Effects of Recombinant Granulocyte-macrophage Colony-stimulating Factor on Bovine Peripheral Blood and Mammary Gland Neutrophil Function in vitro Lorraine M. Sordillo, Guy Afseth, Gerald Davies and Lorne A. Babiuk ABSTRACT
Modulation of peripheral blood and gland neutrophil function following in vitro exposure to recombinant bovine granulocyte-macrophage colony-stimulating factor (rBoGMCSF) was studied. Bovine blood and mammary gland neutrophils were cultured for 9 h in media containing 0.005, 0.05 or 0.5 ug/mL rBoGMCSF. Neutrophils treated with rBoGMCSF exhibited significantly more chemotactic and bactericidal activities and tended to produce more superoxide anion than control cells. The effects of rBoGM-CSF on bovine neutrophil populations appeared to be dose-dependent. The production of superoxide anion and the bactericidal activity of mammary gland neutrophils were consistently higher than blood neutrophils. Only moderate increases in lipopolysaccharide-induced mammary gland neutrophil functions were observed following incubation with rBoGM-CSF which suggests that there may be a threshold of immunomodulation for these prestimulated cells. It may be possible to augment the functional capacity of bovine neutrophil populations in vivo through the therapeutic application of rBoGM-CSF and consequently enhance resistance of dairy cattle to bacterial infections. mammary
RESUME Les variations de la fonction des neutrophiles dans le sang peripherique et dans la glande mammaire a la suite d'une exposition in vitro au facteur recombinant stimulant des colonies de granulocytes et de macrophages bovin
(recombinant bovine granulocyte- glycoproteins that bind to cells by a macrophage colony-stimulating factor common receptor and are produced by (rBoGM-CSF)) furent etudiees. a variety of cells including fibroblasts, Des neutrophiles provenant du sang endothelial cells, macrophages and peripherique et de la glande mammaire T lymphocytes (1). Granulocytede bovins furent cultives pendant macrophage colony-stimulating factor 9 heures dans un milieu contenant (GM-CSF) was first identified by its 0,005, 0,05 ou 0,5 Ag/mL de rBoGM- capacity to induce hematopoietic proCSF. Les neutrophiles traites avec le genitor cells to proliferate and difrBoGM-CSF demontrerent une plus ferentiate into granulocytes and grande activite bactericide et chemo- macrophages (1). Several recent studtactique et eurent tendance a produire ies in humans have shown that GMplus d'anions superoxide que les CSF is not only a hematopoietic cellules temoins. Les effets du rBoGM- growth-inducing molecule, but also CSF sur les populations de neutro- affects a variety of functions of mature philes bovins apparurent comme etant granulocytes and monocytes (2-6). relies 'a la dose. La production d'anions Human GM-CSF affects neutrophils superoxide et l'activite bactericide des directly by decreasing migratory activneutrophiles de la glande mammaire ity, and enhancing phagocytosis, etaient constamment plus elevees que antibody-dependent cellular cytotoxcelles des neutrophiles sanguins. Seule icity, and superoxide anion production une augmentation moderee dans les in response to n-formyl-methionylfonctions induites par les liposac- leucyl-phenylalanine (FMLP) (2-4). charides dans les neutrophiles mam- Monocytes respond to GM-CSF by maires fut observee a la suite de l'incu- induction of macrophage tumoricidal bation avec le rBoGM-CSF, suggerant activity and stimulation of mRNA la possibilite d'un seuil dans l'immu- expression and protein synthesis of nomodulation de ces cellules pre- M-CSF and G-CSF (5,6). The prostimulees. II peut etre possible d'aug- nounced influence of GM-CSF on menter la capacite fonctionnelle des phagocytic cell populations suggest populations de neutrophiles bovins possible clinical applications in the prein vivo par l'usage therapeutique du vention of infectious bacterial diseases rBoGM-CSF et ameliorer par conse- of livestock, such as bovine mastitis. quent la resistance des bovins laitiers The ability of mammary gland aux infections bacteriennes. (Traduit neutrophils to phagocytoze mastitispar Dr Anne Provencher) causing pathogens and suppress bacterial multiplication is critical to the outcome of intramammary infection (7). Although both neutrophils and INTRODUCTION macrophages predominate in mammary Colony stimulating factors are a gland secretions and tissues (8) evigroup of cytokines required for the dence suggests that the antibacterial proliferation and differentiation of a activities of these cells are reduced in variety of hematopoietic stem cells. the presence of mammary secretions These growth factors are distinct due to a lack of energy source, low
Veterinary Infectious Disease Organization, University of Saskatchewan, 124 Veterinary Road, Saskatoon, Saskatchewan S7N OWO. This work was supported by the Natural Sciences and Engineering Research Council of Canada and CIBA-GEIGY Canada Limited, Mississauga, Ontario. Published with the approval of the Director of VIDO as journal series no. 120. Submitted May 6, 1991.
16
Can J Vet Res 1992; 56: 16-21
concentrations of opsonins, and interference by milk constituents (7,8). To overcome the suppressive nature of mammary gland secretions, a relatively large proportion of functionally competent mammary gland neutrophils are required for effective protection against mastitis-causing pathogens (7). The purpose of this study was to evaluate the modulatory effects of rBoGM-CSF on the functional capabilities of bovine peripheral blood and mammary gland neutrophils in vitro. The possible clinical application of rBoGM-CSF as an adjunct to mastitis control is discussed. MATERIALS AND METHODS MAMMARY NEUTROPHIL ISOLATION
purity, and viability was more than 99olo as determined by trypan blue exclusion. IN VITRO TREATMENT OF NEUTROPH I LS
The rBoGM-CSF was synthesized in Escherichia coli by recombinant DNA technology and purified to homogeneity as determined by high-pressure liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described previously (9). The stock solution was supplied as a sterile solution at a concentration of 0.75 mg/mL by CIBA-GEIGY Ltd., Basel, Switzerland (lot number M3RD5 1). Aliquots of the isolated neutrophils were resuspended in antibiotic-free RPMI media containing Soo FBS supplemented with 0.005 yg, 0.05 jtg or 0.5 ug rBoGM-CSF to a final concentration of 1 x 107 cells/ mL. Control cells were resuspended in antibiotic-free RPMI media containing only 5ol FBS. In a preliminary trial designed to establish optimal in vitro conditions, the neutrophil suspensions were incubated for 3, 6, 9 and 12 h at 37°C. Following each incubation, the ability of neutrophils to release superoxide anion was determined. An aliquot of cells also was used to determine viability during the 12 h culture. In the following trial, isolated mammary gland and peripheral neutrophils were incubated for only 9 h before the cells were used in the functional assays including chemotaxis, bactericidal activity, and superoxide anion production.
Neutrophils were isolated from the mammary gland of three nonlactating dairy cows after intramammary injection of 5 Azg of lipopolysaccharide (LPS) in 5 mL of Hanks' balanced salt solution (HBSS). At 15 h after LPS administration, 30 mL of HBSS were injected into the LPS-treated quarter and mammany fluid was collected into a sterile polypropylene tube. Mammary gland neutrophils were filtered through a 30 !tm Nitex filter and purified by Ficoll-Hypaque density centrifugation. After two washings, the cells were resuspended in HBSS containing Soo fetal bovine serum (FBS). The percentage of viable cells was determined by trypan blue exclusion. Mammary neutrophils were cytocentrifuged onto poly-L-lysine coated slides and stained with Wright's CHEMOTAXIS ASSAY stain for differential cell counts. Chemotaxis was assayed in a modified Boyden chamber as previPERIPHERAL BLOOD NEUTROPHIL ously described (10). Neutrophils were ISOLATION pelleted following incubation in Venous blood samples were col- rBoGM-CSF and resuspended in lected in heparinized vacuum tubes HBSS at a concentration of 2 x from three calves (genetically identical 106 cells/mL. A stock solution of the triplets). Blood samples were cen- synthetic chemotactic peptide, FMLP, trifuged at 500 g for 35 min and the was diluted to 10- 8 M in HBSS. plasma and buffy coat layer were dis- Neutrophils were placed in the upper carded. Neutrophils were isolated from chambers and either HBSS alone the remaining red blood cell layer by (estimation of random migration) or adding 10 mL of water for 20 s fol- FMLP (estimation of directed migralowed by the addition of 10 mL of 2 x tion) was pipetted into the lower concentration of HBSS to restore the chambers. The apparatus was incubated isotonicity. The remaining cells were at 37°C for 45 min, followed by immewashed twice with HBSS. The cell diate fixation and staining with Diffisolation procedure yielded a neutro- Quick (Dade Diagnostics, Miami, phil population of more than 90o0o Florida). Migration was estimated by
conventional cell counts (1 1). A chemotactic index was calculated by dividing the distance travelled toward the chemoattractant by the random migration distance. SUPEROXIDE ANION PRODUCTION
Superoxide anion release by neutrophils was measured by the reduction of nitroblue tetrazolium (NBT). The quantitative NBT reduction assay was performed by adding 250 pL of neutrophils (5 x 106 cells/ mL) to 250 uL of preopsonized zymosan (10 mg/L) and 500 t4L of 0.2 mg/mL NBT. The reactants were incubated at 37°C for 30 min and the reaction was stopped with cold 0.1 N hydrochloric acid. The purple formazan formed by the reduced NBT was pelleted by centrifugation and resuspended in 1 mL dimethyl sulfoxide. The suspension was placed in an 80°C water bath for 1 h and the optical density was determined at 580 nm. Each test sample was assayed in triplicate and compared with a control receiving no zymosan activation. The results were expressed as the optical density per 1.25 x 105 neutrophils. BACTERICIDAL ASSAY
Bacterial killing by neutrophils was measured by colorimetric determination of formazan production as described previously (12). Briefly, blood and mammary gland neutrophil bactericidal activity were evaluated using a strain of Staphylococcus aureus isolated from clinical bovine mastitis. Bacteria in logarithmic phase growth were opsonized with antiS. aureus bovine sera (agglutination titer of 1/8). Opsonized S. aureus were incubated in microtiter plates with isolated cells at a ratio of ten bacteria per neutrophil. Following a 1 h incubation, neutrophils were lysed with saponin (0.2 mg/mL) and the tetrazolium compound, 3-[4,5-dimethylthiazol2yl]-2,5-diphenyltetrazolium bromide (MTT) was added to each well (100 /tg/ well). The ability of live S. aureus to reduce MTT to purple formazan was quantitated by measuring optical density at 560 nm. Percent killing of the S. aureus by neutrophils was determined from a standard formazan curve derived by adding MTT to known concentrations of bacteria. 17
STATISTICAL ANALYSES
Control
-
.005 pg/ml
.05 pg/mI
W
.5 pg/ml
0.50
Data were analyzed by least squares analyses of variance using the general linear model procedure. Statistical significance was determined by the Duncan's multiple range test and by pairwise t-test.
0.40
0.30
RESULTS
0 Results from the initial trial indicated that 9 h incubation with rBoGM-CSF resulted in optimal stimulation of peripheral blood neutrophil populations in vitro (Fig. 1). Treatment of neutrophils with 0.005 Ag/mL rBoGM-CSF significantly increased (p < 0.05) the reduction of NBT by reactive oxygen species after 9 h in culture when compared to control cells cultured with HBSS. Although the oxidative metabolism tended to be greater with those cells treated with the higher concentrations (0.05 and 0.5 ug/mL) of rBoGM-CSF for 9 h, there were no significant increases over the control cells. Viability counts remained unchanged for both the control and rBoGM-CSF treated neutrophils throughout the 12 h incubation period (data not shown). The effects of 9 h incubation with increasing doses of rBoGM-CSF on peripheral blood and mammary gland neutrophil function in vitro are summarized in Table I. Treatment with rBoGM-CSF increased the chemotactic and bactericidal capacity of neutrophils obtained from both blood and the mammary gland. The chemotactic index and bactericidal activity of treated neutrophils was enhanced in a dose-dependent manner with a significant (p < 0.05) increase over control neutrophils observed at the highest dose (0.5 gtg/mL). There was no significant effect of rBoGM-CSF on the production of superoxide anion by bovine neutrophils although values tended to be higher following incubation with this cytokine. Comparisons of blood and mammary gland neutrophil function following in vitro stimulation with rBoGM-CSF are summarized in Figs. 2-4. There were no significant differences between mammary gland and blood neutrophils with regard to chemotaxis (Fig. 2). Mammary gland neutrophils consistently produced more superoxide anion 18
0.20
0.10
0.00
6
3
9
12
INCUBATION PERIOD (hours) Fig. 1. Effects of rBoGM-CSF on the reduction of NBT by bovine neutrophils. Blood neutrophils were incubated with increasing doses of rBoGM-CSF over a 12 h period. The cytokine-treated neutrophils were incubated with 0.005, 0.05 or 0.5 Ag/mL of rBoGM-CSF. Control neutrophils were incubated with HBSS. Results are expressed as the mean optical density of formazan at 572 nm ± SEM. *Significantly different from control cells (p < 0.05).
TABLE I. Effects of rBoGM-CSF on bovine peripheral blood and mammary gland neutrophil function in vitro
Chemotactic Bactericidal Production index NBT activity 0.63b ± 0.21 76.72b ± 3.01 0 0.413a ± 0.03 1.lOab ± 0.30 0.498a + 0.04 76.23b ± 2.77 0.005 1.28ab ± 0.42 77.30b ± 2.54 0.05 0.503a + 0.05 1.92a ± 0.26 0.5 0.485a + 0.03 84.01a + 1.82 a,bMeans within each functional assay with different superscripts differ (p < 0.05) GM-CSF (ug/mL)
following zymosan stimulation when compared with neutrophils obtained from blood (Fig. 3). Reduction of NBT by mammary gland neutrophils was significantly (p < 0.05) greater than control cells at 0.5 Ag/mL rBoGM-CSF. Mammary gland neutrophils showed significantly more bactericidal capabilities than blood neutrophils at all rBoGM-CSF concentrations (Fig. 4). DISCUSSION
Bovine mastitis occurs when microorganisms gain entrance to the mammary gland via the teat canal, colonize the duct system and alveoli, and cause an inflammatory response (13). Once bacteria successfully penetrate the teat end opening, a number of humoral
and cellular factors act to provide specific and nonspecific immunity against invading pathogens (13). Mammary gland neutrophils are a major line of defense (7,13). Several studies showed that the effectiveness of neutrophils in preventing or eliminating intramammary infections depends on the promptness and magnitude of the migratory response and the subsequent bactericidal capabilities at the foci of infection (14,15). The ability to augment this important host defense mechanism through the therapeutic administration of GM-CSF may have a pronounced influence on the establishment of intramammary infections. Neutrophils accumulate in milk in response to bacterial infection and can constitute greater than 90070 of the total leukocyte population during inflammation (13). However, there is
2.00 6A
1.50
co 0
1.00
*a
1
_~ Mammary
Blood
2.50
0.50 0.00
0
.005
.5
.05
GM-CSF (ig/ml)
Fig. 2. Effects of increasing doses of rBoGM-CSF on the chemotactic capabilities of neutrophils obtained from blood and the mammary gland. The cytokine-treated neutrophils were incubated for 9 h with 0.005, 0.05 or 0.5 jLg/mL of rBoGM-CSF. Control neutrophils were incubated with HBSS. Results are expressed as the mean chemotaxis index of the cultured neutrophils ± SEM.
0.60 0.50
0.40
_~ Mammary
Blood
0.70
T
0.30
0.20
0.10
T
T
T
0.00 0
.005
.05
.5
GM-CSF (pg/ml) Fig. 3. Effects of increasing doses of rBoGM-CSF on the reduction of NBT by neutrophils obtained from blood and the mammary gland. The cytokine-treated neutrophils were incubated for 9 h with 0.005, 0.05 or 0.5 Ag/mL of rBoGM-CSF. Control neutrophils were incubated with HBSS. Results are expressed as the mean optical density of formazan at 572 nm ± SEM. *Significantly different from peripheral blood neutrophils (p < 0.05).
generally a 24 h time lapse between bacterial entry into the mammary gland and the appearance of sufficient numbers of neutrophils in the milk (7). This delay in neutrophil diapedesis into the mammary gland is associated with development of severe coliform mastitis (14). Results from the present
study showed that in vitro treatment of peripheral blood and mammary gland neutrophils with rBoGM-CSF can significantly increase chemotaxis towards FMLP. The effects of rBoGM-CSF on bovine neutrophils appears to be dosedependent with the greatest increases observed at the highest concentration
of the cytokine for both blood and mammary gland neutrophil populations. Since an early and rapid egression of neutrophils from the blood stream is cricital for the control of new intramammary infections, enhancement of the chemotactic capability of bovine neutrophils may increase the resistance of the mammary gland to invading pathogens. Mammary gland neutrophils function by phagocytozing and killing mastitis-causing pathogens (7,13). In this study, in vitro treatment of bovine neutrophils with rBoGM-CSF tended to increase the reduction of NBT by superoxide anion and significantly increased bactericidal capabilities against S. aureus compared with control cells. Neutrophils produce superoxide anion, hydrogen peroxide, and oxygen radicals during the burst of oxidative metabolism which takes place immediately after phagocytosis (16). This burst of oxidative metabolism is an important step leading to the bactericidal activity of phagocytes and is often used as an indirect measure of the functional capacity of neutrophils. The enhanced reduction of NBT by rBoGM-CSF treated neutrophils suggests that this cytokine is capable of regulating a critical component of the neutrophil's bactericidal capabilities following phagocytosis. Previous studies have shown that S. aureus can survive intracellularly after engulfment by mammary gland neutrophils, resulting in chronic infections upon subsequent release from degenerative cells (7,13,17). Results from this study demonstrated the capability of rBoGM-CSF to enhance the in vitro intracellular killing activity of both blood and mammary gland ne-utrophils against S. aureus. Other researchers have reported similar findings in which the phagocytic and bactericidal capabilities of blood neutrophils were augmented following treatment with rBoGM-CSF (18). These findings suggest that rBoGMCSF may be effective at augmenting boving neutrophil microbicidal functions in vivo and may facilitate the clearance of mastitis-causing pathogens from mammary glands during the initial stages of infection. It is well documented that mammary gland neutrophils have lower phagocytic and bactericidal capabilities than 19
100 -
4
90
*
REFERENCES
Mammary
Blood *
*_
8p" 80
70 60
50 0
.005
GM-CSF
.05
.5
(pg/ml)
Fig. 4. Effects of increasing doses of rBoGM-CSF on the bactericidal capabilities of neutrophils obtained from blood and the mammary gland. The cytokine-treated neutrophils were incubated for 9 h with 0.005, 0.05 or 0.5 Ag/mL of rBoGM-CSF. Control neutrophils were incubated with HBSS. Results are expressed as the mean percent killing of opsonized S. aureus by the cultured neutrophils ± SEM. *Significantly different from peripheral blood neutrophils (p < 0.05).
peripheral blood leukocytes due to the suppressive effects of mammary gland secretions (19). Milk constituents, such as fat and casein, interfere with the ability of neutrophils to phagocytose and kill mastitis-causing organisms (7,19). Diminished neutrophil bactericidal mechanisms were attributed to degranulation of the cells after indiscriminant ingestion of the milk components which deplete the supply of hydrolytic enzymes necessary for killing phagocytosed bacteria (7). In this study, comparisons were made between the functional capabilities of blood neutrophils and those stimulated into the mammary gland after LPS infusion following in vitro treatment with rBoGM-CSF. In contrast to neutrophils isolated from nonstimulated mammary glands (19), cells elicited into the mammary gland with a potent bacterial product had significantly higher bactericidal activity and generally produced more reactive oxygen species than blood neutrophils. Neutrophils pass through gradients of chemoattractants to reach sites of infection and it is hypothesized that cells at the foci of inflammation are in a highly active state because of initial exposure to chemotactic factors (20). It has been reported that both in vivo and in vitro 20
exposure of neutrophils to chemotactic peptides can enhance oxidative metabolism and amplify subsequent bactericidal activities in response to additional stimuli (20,21). These phenomena would explain the enhanced production of superoxide anion and bactericidal activity of LPS induced mammary gland neutrophils observed in the present study. Treatment of mammary gland neutrophils with rBoGM-CSF resulted in only moderate increases in both the reduction of NBT and bactericidal activity over controls, suggesting that there may be a threshold of immunomodulation for these cell populations. Results from this study correspond with others which demonstrate the direct action of rBoGM-CSF on mature bovine neutrophil populations. These findings suggest that rBoGMCSF may have some therapeutic benefits in the control of infectious bacterial diseases of cattle, such as mastitis. Administration of rBoGMCSF in vivo may enhance the recruitment of functionally competent neutrophils to the mammary gland during the early stages of mastitis and prevent the establishment of new intramammary infections in a nonspecific manner.
1. METCALF D. The granulocyte-macrophage colony-stimulating factors. Science 1985; 229: 16-22. 2. GASSON JC, WEISBART RH, KAUFMAN SE, CLARK SC, HEWICK RM, WONG GG, GOLDE DW. Purified human granulocyte-macrophage colony-stimulating factor: direct action on neutrophils. Science 1984; 226: 1339-1342. 3. LOPEZ AF, WILLIAMSON DJ, GAMBLE JR, BEGLEY CG, HARLAN JM, KLEBANOFF SJ, WALTERSDORPH A, WONG GG, CLARK SC, VADAS MA. Recombinant human granulocyte-macrophage colony-stimulating factor stimulates in vitro mature human neutrophil and eosinophil function, surface receptor expression and survival. J Clin Invest 1986; 78: 1220-1228. 4. WEISBART RH, GOLDE DW, CLARK SC, WONG GG, GASSON JC. Human granulocyte-macrophage colony-stimulating factor is a neutrophil activator. Nature 1985;
314: 361-363. 5. GRABSTEIN KH, URDAL DL, URDAL RJ, TUSHINSKI RH, MOCHIZUKI DY, PRICE VL, CANTRELL MA, GILLIS S, CONLON PJ. Induction of macrophage tumoricidal activity by granulocytemacrophage colony-stimulating factor. Science 1986; 236: 506-508. 6. HORIGUCHI J, WARREN MK, KUFE D. Expression of the macrophage specific colony-stimulating factor in human monocytes treated with granulocyte-macrophage colony-stimulating factor. Blood 1987; 69: 1259-1261. 7. PAAPE MJ, WERGIN WP, GUIDRY AJ, PEARSON RE. Phagocytic defense of the ruminant mammary gland. Adv Exp Med Biol 1981; 137: 555-567. 8. SORDILLO LM, NICKERSON SC. Morphologic changes in the bovine mammary gland during involution and lactogenesis. Am J Vet Res 1988; 49: 1112-1120. 9. LEONG SR, FLAGGS GM, LAWMAN MJP, GRAY PW. Cloning and expression of the cDNA for bovine granulocytemacrophage colony-stimulating factor. Vet Immunol Immunopathol 1989; 21: 261-278. 10. FALK W, GOODWIN RH, LEONARD EJ. A 48-well microchemotaxis assembly for rapid and accurate measurement of leukocyte migration. J Immunol Methods 1980; 33: 239-254. 11. ZIGMOND SH, HIRSCH JG. Leukocyte locomotion and chemotaxis: new methods for evaluation and demonstration of a cellderived chemotactic factor. J Exp Med 1973; 137: 387-410. 12. STEVENS MG, KEHRLI ME, CANNING PC. A colorimetric assay for quantitating bovine neutrophil bactericidal activity. Vet Immunol Immunopathol 1991; 28: 45-56. 13. NICKERSON SC. Immune mechanisms of the bovine udder: an overview. J Am Vet Med 1985; 187: 41-47. 14. HILL AW. Factors influencing the outcome of Escherichia coli mastitis in the dairy cow. Res Vet Sci 1981; 31: 107-112. 15. SCHALM OW, LASMANIS J, JAIN NC. Conversion of chronic staphylococcal mastitis to acute gangrenous mastitis after
neutropenia in blood and bone marrow produced by an equine anti-bovine leukocyte serum. Am J Vet Res 1976; 37: 885-890. 16. BADWEY JA, KARNOVSKY ML. Active oxygen species and functions of phagocytic leukocytes. Ann Rev Biochem 1980; 49: 695-726. 17. NEWBOLD FHS, NEAVE FK. The recovery of small numbers of Staphylococcus aureus infused into the bovine teat cistern. J Dairy Res 1965; 32: 157-163.
18. REDDY PG, McVEY DS, CHENGAPPA MM, BLECHA F, MINOCHA HC, BAKER PE. Bovine recombinant granulocytemacrophage colony-stimulating factor enhancement of bovine neutrophil functions in vitro. Am J Vet Res 1990; 51: 1395-1399. 19. WEBER K, PETERHANS E, WYLER R. The chemiluminescent response of bovine polymorphonuclear leukocytes isolated from milk and blood. Vet Immunol Immunopathol 1983; 4: 397-412.
20. ENGLISH D, LUKENS JN. Regulation of neutrophil inflammatory mediator release: chemotactic peptide activation of stimulusdependent cytotoxicity. J Immunol 1983; 130: 850-856. 21. ISSEKUTZ AC, LEE K, BIGGAR WD. Enhancement of human neutrophil bactericidal activity by chemotactic factors. Infect Immun 1979; 24: 295-299.
21
Modulation of peripheral blood and gland neutrophil function following in vitro exposure to recombinant bovine granulocyte-macrophage colony-stimulating factor (rBoGMCSF) was studied. Bovine blood and mammary gland neutrophils were cultured for 9 h in media containing 0.005, 0.05 or 0.5 ug/mL rBoGMCSF. Neutrophils treated with rBoGMCSF exhibited significantly more chemotactic and bactericidal activities and tended to produce more superoxide anion than control cells. The effects of rBoGM-CSF on bovine neutrophil populations appeared to be dose-dependent. The production of superoxide anion and the bactericidal activity of mammary gland neutrophils were consistently higher than blood neutrophils. Only moderate increases in lipopolysaccharide-induced mammary gland neutrophil functions were observed following incubation with rBoGM-CSF which suggests that there may be a threshold of immunomodulation for these prestimulated cells. It may be possible to augment the functional capacity of bovine neutrophil populations in vivo through the therapeutic application of rBoGM-CSF and consequently enhance resistance of dairy cattle to bacterial infections. mammary
RESUME Les variations de la fonction des neutrophiles dans le sang peripherique et dans la glande mammaire a la suite d'une exposition in vitro au facteur recombinant stimulant des colonies de granulocytes et de macrophages bovin
(recombinant bovine granulocyte- glycoproteins that bind to cells by a macrophage colony-stimulating factor common receptor and are produced by (rBoGM-CSF)) furent etudiees. a variety of cells including fibroblasts, Des neutrophiles provenant du sang endothelial cells, macrophages and peripherique et de la glande mammaire T lymphocytes (1). Granulocytede bovins furent cultives pendant macrophage colony-stimulating factor 9 heures dans un milieu contenant (GM-CSF) was first identified by its 0,005, 0,05 ou 0,5 Ag/mL de rBoGM- capacity to induce hematopoietic proCSF. Les neutrophiles traites avec le genitor cells to proliferate and difrBoGM-CSF demontrerent une plus ferentiate into granulocytes and grande activite bactericide et chemo- macrophages (1). Several recent studtactique et eurent tendance a produire ies in humans have shown that GMplus d'anions superoxide que les CSF is not only a hematopoietic cellules temoins. Les effets du rBoGM- growth-inducing molecule, but also CSF sur les populations de neutro- affects a variety of functions of mature philes bovins apparurent comme etant granulocytes and monocytes (2-6). relies 'a la dose. La production d'anions Human GM-CSF affects neutrophils superoxide et l'activite bactericide des directly by decreasing migratory activneutrophiles de la glande mammaire ity, and enhancing phagocytosis, etaient constamment plus elevees que antibody-dependent cellular cytotoxcelles des neutrophiles sanguins. Seule icity, and superoxide anion production une augmentation moderee dans les in response to n-formyl-methionylfonctions induites par les liposac- leucyl-phenylalanine (FMLP) (2-4). charides dans les neutrophiles mam- Monocytes respond to GM-CSF by maires fut observee a la suite de l'incu- induction of macrophage tumoricidal bation avec le rBoGM-CSF, suggerant activity and stimulation of mRNA la possibilite d'un seuil dans l'immu- expression and protein synthesis of nomodulation de ces cellules pre- M-CSF and G-CSF (5,6). The prostimulees. II peut etre possible d'aug- nounced influence of GM-CSF on menter la capacite fonctionnelle des phagocytic cell populations suggest populations de neutrophiles bovins possible clinical applications in the prein vivo par l'usage therapeutique du vention of infectious bacterial diseases rBoGM-CSF et ameliorer par conse- of livestock, such as bovine mastitis. quent la resistance des bovins laitiers The ability of mammary gland aux infections bacteriennes. (Traduit neutrophils to phagocytoze mastitispar Dr Anne Provencher) causing pathogens and suppress bacterial multiplication is critical to the outcome of intramammary infection (7). Although both neutrophils and INTRODUCTION macrophages predominate in mammary Colony stimulating factors are a gland secretions and tissues (8) evigroup of cytokines required for the dence suggests that the antibacterial proliferation and differentiation of a activities of these cells are reduced in variety of hematopoietic stem cells. the presence of mammary secretions These growth factors are distinct due to a lack of energy source, low
Veterinary Infectious Disease Organization, University of Saskatchewan, 124 Veterinary Road, Saskatoon, Saskatchewan S7N OWO. This work was supported by the Natural Sciences and Engineering Research Council of Canada and CIBA-GEIGY Canada Limited, Mississauga, Ontario. Published with the approval of the Director of VIDO as journal series no. 120. Submitted May 6, 1991.
16
Can J Vet Res 1992; 56: 16-21
concentrations of opsonins, and interference by milk constituents (7,8). To overcome the suppressive nature of mammary gland secretions, a relatively large proportion of functionally competent mammary gland neutrophils are required for effective protection against mastitis-causing pathogens (7). The purpose of this study was to evaluate the modulatory effects of rBoGM-CSF on the functional capabilities of bovine peripheral blood and mammary gland neutrophils in vitro. The possible clinical application of rBoGM-CSF as an adjunct to mastitis control is discussed. MATERIALS AND METHODS MAMMARY NEUTROPHIL ISOLATION
purity, and viability was more than 99olo as determined by trypan blue exclusion. IN VITRO TREATMENT OF NEUTROPH I LS
The rBoGM-CSF was synthesized in Escherichia coli by recombinant DNA technology and purified to homogeneity as determined by high-pressure liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described previously (9). The stock solution was supplied as a sterile solution at a concentration of 0.75 mg/mL by CIBA-GEIGY Ltd., Basel, Switzerland (lot number M3RD5 1). Aliquots of the isolated neutrophils were resuspended in antibiotic-free RPMI media containing Soo FBS supplemented with 0.005 yg, 0.05 jtg or 0.5 ug rBoGM-CSF to a final concentration of 1 x 107 cells/ mL. Control cells were resuspended in antibiotic-free RPMI media containing only 5ol FBS. In a preliminary trial designed to establish optimal in vitro conditions, the neutrophil suspensions were incubated for 3, 6, 9 and 12 h at 37°C. Following each incubation, the ability of neutrophils to release superoxide anion was determined. An aliquot of cells also was used to determine viability during the 12 h culture. In the following trial, isolated mammary gland and peripheral neutrophils were incubated for only 9 h before the cells were used in the functional assays including chemotaxis, bactericidal activity, and superoxide anion production.
Neutrophils were isolated from the mammary gland of three nonlactating dairy cows after intramammary injection of 5 Azg of lipopolysaccharide (LPS) in 5 mL of Hanks' balanced salt solution (HBSS). At 15 h after LPS administration, 30 mL of HBSS were injected into the LPS-treated quarter and mammany fluid was collected into a sterile polypropylene tube. Mammary gland neutrophils were filtered through a 30 !tm Nitex filter and purified by Ficoll-Hypaque density centrifugation. After two washings, the cells were resuspended in HBSS containing Soo fetal bovine serum (FBS). The percentage of viable cells was determined by trypan blue exclusion. Mammary neutrophils were cytocentrifuged onto poly-L-lysine coated slides and stained with Wright's CHEMOTAXIS ASSAY stain for differential cell counts. Chemotaxis was assayed in a modified Boyden chamber as previPERIPHERAL BLOOD NEUTROPHIL ously described (10). Neutrophils were ISOLATION pelleted following incubation in Venous blood samples were col- rBoGM-CSF and resuspended in lected in heparinized vacuum tubes HBSS at a concentration of 2 x from three calves (genetically identical 106 cells/mL. A stock solution of the triplets). Blood samples were cen- synthetic chemotactic peptide, FMLP, trifuged at 500 g for 35 min and the was diluted to 10- 8 M in HBSS. plasma and buffy coat layer were dis- Neutrophils were placed in the upper carded. Neutrophils were isolated from chambers and either HBSS alone the remaining red blood cell layer by (estimation of random migration) or adding 10 mL of water for 20 s fol- FMLP (estimation of directed migralowed by the addition of 10 mL of 2 x tion) was pipetted into the lower concentration of HBSS to restore the chambers. The apparatus was incubated isotonicity. The remaining cells were at 37°C for 45 min, followed by immewashed twice with HBSS. The cell diate fixation and staining with Diffisolation procedure yielded a neutro- Quick (Dade Diagnostics, Miami, phil population of more than 90o0o Florida). Migration was estimated by
conventional cell counts (1 1). A chemotactic index was calculated by dividing the distance travelled toward the chemoattractant by the random migration distance. SUPEROXIDE ANION PRODUCTION
Superoxide anion release by neutrophils was measured by the reduction of nitroblue tetrazolium (NBT). The quantitative NBT reduction assay was performed by adding 250 pL of neutrophils (5 x 106 cells/ mL) to 250 uL of preopsonized zymosan (10 mg/L) and 500 t4L of 0.2 mg/mL NBT. The reactants were incubated at 37°C for 30 min and the reaction was stopped with cold 0.1 N hydrochloric acid. The purple formazan formed by the reduced NBT was pelleted by centrifugation and resuspended in 1 mL dimethyl sulfoxide. The suspension was placed in an 80°C water bath for 1 h and the optical density was determined at 580 nm. Each test sample was assayed in triplicate and compared with a control receiving no zymosan activation. The results were expressed as the optical density per 1.25 x 105 neutrophils. BACTERICIDAL ASSAY
Bacterial killing by neutrophils was measured by colorimetric determination of formazan production as described previously (12). Briefly, blood and mammary gland neutrophil bactericidal activity were evaluated using a strain of Staphylococcus aureus isolated from clinical bovine mastitis. Bacteria in logarithmic phase growth were opsonized with antiS. aureus bovine sera (agglutination titer of 1/8). Opsonized S. aureus were incubated in microtiter plates with isolated cells at a ratio of ten bacteria per neutrophil. Following a 1 h incubation, neutrophils were lysed with saponin (0.2 mg/mL) and the tetrazolium compound, 3-[4,5-dimethylthiazol2yl]-2,5-diphenyltetrazolium bromide (MTT) was added to each well (100 /tg/ well). The ability of live S. aureus to reduce MTT to purple formazan was quantitated by measuring optical density at 560 nm. Percent killing of the S. aureus by neutrophils was determined from a standard formazan curve derived by adding MTT to known concentrations of bacteria. 17
STATISTICAL ANALYSES
Control
-
.005 pg/ml
.05 pg/mI
W
.5 pg/ml
0.50
Data were analyzed by least squares analyses of variance using the general linear model procedure. Statistical significance was determined by the Duncan's multiple range test and by pairwise t-test.
0.40
0.30
RESULTS
0 Results from the initial trial indicated that 9 h incubation with rBoGM-CSF resulted in optimal stimulation of peripheral blood neutrophil populations in vitro (Fig. 1). Treatment of neutrophils with 0.005 Ag/mL rBoGM-CSF significantly increased (p < 0.05) the reduction of NBT by reactive oxygen species after 9 h in culture when compared to control cells cultured with HBSS. Although the oxidative metabolism tended to be greater with those cells treated with the higher concentrations (0.05 and 0.5 ug/mL) of rBoGM-CSF for 9 h, there were no significant increases over the control cells. Viability counts remained unchanged for both the control and rBoGM-CSF treated neutrophils throughout the 12 h incubation period (data not shown). The effects of 9 h incubation with increasing doses of rBoGM-CSF on peripheral blood and mammary gland neutrophil function in vitro are summarized in Table I. Treatment with rBoGM-CSF increased the chemotactic and bactericidal capacity of neutrophils obtained from both blood and the mammary gland. The chemotactic index and bactericidal activity of treated neutrophils was enhanced in a dose-dependent manner with a significant (p < 0.05) increase over control neutrophils observed at the highest dose (0.5 gtg/mL). There was no significant effect of rBoGM-CSF on the production of superoxide anion by bovine neutrophils although values tended to be higher following incubation with this cytokine. Comparisons of blood and mammary gland neutrophil function following in vitro stimulation with rBoGM-CSF are summarized in Figs. 2-4. There were no significant differences between mammary gland and blood neutrophils with regard to chemotaxis (Fig. 2). Mammary gland neutrophils consistently produced more superoxide anion 18
0.20
0.10
0.00
6
3
9
12
INCUBATION PERIOD (hours) Fig. 1. Effects of rBoGM-CSF on the reduction of NBT by bovine neutrophils. Blood neutrophils were incubated with increasing doses of rBoGM-CSF over a 12 h period. The cytokine-treated neutrophils were incubated with 0.005, 0.05 or 0.5 Ag/mL of rBoGM-CSF. Control neutrophils were incubated with HBSS. Results are expressed as the mean optical density of formazan at 572 nm ± SEM. *Significantly different from control cells (p < 0.05).
TABLE I. Effects of rBoGM-CSF on bovine peripheral blood and mammary gland neutrophil function in vitro
Chemotactic Bactericidal Production index NBT activity 0.63b ± 0.21 76.72b ± 3.01 0 0.413a ± 0.03 1.lOab ± 0.30 0.498a + 0.04 76.23b ± 2.77 0.005 1.28ab ± 0.42 77.30b ± 2.54 0.05 0.503a + 0.05 1.92a ± 0.26 0.5 0.485a + 0.03 84.01a + 1.82 a,bMeans within each functional assay with different superscripts differ (p < 0.05) GM-CSF (ug/mL)
following zymosan stimulation when compared with neutrophils obtained from blood (Fig. 3). Reduction of NBT by mammary gland neutrophils was significantly (p < 0.05) greater than control cells at 0.5 Ag/mL rBoGM-CSF. Mammary gland neutrophils showed significantly more bactericidal capabilities than blood neutrophils at all rBoGM-CSF concentrations (Fig. 4). DISCUSSION
Bovine mastitis occurs when microorganisms gain entrance to the mammary gland via the teat canal, colonize the duct system and alveoli, and cause an inflammatory response (13). Once bacteria successfully penetrate the teat end opening, a number of humoral
and cellular factors act to provide specific and nonspecific immunity against invading pathogens (13). Mammary gland neutrophils are a major line of defense (7,13). Several studies showed that the effectiveness of neutrophils in preventing or eliminating intramammary infections depends on the promptness and magnitude of the migratory response and the subsequent bactericidal capabilities at the foci of infection (14,15). The ability to augment this important host defense mechanism through the therapeutic administration of GM-CSF may have a pronounced influence on the establishment of intramammary infections. Neutrophils accumulate in milk in response to bacterial infection and can constitute greater than 90070 of the total leukocyte population during inflammation (13). However, there is
2.00 6A
1.50
co 0
1.00
*a
1
_~ Mammary
Blood
2.50
0.50 0.00
0
.005
.5
.05
GM-CSF (ig/ml)
Fig. 2. Effects of increasing doses of rBoGM-CSF on the chemotactic capabilities of neutrophils obtained from blood and the mammary gland. The cytokine-treated neutrophils were incubated for 9 h with 0.005, 0.05 or 0.5 jLg/mL of rBoGM-CSF. Control neutrophils were incubated with HBSS. Results are expressed as the mean chemotaxis index of the cultured neutrophils ± SEM.
0.60 0.50
0.40
_~ Mammary
Blood
0.70
T
0.30
0.20
0.10
T
T
T
0.00 0
.005
.05
.5
GM-CSF (pg/ml) Fig. 3. Effects of increasing doses of rBoGM-CSF on the reduction of NBT by neutrophils obtained from blood and the mammary gland. The cytokine-treated neutrophils were incubated for 9 h with 0.005, 0.05 or 0.5 Ag/mL of rBoGM-CSF. Control neutrophils were incubated with HBSS. Results are expressed as the mean optical density of formazan at 572 nm ± SEM. *Significantly different from peripheral blood neutrophils (p < 0.05).
generally a 24 h time lapse between bacterial entry into the mammary gland and the appearance of sufficient numbers of neutrophils in the milk (7). This delay in neutrophil diapedesis into the mammary gland is associated with development of severe coliform mastitis (14). Results from the present
study showed that in vitro treatment of peripheral blood and mammary gland neutrophils with rBoGM-CSF can significantly increase chemotaxis towards FMLP. The effects of rBoGM-CSF on bovine neutrophils appears to be dosedependent with the greatest increases observed at the highest concentration
of the cytokine for both blood and mammary gland neutrophil populations. Since an early and rapid egression of neutrophils from the blood stream is cricital for the control of new intramammary infections, enhancement of the chemotactic capability of bovine neutrophils may increase the resistance of the mammary gland to invading pathogens. Mammary gland neutrophils function by phagocytozing and killing mastitis-causing pathogens (7,13). In this study, in vitro treatment of bovine neutrophils with rBoGM-CSF tended to increase the reduction of NBT by superoxide anion and significantly increased bactericidal capabilities against S. aureus compared with control cells. Neutrophils produce superoxide anion, hydrogen peroxide, and oxygen radicals during the burst of oxidative metabolism which takes place immediately after phagocytosis (16). This burst of oxidative metabolism is an important step leading to the bactericidal activity of phagocytes and is often used as an indirect measure of the functional capacity of neutrophils. The enhanced reduction of NBT by rBoGM-CSF treated neutrophils suggests that this cytokine is capable of regulating a critical component of the neutrophil's bactericidal capabilities following phagocytosis. Previous studies have shown that S. aureus can survive intracellularly after engulfment by mammary gland neutrophils, resulting in chronic infections upon subsequent release from degenerative cells (7,13,17). Results from this study demonstrated the capability of rBoGM-CSF to enhance the in vitro intracellular killing activity of both blood and mammary gland ne-utrophils against S. aureus. Other researchers have reported similar findings in which the phagocytic and bactericidal capabilities of blood neutrophils were augmented following treatment with rBoGM-CSF (18). These findings suggest that rBoGMCSF may be effective at augmenting boving neutrophil microbicidal functions in vivo and may facilitate the clearance of mastitis-causing pathogens from mammary glands during the initial stages of infection. It is well documented that mammary gland neutrophils have lower phagocytic and bactericidal capabilities than 19
100 -
4
90
*
REFERENCES
Mammary
Blood *
*_
8p" 80
70 60
50 0
.005
GM-CSF
.05
.5
(pg/ml)
Fig. 4. Effects of increasing doses of rBoGM-CSF on the bactericidal capabilities of neutrophils obtained from blood and the mammary gland. The cytokine-treated neutrophils were incubated for 9 h with 0.005, 0.05 or 0.5 Ag/mL of rBoGM-CSF. Control neutrophils were incubated with HBSS. Results are expressed as the mean percent killing of opsonized S. aureus by the cultured neutrophils ± SEM. *Significantly different from peripheral blood neutrophils (p < 0.05).
peripheral blood leukocytes due to the suppressive effects of mammary gland secretions (19). Milk constituents, such as fat and casein, interfere with the ability of neutrophils to phagocytose and kill mastitis-causing organisms (7,19). Diminished neutrophil bactericidal mechanisms were attributed to degranulation of the cells after indiscriminant ingestion of the milk components which deplete the supply of hydrolytic enzymes necessary for killing phagocytosed bacteria (7). In this study, comparisons were made between the functional capabilities of blood neutrophils and those stimulated into the mammary gland after LPS infusion following in vitro treatment with rBoGM-CSF. In contrast to neutrophils isolated from nonstimulated mammary glands (19), cells elicited into the mammary gland with a potent bacterial product had significantly higher bactericidal activity and generally produced more reactive oxygen species than blood neutrophils. Neutrophils pass through gradients of chemoattractants to reach sites of infection and it is hypothesized that cells at the foci of inflammation are in a highly active state because of initial exposure to chemotactic factors (20). It has been reported that both in vivo and in vitro 20
exposure of neutrophils to chemotactic peptides can enhance oxidative metabolism and amplify subsequent bactericidal activities in response to additional stimuli (20,21). These phenomena would explain the enhanced production of superoxide anion and bactericidal activity of LPS induced mammary gland neutrophils observed in the present study. Treatment of mammary gland neutrophils with rBoGM-CSF resulted in only moderate increases in both the reduction of NBT and bactericidal activity over controls, suggesting that there may be a threshold of immunomodulation for these cell populations. Results from this study correspond with others which demonstrate the direct action of rBoGM-CSF on mature bovine neutrophil populations. These findings suggest that rBoGMCSF may have some therapeutic benefits in the control of infectious bacterial diseases of cattle, such as mastitis. Administration of rBoGMCSF in vivo may enhance the recruitment of functionally competent neutrophils to the mammary gland during the early stages of mastitis and prevent the establishment of new intramammary infections in a nonspecific manner.
1. METCALF D. The granulocyte-macrophage colony-stimulating factors. Science 1985; 229: 16-22. 2. GASSON JC, WEISBART RH, KAUFMAN SE, CLARK SC, HEWICK RM, WONG GG, GOLDE DW. Purified human granulocyte-macrophage colony-stimulating factor: direct action on neutrophils. Science 1984; 226: 1339-1342. 3. LOPEZ AF, WILLIAMSON DJ, GAMBLE JR, BEGLEY CG, HARLAN JM, KLEBANOFF SJ, WALTERSDORPH A, WONG GG, CLARK SC, VADAS MA. Recombinant human granulocyte-macrophage colony-stimulating factor stimulates in vitro mature human neutrophil and eosinophil function, surface receptor expression and survival. J Clin Invest 1986; 78: 1220-1228. 4. WEISBART RH, GOLDE DW, CLARK SC, WONG GG, GASSON JC. Human granulocyte-macrophage colony-stimulating factor is a neutrophil activator. Nature 1985;
314: 361-363. 5. GRABSTEIN KH, URDAL DL, URDAL RJ, TUSHINSKI RH, MOCHIZUKI DY, PRICE VL, CANTRELL MA, GILLIS S, CONLON PJ. Induction of macrophage tumoricidal activity by granulocytemacrophage colony-stimulating factor. Science 1986; 236: 506-508. 6. HORIGUCHI J, WARREN MK, KUFE D. Expression of the macrophage specific colony-stimulating factor in human monocytes treated with granulocyte-macrophage colony-stimulating factor. Blood 1987; 69: 1259-1261. 7. PAAPE MJ, WERGIN WP, GUIDRY AJ, PEARSON RE. Phagocytic defense of the ruminant mammary gland. Adv Exp Med Biol 1981; 137: 555-567. 8. SORDILLO LM, NICKERSON SC. Morphologic changes in the bovine mammary gland during involution and lactogenesis. Am J Vet Res 1988; 49: 1112-1120. 9. LEONG SR, FLAGGS GM, LAWMAN MJP, GRAY PW. Cloning and expression of the cDNA for bovine granulocytemacrophage colony-stimulating factor. Vet Immunol Immunopathol 1989; 21: 261-278. 10. FALK W, GOODWIN RH, LEONARD EJ. A 48-well microchemotaxis assembly for rapid and accurate measurement of leukocyte migration. J Immunol Methods 1980; 33: 239-254. 11. ZIGMOND SH, HIRSCH JG. Leukocyte locomotion and chemotaxis: new methods for evaluation and demonstration of a cellderived chemotactic factor. J Exp Med 1973; 137: 387-410. 12. STEVENS MG, KEHRLI ME, CANNING PC. A colorimetric assay for quantitating bovine neutrophil bactericidal activity. Vet Immunol Immunopathol 1991; 28: 45-56. 13. NICKERSON SC. Immune mechanisms of the bovine udder: an overview. J Am Vet Med 1985; 187: 41-47. 14. HILL AW. Factors influencing the outcome of Escherichia coli mastitis in the dairy cow. Res Vet Sci 1981; 31: 107-112. 15. SCHALM OW, LASMANIS J, JAIN NC. Conversion of chronic staphylococcal mastitis to acute gangrenous mastitis after
neutropenia in blood and bone marrow produced by an equine anti-bovine leukocyte serum. Am J Vet Res 1976; 37: 885-890. 16. BADWEY JA, KARNOVSKY ML. Active oxygen species and functions of phagocytic leukocytes. Ann Rev Biochem 1980; 49: 695-726. 17. NEWBOLD FHS, NEAVE FK. The recovery of small numbers of Staphylococcus aureus infused into the bovine teat cistern. J Dairy Res 1965; 32: 157-163.
18. REDDY PG, McVEY DS, CHENGAPPA MM, BLECHA F, MINOCHA HC, BAKER PE. Bovine recombinant granulocytemacrophage colony-stimulating factor enhancement of bovine neutrophil functions in vitro. Am J Vet Res 1990; 51: 1395-1399. 19. WEBER K, PETERHANS E, WYLER R. The chemiluminescent response of bovine polymorphonuclear leukocytes isolated from milk and blood. Vet Immunol Immunopathol 1983; 4: 397-412.
20. ENGLISH D, LUKENS JN. Regulation of neutrophil inflammatory mediator release: chemotactic peptide activation of stimulusdependent cytotoxicity. J Immunol 1983; 130: 850-856. 21. ISSEKUTZ AC, LEE K, BIGGAR WD. Enhancement of human neutrophil bactericidal activity by chemotactic factors. Infect Immun 1979; 24: 295-299.
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