ABSTRACT. Pentoxifylline has been reported to improve peripheral vascular circulation by altering the flow properties of blood. To determine if the hemorrheo-.
Effects of Pentoxifylline on Equine Neutrophil Function and Flow Properties Douglas J. Weiss, Raymond J. Geor, Steven M. Burris and Clark M. Smith II
ABSTRACT
Pentoxifylline has been reported to improve peripheral vascular circulation by altering the flow properties of blood. To determine if the hemorrheological effects of pentoxifylline were mediated by alterations in neutrophil function and/or flow properties, we evaluated the drug's effects on equine neutrophils in vitro. Pentoxifylline, at a concentration of 1 x 10-1 M, but not at concentrations of 1 x 10-6 M to 1 x 10-2 M, markedly suppressed neutrophil superoxide production, zymosan phagocytosis and adherence to nylon wool. Pentoxifylline failed to improve neutrophil filterability through 3 1t polycarbonate filters at any concentration tested. We conclude that equine neutrophil function and flow properties are unlikely to be affected by pentoxifylline concentrations achievable in vivo. RESUME
Il a ete rapporte que la pentoxifylline ameliore la circulation sanguine peripherique en alterant les proprietes du flux sanguin. Afin d'evaluer si les effets de Ia pentoxifylline sont medies par une alteration de la fonction des neutrophiles ou une alteration des propriet6s du flux sanguin, nous avons evalue, in vitro, les effets de ce medicament sur les neutrophiles equins. La pentoxifylline, a une concentration de 1 x 10-1 M, mais pas A des concentrations variant de 1 x 10-6 M a 1 x 10-2 M, diminue par les neutrophiles la production de superoxide, la capacite de phagocytose du zymosan et l'adherence a la laine de nylon. La pentoxifylline n'a pas ameliore la filtrabilite des neutrophiles A travers des filtres de 3,u en polycarbonate. Des
resultats de cette etude, nous pouvons conclure qu'il est peu probable que la fonction et les proprintes du flux des neutrophiles equins soient affectees in vivo par la pentoxifylline. (Traduit par Dr Jean-Pierre Lavoie)
INTRODUCTION
Pentoxifylline (Navicon, Sanofi Animal Health Inc., Overland, Kansas) is a methylxanthine derivative which has been used for treatment of navicular disease in horses (1). Navicular disease may result from ischemia induced by thrombosis of the navicular arteries and perhaps periosteal arteries (2). Studies in humans and laboratory animals indicate that the clinical benefit of pentoxifylline may be due to improved microvascular blood flow (3,4,5,6). Improved microvascular blood flow has been variously ascribed to increased red blood cell (RBC) deformability (3,4), increased neutrophil deformability or altered neutrophil function (5,6). We have recently shown that pentoxifylline increases RBC deformability when administered orally to horses at 3 g twice a day (7). The purpose of this study was to further investigate the mechanisms of action of pentoxifylline in horses by assessing the in vitro effects of pentoxifylline on equine neutrophil function and flow properties through polycarbonate filters. MATERIALS AND METHODS BLOOD COLLECTION AND PROCESSING
Whole blood, anticoagulated with tripotassium EDTA or lithium heparin was obtained from three healthy
thoroughbred horses recently retired from competitive racing. Blood samples were processed and analyzed within 4 h of sample collection. For superoxide production, zymosan phagocytosis and neutrophil filterability assays, neutrophils were first isolated by Percoll (Sigma Chemical Co., St. Louis, Missouri) density gradient centrifugation (8). After centrifugation, neutrophils were washed three times in Dulbecco's phosphatebuffered saline (Gibco, Grand Island, New York). Neutrophil purity exceeded 98% and viability, as determined by trypan blue exclusion, was greater than 95%. For neutrophil filtration studies, neutrophils were evaluated for evidence of activation by placing in a hemocytometer and examining by light microscopy. Microscopic evidence of activation included pseudopod formation and membrane ruffling. Samples were used only if greater than 950% of the neutrophils were spherical. NEUTROPHIL SUPEROXIDE PRODUCTION ASSAY
Two hundred microliters of the neutrophil suspension (2 x 107 cells/mL) was incubated with 200 uL of Hanks solution (Gibco) or Hanks solution containing pentoxifylline at final concentrations ranging from 1 x 10-6 M to 1 x 10- M. Samples were incubated at 37°C for 1 h and superoxide production was assayed by determination of superoxide dismutaseinhibitable reduction of ferricytochrome C (9). Samples were analyzed in triplicate and data from three horses were used for statistical analysis. ZYMOSAN PHAGOCYTOSIS ASSAY
The capacity of neutrophils to phagocytize opsonized zymosan particles was determined as previously described (9). In this test. 250 yL of
Department of Veterinary PathoBiology (Weiss), Department of Population Sciences (Geor) and Department of Pediatrics (Burris, Smith II), University of Minnesota, St. Paul, Minnesota 55108. Submitted February 11, 1992.
Can J Vet Res 1992; 56: 313-317
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neutrophil suspension (2 x 107 cells/mL) was incubated for 1 h with 250 IuL of Hanks solution with and without pentoxifylline at final concentrations ranging from 1 x 10-6 M to 1 x 10-1 M. After incubation, 0.1 mL of zymosan (5 mg/mL) opsonized with autologous plasma, was added. At 1, 5 and 10 min, 0.1 mL of the neutrophilzymosan suspension was removed and placed in 0.1 mL of 0.25 M N-ethyl maleimide. Thereafter, 20 mL of 0.25 M sucrose solution, containing 0.1 mM EDTA, 5 ltM colchicine and 1.0 % bovine serum albumin was added to disrupt neutrophil clusters. Cytocentrifuge slide preparations were made and stained with Diff-Quik stain (American Scientific Products, McGaw Park, Illinois). This stain gave excellent definition of zymosan particles. Slides were examined by light microscopy under 1000 x magnification. The number of zymosan particles within the circumference of the cells and in the same field of focus as the cells were enumerated. All samples were performed in duplicate and data from three horses, were analyzed. NEUTROPHIL ADHERENCE ASSAY
Neutrophil adherence to nylon wool was determined based on modifications of previously described procedures (9,10). These procedures varied in the amount of nylon wool used in the assays. Three mL of heparinized whole blood was incubated for 1 h at 37°C with 0, 1 x 10-6, 1 x 10-5, 1 x 10-4, 1 X 10-3, 1 X 10-2 and 1 x 10-1 M pentoxifylline. Before and after incubation, total leukocyte counts were determined using an electronic cell counter and blood smears were made for determination of differential leukocyte counts. Adherence columns were prepared by packing 1.0 mL syringe casings with 40 or 100 mg of washed nylon wool (Polysciences Inc., Warrington, Pennsylvania). The wool was packed to the 0.4 mL mark on the syringe casing. One mL of blood was applied to the top of the column and allowed to filter for 10 min at room temperature. The percentage of neutrophils adhering to the column was determined by comparing total neutrophil counts before and after filtration. All samples were tested in duplicate and data from three horses were analyzed.
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Fig. 1. Effect of pentoxifylline (PTX) on superoxide production by equine neutrophils. Asterisk indicates statistically significant difference from control values (p < 0.01) as determined by analysis of variance. NEUTROPHIL FILTERABILITY ASSAY
Neutrophils (6 x 106/mL) were incubated for 1 h with an equal volume of Dulbecco's solution containing 0 to 1 x 10-1 M pentoxifylline. After incubation, the pressure resulting from filtering neutrophils through polycarbonate filters was determined (6). The filtration device consisted of a Harvard syringe pump (Millis, Massachusetts), a Statham transducer and a polygraph recorder. Neutrophil suspensions were pumped at a constant flow rate (1.1 mL/min) through a 13 mm polycarbonate filter (Nucleopore Corp., Pleasanton, California). Initial studies, using Hemafil filters with an average pore diameter of 5 , resulted in negligible pressure rises. Substitution of filters with an average pore diameter of 3 A resulted in reproducible pressure curves. Pressure measurements were determined on the pump side of the filter. A pressure curve was generated
which was characterized by an initial sharp pressure rise which plateaued within the first two seconds and a secondary less rapid pressure rise. The
ratio of the pressure at the plateau of the initial pressure curve (Pi) and the pressure generated by the buffer alone (Po) was enumerated for each sample. The Pi/Po has previously been shown to reflect the resistance of cells to filtration whereas the secondary pressure rise reflects the plugging of the pores in the filter (6). The Pi/Po for each neutrophil suspension was measured in triplicate or quadruplicate and average values calculated. STATISTICAL ANALYSIS
Data were analyzed by one-way analysis of variance. If the F ratio was significant, the means of interest were compared using Scheffe's F test. Significance was reported at the p
