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Oxygen radicals and nitric oxide production by turkey respiratory macrophages
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Pergamon
Developmental and Comparative Immunology\ Vol[ 11\ No[ 3\ pp[ 396Ð305\ 0887 Þ 0887 Elsevier Science Ltd[ All rights reserved Printed in Great Britain 9034Ð294X:87 ,08[99¦9[99
PII] S9034Ð294X"87#99908Ð5
OXYGEN RADICALS AND NITRIC OXIDE PRODUCTION BY TURKEY RESPIRATORY MACROPHAGES Anne E. F. Van Nerom, Richard Ducatelle and Freddy Haesebrouck Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, University of Ghent, Ghent Belgium "Received September 0886^ Accepted January 0887#
Abstract*The in~uence of different induction protocols on the recovery of elicited turkey res! piratory macrophages "RM#\ and on their oxy! genation activity and nitric oxide "NO# production was examined[ RM were induced in three week old speci_c pathogen free turkeys with Sephadex G!49\ Thioglycollate broth\ and an emulsion of incomplete Freund|s adjuvant "IFA#\ supplemented either with Mycoplasma hyorhinis grown in Modi_ed Channock broth "IFA!M[ hyorhinis# or with Modi_ed Channock broth "IFA!Broth#[ The RM were recovered by lavage of the lungs and air sacs and were puri_ed by centrifugation through a Percoll suspension[ Their oxygenation activity was evaluated in lum! inol!enhanced chemiluminescence assays\ fol! lowing stimulation with Zymosan A[ The NO production was evaluated by incubating the RM with lipopolysaccharide "LPS# from Salmonella enteritidis for 13 or 37 hours[ The number of recovered RM was slightly\ but not signi_cantly lower for Sephadex G!49 and IFA!Broth than for Thioglycollate broth and IFA!M[ hyorhinis[ RM elicited with Sephadex G!49 and IFA!Broth showed a signi_cantly higher oxidative burst response to Zymosan A\ compared to the Thi! oglycollate and IFA!M[ hyorhinis elicited RM[ Although all elicited RM showed a high NO pro!
Address correspondence to Anne Van Nerom\ Department of Pathology\ Bacteriology and Avian Diseases\ Faculty of Veterinary Medi! cine\ Salisburylaan\ 022\ B!8719 Merelbeke\ Belgium[ Tel] 99 21 "9# 8 153 63 20^ Fax] 99 21 "9# 8 153 63 83^ E!mail] anne[vanner! omÝrug[ac[be
duction upon stimulation with LPS\ no signi_cant differences were seen in the NO production of the RM obtained following the different induction treatments[ Our results point out that care should be taken when applying elicited RM for in vitro assays\ as distinct levels of oxygenation activity were obtained using different induction protocols[ Þ 0887 Elsevier Science Ltd[ All rights reserved Keywords*Respiratory burst^ NO^ Res! piratory macrophages^ Turkeys^ Chemi! luminescence[
Nomenclature CL CLP cpm DMEM HBSS IFA LPS NO RM SPF
chemiluminescence^ chemiluminigenic probe^ counts per minute^ Dulbecco|s Modi_ed Eagle|s Medium^ Hank|s Balanced Salt Solution^ incomplete Freund|s adjuvant^ lipopolysaccharide^ nitric oxide^ respiratory macrophages^ speci_c pathogen free[
Introduction Monocytes and macrophages exert a key role in speci_c and non!speci_c host defence\ including in the respiratory tract "0#\ which represents a primary portal of entry for a large number of pathogens[ In contrast to the respiratory tract of mammals\ where numerous resident pul!
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monary macrophages exert the important function of non!speci_c _rst!line defence\ only very low numbers of resident res! piratory macrophages are retrievable in avian species "1\ 2#[ In order to enable research on local tis! sue macrophages in avian species\ di}erent investigators have applied induction pro! tocols for the attraction of macrophages into the respiratory tract "2# and peritoneal space "3\ 4#[ The necessity of inducing mac! rophages is due to the very scarce amount of resident macrophages at those sites in birds[ The underlying assumption is that those elicited macrophages resemble and represent a similar cell population as would be attracted under infectious conditions[ Any injected substance\ however\ evokes a local in~ammatory response\ involving the attraction of exudate cells and the release of cytokines[ The characteristics of this in~ammatory response might therefore in~uence the properties of the induced exudate cells\ in particular the macro! phages[ A major function of macrophages is the respiratory burst\ where toxic oxygen products\ such as superoxide\ hydrogen peroxide\ singlet oxygen and hydroxyl rad! icals are released\ resulting in oxidative microbicidal and cytocidal properties "5Ð 7#[ This respiratory burst is triggered by the NADPH oxidase system\ which is acti! vated following the perturbation of the plasma membrane during phagocytosis or following the interaction between the cell surface and a variety of agents "5#[ Inter! action of the generated oxygenating agents with certain substrates\ results in the for! mation of electronically excited oxy! genation products[ As these products return to their ground state\ photon emis! sion is responsible for the phenomenon of chemiluminescence "CL#[ This CL can be ampli_ed by addition of luminescent sub! strates such as luminol and lucigenin "6Ð 09#\ which provides a sensitive tool for examining the oxidative activity of phago! cytic leukocytes[ Recently we dem! onstrated respiratory burst!dependent
A. E. F. Van Nerom et al.
activity in turkey monocytes towards di}erent soluble and particulate stimuli "00#[ Another important defence mechanism of mononuclear phagocytes constitutes the L!arginine dependent production of nitric oxide "NO# "01#[ Recent investigations demonstrated that activated turkey bone marrow mononuclear cells "02# and turkey monocytes "unpublished results# were cap! able of producing NO\ upon stimulation with lipopolysaccharide "LPS#[ As nitric oxide is a major e}ector molecule for the microbicidal and cytotoxic response of the rodent macrophages "04Ð06#\ these capacities probably also play a major role in the host defence of turkeys[ Both the respiratory burst!dependent and nitric oxide!dependent activity of mononuclear phagocytes can be modi_ed by cytokines "0\ 07\ 08#[ Thus\ the proper! ties of elicited respiratory macrophages might also be modi_ed by cytokines in vivo\ depending on the induction protocol applied[ Therefore\ the aim of the present study was to investigate whether di}erent pro! tocols\ used for the induction of turkey RM\ modi_ed the respiratory burst!depen! dent and nitric oxide!dependent activity of these macrophages in vitro[ The res! piratory burst!dependent activity of the elicited RM was evaluated by measuring their oxidative response upon stimulation with Zymosan A in a luminol!enhanced CL assay[ The production of NO was assessed following stimulation with LPS of Salmonella enteritidis[ Both Zymosan A and LPS of Salmonella enteritidis have been demonstrated to be a strong stimulus for the respiratory burst "00# and the NO production "unpublished results# in turkey monocytes\ respectively[
Material and Methods Poults Speci_c pathogen free "SPF# turkey eggs "obtained from the Centre National d|
Oxidative and NO activity of turkey respiratory macrophages
Etude Veterinaire et Alimentaire\ CNEVA\ Ploufragan\ France# were hat! ched in isolation[ The poults were reared in isolation units[ Food and water were available ad libitum[
Induction of Respiratory Macropha`es "RM# At three weeks of age the turkeys were divided into 3 groups of 7 to 09 birds and transabdominaly injected into each abdominal air sac with one millilitre of one of the substances given below[ , In group 0\ Sephadex G!49 was used to induce RM[ A 2) "w:v# suspension of Sephadex G!49 "Pharmacia\ Uppsala\ Sweden# was prepared as described by Sabet et al[ "3#\ with the exception that phosphate bu}ered saline was replaced by Hank|s Balanced Salt Solution "HBSS^ Gibco\ Merelbeke\ Belgium#[ The lavage of the lungs and air sacs was carried out 2 days following the injec! tion[ , Turkeys of group 1 received 09) Thi! oglycollate "Thioglycollate medium USP\ Conforma\ Destelbergen\ Belgium# in endotoxin!free water for perfusion "PO25\ Conforma\ Destel! bergen\ Belgium#[ They were lavaged 3 days post injection "p[i[#[ , The turkeys of group 2 were injected with an emulsion of incomplete Freund|s adjuvant "IFA#\ supplemented with Mycoplasma hyorhinis suspended in Modi_ed Channock broth[ This IFA!M[ hyorhinis emulsion was prepared as follows[ A cell culture adapted strain of Mycoplasma hyorhinis was grown in Modi_ed Channock Medium without thallium acetate "J[ M[ Bradbury\ per! sonal communication#[ This strain was broth to broth subcultured three times and subsequently stored in aliquots at −69>C[ A viability count was carried out on one vial of this stock\ giving 0[46×096 CFU:ml[ For the inoculum
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preparation\ another vial was thawed and diluted in a 0:1 "v:v# emulsion of incomplete Freund|s adjuvant "Difco\ Detroit\ USA# in sterile HBSS\ to pro! vide inocula containing approximately 0×095 CFU:ml[ The lavage of the lungs and air sacs was carried out 6 days fol! lowing the injection[ , The turkeys of group 3 received an injec! tion of IFA emulsion supplemented with Modi_ed Channock broth "IFA!broth#[ A 0:1 "v:v# emulsion of incomplete Freund|s adjuvant in sterile HBSS was prepared with 5[24) "v:v# Modi_ed Channock Medium without thallium acetate\ i[e[ corresponding to the amount administered to the IFA!M[ hyorhinis inoculated turkeys[ The lungs and air sacs of these turkeys were also lavaged 6 days p[i[
Collection of Respiratory Macropha`es Respiratory macrophages were collected according to the procedure of Ficken et al[ "2#\ with some modi_cations[ Turkeys were euthanized and the trachea was exposed by a ventral midline skin incision[ A needle connected to a syringe was inserted into the trachea\ which was clasped[ Sixty millilitre lavage ~uid\ consisting of HBSS without calcium and magnesium "HBSS w:o Ca:Mg^ Gibco\ Merelbeke\ Belgium#\ sup! plemented with 099 mg:ml streptomycin\ 099 mg:ml vancomycin and 14 U:ml hep! arin "Leo pharmaceutical products\ Vil! voorde\ Belgium# was injected into the lungs and air sacs[ The lavage ~uid was withdrawn by gravity ~ow while the abdo! men and thorax were gently massaged[ The collected ~uid was centrifuged for 09 min at 0929 g\ 3>C[ The obtained pellet was resuspended in 2 ml HBSS w:o Ca:Mg\ supplemented with 099 mg:ml streptomycin and 099 mg:ml vancomycin\ and laid upon 2 ml Percoll suspension "density 0[968^ 299 mOsmol:kg H1O#\ composed of 7[7) "v:v# 09×concentrated Hanks| Balanced
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Salt Solution "09×HBSS^ Gibco\ Merel! beke\ Belgium# and 45[7) "v:v# Percoll stock suspension "Pharmacia Biotech\ Roosendael\ the Netherlands# in a 01[4 ml conical centrifuge tube[ Following cen! trifugation at 0629 g for 29 minutes at 3>C\ the cells on top of the Percoll solution were collected and washed once with sterile HBSS w:o Ca:Mg[ The cells were resus! pended at a concentration of 0×095 cells:ml\ in leukocyte medium\ com! posed of Dulbecco|s Modi_ed Eagle|s Medium "DMEM^ endotoxin level] 9[94 ng:ml^ Gibco\ Merelbeke\ Belgium#\ supplemented with 099 mg:ml strep! tomycin\ 099 mg:ml vancomycin\ 0) sodium pyruvate "Gibco\ Merelbeke\ Belgium#\ 0) L!glutamine "Gibco\ Mer! elbeke\ Belgium# and 09) low endotoxin myoclone super plus bovine serum "endo! toxin level ³0 ng:ml^ Gibco\ Merelbeke\ Belgium#[ The purity of the macrophage suspensions was assessed by microscopic examination of Haemacolorþ "Merck\ Darmstadt\ Germany# stained smears[ The viability was determined by exclusion of trypan blue[ Cells were checked for per! oxidase activity by staining _xed smears with DAB "2\2?!diaminobenzidine tetra! chloride tablets\ Sigma Biosciences\ St[ Louis\ USA#\ prepared as recommended by the manufacturer\ and hematoxylin[
Tri``erin` A`ent for Chemiluminescence Zymosan A was applied as a triggering agent in the chemiluminescence assays[ A suspension of 9[14 g) Zymosan A in HBSS was heated for 29 minutes in a water bath at 89>C[ After cooling\ the suspension was aliquoted and stored at −69>C[ The _nal assay concentration of Zymosan A was 8[5 mg)[
A. E. F. Van Nerom et al.
piratory macrophages\ luminol was applied as chemiluminigenic probe "CLP# "Sigma Chemical Co[\ St[ Louis\ USA#[ Luminol was dissolved in DMSO to give a concentration of 093 mM\ and was stored in small aliquots at −69>C[ Prior to use\ the luminol stock solution was diluted in HBSS\ and 099 ml was added to the cuvet! tes[ Final assay concentration of luminol was 199 mM[
Chemiluminescence Assay The CL assays were performed in poly! styrene biolum cuvettes "Lumac\ Landg! raaf\ the Netherlands# at 26>C\ using a six! channel luminometer "Lumicon\ Hamil! ton\ Bonaduz\ Switzerland#[ The cuvettes were not stirred during the assay[ Mea! suring of the CL reactions of isolated res! piratory macrophages "RM# was performed as follows[ The cuvettes were supplied with 499 ml RM suspension "4×094 RM# and incubated in 4) CO1 at 26>C for one hour\ to allow adherence of the cells[ After incubation\ the supernatant was replaced by 399 ml HBSS and 099 ml luminol solution[ The cuvettes were placed in the luminometer and the spontaneous CL "cells without stimulating agent# was recorded for at least 09 min[ The CL was measured as counts per minute "cpm#[ Each cuvette was read for 09 seconds per minute[ After stabilisation of the spon! taneous CL\ the reaction was started by the addition of 19 ml Zymosan A per cuvette[ The oxidative activity of the RM was recorded for one hour[ Data were cor! rected for spontaneous CL[ The RM of all turkeys were tested for CL and a minimum of two measurements were done per turkey[
Luminescent Substrate
Stimulation of the NO Production in Turkey RM
For the evaluation of the chemi! luminescence response of turkey res!
The presence of NO in the supernatants of the elicited respiratory macrophages was
Oxidative and NO activity of turkey respiratory macrophages
determined by measuring the amount of nitrite\ a metabolic product of NO[ There! fore\ RM were seeded in 85!well culture plates at 0[4×094 RM per well in 049 ml leukocyte medium[ The plates were incu! bated in 4) CO1 at 26>C for one hour\ to allow adherence of the cells[ After incu! bation\ the RM were stimulated by the addition of 09 ml:well of LPS from Sal! monella enteritidis "Sigma\ Chemical Co[\ St[ Louis\ USA# diluted in leukocyte medium\ to give a _nal LPS concentration of 09 mg:ml[ Unstimulated control RM received 09 ml leukocyte medium per well[ The plates were subsequently reincubated in 4) CO1 at 26>C[ Supernatants were tested for the presence of nitrite after 13 or 37 hours of incubation with LPS\ as described "19#[ Brie~y\ 099 ml of the super! natant from each well was transferred to a new 85!well plate\ which was stored at −19>C until further testing[ Plates were thawed and 099 ml Griess reagent "0:0 of 9[0) N!0!naphthylethylenediamine dihy! drochloride and 0) sulphanilamide in 4) H2PO3^ Sigma Chemical Co[\ St[ Louis\ USA# was added per well[ The absorption "OD 449 nm# was measured after an incu! bation of 04 minutes at room temperature\ using a microplate reader "Titertek Mul! tiscan Plus\ TechGen\ Brussels\ Belgium#[ The OD 449 was converted to nitrite con! centration "mM# using a sodium nitrite standard curve in leukocyte medium[ Depending on the number of recovered RM per turkey\ the RM of 3 to 6 turkeys per induction treatment group were assessed for the production of nitric oxide[ Tests were done on 1 to 6 wells per turkey[
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obtained for each treatment were pooled[ Comparison of the mean peak CL was per! formed with Sche}e|s pairwise comparison of means[ For the evaluation of the NO production\ the mean nitrite con! centrations from the wells "n 1 to 6# of each turkey were calculated and analysed with one!way analysis of variances and Sche}e|s pairwise comparison of means[ The mean yield of RM elicited by the di}erent induction treatment groups was compared with one!way analysis of vari! ances and Sche}e|s pairwise comparison of means[
Results Respiratory Macropha`es The induction treatments with Sephadex G!49\ Thioglycollate broth\ IFA!Broth and IFA!M[ hyorhinis\ resulted in a mean recovery of 5[5621[86 "09#\ 09[0822[91 "09#\ 3[2120[30 "7# and 01[4323[78 "8# respiratory macrophages\ respectively\ representing the mean recovery "×095# per turkey 2 s[e[m[ of the number of turkeys "n# applied per induction treatment group[ Though the number of elicited RM was slightly higher for the induction treatments Thioglycollate broth and IFA!M[ hyorhi! nis\ these di}erences were not signi_cant\ because of the high variability within treat! ment groups[ The viability of the RM exceeded 87)[ Some erythrocytes "³2)# were present in a small number of lavages[ No peroxidase activity was seen upon per! oxidase staining[
Statistical Analysis Chemiluminescence For the evaluation of the oxygenation activity\ peak CL velocity was transformed to natural logs\ and was compared with one!way analysis of variances[ As there were no signi_cant di}erences in peak CL for RM from di}erent turkeys within the induction treatment groups\ the CL results
The luminol!enhanced respiratory burst response of turkey RM upon stimulation with Zymosan A is given in Fig[ 0[ Each CL velocity curve represents a single rep! resentative response of RM elicited with the di}erent induction protocols[ The tem!
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A. E. F. Van Nerom et al.
NO Production
Figure 1. Chemiluminescent velocity curves of turkey respiratory macrophages, stimulated with Zymosan A, in the presence of luminol. Luminol-enhanced chemiluminescent velocity curves of Zymosan A stimulated turkey respiratory macrophages induced with Sephadex G-50 (———), Thioglycolate (—R—), incomplete Freunds’ adjuvant supplemented with broth ( · · · ) and incomplete Freunds’ adjuvant supplemented with Mycoplasma hyorhinis = –). The chemiluminescent velocity curves (–— represent the counts per minute emitted per cuvette from single representative CL assays.
poral pattern of the CL responses of the Sephadex G!49 and IFA!broth induced RM on the one hand\ and of the Thi! oglycollate broth and IFA!M[ hyorhinis induced RM on the other hand were nearly identical[ Stimulation of the Sephadex G! 49 and IFA!broth elicited RM with Zymo! san A induced a short lag!period\ followed by a rapid acceleration of the photon emis! sion[ The short lag!period was also seen in the Zymosan A stimulated Thioglycollate broth and IFA!M[ hyorhinis induced RM\ but the following responses were less accel! erated[ The peak values and time to peak of the CL responses\ obtained after stimulation of the turkey RM are presented in Table 0[ The peak values obtained by the Sephadex G!49 and IFA!broth induced RM were sig! ni_cantly higher "p³9[94# than the peak values obtained by the Thioglycollate broth and IFA!M[ hyorhinis induced RM[ The time needed to reach maximum vel! ocity was similar for all induced RM[
Incubation of the RM with LPS from Salmonella enteritidis elicited considerably the NO production\ as determined by mea! suring the amount of nitrite[ The nitrite concentrations present in the supernatants of the RM after 13 or 37 hours of incu! bation with LPS\ are shown in Tables 1 and 2\ respectively[ When comparing the NO production by the RM obtained fol! lowing the di}erent induction protocols\ no signi_cant di}erences were observed in the nitrite concentrations either at 13\ or at 37 hours of incubation with LPS[ Unstimulated RM produced only low levels of NO[ No signi_cant di}erences were observed between the mean nitrite concentrations present in the supernatants of unstimulated RM obtained following the di}erent induction protocols either at 13\ or at 37 hours of incubation[ Hence\ the mean nitrite concentration 2 s[e[m[ in all unstimulated RM were calculated\ giving 3[73 mM20[95 "10# and 6[85 mM21[51 "06# after 13 and 37 hours of incubation\ respectively\ with "n# num! ber of turkeys tested[
Discussion In the present study we applied di}erent protocols for the induction of turkey res! piratory macrophages\ aiming to evaluate the e}ect of the applied substances on the respiratory burst!dependent and nitric oxide!dependent activity of these mac! rophages in vitro[ Due to the absence of signi_cant numbers of resident RM in poultry "1\ 2#\ turkeys are particularly con! venient for testing these activities in induced RM[ Indeed\ following an appro! priate induction treatment\ an hom! ogenous macrophage population is recovered from the turkey air sacs\ and no interfering by resident macrophages will occur[ In contrast\ the induction of per! itoneal macrophages in mice e[g[ results in a mixture of exudate and resident mac!
Oxidative and NO activity of turkey respiratory macrophages
413
Table 1. Results of the chemiluminescence response of turkey respiratory macrophages after stimulation with Zymosan A, in the presence of luminol. Peak value (×103 counts per minute) and time to peak (minutes) of respiratory macrophages obtained following induction with Sephadex Peak value Time to peak
Thioglycolate
IFA-broth
IFA-M. hyorh.
38.7426.62 (20)*a 19.3722.03 (22)b 42.0424.62 (18)a 21.3922.26 (20)b 2521.3 2420.9 2620.9 2220.8
* Mean counts per minute (×103) per 5×105 respiratory macrophages 2 s.e.m. (n), where (n) represents the number of CL assays from 8 to 10 turkeys per induction treatment group, tested at least in duplicate. Different letters (a,b) represent significant differences for peak values among the respiratory macrophages obtained by different induction treatments (pð0.05).
Table 2. Results of the in vitro nitric oxide (NO) production by turkey respiratory macrophages after incubation with lipopolysaccharide (LPS) for 24 hours. Nitrite accumulation (mM) in the supernatants of 24 hour LPS (10 mg/ml) stimulated respiratory macrophages* obtained following induction with Sephadex
Thioglycolate
IFA-broth
IFA-M. hyorh.
37.6822.24 (6)† 22.7020.81 (6) 25.9820.40 (6) 31.7320.71 (6)
43.2921.78 (6) 12.4620.37 (6) 20.6120.00 (3) 53.1420.32 (3) 19.382.058 (6) 27.6820.33 (6) 46.1721.90 (6)
43.2221.85 (3) 16.5120.62 (3) 31.1221.73 (3) 27.9821.24 (3)
18.0620.36 (3) 30.7522.01 (3) 26.1120.84 (3) 43.4920.60 (3) 31.66291 (3) 31.57278 (3)
29.5523.32 (4)‡
31.8225.90 (7)
29.7125.49 (4)
28.6424.11 (6)
*
Seeded in 96-well culture plates (106 cells/ml; 150 ml/well. †Mean per turkey 2 s.e.m. (number of wells per turkey). ‡Mean per group of induction treatment 2 s.e.m. (number of turkeys tested per induction treatment group).
Table 3. Results of the in vitro nitric oxide (NO) production by turkey respiratory macrophages after incubation with lipopolysaccharide (LPS) for 48 hours. Nitrite accumulation (mM) in the supernatants of 48 hour LPS (10 mg/ml) stimulated respiratory macrophages* obtained following induction with
*
Sephadex
Thioglycolate
IFA-broth
IFA-M. hyorh.
76.2025.14 (6)† 45.0420.99 (7) 46.7420.90 (6) 53.0721.01 (6)
80.9823.46 (6) 22.1820.71 (6) 34.2620.89 (3) 62.1721.02 (2)
65.8320.54 (3) 27.4320.27 (3) 57.0022.96 (3) 41.3120.91 (3)
31.9420.91 (3) 42.1721.14 (3) 72.9720.39 (3) 64.8721.10 (3) 60.4122.30 (3)
55.2627.19 (4)‡
49.89213.32 (4)
47.8928.50 (4)
54.4727.57 (5)
Seeded in 96-well culture plates (106 cells/ml; 150 ml/well). †Mean per turkey 2 s.e.m. (number of wells per turkey). ‡Mean per group of induction treatment 2 s.e.m. (number of turkeys tested per induction treatment group).
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rophages\ which may both possess di}erent capacities[ The applied induction treatments were chosen because Sephadex "3\ 4# and Thi! oglycollate broth "10# have been applied for the induction of peritoneal exudate cells in chickens\ and incomplete Freund|s adju! vant has been used for the induction of turkey RM "2#[ Previously we were able to activate turkey monocyte!derived mac! rophages in vitro following the incubation with Mycoplasma hyorhinis "03#\ resulting in a rise in NO production[ We now wanted to investigate whether this increased activity could also be induced in vivo[ Therefore\ we supplemented the IFA emul! sion used by Ficken et al[ "2# with a sus! pension of M[ hyorhinis[ As this M[ hyorhinis culture was grown in Modi_ed Channock Broth\ the control IFA emul! sion was supplemented with an equivalent amount of this broth[ We adopted the time interval between the induction and the col! lection of the RM induced with Sephadex G!49 "3# and IFA emulsion "4# from the mentioned reports\ and the interval of 3 days for Thioglycollate broth\ as applied at our laboratory[ To be able to compare the oxidative capacity of the RM induced following the di}erent protocols\ the CL assays were per! formed with a constant number of RM\ a _xed quantity of Zymosan A and a con! stant _nal assay concentration of luminol[ The only remaining variable is thus the source of RM[ As the kinetic pattern is mainly determined by the chem! iluminigenic probe "CLP# and the stimulus applied "7#\ their standard application accounts for the similarity of the chem! iluminescent velocity curves obtained for all RM[ The di}erent induction protocols\ however\ did not result in a comparable oxidative activity by the RM\ as the peak CL reached by the Sephadex G!49 and IFA!broth induced RM were signi_cantly higher "p³9[94# than the peak values reached by the Thioglycollate broth and IFA!M[ hyorhinis induced RM[ It is poss! ible that the di}erences in interval between
A. E. F. Van Nerom et al.
the time of induction and the lavage of lungs and air sacs\ might have in~uenced the observed di}erences in peak velocity "11#[ However\ though both the IFA!broth and IFA!M[ hyorhinis induced RM were collected 6 days after induction\ the CL response by the IFA!broth induced RM was signi_cantly higher than the CL response by the IFA!M[ hyorhinis induced RM[ This demonstrates that both induced in~ammatory responses alter the oxidative capacity of the RM di}erently[ It is poss! ible however\ that a higher dose of Myco! plasma might have induced di}erent responses\ as seen with Pasteurella mul! tocida "12#[ Since we used the same CL assay con! ditions for RM as for turkey monocytes "00#\ the respiratory burst responses of both cell types can be compared[ The res! piratory burst responses in the RM were higher than in the monocytes\ which was expected\ as the oxidative capacity is more elevated in in~ammatory macrophages "07#[ Because of this higher response\ the number of RM per cuvette was reduced in comparison to the number of monocytes applied previously "00#[ When corrected for the di}erences in the number of phago! cytes per CL assay\ the respiratory burst response of RM is approximately 5[4 fold "Thioglycollate broth and IFA!M[ hyorhi! nis# and 02 fold "IFA!broth and Sephadex# higher than that of monocytes[ As shown previously\ turkey monocytes lack peroxidase activity "00#[ Similarly\ in this study\ no peroxidase activity could be detected in the elicited RM following per! oxidase staining[ Because luminol! enhanced CL primarily results from the peroxidase activity "8#\ it is the relative ine.cient peroxidase!independent dioxy! genation of luminol that may account for the observed CL responses "6\ 8#[ Thus\ similar to the observations with turkey monocytes "00#\ it is probable that luci! genin!enhanced assays would result in much higher responses\ as the lucigenin! enhanced CL is only related to the presence of O− 1 [ However\ in this comparative ž
Oxidative and NO activity of turkey respiratory macrophages
study\ we applied luminol as CLP\ because of the potentially poisonous properties of lucigenin[ Whereas the oxygenation activity of the elicited RM was dependent of the induc! tion protocol applied\ this was not the case for the production of NO[ All RM\ irres! pective of the induction protocols applied\ showed a high production of NO following the incubation with LPS from S[ enteritidis[ This production of NO lies in the range of the amount of NO produced by turkey bone marrow macrophages activated in vitro with recombinant turkey interferon "02#[ Moreover\ the amount of nitrite found in the supernatants of the LPS! stimulated RM is comparable to the nitrite concentrations connected with the NO! dependent microbicidal activity of murine macrophages "13Ð15#[ Compared to the NO production by LPS stimulated turkey monocytes\ the obtained NO production by RM is about 19 fold higher after 13 hours\ and about 06 fold higher after 37 hours of incubation with LPS "data not shown#[ In this study we demonstrated that the use of the di}erent protocols for the induc! tion of RM speci_cally modi_es the res! piratory burst!dependent activity of these
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macrophages in vitro[ Also\ the elicited RM responded with a high NO production upon stimulation with LPS\ which dis! tinguishes them from monocytes[ Finally\ our results point out that care should be taken when applying induced RM\ under the assumption that those elicited mac! rophages resemble and represent a similar cell population as the one that would be attracted under infectious conditions[ For\ under our experimental conditions\ the addition of M[ hyorhinis to the induction treatment signi_cantly decreased the res! piratory burst response of the elicited RM[ Thus\ the properties of an infection! induced macrophage population should _rst be tested\ before extrapolating the results obtained by induced RM\ as both in~ammatory responses might in~uence these properties di}erently[
Acknowled`ements*The authors which to thank Mr[ K[ Pattyn\ Mr[ L[ Hauttekeete and Miss K[ Tilmant for their assistance[ This work was supported by a grant of the Flemish Insti! tute for the promotion of scienti_c!tech! nological research in the industry "IWT\ Brussels\ Belgium#[
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6[ 7[
8[
09[
Immun[ Microbiol[ Infect[ Dis\ 0874\ 7"1#\ 076Ð 193[ Allen\ C[ R[ Role of oxygen in phagocyte microbicidal action[ Environ[ Health Perspect\ 0883\ 091 "Suppl[ 09#\ 190Ð197[ Allen\ C[ R[ Biochemiexcitation] chemi! luminescence and the study of biological oxy! genation reactions[ In Chemical and biolo`ical `eneration of excited states[ Academic Press\ Inc[\ 0871\ pp[ 298Ð233[ Allen\ C[ R[ Phagocytic leukocyte oxygenation activities and chemiluminescence] a kinetic approach to analysis[ Meth[ Enzymol\ 0875\ 022\ 338Ð382[ Faulkner\ K[ and Fridovich\ I[ Luminol and luci! genin as detectors for O− 1 [ Free Radical Biol[ Med\ 0882\ 04\ 336Ð340[ Van Nerom\ A[\ Desmidt\ M[\ Ducatelle\ R[ and Haesebrouck\ F[ Lucigenin and luminol enhanced chemiluminescence in turkey mono! cytes[ J[ Biolum[ Chemilum[\ 0886\ 01\ 196Ð103[ ž
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416 01[ Moncada\ S[\ Palmer\ R[ M[ J[ and Higgs\ E[ A[ Nitric oxide] physiology\ pathophysiology\ and pharmacology[ Pharmacol[ Rev\ 0880\ 31\ 098Ð 031[ 02[ Suresh\ M[\ Karaca\ K[\ Foster\ D[ and Sharma\ J[ M[ Molecular and functional characterization of turkey interferon[ J[ Virol\ 0884\ 58\ 7048Ð 7052[ 03[ Van Nerom\ A[\ Desmidt\ M[\ Ducatelle\ R[ and Haesebrouck\ F[ Interaction between an avian Chlamydia psittaci strain and activated monocyte derived macrophages from turkeys[ In Proc[ Joint meetin` ESVP + GTP\ Ghent\ Belgium\ 13Ð17 September\ 0885\ pp[ 036[ 04[ Green\ S[ J[\ Meltzer\ M[ S[\ Hibbs\ J[ B[ Jr[ and Nacy\ C[ A[ Activated macrophages destroy intracellular Leishmania major amastigotes by an L!arginine!dependent killing mechanism[ J[ Immunol\ 0889\ 033\ 167Ð172[ 05[ Flesch\ I[ E[ A[ and Kaufmann\ S[ H[ E[ Mech! anisms involved in mycobacterial growth inhi! bition by gamma interferon!activated bone marrow macrophages] role of reactive nitrogen intermediates[ Infect[ Immun\ 0880\ 48\ 2102Ð 2107[ 06[ Hibbs\ J[ B[ Jr[\ Vavrin\ Z[ and Taintor\ R[ R[ L! Arginine is required for expression of the acti! vated macrophage e}ector mechanism causing selective metabolic inhibition in target cells[ J[ Immunol\ 0876\ 027\ 449Ð454[ 07[ Adams\ D[ O[ and Hamilton\ T[ H[ The cell biology of macrophage activation[ Ann[ Rev[ Immunol\ 0873\ 1\ 172Ð207[ 08[ Nussler\ A[ K[ and Billiar\ T[ R[ In~ammation\ immunoregulation\ and inducible nitric oxide synthase[ J[ Leukoc[ Biol\ 0882\ 43\ 060Ð067[
A. E. F. Van Nerom et al. 19[ Green\ L[ C[\ Wagner\ D[ A[\ Glogowiski\ J[\ Skipper\ P[ L[\ Wishnok\ J[ S[ and Tannenbaum\ S[ R[ Analysis of nitrate\ nitrite\ and ð04NŁ nitrate in biological ~uids[ Anal[ Biochem\ 0871\ 015\ 020Ð027[ 10[ Dijkmans\ R[\ Creemers\ J[ and Billiau\ A[ Chicken macrophage activation by interferon] do birds lack the molecular homologue of mamalian interferon!g< Vet[ Immunol[ Immunopathol\ 0889\ 15\ 208Ð221[ 11[ Golemboski\ K[ A[\ Whelan\ J[\ Shaw\ S[\ Kinsella\ J[ E[ and Dietert\ R[ R[ Avian in~am! matory macrophage function] shifts in ara! chidonic acid metabolism\ respiratory burst\ and cell!surface phenotype during the response to Sephadex[ J[ Leukoc[ Biol\ 0889\ 37\ 384Ð490[ 12[ Toth\ T[ E[ and Siegel\ P[ B[ Cellular defence of the avian respiratory system] dose!response relationship and duration of response in intra! tracheal stimulation of avian respiratory phago! cytes by a Pasteurella multocida bacterin[ Avian Dis[\ 0882\ 26\ 645!651[ 13[ James\ S[ L[ and Glaven\ J[ Macrophage cyto! toxicity against schistosomula of Schistosoma mansoni involves arginine!dependent production of reactive nitrogen intermediates[ J[ Immunol\ 0878\ 032\ 3197Ð3101[ 14[ Green\ S[ J[\ Meltzer\ M[ S[\ Hibbs\ J[ B[ and Nacy\ C[ A[ Activated macrophages destroy intracellular Leishmania major amastigotes by an L!arginine!dependent killing mechanism[ J[ Immunol\ 0889\ 033\ 167Ð172[ 15[ Park\ J[ and Rikihisa\ Y[ L!arginine!dependent killing of intracellular Ehrlichia risticii by mac! rophages treated with gamma interferon[ Infect[ Immun\ 0881\ 59\ 2493Ð2497[
Pergamon
Developmental and Comparative Immunology\ Vol[ 11\ No[ 3\ pp[ 396Ð305\ 0887 Þ 0887 Elsevier Science Ltd[ All rights reserved Printed in Great Britain 9034Ð294X:87 ,08[99¦9[99
PII] S9034Ð294X"87#99908Ð5
OXYGEN RADICALS AND NITRIC OXIDE PRODUCTION BY TURKEY RESPIRATORY MACROPHAGES Anne E. F. Van Nerom, Richard Ducatelle and Freddy Haesebrouck Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, University of Ghent, Ghent Belgium "Received September 0886^ Accepted January 0887#
Abstract*The in~uence of different induction protocols on the recovery of elicited turkey res! piratory macrophages "RM#\ and on their oxy! genation activity and nitric oxide "NO# production was examined[ RM were induced in three week old speci_c pathogen free turkeys with Sephadex G!49\ Thioglycollate broth\ and an emulsion of incomplete Freund|s adjuvant "IFA#\ supplemented either with Mycoplasma hyorhinis grown in Modi_ed Channock broth "IFA!M[ hyorhinis# or with Modi_ed Channock broth "IFA!Broth#[ The RM were recovered by lavage of the lungs and air sacs and were puri_ed by centrifugation through a Percoll suspension[ Their oxygenation activity was evaluated in lum! inol!enhanced chemiluminescence assays\ fol! lowing stimulation with Zymosan A[ The NO production was evaluated by incubating the RM with lipopolysaccharide "LPS# from Salmonella enteritidis for 13 or 37 hours[ The number of recovered RM was slightly\ but not signi_cantly lower for Sephadex G!49 and IFA!Broth than for Thioglycollate broth and IFA!M[ hyorhinis[ RM elicited with Sephadex G!49 and IFA!Broth showed a signi_cantly higher oxidative burst response to Zymosan A\ compared to the Thi! oglycollate and IFA!M[ hyorhinis elicited RM[ Although all elicited RM showed a high NO pro!
Address correspondence to Anne Van Nerom\ Department of Pathology\ Bacteriology and Avian Diseases\ Faculty of Veterinary Medi! cine\ Salisburylaan\ 022\ B!8719 Merelbeke\ Belgium[ Tel] 99 21 "9# 8 153 63 20^ Fax] 99 21 "9# 8 153 63 83^ E!mail] anne[vanner! omÝrug[ac[be
duction upon stimulation with LPS\ no signi_cant differences were seen in the NO production of the RM obtained following the different induction treatments[ Our results point out that care should be taken when applying elicited RM for in vitro assays\ as distinct levels of oxygenation activity were obtained using different induction protocols[ Þ 0887 Elsevier Science Ltd[ All rights reserved Keywords*Respiratory burst^ NO^ Res! piratory macrophages^ Turkeys^ Chemi! luminescence[
Nomenclature CL CLP cpm DMEM HBSS IFA LPS NO RM SPF
chemiluminescence^ chemiluminigenic probe^ counts per minute^ Dulbecco|s Modi_ed Eagle|s Medium^ Hank|s Balanced Salt Solution^ incomplete Freund|s adjuvant^ lipopolysaccharide^ nitric oxide^ respiratory macrophages^ speci_c pathogen free[
Introduction Monocytes and macrophages exert a key role in speci_c and non!speci_c host defence\ including in the respiratory tract "0#\ which represents a primary portal of entry for a large number of pathogens[ In contrast to the respiratory tract of mammals\ where numerous resident pul!
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monary macrophages exert the important function of non!speci_c _rst!line defence\ only very low numbers of resident res! piratory macrophages are retrievable in avian species "1\ 2#[ In order to enable research on local tis! sue macrophages in avian species\ di}erent investigators have applied induction pro! tocols for the attraction of macrophages into the respiratory tract "2# and peritoneal space "3\ 4#[ The necessity of inducing mac! rophages is due to the very scarce amount of resident macrophages at those sites in birds[ The underlying assumption is that those elicited macrophages resemble and represent a similar cell population as would be attracted under infectious conditions[ Any injected substance\ however\ evokes a local in~ammatory response\ involving the attraction of exudate cells and the release of cytokines[ The characteristics of this in~ammatory response might therefore in~uence the properties of the induced exudate cells\ in particular the macro! phages[ A major function of macrophages is the respiratory burst\ where toxic oxygen products\ such as superoxide\ hydrogen peroxide\ singlet oxygen and hydroxyl rad! icals are released\ resulting in oxidative microbicidal and cytocidal properties "5Ð 7#[ This respiratory burst is triggered by the NADPH oxidase system\ which is acti! vated following the perturbation of the plasma membrane during phagocytosis or following the interaction between the cell surface and a variety of agents "5#[ Inter! action of the generated oxygenating agents with certain substrates\ results in the for! mation of electronically excited oxy! genation products[ As these products return to their ground state\ photon emis! sion is responsible for the phenomenon of chemiluminescence "CL#[ This CL can be ampli_ed by addition of luminescent sub! strates such as luminol and lucigenin "6Ð 09#\ which provides a sensitive tool for examining the oxidative activity of phago! cytic leukocytes[ Recently we dem! onstrated respiratory burst!dependent
A. E. F. Van Nerom et al.
activity in turkey monocytes towards di}erent soluble and particulate stimuli "00#[ Another important defence mechanism of mononuclear phagocytes constitutes the L!arginine dependent production of nitric oxide "NO# "01#[ Recent investigations demonstrated that activated turkey bone marrow mononuclear cells "02# and turkey monocytes "unpublished results# were cap! able of producing NO\ upon stimulation with lipopolysaccharide "LPS#[ As nitric oxide is a major e}ector molecule for the microbicidal and cytotoxic response of the rodent macrophages "04Ð06#\ these capacities probably also play a major role in the host defence of turkeys[ Both the respiratory burst!dependent and nitric oxide!dependent activity of mononuclear phagocytes can be modi_ed by cytokines "0\ 07\ 08#[ Thus\ the proper! ties of elicited respiratory macrophages might also be modi_ed by cytokines in vivo\ depending on the induction protocol applied[ Therefore\ the aim of the present study was to investigate whether di}erent pro! tocols\ used for the induction of turkey RM\ modi_ed the respiratory burst!depen! dent and nitric oxide!dependent activity of these macrophages in vitro[ The res! piratory burst!dependent activity of the elicited RM was evaluated by measuring their oxidative response upon stimulation with Zymosan A in a luminol!enhanced CL assay[ The production of NO was assessed following stimulation with LPS of Salmonella enteritidis[ Both Zymosan A and LPS of Salmonella enteritidis have been demonstrated to be a strong stimulus for the respiratory burst "00# and the NO production "unpublished results# in turkey monocytes\ respectively[
Material and Methods Poults Speci_c pathogen free "SPF# turkey eggs "obtained from the Centre National d|
Oxidative and NO activity of turkey respiratory macrophages
Etude Veterinaire et Alimentaire\ CNEVA\ Ploufragan\ France# were hat! ched in isolation[ The poults were reared in isolation units[ Food and water were available ad libitum[
Induction of Respiratory Macropha`es "RM# At three weeks of age the turkeys were divided into 3 groups of 7 to 09 birds and transabdominaly injected into each abdominal air sac with one millilitre of one of the substances given below[ , In group 0\ Sephadex G!49 was used to induce RM[ A 2) "w:v# suspension of Sephadex G!49 "Pharmacia\ Uppsala\ Sweden# was prepared as described by Sabet et al[ "3#\ with the exception that phosphate bu}ered saline was replaced by Hank|s Balanced Salt Solution "HBSS^ Gibco\ Merelbeke\ Belgium#[ The lavage of the lungs and air sacs was carried out 2 days following the injec! tion[ , Turkeys of group 1 received 09) Thi! oglycollate "Thioglycollate medium USP\ Conforma\ Destelbergen\ Belgium# in endotoxin!free water for perfusion "PO25\ Conforma\ Destel! bergen\ Belgium#[ They were lavaged 3 days post injection "p[i[#[ , The turkeys of group 2 were injected with an emulsion of incomplete Freund|s adjuvant "IFA#\ supplemented with Mycoplasma hyorhinis suspended in Modi_ed Channock broth[ This IFA!M[ hyorhinis emulsion was prepared as follows[ A cell culture adapted strain of Mycoplasma hyorhinis was grown in Modi_ed Channock Medium without thallium acetate "J[ M[ Bradbury\ per! sonal communication#[ This strain was broth to broth subcultured three times and subsequently stored in aliquots at −69>C[ A viability count was carried out on one vial of this stock\ giving 0[46×096 CFU:ml[ For the inoculum
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preparation\ another vial was thawed and diluted in a 0:1 "v:v# emulsion of incomplete Freund|s adjuvant "Difco\ Detroit\ USA# in sterile HBSS\ to pro! vide inocula containing approximately 0×095 CFU:ml[ The lavage of the lungs and air sacs was carried out 6 days fol! lowing the injection[ , The turkeys of group 3 received an injec! tion of IFA emulsion supplemented with Modi_ed Channock broth "IFA!broth#[ A 0:1 "v:v# emulsion of incomplete Freund|s adjuvant in sterile HBSS was prepared with 5[24) "v:v# Modi_ed Channock Medium without thallium acetate\ i[e[ corresponding to the amount administered to the IFA!M[ hyorhinis inoculated turkeys[ The lungs and air sacs of these turkeys were also lavaged 6 days p[i[
Collection of Respiratory Macropha`es Respiratory macrophages were collected according to the procedure of Ficken et al[ "2#\ with some modi_cations[ Turkeys were euthanized and the trachea was exposed by a ventral midline skin incision[ A needle connected to a syringe was inserted into the trachea\ which was clasped[ Sixty millilitre lavage ~uid\ consisting of HBSS without calcium and magnesium "HBSS w:o Ca:Mg^ Gibco\ Merelbeke\ Belgium#\ sup! plemented with 099 mg:ml streptomycin\ 099 mg:ml vancomycin and 14 U:ml hep! arin "Leo pharmaceutical products\ Vil! voorde\ Belgium# was injected into the lungs and air sacs[ The lavage ~uid was withdrawn by gravity ~ow while the abdo! men and thorax were gently massaged[ The collected ~uid was centrifuged for 09 min at 0929 g\ 3>C[ The obtained pellet was resuspended in 2 ml HBSS w:o Ca:Mg\ supplemented with 099 mg:ml streptomycin and 099 mg:ml vancomycin\ and laid upon 2 ml Percoll suspension "density 0[968^ 299 mOsmol:kg H1O#\ composed of 7[7) "v:v# 09×concentrated Hanks| Balanced
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Salt Solution "09×HBSS^ Gibco\ Merel! beke\ Belgium# and 45[7) "v:v# Percoll stock suspension "Pharmacia Biotech\ Roosendael\ the Netherlands# in a 01[4 ml conical centrifuge tube[ Following cen! trifugation at 0629 g for 29 minutes at 3>C\ the cells on top of the Percoll solution were collected and washed once with sterile HBSS w:o Ca:Mg[ The cells were resus! pended at a concentration of 0×095 cells:ml\ in leukocyte medium\ com! posed of Dulbecco|s Modi_ed Eagle|s Medium "DMEM^ endotoxin level] 9[94 ng:ml^ Gibco\ Merelbeke\ Belgium#\ supplemented with 099 mg:ml strep! tomycin\ 099 mg:ml vancomycin\ 0) sodium pyruvate "Gibco\ Merelbeke\ Belgium#\ 0) L!glutamine "Gibco\ Mer! elbeke\ Belgium# and 09) low endotoxin myoclone super plus bovine serum "endo! toxin level ³0 ng:ml^ Gibco\ Merelbeke\ Belgium#[ The purity of the macrophage suspensions was assessed by microscopic examination of Haemacolorþ "Merck\ Darmstadt\ Germany# stained smears[ The viability was determined by exclusion of trypan blue[ Cells were checked for per! oxidase activity by staining _xed smears with DAB "2\2?!diaminobenzidine tetra! chloride tablets\ Sigma Biosciences\ St[ Louis\ USA#\ prepared as recommended by the manufacturer\ and hematoxylin[
Tri``erin` A`ent for Chemiluminescence Zymosan A was applied as a triggering agent in the chemiluminescence assays[ A suspension of 9[14 g) Zymosan A in HBSS was heated for 29 minutes in a water bath at 89>C[ After cooling\ the suspension was aliquoted and stored at −69>C[ The _nal assay concentration of Zymosan A was 8[5 mg)[
A. E. F. Van Nerom et al.
piratory macrophages\ luminol was applied as chemiluminigenic probe "CLP# "Sigma Chemical Co[\ St[ Louis\ USA#[ Luminol was dissolved in DMSO to give a concentration of 093 mM\ and was stored in small aliquots at −69>C[ Prior to use\ the luminol stock solution was diluted in HBSS\ and 099 ml was added to the cuvet! tes[ Final assay concentration of luminol was 199 mM[
Chemiluminescence Assay The CL assays were performed in poly! styrene biolum cuvettes "Lumac\ Landg! raaf\ the Netherlands# at 26>C\ using a six! channel luminometer "Lumicon\ Hamil! ton\ Bonaduz\ Switzerland#[ The cuvettes were not stirred during the assay[ Mea! suring of the CL reactions of isolated res! piratory macrophages "RM# was performed as follows[ The cuvettes were supplied with 499 ml RM suspension "4×094 RM# and incubated in 4) CO1 at 26>C for one hour\ to allow adherence of the cells[ After incubation\ the supernatant was replaced by 399 ml HBSS and 099 ml luminol solution[ The cuvettes were placed in the luminometer and the spontaneous CL "cells without stimulating agent# was recorded for at least 09 min[ The CL was measured as counts per minute "cpm#[ Each cuvette was read for 09 seconds per minute[ After stabilisation of the spon! taneous CL\ the reaction was started by the addition of 19 ml Zymosan A per cuvette[ The oxidative activity of the RM was recorded for one hour[ Data were cor! rected for spontaneous CL[ The RM of all turkeys were tested for CL and a minimum of two measurements were done per turkey[
Luminescent Substrate
Stimulation of the NO Production in Turkey RM
For the evaluation of the chemi! luminescence response of turkey res!
The presence of NO in the supernatants of the elicited respiratory macrophages was
Oxidative and NO activity of turkey respiratory macrophages
determined by measuring the amount of nitrite\ a metabolic product of NO[ There! fore\ RM were seeded in 85!well culture plates at 0[4×094 RM per well in 049 ml leukocyte medium[ The plates were incu! bated in 4) CO1 at 26>C for one hour\ to allow adherence of the cells[ After incu! bation\ the RM were stimulated by the addition of 09 ml:well of LPS from Sal! monella enteritidis "Sigma\ Chemical Co[\ St[ Louis\ USA# diluted in leukocyte medium\ to give a _nal LPS concentration of 09 mg:ml[ Unstimulated control RM received 09 ml leukocyte medium per well[ The plates were subsequently reincubated in 4) CO1 at 26>C[ Supernatants were tested for the presence of nitrite after 13 or 37 hours of incubation with LPS\ as described "19#[ Brie~y\ 099 ml of the super! natant from each well was transferred to a new 85!well plate\ which was stored at −19>C until further testing[ Plates were thawed and 099 ml Griess reagent "0:0 of 9[0) N!0!naphthylethylenediamine dihy! drochloride and 0) sulphanilamide in 4) H2PO3^ Sigma Chemical Co[\ St[ Louis\ USA# was added per well[ The absorption "OD 449 nm# was measured after an incu! bation of 04 minutes at room temperature\ using a microplate reader "Titertek Mul! tiscan Plus\ TechGen\ Brussels\ Belgium#[ The OD 449 was converted to nitrite con! centration "mM# using a sodium nitrite standard curve in leukocyte medium[ Depending on the number of recovered RM per turkey\ the RM of 3 to 6 turkeys per induction treatment group were assessed for the production of nitric oxide[ Tests were done on 1 to 6 wells per turkey[
411
obtained for each treatment were pooled[ Comparison of the mean peak CL was per! formed with Sche}e|s pairwise comparison of means[ For the evaluation of the NO production\ the mean nitrite con! centrations from the wells "n 1 to 6# of each turkey were calculated and analysed with one!way analysis of variances and Sche}e|s pairwise comparison of means[ The mean yield of RM elicited by the di}erent induction treatment groups was compared with one!way analysis of vari! ances and Sche}e|s pairwise comparison of means[
Results Respiratory Macropha`es The induction treatments with Sephadex G!49\ Thioglycollate broth\ IFA!Broth and IFA!M[ hyorhinis\ resulted in a mean recovery of 5[5621[86 "09#\ 09[0822[91 "09#\ 3[2120[30 "7# and 01[4323[78 "8# respiratory macrophages\ respectively\ representing the mean recovery "×095# per turkey 2 s[e[m[ of the number of turkeys "n# applied per induction treatment group[ Though the number of elicited RM was slightly higher for the induction treatments Thioglycollate broth and IFA!M[ hyorhi! nis\ these di}erences were not signi_cant\ because of the high variability within treat! ment groups[ The viability of the RM exceeded 87)[ Some erythrocytes "³2)# were present in a small number of lavages[ No peroxidase activity was seen upon per! oxidase staining[
Statistical Analysis Chemiluminescence For the evaluation of the oxygenation activity\ peak CL velocity was transformed to natural logs\ and was compared with one!way analysis of variances[ As there were no signi_cant di}erences in peak CL for RM from di}erent turkeys within the induction treatment groups\ the CL results
The luminol!enhanced respiratory burst response of turkey RM upon stimulation with Zymosan A is given in Fig[ 0[ Each CL velocity curve represents a single rep! resentative response of RM elicited with the di}erent induction protocols[ The tem!
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A. E. F. Van Nerom et al.
NO Production
Figure 1. Chemiluminescent velocity curves of turkey respiratory macrophages, stimulated with Zymosan A, in the presence of luminol. Luminol-enhanced chemiluminescent velocity curves of Zymosan A stimulated turkey respiratory macrophages induced with Sephadex G-50 (———), Thioglycolate (—R—), incomplete Freunds’ adjuvant supplemented with broth ( · · · ) and incomplete Freunds’ adjuvant supplemented with Mycoplasma hyorhinis = –). The chemiluminescent velocity curves (–— represent the counts per minute emitted per cuvette from single representative CL assays.
poral pattern of the CL responses of the Sephadex G!49 and IFA!broth induced RM on the one hand\ and of the Thi! oglycollate broth and IFA!M[ hyorhinis induced RM on the other hand were nearly identical[ Stimulation of the Sephadex G! 49 and IFA!broth elicited RM with Zymo! san A induced a short lag!period\ followed by a rapid acceleration of the photon emis! sion[ The short lag!period was also seen in the Zymosan A stimulated Thioglycollate broth and IFA!M[ hyorhinis induced RM\ but the following responses were less accel! erated[ The peak values and time to peak of the CL responses\ obtained after stimulation of the turkey RM are presented in Table 0[ The peak values obtained by the Sephadex G!49 and IFA!broth induced RM were sig! ni_cantly higher "p³9[94# than the peak values obtained by the Thioglycollate broth and IFA!M[ hyorhinis induced RM[ The time needed to reach maximum vel! ocity was similar for all induced RM[
Incubation of the RM with LPS from Salmonella enteritidis elicited considerably the NO production\ as determined by mea! suring the amount of nitrite[ The nitrite concentrations present in the supernatants of the RM after 13 or 37 hours of incu! bation with LPS\ are shown in Tables 1 and 2\ respectively[ When comparing the NO production by the RM obtained fol! lowing the di}erent induction protocols\ no signi_cant di}erences were observed in the nitrite concentrations either at 13\ or at 37 hours of incubation with LPS[ Unstimulated RM produced only low levels of NO[ No signi_cant di}erences were observed between the mean nitrite concentrations present in the supernatants of unstimulated RM obtained following the di}erent induction protocols either at 13\ or at 37 hours of incubation[ Hence\ the mean nitrite concentration 2 s[e[m[ in all unstimulated RM were calculated\ giving 3[73 mM20[95 "10# and 6[85 mM21[51 "06# after 13 and 37 hours of incubation\ respectively\ with "n# num! ber of turkeys tested[
Discussion In the present study we applied di}erent protocols for the induction of turkey res! piratory macrophages\ aiming to evaluate the e}ect of the applied substances on the respiratory burst!dependent and nitric oxide!dependent activity of these mac! rophages in vitro[ Due to the absence of signi_cant numbers of resident RM in poultry "1\ 2#\ turkeys are particularly con! venient for testing these activities in induced RM[ Indeed\ following an appro! priate induction treatment\ an hom! ogenous macrophage population is recovered from the turkey air sacs\ and no interfering by resident macrophages will occur[ In contrast\ the induction of per! itoneal macrophages in mice e[g[ results in a mixture of exudate and resident mac!
Oxidative and NO activity of turkey respiratory macrophages
413
Table 1. Results of the chemiluminescence response of turkey respiratory macrophages after stimulation with Zymosan A, in the presence of luminol. Peak value (×103 counts per minute) and time to peak (minutes) of respiratory macrophages obtained following induction with Sephadex Peak value Time to peak
Thioglycolate
IFA-broth
IFA-M. hyorh.
38.7426.62 (20)*a 19.3722.03 (22)b 42.0424.62 (18)a 21.3922.26 (20)b 2521.3 2420.9 2620.9 2220.8
* Mean counts per minute (×103) per 5×105 respiratory macrophages 2 s.e.m. (n), where (n) represents the number of CL assays from 8 to 10 turkeys per induction treatment group, tested at least in duplicate. Different letters (a,b) represent significant differences for peak values among the respiratory macrophages obtained by different induction treatments (pð0.05).
Table 2. Results of the in vitro nitric oxide (NO) production by turkey respiratory macrophages after incubation with lipopolysaccharide (LPS) for 24 hours. Nitrite accumulation (mM) in the supernatants of 24 hour LPS (10 mg/ml) stimulated respiratory macrophages* obtained following induction with Sephadex
Thioglycolate
IFA-broth
IFA-M. hyorh.
37.6822.24 (6)† 22.7020.81 (6) 25.9820.40 (6) 31.7320.71 (6)
43.2921.78 (6) 12.4620.37 (6) 20.6120.00 (3) 53.1420.32 (3) 19.382.058 (6) 27.6820.33 (6) 46.1721.90 (6)
43.2221.85 (3) 16.5120.62 (3) 31.1221.73 (3) 27.9821.24 (3)
18.0620.36 (3) 30.7522.01 (3) 26.1120.84 (3) 43.4920.60 (3) 31.66291 (3) 31.57278 (3)
29.5523.32 (4)‡
31.8225.90 (7)
29.7125.49 (4)
28.6424.11 (6)
*
Seeded in 96-well culture plates (106 cells/ml; 150 ml/well. †Mean per turkey 2 s.e.m. (number of wells per turkey). ‡Mean per group of induction treatment 2 s.e.m. (number of turkeys tested per induction treatment group).
Table 3. Results of the in vitro nitric oxide (NO) production by turkey respiratory macrophages after incubation with lipopolysaccharide (LPS) for 48 hours. Nitrite accumulation (mM) in the supernatants of 48 hour LPS (10 mg/ml) stimulated respiratory macrophages* obtained following induction with
*
Sephadex
Thioglycolate
IFA-broth
IFA-M. hyorh.
76.2025.14 (6)† 45.0420.99 (7) 46.7420.90 (6) 53.0721.01 (6)
80.9823.46 (6) 22.1820.71 (6) 34.2620.89 (3) 62.1721.02 (2)
65.8320.54 (3) 27.4320.27 (3) 57.0022.96 (3) 41.3120.91 (3)
31.9420.91 (3) 42.1721.14 (3) 72.9720.39 (3) 64.8721.10 (3) 60.4122.30 (3)
55.2627.19 (4)‡
49.89213.32 (4)
47.8928.50 (4)
54.4727.57 (5)
Seeded in 96-well culture plates (106 cells/ml; 150 ml/well). †Mean per turkey 2 s.e.m. (number of wells per turkey). ‡Mean per group of induction treatment 2 s.e.m. (number of turkeys tested per induction treatment group).
414
rophages\ which may both possess di}erent capacities[ The applied induction treatments were chosen because Sephadex "3\ 4# and Thi! oglycollate broth "10# have been applied for the induction of peritoneal exudate cells in chickens\ and incomplete Freund|s adju! vant has been used for the induction of turkey RM "2#[ Previously we were able to activate turkey monocyte!derived mac! rophages in vitro following the incubation with Mycoplasma hyorhinis "03#\ resulting in a rise in NO production[ We now wanted to investigate whether this increased activity could also be induced in vivo[ Therefore\ we supplemented the IFA emul! sion used by Ficken et al[ "2# with a sus! pension of M[ hyorhinis[ As this M[ hyorhinis culture was grown in Modi_ed Channock Broth\ the control IFA emul! sion was supplemented with an equivalent amount of this broth[ We adopted the time interval between the induction and the col! lection of the RM induced with Sephadex G!49 "3# and IFA emulsion "4# from the mentioned reports\ and the interval of 3 days for Thioglycollate broth\ as applied at our laboratory[ To be able to compare the oxidative capacity of the RM induced following the di}erent protocols\ the CL assays were per! formed with a constant number of RM\ a _xed quantity of Zymosan A and a con! stant _nal assay concentration of luminol[ The only remaining variable is thus the source of RM[ As the kinetic pattern is mainly determined by the chem! iluminigenic probe "CLP# and the stimulus applied "7#\ their standard application accounts for the similarity of the chem! iluminescent velocity curves obtained for all RM[ The di}erent induction protocols\ however\ did not result in a comparable oxidative activity by the RM\ as the peak CL reached by the Sephadex G!49 and IFA!broth induced RM were signi_cantly higher "p³9[94# than the peak values reached by the Thioglycollate broth and IFA!M[ hyorhinis induced RM[ It is poss! ible that the di}erences in interval between
A. E. F. Van Nerom et al.
the time of induction and the lavage of lungs and air sacs\ might have in~uenced the observed di}erences in peak velocity "11#[ However\ though both the IFA!broth and IFA!M[ hyorhinis induced RM were collected 6 days after induction\ the CL response by the IFA!broth induced RM was signi_cantly higher than the CL response by the IFA!M[ hyorhinis induced RM[ This demonstrates that both induced in~ammatory responses alter the oxidative capacity of the RM di}erently[ It is poss! ible however\ that a higher dose of Myco! plasma might have induced di}erent responses\ as seen with Pasteurella mul! tocida "12#[ Since we used the same CL assay con! ditions for RM as for turkey monocytes "00#\ the respiratory burst responses of both cell types can be compared[ The res! piratory burst responses in the RM were higher than in the monocytes\ which was expected\ as the oxidative capacity is more elevated in in~ammatory macrophages "07#[ Because of this higher response\ the number of RM per cuvette was reduced in comparison to the number of monocytes applied previously "00#[ When corrected for the di}erences in the number of phago! cytes per CL assay\ the respiratory burst response of RM is approximately 5[4 fold "Thioglycollate broth and IFA!M[ hyorhi! nis# and 02 fold "IFA!broth and Sephadex# higher than that of monocytes[ As shown previously\ turkey monocytes lack peroxidase activity "00#[ Similarly\ in this study\ no peroxidase activity could be detected in the elicited RM following per! oxidase staining[ Because luminol! enhanced CL primarily results from the peroxidase activity "8#\ it is the relative ine.cient peroxidase!independent dioxy! genation of luminol that may account for the observed CL responses "6\ 8#[ Thus\ similar to the observations with turkey monocytes "00#\ it is probable that luci! genin!enhanced assays would result in much higher responses\ as the lucigenin! enhanced CL is only related to the presence of O− 1 [ However\ in this comparative ž
Oxidative and NO activity of turkey respiratory macrophages
study\ we applied luminol as CLP\ because of the potentially poisonous properties of lucigenin[ Whereas the oxygenation activity of the elicited RM was dependent of the induc! tion protocol applied\ this was not the case for the production of NO[ All RM\ irres! pective of the induction protocols applied\ showed a high production of NO following the incubation with LPS from S[ enteritidis[ This production of NO lies in the range of the amount of NO produced by turkey bone marrow macrophages activated in vitro with recombinant turkey interferon "02#[ Moreover\ the amount of nitrite found in the supernatants of the LPS! stimulated RM is comparable to the nitrite concentrations connected with the NO! dependent microbicidal activity of murine macrophages "13Ð15#[ Compared to the NO production by LPS stimulated turkey monocytes\ the obtained NO production by RM is about 19 fold higher after 13 hours\ and about 06 fold higher after 37 hours of incubation with LPS "data not shown#[ In this study we demonstrated that the use of the di}erent protocols for the induc! tion of RM speci_cally modi_es the res! piratory burst!dependent activity of these
415
macrophages in vitro[ Also\ the elicited RM responded with a high NO production upon stimulation with LPS\ which dis! tinguishes them from monocytes[ Finally\ our results point out that care should be taken when applying induced RM\ under the assumption that those elicited mac! rophages resemble and represent a similar cell population as the one that would be attracted under infectious conditions[ For\ under our experimental conditions\ the addition of M[ hyorhinis to the induction treatment signi_cantly decreased the res! piratory burst response of the elicited RM[ Thus\ the properties of an infection! induced macrophage population should _rst be tested\ before extrapolating the results obtained by induced RM\ as both in~ammatory responses might in~uence these properties di}erently[
Acknowled`ements*The authors which to thank Mr[ K[ Pattyn\ Mr[ L[ Hauttekeete and Miss K[ Tilmant for their assistance[ This work was supported by a grant of the Flemish Insti! tute for the promotion of scienti_c!tech! nological research in the industry "IWT\ Brussels\ Belgium#[
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