SHORT COMMUNICATIONS Oxygen Radical Production by Avian Leukocytes Peter Conlon, Dale Smith and Todd Gowlett

ABSTRACT

Oxygen radical production by heterophils of red-tailed hawks and chickens, and by neutrophils of calves, was evaluated in a chemiluminescence microassay. Leukocytes were isolated by centrifugation of blood in capillary tubes and then challenged with opsonized zymosan in the presence of luminol. Avian heterophils produced significantly fewer oxygen radicals than did bovine neutrophils. RESUME La production de radicaux d'oxygene par des heterophiles de poulets et de faucons a queue rouge ainsi que par des neutrophiles de veaux a ete mesuree par une microtechnique se servant

de la chimiluminescence. Les leucocytes ont ete obtenus apres centrifugation des echantillons sanguins dans des tubes capillaires et par la suite mis en presence de zymosan opsonise en presence de luminol. Les resultats obtenus demontrent que les heterophiles produisent significativement moins de radicaux d'oxygene que les neutro-

philes. (Traduitpar Dr Pascal Dubreuil) Mammalian neutrophils which encounter foreign particles undergo a

''respiratory burst" during which they glucose and oxygen and produce oxygen radicals (OR; superoxide, hydrogen peroxide, hydroxyl radical and singlet oxygen) (1). The bactericidal capacity of the cells is closely tied to the production of these chemicals, and evaluation of this capacity by consume

luminol-dependent chemiluminescence assays has become well-accepted (2,3). Although mammalian neutrophil function has been intensively examined in this manner, that of the avian heterophil has not. This paper describes the application of a luminol-dependent chemiluminescence microassay to avian heterophils and bovine neutrophils. Its requirement for only small amounts of blood makes it ideal for evaluating cells from birds, where total blood volume is not great, and where there may be difficulty in obtaining blood samples. Also, since purification of the blood to obtain a pure heterophil or neutrophil fraction is not necessary, time is saved, and the use of gradient centrifugation, which may be disruptive to cell function, is avoided. Five mature White Leghorn chickens, five mature red-tailed hawks (Buteo jamaicensis) and five Holstein calves (4-5 months of age) were used in the experiments. All were handled according to the guidelines of the Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care. The hawks were sedated with a mixture of xylazine (Rompun; Haver, Etobicoke, Ontario; 0.8 mg/kg) and ketamine (Ketaset; Austin Laboratories, Joliette, Quebec; 7.2 mg/kg) given intramuscularly before blood sampling. Five mL syringes were flushed with heparin, and 4 mL of blood were removed from the deep ulnar vein in the birds and the jugular vein in the calves. A complete blood count and a differential examination of Wright'sstained smears were done on each

sample in order to determine the total and relative numbers of leukocytes present, and these values were used to calculate the total number of cells and the relative numbers of each cell type in the buffy coat preparation. Similar to the method of Thomas et al (4), for each subject 100 gL of blood were drawn up into each of six 140 ytL heparinized capillary tubes (Type D551, Radiometer, Copenhagen). The tubes were sealed and kept on ice until centrifuged for 20 min at 500 g. After centrifugation, and without disturbing the easily visible buffy coat, the plasma was removed with a 10 ,uL Hamilton syringe and discarded. Using a three-sided file, a notch was scored in the tube 0.5 mm below the buffy coat, the tube broken at this point and the section of tube containing the erythrocytes discarded. The buffy coat in the remaining section was ejected into a cuvette containing 200 ltL of Hanks' balanced salt solution (HBSS), pH 7.4, at 37°C, by inserting the end of the capillary tube into the end of a plastic transfer pipette and repeatedly flushing the capillary tube. Zymosan A (30 mg) was washed in 3 mL HBSS, centrifuged and resuspended in 2.1 mL HBSS and 900 AL plasma taken from one of the subjects of each species chosen at random. This mixture was incubated in a shaking water bath for 30 min at 37°C, then washed with and resuspended in HBSS to produce a final concentration of 10 mg/mL. Luminol (13.25 mg) was dissolved in 10 mL dimethyl sulfoxide (DMSO) to produce a 5 x 10-2 M stock solution, which was diluted in HBSS to produce a working solution

Department of Biomedical Sciences (Conlon, Gowlett) and Department of Pathology (Smith), Ontario Veterinary College, University of Guelph, Guelph, Ontario NIG 2W1. Submitted August 17, 1990.

Can J Vet Res 1991; 55: 193-195

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numbers most closely correlated with OR production are the mature cells, regardless of species. Cell size differences among avian Mature cells* and mammalian species may have con+ band cells Mature cells* Mature cells tributed to the increased OR genera+ monocytes + band cells only* Species the bovine neutrophils. Avian tion by 3.36 ± 1.23BC 4.48 ± 1.54B 4.48 ± 1.54B Chickens heterophils are approximately 8 itm in 8.37 ± 3.95AC 8.72 ± 4.06A 8.72 ± 4.06A Hawks diameter (5), while those of cattle ± 15.50AB ± 31.69 33.36 12.94AB 34.89 ± 14.86AB Calves 10-15 zm and average from range Within each column, means with the same superscript are significantly different from each other 11.5 Am (6). Comparing the surface (p < 0.05) area of two spheres of these sizes *Mature neutrophils or heterophils reveals that a sphere of 8 ltm in diameter has an area of 201.1 Am2, while one of 11.5 Am diameter has an area 415.5 Am2. Therefore, although of in TABLE II. Pearson's correlation coefficients of peak chemiluminescence and cell numbers chickens, red-tailed hawks and calves after stimulation with opsonized zymosan in the presence some of the increased activity of the of luminol bovine cells may be due to a potentially greater surface area, this would probMature cells Band cells Monocytes ably account for a doubling of OR only* Species generating capacity at most, rather 0.49 0.51 0.98 Chickens 0.39 0.14 0.80 than an eightfold increase as we found. Hawks 0.48 0.76 0.90 Calves To our knowledge, the evaluation of oxidative function in avian leukocytes *Mature neutrophils or heterophils by a chemiluminescence assay has not been previously reported. Penniall and Spitznagel (7) showed that chicken heterophils undergo a "respiratory of 5 x 10-5 M. All reagents were by comparing cell numbers with burst" during phagocytosis, but they concluded that the cells do not produce obtained from Sigma Chemical Co., chemiluminescence production. The mature heterophils of both hydrogen peroxide. The contradictory St. Louis, Missouri. Immediately after each buffy coat species of birds produced significantly finding of our work may be due to a was suspended in HBSS, 200 ,L of less CL than did the mature neutro- lower sensitivity of the assays used luminol, 50 yL of opsonized zymosan phils of calves (Table I). When band previously (formate reduction and and a stirring bar were added to the cells were included with neutrophils for scopoletin technique). The dearth of studies evaluating cuvette, which was placed in a dual the calves, similar differences among channel integrating luminometer species remained. When monocytes avian leukocyte oxidative function (Model 460, Chrono-Log Corp., were included, however, there was sig- may, in part, have been due to the Pittsburgh, Pennsylvania). The mix- nificantly more CL produced by the relatively large volumes of blood pretures were kept at 37°C and stirred at hawk cells than by the chicken cells, viously needed to produce pure phago300 rpm by means of a magnetic stir- while the calves still produced the cytic cell preparations. Although a rer. In order to minimize the effects of most. Correlation coefficients revealed microassay for whole blood has been time, the samples from all the subjects that, for each species, the peak chemi- previously described (8), high concenwere randomized in the order in which luminescence production was most trations of erythrocytes can cause they were run throughout the day. The closely correlated with mature cell quenching of chemiluminescence, so separation of those cells having peak chemiluminescence (CL) produc- numbers (Table II). The CL assay employed in this study oxidative capacity is important tion was determined automatically by the luminometer and calculated as proved to be a relatively simple way to for accurate measurement of OR evaluate the OR production of leuko- production. millivolts/105 cells. Although hydrogen peroxide is By means of a statistical analysis cytes from two species of birds and programme (SAS, Cary, North from calves. From our results it is somewhat bactericidal alone, its Carolina), comparison of oxygen apparent that there is a marked dif- efficacy is greatly increased through radical production among species was ference in the capacity for OR genera- the action of myeloperoxidase. In performed by one-way analysis of tion by leukocytes from birds and mammalian cells, myeloperoxidase variance (ANOVA). If any significant calves. On a per 105 cells basis, the catalyses the oxidation of halide ions differences were found, specific dif- mature neutrophils of calves produced to hypohalite ions by hydrogen peroxferences between group pairs were approximately eight times more chemi- ide. Chloride ion is the probable physdetermined by least significant dif- luminescence than did the heterophils iological substrate, since it is the most ferences. The probability level for of chickens and approximately four abundant halide in cells (9). Comparisignificance was p < 0.05. Pearson's times that of hawks. Correlation coef- son of the efficacy of bacterial killing correlation coefficients were calculated ficients demonstrated that the cell in the presence and absence of myeloTABLE I. Peak chemiluminescence produced by heterophils or neutrophils, and by monocytes of chickens, red-tailed hawks and calves after stimulation with opsonized zymosan in the presence of luminol. Results are the mean and standard deviation of six replicates on each of five subjects/ species and are expressed as millivolts/105 cells

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peroxidase has been carried out, and hydrogen peroxide concentrations of 0.5 mM are needed in the absence of the enzyme, as compared to 10 /AM in the presence of the enzyme (10). Myeloperoxidase has not been detected in avian heterophils (11,12). This absence may be either the result of the cells normally not producing as much hydrogen peroxide as do corresponding mammalian cells (as was demonstrated in this study), thus reducing the necessity for production of the enzyme, or it may be the cause of an evolutionary adaptation which has resulted in the cells generating less hydrogen peroxide than do mammalian cells, since the "potentiating" enzyme is absent. However, these less active oxygen-dependent mechanisms in avian heterophils do not seem to put the cells at a disadvantage compared to their mammalian counterparts since the former have been shown to readily phagocytize and kill yeasts and both gram-negative and gram-positive bacteria (13). Using the assay technique described in this paper, the immunological and pharmacological investigation of the

bactericidal function of avian heterophils, and also of the phagocytic cells of other species, will be facilitated. ACKNOWLEDGMENTS

The authors thank Karen Machin for her excellent technical assistance. REFERENCES 1. FANTONE JC, WARD PA. Role of oxygen-derived free radicals and metabolites in leukocyte-dependent inflammatory reactions. Am J Pathol 1982; 107: 398-417. 2. ALLEN RC, STJERNHOLM RL, STEELE RH. Evidence of the generation of an electronic excitation state in human polymorphonuclear leukocytes and its participation in bactericidal activity. Biochem Biophys Res Commun 1972; 47: 679-684. 3. EASMON CSF, COLE PJ, WILLIAMS AJ, HASTINGS M. The measurement of opsonic and phagocytic function by luminoldependent chemiluminescence. Immunology 1980; 41: 67-74. 4. THOMAS VL, SANFORD BA, DRISCOLL MS, CASTO DT, RAMAMURTHY RS. Luminol-dependent chemiluminescence microassay for phagocytic function. J Immunol Methods 1988; 111: 227-232. 5. MONTALI RJ. Comparative pathology of inflammation in the higher vertebrates (rep-

tiles, birds and mammals). J Comp Pathol 1988; 99: 1-26. 6. JAIN NC. Schalm's Veterinary Hematology, 4th ed. Philadelphia: Lea & Febiger, 1986: 195. 7. PENNIALL R, SPITZNAGEL JK. Chicken neutrophils: Oxidative metabolism in phagocytic cells devoid of myeloperoxidase. Proc Natl Acad Sci USA 1975; 72: 5012-5015. 8. TONO-OKA T, UENO N, MATSUMOTO T, OHKAWA M, MATSUMOTO S. Chemiluminescence of whole blood. 1. A simple and rapid method for the estimation of phagocytic function of granulocytes and opsonic activity in whole blood. Clin Immunol Immunopathol 1983; 26: 66-75. 9. KLEBANOFF SJ. Myeloperoxidase-halidehydrogen peroxide antibacterial system. J Bacteriol 1968; 95: 2131-2138. 10. McRIPLEY RJ, SBARRA AJ. Role of the phagocyte in host-parasite interactions. XII. Hydrogen peroxide-myeloperoxidase bactericidal system in the phagocyte. J Bacteriol 1967; 94: 1425-1430. 11. DEIN FL. Laboratory Manual of Avian Hematology. East Northport, New York: Association of Avian Veterinarians, 1984. 12. TOPP RC, CARLSON HC. Studies on avian heterophils, II. Histochemistry. Avian Dis 1972; 16: 369-373. 13. BRUNE K, LEFFELL MS, SPITZNAGEL JK. Microbicidal activity of peroxidaseless chicken heterophile leukocytes. Infect Immun 1972; 5: 283-287.

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Oxygen radical production by avian leukocytes.

Oxygen radical production by heterophils of red-tailed hawks and chickens, and by neutrophils of calves, was evaluated in a chemiluminescence microass...
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