Biotherapy 3:233 239, 199[. ~ 1991 Kluwer Academic Publishers. Printed in the Netherlands.
In vitro modulation of canine polymorphonuclear leukocyte function by
granulocyte-macrophage colony stimulating factor Michele M. D'Alesandro, Dale F. Gruber, Kevin P. O'Halloran & Thomas J. MacVittie Armed Forces Radiobiology Research Institute, Experimental Hematology Department, Bethesda, Maryland 20814, USA Received 26 March 1990: accepted 5 April 1990
Key words: chemotaxis, flow cytometry, granulocyte-macrophage colony stimulating factor, membrane potential, oxidative burst, polymorphonuclear leukocyte Abstract
Granulocyte-macrophage colony stimulating factor (GMCSF) promotes the growth of granulocytes and macrophages from undifferentiated bone marrow cells and modulates the oxidative responses of polymorphonuctear leukocytes (PMN) to endogenous chemoattractants. We found that, in vitro, naturally occurring glycolsylated human GMCSF does not disturb the resting canine PMN membrane potential, may attentuate PMN oxidative responses to PMA, and is, to a small degree, chemotaxigenic. GMCSF, however, inhibits PMN chemotaxis to zymosanactivated plasma (ZAP). Compared to temperature controls, GMCSF (1-100 U/ml) produced up to 1.5-fold increases in H202 production after 15 minutes, while phorbol myristate acetate (PMA) treated cells increased H202 production 8-12-fold after 15 minutes. Preincubation of cells with GMCSF ( 1- 100 U/ml) prior to PMA stimulation significantly reduced the H202 levels induced by PMA. H202 production was inhibited up to 15% after 15 minutes of GMCSF preincubation and up to 40% after 60 minutes of preincubation. As a chemotaxigenic agent, GMCSF (10-1000U/ml) was able to elicit 49%-102% increases in quantitative cellular migration, compared to random migration. Total cellular chemotaxis to GMCSF was < 30% of the response to ZAP. Preincubation of PMNs with GMCSF for 15 minutes significantly inhibited ZAP-induced cellular migration. Human GMCSF does not appear to activate canine PMN in vitro and may actually down-regulate PMN inflammatory responses.
Abbreviations" FL: fluorescence intensity; GMCSF: granulocyte-macrophage colony-stimulating factor; HBSS: Hanks' balanced salt solution; H202: hydrogen peroxide; MP: membrane potential; PMA: phorbol myristate acetate; PMN: polymorphonuclear leukocyte. Supported by the Armed Forces Radiobiology Research Institute, Defense Nuclear Agency, under work unit No, 00082. Views presented in this paper are those of the authors: no endorsement by the Defense Nuclear Agency has been given or should be inferred. Research was conducted according to the principles enunciated in the "Guide for the Care and Use of Laboratory Animals'" prepared by the Institute of Laboratory Animal Resources, National Research Council.
T-lymphocyte derived granulocyte-macrophage colony stimulating factor (GMCSF) reportedly demonstrates numerous inflammatory bioregulatory properties. The 22 Kd gly-
234 coprotein inhibits polymorphonuclear leukocyte (PMN) migration , stimulates phagocytosis of bacteria , and primes PMNs for enhanced oxidative responses to fmlp, LTB4, or C5a desArg . In addition to GMCSF, endotoxin , low levels of fmlp [ 5], and B-cell derived factor  also prime PMNs for enhanced oxidative responses, suggesting the possibility of multiple priming mechanisms. Priming may enhance normal oxidative burst levels to those necessary for a competent response during an inflammatory event. Exogenous administration of GMCSF stimulates macrophage, granulocyte, and eosinophil colony formation in human bone marrow cell cultures  and granulocyte, macrophage, erythroid, and mixed colonies in nonhuman primates [8, 9]. The ability of GMCSF to accelerate the growth and maturation of marrow cells and activate mature PMNs becomes an important clinical factor, and may be related to the incidence/severity of infection and related neutropenia [9, 10]. In light of both quantitative and qualitative effects, GMCSF may have potential therapeutic application in the treatment of infection or trauma. In this report, we examine the in vitro ability of glycosylated human GMCSF to directly or indirectly influence mobility, oxidative function, and resting membrane potentials of isolated canine peripheral blood PMNs.
Materials and methods
Reagents. Phorbol myristate acetate (PMA), zymosan, dimethylsulfoxide (Sigma Chemical Company, St. Louis, MO); Hanks' balanced salt solution (HBSS), phosphate buffered saline (PBS), trypan blue (Grand Island Biological Company, Grand Island, NY); dichlorofluorescein diacetate (DCFH-DA) (Eastman Kodak Company, Rochester, NY); ammonium chloride (Fisher Scientific, Silver Spring, MD); dipentyloxocarbocyanine ( DiOCs(3)) (Molecular Probes, Junction City, OR). Stock solutions of DCFH-DA (5 mmol/
L) and DiOCs(3) (1 mmol/L) were stored in absolute ethanol at -70°C. PMA was dissolved in dimethylsulfoxide (10 mmol/L) and stored at -70°C. Fetal bovine serum (Hyclone Labs, Logan, UT) was heat-inactivated (56°C, 60 minutes) and filtered (0.45 um filter) before use. Human GMCSF (Genzyme, Boston, MA), specific activity 2200 CFU/ug, was, by manufacturer specifications, free of detectable levels of IL-1, IL-2, and interferon. Animal model. Six Hra purpose-bred beagles (1-2 years old, 10-12 kg) were quarantined on arrival and screened for evidence of disease. Animals were kenneled in an AAALAC accredited facility and provided commercial dog chow and tap water ad libitum. Holding rooms were maintained at 21 _+ I°C with 50% _+ 10% relative humidity. Animals were housed on a 12-hour light/dark full spectrum lighting cycle with no twilight. P M N isolation. Peripheral blood was drawn one time from the lateral saphenous vein into syringes containing preservative-free heparin (10 U/ml). After phlebotomy, animals were returned to the general research colony. Blood was washed free of plasma with HBSS without Ca + + and Mg ++ (400 x g, 10 minutes, 20°C) and contaminating red blood cells (RBC) were lysed with 0.83% ammonium chloride (10 minutes, 4°C). Leukocytes were pelleted and minimally resuspended in PBS supplemented with 0.2% heat-inactivated fetal bovine serum and analyzed. Chemotaxis. PMN chemotaxis was evaluated by quantitating cells that migrated through a 10 um thick polycarbonate membrane separating upper and lower wells of a microchemotaxis chamber assembly (Neuro Probe, Bethesda, MD). Lower wells contained aliquots of zymosan activated plasma (1:100 dilution)  or media. Upper wells contained cell concentrations which were corrected by differential cell analysis to reflect the addition of 105 PMNs. Chambers were incu-
235 bated at 37°C for 1 hour. Membranes were removed, fixed in 100% methanol for 1 minute, and stained with Diff-Quik (Fisher Scientific, Silver Spring, MD). Seven highpower ( 1 0 0 x ) microscope fields (HPF) across the diameter of the well were quantitated for cells that migrated through the filter. Data are expressed as the mean number of migrated cells/HPF/hour.
Membrane potential changes were determined by measuring intracellular concentrations of DiOCs(3). PMNs (106cells/ml) in HBSS supplemented with 1 mg/ml glucose were incubated with 10 s M DiOCs(3) for 10 minutes at 37 ~C. Intracellular fluorescence intensity of cells incubated in this manner decreased as the membrane depolarized. Cells were stimulated with PMA (100 ng/ml) or G M C S F (0.1-1000 U/ml) for 7 minutes at 37°C and changes in membrane potential were measured by flow cytometry using a FACS A N A L Y Z E R interfaced to a C O N S O R T 30 data acquisition analyzer (Becton Dickinson, Sunnyvale, CA). PMNs were distinguished and gated based on coulter volume and right-angle light-scatter properties.
Statistical analysis. All data are presented as the mean + standard error. Statistical differences were determined using the Student's t-test. P values