Taurine chloramine down-regulates the generation of murine neutrophil inflammatory mediators

Ž .Immunopharmacology 40 1998 27–38

Taurine chloramine down-regulates the generation of murineneutrophil inflammatory mediators

Janusz Marcinkiewicz a,), Agnieszka Grabowska a, Joanna Bereta b,Krzysztof Bryniarski a, Bernadeta Nowak a

a Department of Immunology, Jagiellonian UniÕersity Medical College, ul. Czysta 18, 31-121 Cracow, Polandb Institute of Molecular Biology of Jagiellonian UniÕersity, Cracow, Poland

Received 29 January 1998; revised 4 May 1998; accepted 11 May 1998

Abstract

Ž .We previously reported that taurine chloramine TauCl , a product of activated neutrophils, inhibits the generation ofŽ .macrophage inflammatory mediators such as nitric oxide NO , TNF-a , and PGE . Taurine, the most abundant free amino2

Ž .acid in the cytosol of neutrophils, is chlorinated to form TauCl by the halide-dependent myeloperoxidase MPO system.Under physiological conditions, TauCl reduces HOCl toxicity. In this study, we investigated the influence of TauCl ongeneration of oxygen free radicals, cytokines and eicosanoids by activated murine peritoneal neutrophils. We found thatTauCl, but not taurine alone, inhibited the production of NO, prostaglandin E , interleukin-6 and tumor necrosis factor-a , in2

a dose-dependent manner. In contrast, the products of the respiratory burst, as measured by luminol-dependent chemilumi-Ž .nescence LCL , were reduced by both taurine and TauCl. However, taurine affected LCL at higher concentrations and to a

lesser extent than TauCl. The results of these studies suggest that TauCl decreases production of tissue-damaginginflammatory mediators and may regulate the balance between protective, microbicidal and toxic effect of neutrophils.q 1998 Elsevier Science B.V. All rights reserved.

Keywords: Taurine chloramine; Neutrophils; Nitric oxide; Reactive oxygen intermediates; Tumor necrosis factor-a

1. Introduction

Ž .Neutrophils PMN , the major cells of acute in-flammation, play an important role in host defence

Abbreviations: Tau: taurine; TauCl: taurine monochloramine;TNF-a: tumor necrosis factor-a; IFN-g: interferon-g; IL-6: inter-leukin-6; PGE : prostaglandin E ; iNOS: inducible nitric oxide2 2

synthase; NO: nitric oxide; ROI: reactive oxygen intermediates;LPS: lipopolysaccharide; PMN: polymorphonuclear leukocytes;MPO: myeloperoxidase; CL: chemiluminescence; SOD: superox-ide dismutase; ABAH: 4-aminobenzoyl hydrazine

) Corresponding author. Tel.: q48 12 633 94 31; fax: q48 12633 94 31

against various infectious agents but, paradoxically,Žare also involved in damage of host tissues Smith,

.1994; Lloyd and Oppenheim, 1992 . During phago-cytosis, neutrophils are activated and microbicidal

Ž .agents such as reactive oxygen intermediates ROIŽ .and reactive nitrogen intermediates RNI are gener-Žated intracellularly to kill pathogens Klebanoff and

.Hamon, 1992; Thomas, 1979; Ding et al., 1988 .However, tissue damage may occur when neutrophil

Žcytotoxic microbicidal products e.g., HO, HOCl,.NO are released extracellularly to such extent that

Žhost defences are locally overwhelmed Cantin, 1994;.Claesson et al., 1996 . Moreover, activated neu-

0162-3109r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.Ž .PII: S0162-3109 98 00023-X

( )J. Marcinkiewicz et al.r Immunopharmacology 40 1998 27–3828

trophils release a number of proinflammatory media-Ž .tors including cytokines TNF-a , IL-1, IL-6 and

Ž .eicosanoids PGE , LTB which also contribute to2 4Žthe host tissue injury Smith, 1994; Rappole and

.Werb, 1988 . This pathological process may be at-tenuated by several regulatory mechanisms. For ex-ample, neutrophils contain large reserves of endoge-nous antioxidants such as glutathione, ascorbate and

Ž .taurine Learn et al., 1990; Voetman et al., 1980 .Ž .Taurine, 2-aminoethanesulfonate Tau , was re-

ported to be the most abundant free amino acid inthe cytosol of leukocytes, especially in neutrophilsŽ . Ž10–30 mM Fukkuda et al., 1982; Wright et al.,

.1986 . It was proposed that Tau acts as a scavengerŽ .for hypochlorous acid HOCl , a microbicidal agent

produced by the myeloperoxidase–hydrogen perox-Ž y.ide–chloride MPO–H O –Cl system of activated2 2

Ž .monocytes and neutrophils Wright et al., 1986 . Thereaction of Tau with HOCl forms taurine chloramineŽ .TauCl , the long-lived oxidant, which is less reac-

Žtive and much less toxic than HOCl Zgliczynski et.al., 1971; Marquez and Dunford, 1994 . Therefore,

Tau in the cytosol of neutrophils may protect thesecells against attack by chlorinated oxidants that es-cape from phagolysosomes. In addition, Tau releasedextracellularly may protect the tissue at a site of

Ž .inflammation Cantin, 1994 . TauCl itself, however,may exert prolonged oxidative and chlorinating ef-fects long after the initiation of the respiratory burst

Žand at some distance from the cell of origin Zglic-.zynski et al., 1971 . Recently, a few studies have

suggested that TauCl could act to down-regulate theŽinflammatory response Park et al., 1993;.Marcinkiewicz et al., 1995a,b . For example, TauCl,

at non-toxic concentrations, inhibits in vitro the pro-duction of various macrophage inflammatory media-

Ž .tors such as TNF-a , nitric oxide NO and prosta-Žglandins Marcinkiewicz et al., 1995a,b; Park et al.,

.1995 . TauCl chloramine is transported intomacrophages and if it is present either before orduring activation, production of the above inflamma-tory mediators is inhibited. In contrast to the well-documented suppression of macrophage functions byTauCl, much less is known about how this agentinfluences neutrophils, the cells which closely coop-erate with macrophages at the site of inflammation.

The aim of this study was to determine whetherTauCl and its precursor Tau, can down-regulate the

generation of microbicidal and proinflammatoryagents by neutrophils, and thus minimise host tissuedamage by these cells.

2. Materials and methods

2.1. Synthesis of TauCl

TauCl was prepared in our laboratory from NaOClŽ .Aldrich, Steinheim, Germany dissolved in 0.2 M

Ž . Žphosphate buffer pH 7.4–7.5 and Tau Sigma, St.. ŽLouis, MO , as described previously Marcinkiewicz

.et al., 1995a,b .Each preparation of TauCl was monitored by UV

Ž .absorption 200 to 400 nm to confirm the presenceŽ .of monochloramine TauCl and the absence of

Ž . ydichloramine TauCl and unreacted HOClrOCl .2

The concentration of TauCl was determined by mea-Ž . Žsuring its absorption at 252 nm ´ s415 ThomasM

. Žet al., 1986 . Stock solutions of Tau and TauCl 10.mM were kept at 48C for a maximum period of 4

days before use.

2.2. Mice

Inbred CBArJ male mice from the breeding unit,Department of Immunology, Jagiellonian UniversityMedical College, Cracow, Poland, were used be-tween 6 and 8 weeks of age.

2.3. Cells

Isolation of neutrophils. Peritoneal mouse neu-Ž .trophils PMN were elicited by intraperitoneal injec-

Ž .tion of 2 ml of Markol 52 oil Exon, USA . CellsŽwere collected 12 h later Middleton and Campbell,

.1989 by washing out the peritoneal cavity with 5 mlŽ .of PBS phosphate buffer solution containing 5 U

Ž .heparinrml Polfa, Warsaw, Poland . Cells werecentrifuged and red blood cells were lysed by os-motic shock using distilled water; osmolarity wasrestored by adding 2= concentrated PBS. The cellscollected were at least 90–95% neutrophils, as as-sessed by microscopic examination of cytocentrifugepreparations fixed in methanol and stained with May

Ž .GrunwaldrGiemsa Merck, UK . The presence ofŽ .macrophages 3–7% was judged by cytochemical

( )J. Marcinkiewicz et al.r Immunopharmacology 40 1998 27–38 29

demonstration of non-specific esterase positiveŽmononuclear cells, using a-naphthyl acetate Yam et

.al., 1971 .

2.4. Cell culture

2.4.1. ActiÕation of the cellsNeutrophils were cultured in 96-well flat-bottom

culture plates at 2=105 cellsrwell in RPMI 1640medium supplemented with 10% foetal calf serumŽ . Ž .FCS both Gibco, Grand Island, NY at 378C in anatmosphere of 5% CO . Cells were activated with2

Ž .IFN-g 20 Urml Sigma and LPS 100 ngrmlŽ .Escherichia coli 0111 B:4, Sigma . Activated cells

Žwere incubated with either Tau or TauCl 0.03–0.3.mM , if not otherwise stated. After 24 h, super-

natants were collected and frozen at y208C forNOy, IL-6, TNF-a , and PGE assays.2 2

Neutrophil viability was routinely monitored bycellular exclusion of trypan blue.

2.5. Chemiluminescence assay

Neutrophils were placed in 96 flat-bottom blackŽ . 5plates Nunc, Roskilde, Denmark , 5 = 10

cellsrwell in RPMIq10% FCS. After a dark adap-tation period of 40 min at 378C, plates were trans-

Ž .ferred to a Lucy 1 Anthos, Salzburg, Austria lumi-nometer immediately after the addition of zymosan

Ž . Žopsonized with mouse serum Sigma 10.particlesrcell , and photon emission over 57 min

was measured. In some experiments, the followingagents were added to cells at the onset of dark

Ž .adaptation: luminol Sigma , Tau, TauCl, 4-amino-Ž .benzoyl hydrazine ABAH —an inhibitor of

Ž . Ž . Žmyeloperoxidase MPO Aldrich Kettle et al.,. Ž . Ž .1995 , and superoxide dismutase SOD Sigma . In

other experiments, cells were preincubated with theŽpotential inhibitors of chemiluminescence 378C, 40

.min , washed with PBS, then luminol and zymosanwere added, and photon emission was measured.Each type of experiment was run in duplicate.

2.6. The MPO actiÕity

Myeloperoxidase activity was measured using theŽ .technique described by Bradley et al. 1982 . Briefly,

MPO was extracted from cells by suspending them

in ice-cold 0.5% hexadecyltrimethylammonium bro-Ž . Ž .mide HTAB Sigma in 50 mM potassium phos-

phate buffer, pH 6.0. The cells were freeze-thawedthree times, and dispersed by shaking on a vortex.Suspensions were centrifuged at 40 000=g for 15

Ž .min at 48C. Aliquots 0.1 ml of the supernatant weremixed with 1.4 ml of 50 mM phosphate buffer, pH6.0, containing 0.167 mgrml o-dianisidine dihydro-

Ž .chloride Sigma and 0.0005% hydrogen peroxideŽ .Sigma and 200 ml of the mixture were placed in a96-well flat-bottom plate. The absorbance was mea-sured at 460 nm using a Sumal-PE2 spectrophotom-eter after 20 min of incubation at 258C. MPO activity

Žwas calculated from a MPO Calbiochem, San Diego,.CA standard curve and expressed in units. One unit

of MPO activity was defined as that degrading 1 mMof hydrogen peroxide per minute at 258C. Eachsample was measured in duplicate.

( y)2.7. Nitrite NO determination2

Nitric oxide, quantified by the accumulation ofnitrite as a stable end product was determined by a

Ž .microplate assay Ding et al., 1988 . Briefly, 100 mlsamples were removed from supernatants and incu-

Žbated with an equal volume of Griess reagent 1%sulphanilamider0.1%, N-1-naphthylenediamine di-

.hydrochlorider2.5% H PO at room temperature3 4

for 10 min. The absorbance at 550 nm was measuredwith a microplate reader. Nitrite concentration wascalculated from a sodium nitrite standard curve.

2.8. The RNA preparation and Northern blot analy-sis

Cells were cultured in 94 mm tissue culture platesŽ 7 .5=10 cellsrplate . After 4.5 h of incubation ofcells with the indicated factors, total RNA fromnon-adherent cells was prepared using the phenol

Ž .extraction method Rose-John et al., 1988 . RNAŽ .samples 5 mg were separated electrophoretically in

1% agarose gel under denaturating conditionsŽ .Sambrook et al., 1989 . RNA was then capillary

Žtransferred to Hybond-N membranes Amersham,.UK according to the manufacturer’s instruction. The

blots were baked at 808C for 2 h, prehybridizedovernight, and hybridised to cDNA probes specific


Žfor the murine macrophages iNOS a gift from Drs..Q.W. Xie and C. Nathan and for 18S rRNA. The

probes were labelled by the Random Primers DNAŽ .Labelling System Amersham, UK . Hybridisations

were performed at 688C for 24 h. Non-specificallybound radioactivity was removed by washing the

Ž .blots in 2= standard saline citrate SSC at roomtemperature, followed by two subsequent washes in

Ž .2= SSC, 0.1% sodium dodecyl sulphate SDS at688C for 30 min each. The blots were then subjectedto autoradiography using intensifying screens.

2.9. Cytokine assays

2.9.1. The IL-6 and TNF-a bioassaysLevels of IL-6 and TNF-a in the supernatants

were measured using specific indicator cell lines andtesting cell viability with the mitochondrial indicator

Ždye, MTT, as described previously Marcinkiewicz.et al., 1995a . IL-6 bioactivity was analysed by

measuring growth of the B9 hybridoma cell line. TheTNF-a level was measured using the L-929 fibrob-

Ž .last cytotoxicity assay. Mouse rIL-6 Sigma andŽmouse recombinant TNF-a Genzyme, Cambridge,

.MA were used as standards, respectively.

2.9.2. The IL-6 and TNF-a immunoassaysInterleukin-6 was measured using a capture

Ž .ELISA, with microtiter plates Corning, NY coatedwith the rat monoclonal antibody against murine

Ž .IL-6 Genzyme , and the biotinylated rat monoclonalŽantibody against murine IL-6 Pharmingen, San

.Diego, CA , and horseradish peroxidase streptavidinŽ .Vector Laboratories, Burlingame, CA and o-phen-

Ž .ylenediamine and H O both Sigma as substrates2 2Ž .Starnes et al., 1990 . The reaction was stopped with3 M H SO and plates were read at 492 nm in a2 4

96-well plate reader. Recombinant mouse IL-6Ž .PeproTech, Rocky Hill, NY was used as a stan-dard.

TNF-a was measured using a capture ElisaŽ .Abrams et al., 1992 , with microtiter plates coatedwith the hamster monoclonal antibody against murine

Ž .TNF-a Genzyme , and a rabbit polyclonal antibodyŽ .against murine TNF-a Genzyme , peroxidase-con-Ž .jugated anti-rabbit IgG Sigma and with o-phenyl-Ž .enediamine and H O both Sigma as substrates.2 2

The reaction was stopped with 3 M H SO and2 4

plates were read at 492 nm in a 96-well plate reader.Ž .Recombinant murine TNF-a Genzyme was used as

a standard.

Ž 5 .Fig. 1. Influence of Tau and TauCl on luminol-dependent chemiluminescence. Neutrophils 5=10 rwell were dark-adapted in theŽ .presence of: luminol alone control , luminol and Tau, or luminol and TauCl. The chemiluminescence reaction was recorded immediately

after stimulation with zymosan over 3420 s. The figure shows the results of one of five separate experiments.


2.10. Determination of prostaglandin E2

Prostaglandin E was determined by using a2Ž .prostaglandin E EIA Kit Cayman Chemical, MI .2

2.11. Statistical analysis

Results are expressed as mean"SEM. The statis-tical significance of differences between experimen-tal groups was analysed using a factorial ANOVAŽ .Microsoft Excel followed by Scheffe’s test, if ap-propriate. The differences were regarded as signifi-cant for P-0.05.

3. Results

3.1. Influence of TauCl and Tau on neutrophil lumi-( )nol-dependent chemiluminescence LCL

Upon addition of zymosan, neutrophil LCL in-creased light emission 25 times over baseline, reach-

Table 1Effect of Tau and TauCl on myeloperoxidase activity

cw xPMNs MPO activity %a y1 6 bincubated with 10 unitsr10 cells

Ž .1 Control 2.92"0.34 100Ž . Ž .2 Tau 1 mM 3.20"0.33 109Ž . Ž .3 TauCl 1 mM 2.36"0.44 80Ž . Ž .4 TauCl 0.5 mM 2.76"0.47 94Ž . Ž .5 TauCl 0.25 mM 2.97"0.45 102

a PMNs were incubated either with Tau or TauCl. After 1 h, cellswere washed out and MPO was extracted as described in Section2.bActivity of MPO is expressed as 10y1 units of MPO per 106

cells. The limit of detection was 0.001 U of MPO. Each value isthe mean"SEM of four independent experiments performed induplicate.cActivity of MPO is expressed as a percentage of the controlgroup.

ing a maximum about 12 min after application of thestimulus. When either Tau or TauCl were added tothe reaction mixture, a dose-dependent decrease in

Ž .Fig. 2. Influence of Tau and TauCl on luminol-dependent chemiluminescence LCL from neutrophils in the presence of ABAH and SOD.Ž 5 . Ž . Ž .Neutrophils 5=10 rwell were preincubated in the dark 378C with luminol and the following reagents: taurine Tau, 1 mM , taurineŽ . Ž . Ž .chloramine TauCl, 1 mM , superoxide dismutase SOD, 300 Urml , 4-aminobenzoyl hydrazine ABAH, 50 mM . After 40 min, cells were

stimulated with zymosan, and immediately transferred to the luminometer and photon emission over 3420 s was measured. LCL fromŽ . 6 6non-stimulated and control neutrophils stimulated with zymosan was 2.7"1.4=10 and 72.9"7.6=10 counts over 3420 s,

respectively. Values are expressed as a percentage of control and represent the mean"SEM of four separate experiments. )) P-0.001TauqSOD vs. Tau; TauqABAH vs. Tau; SOD vs. control; ABAH vs. control.


Fig. 3. Inhibition of nitric oxide production by TauCl. NeutrophilsŽ 5 .2=10 rwell were stimulated with LPS and IFN-g in the pres-

Ž . Ž .ence of different concentrations of TauCl – B – or Tau – l – .Ž .L-NMMA – ' – , an inhibitor of iNOS synthase, was used as a

specific inhibitor of nitric oxide generation. After 24 h, super-natants were collected and NOy was measured as described in2

Section 2. Values are expressed as mean"SEM of five separateexperiments performed in triplicate. The production of NOy by2

Žnon-stimulated neutrophils was below the limit of detection 2.mM . ) P -0.05; )) P -0.001.

LCL was observed for both reagents. At a concentra-Ž .tion of 1 mM, the inhibitory activity of TauCl 85%

Ž .was significantly P-0.05 higher than activity ofŽ . Ž .Tau 43% Fig. 1 . To determine the duration of this

effect, PMN were pretreated with either Tau orŽTauCl for 1 h. Pretreatment of PMN with TauCl 1

.mM , followed by stimulation with zymosan, re-duced LCL, even in the absence of TauCl. In con-

Ž .trast, the effect of Tau 1 mM was short-lived andŽ .reversible data not shown .

The influence of TauCl and Tau on LCL in theŽ .presence of either superoxide dismutase SOD or

ABAH, a specific inhibitor of MPO, was also tested.

Ž .SOD 300 Urml , which almost completely destroysŽ y.superoxide anion O , inhibited LCL by 75% as2

compared to the control values. ABAH, at a concen-tration of 50 mM which inhibits MPO activity by98% but does not affect the reaction of HOCl with

Ž .Tau, was used Kettle et al., 1995 . As shown in Fig.2, the reduction of LCL by Tau was significantlyŽ .P-0.05 enhanced by both ROI inhibitors. TauClshowed synergistic inhibitory activity against LCL

Ž .only in combination with SOD 97% vs. 83% . Incontrast, the reduction of light emission by TauCl

Ž .alone 85% was similar to that shown by ABAHŽ . Ž .and TauCl together 83% Fig. 2 .

Neither Tau nor TauCl affected the uptake ofŽ .opsonized zymosan data not shown .

3.2. Effect of Tau and TauCl on MPO actiÕity

As shown in Table 1, Tau did not affect theactivity of MPO after 60 min incubation time. TauCl,

Fig. 4. Time course of inhibition of NO and TNF-a production byŽ 5 .TauCl. Neutrophils 2=10 rwell were stimulated with LPS and

Ž . Ž .IFN-g control group . TauCl 300 mM was added to the cellculture at different times after stimulation. Twenty-four hoursafter IFN-g and LPS addition, supernatants were collected fromall cultures and concentrations of NOy and TNF-a were mea-2

Žsured. Values are expressed as a percentage of control cells atcontrol group released 39"5 mM NOy; and 290"18 Urml2

.TNF-a and represent the mean"SEM of three separate experi-ments.


Table 2Time-dependent effects of TauCl exposure on production of inflammatory mediators by neutrophils

AaStimulated PMNs The release

y bw xincubated with of NO mM2

0–8 8–24 h 0–8 8–24 h

– – 0 34"4TauCl – 0 ))0– TauCl 0 29"3

BaStimulated PMNs The release The release The release

b b bw x w x w xincubated with of TNF-a Urml of IL-6 pgrml of PGE ngrml2

0–4 4–24 h 0–4 4–24 h 0–4 4–24 h 0–4 4–24 h

– – 177"34 78"16 3.5"0.5 3.3"0.4 9"1.7 18.8"2.4TauCl – )38"13 )13"5 )0.1"0.1 )0.3"0.1 ))0.7"0.06 ))1.8"0.4– TauCl 178"29 67"17 3.5"0.7 3.1"0.3 8.7"1.1 16.2"2.1

a Ž 5 . Ž . Ž . Ž .PMNs 2=10 rwell were incubated with LPSrIFN-g and TauCl 0.3 mM for 8 h A , or for 4 h B . After the incubation, cells wereŽ .washed and fresh medium without or with TauCl 0.3 mM was added. After an additional incubation, supernatants were collected and

frozen until tested.bResults are expressed as the mean"SEM of three separate experiments.) P-0.05.)) P-0.001.

Ž .only at the highest used concentration 1 mM , pro-Ž .duced a decrease of MPO activity 20% . However,

the effect was not statistically significant.

3.3. Influence of TauCl on NO generation

In this study, exposure of neutrophils to IFN-gŽand LPS resulted in the generation of nitrite 46"7

5 .mM, 2=10 cellsrwell , the end product of NOoxidation. The release of NOy from non-activated2

Ž .cells was negligible 0–4 mM .As shown in Fig. 3, TauCl inhibited the release of

NOy in a dose-dependent manner. The highest tested2Ž .concentration of TauCl 0.3 mM completely abro-

gated the generation of nitrite. Fifty percent inhibi-Ž . ytion IC of NO release was observed at approxi-50 2

mately 150 mM TauCl. Tau did not affect NOgeneration.

3.4. Time course of inhibition of NO generation byTauCl

To determine whether the inhibition of NOy re-2

lease from activated PMN by TauCl results from

inhibition of iNOS expression, similarly to its influ-Žence on NO generation in macrophages Park et al.,

.1993; Marcinkiewicz et al., 1995b or whether itresults from inhibition of NO synthase activity, we

Fig. 5. Northern blot analysis of iNOS mRNA in neutrophils.Neutrophils 5=107rgroup were incubated for 4.5 h with medium

Ž . Ž .alone lane A , with IFN-g and LPS lane B , IFN-g and LPS inŽ . Ž .the presence of Tau 0.3 mM lane C , and IFN-g and LPS in theŽ . Ž .presence of TauCl 0.3 mM lane D . The loading of wells with

RNA samples was visualised by hybridisation with a 18S rRNA-specific probe.



added TauCl to the culture of activated neutrophils atdifferent points of time. As shown in Fig. 4, TauClrevealed its full inhibitory effect only when added tothe cells at the time of activation or within 2 h after

Žthe stimulation with IFN-g and LPS inhibition by.95% . TauCl added 8 h after the activation inhibited

y ŽNO release by 52%, and when added after 22 h 22.h before the collection of supernatants , it did not

affect the level of nitrites. More convincing resultswere obtained when PMN were washed out 8 h afterthe activation, TauCl was added in the absence ofIFN-g and LPS, and NOy was then measured 24 h2

Ž .later Table 2A . This is of particular interest be-cause 6–8 h are required for generation of iNOS.TauCl added after removing the stimulators only

Žslightly inhibited NO production inhibition of nitrite.by 15% . In contrast, TauCl added to the cells

simultaneously with IFN-g and LPS and then re-moved from the culture together with the stimulators8 h later, completely inhibited the release of nitritesŽ .Table 2A .

We also investigated the effect of TauCl on NOgeneration when it was incubated with PMN and

Ž .removed just before activation. TauCl 0.3 mMpreincubated with the cells for 60 min and thenremoved, inhibited NO generation by 65%.

3.5. Inhibition of iNOS mRNA leÕels by TauCl

Northern blot analysis demonstrated that the inhi-bition of nitriternitrate accumulation by TauCl inthe culture medium of stimulated neutrophils is ac-companied by a reduced level of iNOS mRNA. Asshown in Fig. 5, TauCl at the concentration of 0.3mM completely inhibited expression of iNOS mRNAstimulated by IFN-g and LPS.

3.6. Influence of TauCl on the production of cy-tokines and PGE by neutrophils2

Stimulation of neutrophils in vitro with IFN-g andLPS resulted in a pronounced release of various

inflammatory mediators including TNF-a , IL-6 andŽPGE as measured by immunoassays Fig. 6A,B and2

.C . The activity of TNF-a and IL-6 was confirmedŽ .by bioassays data not shown . Unstimulated PMN

released 10–15% of the stimulated cell production ofcytokines and PGE .2

Tau did not influence the production of thesemediators, whilst TauCl inhibited the release of bothcytokines and PGE in a dose-dependent manner2Ž .Fig. 6 . The IC of TauCl for both cytokines was50

approximately 200 mM, and for PGE was 150 mM.2

At the highest tested concentration of 300 mM,TauCl reduced the production of all tested mediatorsto the level of unstimulated cells.

As shown in Fig. 4, TauCl completely inhibitedthe release of TNF-a only when added to the cellssimultaneously with IFN-g and LPS. TauCl added 4h after the stimulation inhibited TNF-a accumula-tion only by 43%. After an additional 4 h, the

Žintroduction of TauCl was ineffective inhibition of.TNF-a by 15–20% .

These results correlated with the kinetics of TNF-asecretion, levels of which were maximal 2–4 h after

Ž .stimulation Marcinkiewicz et al., 1995a .However, when PMNs were washed out 4 h after

the activation to remove the stimulators and thenTauCl was added, no inhibition of the release of

Žeither TNF-a , IL-6 or PGE was observed Table2.2B .

4. Discussion

At a site of inflammation, activated neutrophilsgenerate a variety of chloramines which are productsof the reaction of HOCl with primary and secondary

Ž .amines Zgliczynski et al., 1971 . The lipophilicchloramines, like NH Cl, are membrane permeant2

and can be highly toxic, whereas the hydrophilicTauCl is actively transported into phagocytic cells by

Žan uptake system and is much less cytotoxic Park et

Ž 5 .Fig. 6. Taurine monochloroamine inhibits the release of TNF-a , IL-6 and PGE from neutrophils. Neutrophils 2=10 rwell were2Ž . Ž .stimulated with LPS and IFN-g in the presence of different concentrations of TauCl – B – or Tau – l – . After 24 h, supernatants were

Ž . Ž . Ž .collected and TNF-a A , IL-6 B and PGE C were measured by ELISA. Values are expressed as mean"SEM of five experiments2

performed in triplicate. The limit of detection was 10 Urml of TNF-a , 30 pqrml of PGE , and 60 pqrml of IL-6. Non-stimulated2

neutrophils produced: 15"5 Urml of TNF-a , 0.8"0.2 ngrml of IL-6, and 2.7"0.4 ngrml of PGE .2


.al., 1993; Tatsumi and Flies, 1994 . Recently, it hasbeen demonstrated that TauCl can decrease produc-tion of tissue-damaging inflammatory mediators, aswell as simply representing the end-product of chlo-

Žride scavenging pathways Park et al., 1993;.Marcinkiewicz et al., 1995a,b; Park et al., 1995 .

Thus, TauCl represents a neutrophil-derived media-tor with a direct modulatory effect on macrophagefunction. The high physiological concentration ofTau and the stability of TauCl strengthens the poten-

Žtial impact of this chlorinated amine in vivo Learn.et al., 1990; Weiss et al., 1982 . TauCl inhibits

production of NO, TNF-a and PGE by activated2

macrophages through different mechanisms, with anŽIC value between 0.3 and 0.5 mM Park et al.,50

.1993; Marcinkiewicz et al., 1995b . TauCl inhibitstranscription of the iNOS gene, or some earlierevents in the signal transduction pathway. In con-trast, TauCl appears to suppress the translation ofTNF-a mRNA and affects the post-transcriptional

Žregulation of COX-2 the inducible form of cyclo-.oxygenase protein expression which results in a

decreased release of TNF-a and PGE molecules,2Ž .respectively Park et al., 1995; Quinn et al., 1996 .

Neutrophils, as well as macrophages, contribute tohost tissue damage in many inflammatory conditions.Moreover, neutrophils themselves may become tar-

Ž .gets of their own cytotoxic activities Smith, 1994 .For example, stimulated neutrophils undergo a pro-gressive MPO-dependent inhibition of oxygen and

Ž .glucose metabolism Wright et al., 1986 .The present study examined the possibility that

TauCl is also a modulator of neutrophil functions.Our results demonstrate that TauCl inhibits the neu-trophil-dependent secretion of NO, inflammatory cy-

Ž .tokines TNF-a , IL-6 and prostaglandin E , with an2

IC value of less than 200 mM. Thus, TauCl can act50

on neutrophils at lower concentrations than onŽ .macrophages IC s300–500 mM , perhaps as a50

result of endogenous chloramines generated by stim-ulated PMNs. The maximum inhibitory effect wasobserved when TauCl was added simultaneously withthe stimulatory agents. These data, along with thefinding that TauCl reduces the levels of iNOS mRNAand the level of iNOS protein, suggest that TauClinhibits iNOS gene expression orrand strongly de-creases iNOS mRNA stability. It is unlikely that theenzyme activity is affected, since TauCl added to the

culture at the time when iNOS is already formed didnot influence the accumulation of nitrites. The mech-anisms of inhibition of NO synthesis seem to besimilar in neutrophils and macrophages since thedata presented above are in agreement with theprevious study of the influence of TauCl on NO

Ž .generation in RAW 264.7 cells Park et al., 1995Žand in mouse peritoneal macrophages Marcinkie-

.wicz et al., 1995b .Along with the strong inhibitory activity of TauCl

on NO generation, we have shown inhibition ofTNF-a , IL-6 and PGE . The maximum inhibitory2

effect was observed when TauCl was added at thetime of stimulation. It is of interest that TauClreduces the production of all mediators tested to thelevel observed in non-stimulated PMNs, perhapssuggesting that TauCl interferes with the generationof these mediators, but is not able to inhibit theactivity of already existing proteins.

Our results confirm the hypothesis that TauCl, aproduct of reaction of Tau and HOCl, down-regu-lates the release of inflammatory mediators gener-

Ž .ated by activated phagocytes Marcinkiewicz, 1997 .This idea is also supported by Tatsumi and FliesŽ .1994 who have shown that neutrophil-dependentenhancement of endothelium permeability is attenu-ated by TauCl. In all these situations, Tau alone wasnot effective. However, Tau itself may act as ananti-inflammatory agent by scavenging HOCl, themicrobicidal and tissue-damaging product of the

ŽMPO–H O –halide system Cantin, 1994; Wright et2 2.al., 1986; Stapelton et al., 1997 . HOCl, as well as

Ž y.superoxide anion O , contributes to LCL of neu-2Žtrophils Albrecht and Jungi, 1993; Jungi and Peter-.hans, 1988 . In this study, we reported that both Tau

and TauCl reduced LCL in a dose-dependent man-ner. A similar observation has been described re-

Ž .cently by Kim et al. 1996 , who showed that Tauand TauCl inhibited Oy production by stimulated2

PMN, with Tau being effective at higher concentra-tions then TauCl. These authors claimed that theeffects of Tau on Oy production could be attributed2

to the in vitro formation of TauCl catalysed byPMN-associated halide-dependent MPO. Our resultsdo not confirm this suggestion. In our system, TauClinhibited LCL by mechanisms distinct from those ofTau, as indicated by the differential effects of ROIinhibitors and scavengers. It is also unlikely that


TauCl, formed in vitro from exogenous Tau, is re-sponsible for the inhibition of LCL, since the combi-nation of Tau and an inhibitor of MPO gave asynergistic effect. Further investigations are neces-sary to resolve this problem in detail.

The data presented above strongly suggest that theproducts of the MPOrchlorination pathway, espe-cially Tau, can act as immunological modifiers. Earlyin the acute phase of inflammation, the dominantcomponents of the pathway may be HOCl and otherhighly reactive and cytotoxic chlorine-derived oxi-dants, which will contribute to the overallneutrophil-dependent inflammatory reaction. How-ever, when the acute response continues, the longer-lived products of the MPOrchloride pathway willbegin to dominate. Chlorinated antigens will be takenup by local antigen presenting cells, and contributeto an enhanced activation of antigen-specific T cellsŽ .Marcinkiewicz et al., 1991, 1992 . Simultaneously,TauCl levels will build up, and this compound willdown-regulate the production of neutrophilrmacro-phage inflammatory mediators. TauCl may thereforeplay a role in maintaining the delicate balance be-tween mounting an effective immune response onone hand, and minimising the destructive effect ofthe inflammatory cells on the other.

Acknowledgements

We express our gratitude to Ms. Malgorzata Bobekfor technical assistance. This work was supported bya grant from Committee of Scientific ResearchŽ .Warsaw, Poland and a grant from the WellcomeTrust Foundation.

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Taurine chloramine down-regulates the generation of murine neutrophil inflammatory mediators