Fig. 1). An approximate 4-fold increase over basal release was observed with 10 PM A23187. In the presence of 10 PM A23187, timeresponse experiments (Fig. 2) indicated maximal LTB4 was synthesized by 10 min. Leukotriene B4 concentrations did not decrease or increase through 55 min of incubation at 37°C. In addition, the basal LTB4 release did not change over 55 min. High LTB4 concentrations were synthesized and released from canine PMNs at both 25°C and 37°C. However, greater amounts of LTB4 were synthesized by canine PMNs at 37°C (Fig. 3).

Time-response experiments To characterize the time pattern of LTB4 synthesis and release from canine PMNs, the PMN cell suspensions were incubated with 10 PM A23187 at 37°C for 1, 3, 5, 7, 10, 15, 20, 25, 30, 45, 50, or 55 min. Additional cells were incubated for the same time periods without the calcium ionophore to determine if a change in the basal release of LTB4 occurred over the incubation period. Temperature-response experiments To characterize the effects of temperature on the release of LTB4 from canine PMNs, the PMN cell



.1 1 A23187 (uM)


Fig. 1 Dose-response effect of A23187 on LTB, synthesis in canine polymorphonuclear leukocytes. Canine polymorphonuclear leukocytes were incubated with the indicated concentrations of A23187 as described in the Methods section. The values are the means + SEM of three determinations. The data are representative of three separate experiments giving qualitatively similar results.




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Fig. 2 Time-response effect of A23187-induced LTB, synthesis in canine polymorphonuclear leukocytes. Canine polymo~honuclear leukocytes were incubated with 10 FM A23187 (+) and without A23187 (0) for the indicated times as described in the Methods section. The values are the means rt SEM of three determinations. The data are representative of three separate experiments giving qualitatively similar results

~-Lipoxygenase Inhibitor


Fig. 4 Dose-response effects of BWA4C and BW755C on A23187-induced LTB, synthesis in canine polymorphonuclear leukocytes. Canine polymorphonuciear leukocytes were incubated with the indicated concentrations of BWA4C (0) and BW755C (+) as described in the Methods section. The values are the means + SEM of three determinations. The data are representative of three separate experiments giving qualitatively similar results.






37 25 Temperature (OC1

Ftg. 3 Temperature-response effect of A23187-induced LTB, synthesis in canine poIymo~honuclear leukocytes. Canine poiymorphonuclear leukocytes were incubated with 10 I.LM A23187 at the indicated temperatures as described in the Methods section. The values are the means + SEM of three determinations. The data are representative of three separate experiments giving qualitatively similar results.

Inhibition studies Incubation of canine PMNs with BWA4C and BW755C resutted in dose-response inhibition of LTB, synthesis (Fig. 4). BWA4C had an approximate ICso = 0.1 FM for inhibition of LTB4 synthesis in canine PMNs. BW755C was also able to inhibit LTB4 synthesis in canine PMNs, but was less effective as reflected by an approximate ICsO = 10 PM. In addition, total inhibition of ionophoreinduced LTB4 synthesis was achieved with 1 mM BWA4C, but not with 1 mM BW755C.

In veterinary clinical medicine small animal practioners are faced with a variety of disease categories some of which are similar and some dissimilar to conditions presented to human medical practioners. However, stages of some of the diseases specific to small pet animals, such as parasitic heartworm disease in dogs and viral feline infectious peritonitis in cats are characterized by frank inflammation, involvement of PMNs, and pulmonary edema or ascites. Interest in the pharmacological management of inflammatory diseases initiated this investigation into the capabilities of small pet animal PMNs to synthesize large quantities of LTB4, a known promoter of vascular permeability, edema formation and immune modulation. In addition, it was of interest to characterize the capability of known lipoxygenase inhibitors to block LTB4 synthesis in PMNs from small pet animals. We focused on LTB4 synthesis in PMNs obtained from healthy dogs in this study. The results from this study indicate canine PMNs readily synthesize and release large quantities of LTB4 following stimulation with low micromolar concentrations of calcium ionophore A23187. The data from this investigation and others (2, 6, 16-18) indicate 5-lipoxygenase is activated by calcium. We have indirectly shown a dose-response relationship between 5-lipoxygenase stimulation in canine neutrophils and intracellular calcium concentration. A dose-response increase in the calcium ionophore concentration resulted in a parallel dose-response increase in 5-lipoxygenase activity as reflected by increased LTB4 concentrations (Fig. 1). A basal

LTB, and Canine Polymorphonuclear Leukocytes

PMNs was observed in all our experiments. This release probably resulted from low concentrations of endogenous calcium or small amounts of calcium entering the cells from the calcium enriched incubation media. The data obtained from the time-response experiments (Fig. 2) indicated that large concentrations of LTB4 are probably not stored in canine PMNs. Under optimal experimental conditions, it took 10 min for maximal synthesis and release of LTB4 to occur. This indicated the processes of phospholipase A2 activation with subsequent liberation of arachidonic acid and further metabolism by 5 lipoxygenase and leukotriene A4 hydrolase had to occur for LTB4 to eventually be released from canine PMNs. Similar results have been reported with human PMNs (2, 6, 16). The data obtained from the A23187 doseresponse experiments indicated the free intracelluar calcium concentrations in canine PMNs is not sufficient to maximally stimulate LTB4 synthesis. The observed dose-reponse relationship between calcium concentration and LTB4 synthesis ionophore (Fig. 3) indicated the calcium requirement for activation of the lipoxygenase pathway in canine PMNs. Since both phospholipase A2 and 5have been lipoxygenase shown to be calcium-dependent enzymes in human neutrophils (14), it was not surprising that a similar relationship was shown to be present in canine PMNS. Once we had characterized the synthesis of LTB4 in canine PMNs it was of interest to determine the relative ability of two known lipoxygenase inhibitors to block LTB4 synthesis in canine PMNs. BW755C is a phenyl pyrazoline compound with known antioxidant activity (19). The 5-lipoxygenase inhibitory activity of BW755C has been characterized using human and rodent lipoxygenase sources (1,20, 21). However, BW755C is also an equipotent inhibitor of the cyclooxygenase pathway of arachidonic acid metabolism. BW755C has been shown to decrease both thromboxane and leukotriene synthesis in the same relative amounts after oral administration (19, 22). In comparison, BWA4C is an acetohydroxamic acid that has been reported to be more active than BW755C in inhibiting human PMN 5-lipoxygenase (19, 22). BWA4C can inhibit cyclooxygenase, but at equimolar concentrations BWA4C inhibits 5lipoxygenase to a greater degree. Of futher interest is the observation that BWA4C has a better oral bioavailability than BW755C (19, 22). The results from our dose-repsonse inhibition experiments indicated that both BW755C and BWA4C inhibited 5-lipoxygenase activity in canine PMNs. BWA4C inhibited canine 5-lipoxygenase with an approximate I& = 0.1 PM. In comparison, BW755C inhibited canine 5-lipoxygenase with an


of LTB4 from nonstimulated


approximate ICss = 10 PM. These data indicated that under the in vitro experimental conditions used in this study BWA4C had a much greater efficacy towards inhibiting canine PMN 5-lipoxygenase. These data are in general agreement with Tateson et al (22) who reported an ICH, = 0.04 PM for BWA4C and an ICs0 = 5.4 PM for BW755C to inhibit human PMN 5-lipoxygenase in vitro. In addition, rat PMN 5-lipoxygenase has been reported to be inhibited by BW755C with an ICsa = 43 PM (1). In general, little attention has been paid to leukotriene synthesis, metabolism, and receptor interactions with canine PMNs. Previous studies have shown canine PMNs have the capability to generate LTB4 when the cells were preincubated with uM concentrations of arachidonic acid or [14C]arachidonic acid (23, 24). Thomsen et al (24) showed Beagle PMNs synthesized LTI34 in response to stimulation with interleukin-8. Thomsen et al (24) also demonstrated that Beagle PMNs could metabolize [“%Z]-arachidonic acid into 20-OH-[14C]-LTB4 and 20-COOH-[‘4C]-LTB4 which is a common metabolic pathway for LTB4 in human PMNs (25-27). In regards to decreasing leukotriene synthesis in canine PMNs, Bednar et al (23) used 48-94 PM BW755C to inhibit arachidonic acid metabolism in the PMNs of an unidentified breed of dog. We believe the 5-lipoxygenase inhibition data reported in the present study, using Greyhound PMNs, are the first dose-response inhibition studies using canine PMNs to compare the efficacy of different molecular classes of 5-lipoxygenase inhibitors. Gruber et al (28) using Beagles and Strom and Thomsen (29) using an unidentified breed of dog have shown canine PMNs have extracellular receptors for LTB4. Both research groups demonstrated exogenously added LTB4 induced a degranulation response and chemotaxis of canine PMNs. The results from their studies indicated the LTB4 receptors on canine PMNs are coupled to intracellular events that modulate PMN function. Taken together, the results from the present study and from others indicate the physiology of LTB4 in canine PMNs is similar to that in human PMNs. Canine PMNs are suitable for use in the study of basic leukotriene physiology or for use in the pharmacological screening of novel 5-lipoxygenase inhibitors directed for veterinary or human use.

Acknowledgements The experiments reported in this paper were approved by the Auburn University Committee on Animal Welfare (PRN No 9305-R-085). Supported by a grant from the Scott-Ritchey Center for Research on companion Animal Diseases.


Prostaghmdins Leukotrienes

and Essential Fatty Acids

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Characterization of leukotriene B4 synthesis in canine polymorphonuclear leukocytes.

The synthesis and release of leukotriene B4 (LTB4) from canine polymorphonuclear leukocytes (PMNs) was characterized in terms of incubation time, temp...
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