Is there a role of taurine bromamine in inflammation? Interactive effects with nitrite and hydrogen peroxide

Article abstract of DOI:10.1007/s00011-004-1322-9

Objective and Design: The myeloperoxidase system of neutrophils generates chlorinating and brominating oxidants in vivo. The major haloamines of the system are taurine chloramine (TauCl) and taurine bromamine (TauBr). It has been demonstrated in vitro that TauCl exerts both antiinflammatory and anti-bacterial properties. Much less is known about TauBr. The present study was conducted to compare bactericidal and immunoregulatory capacity of TauBr with that of the major chlorinating oxidants: HOCl and TauCl. Moreover, the effect of nitrites and H₂O₂ on TauBr activity was investigated. Materials: TauBr was prepared by reaction of HOBr with taurine. The reaction was monitored by UV absorption spectra. Methods: Bactericidal activity of TauBr, TauCl and HOCl was tested by incubation of E. coli with the compounds and determined by the pour-plate method. To test the anti-inflammatory activity the compounds were incubated with LPS and IFN-γ stimulated murine peritoneal macrophages. The production of following mediators was measured: nitrites by Griess reaction; TNF-α, IL-6, IL-10, IL-12p40 using capture ELISA. In some experiments the compounds were incubated with either nitrites or H ₂O₂. Results: In our experimental set-up TauBr and HOCl exerted strong bactericidal effects on E. coli (MBC = 110 μM and 8 μM, respectively), while TauCl (< 1000 μM) did not kill test bacteria. However, both, TauBr and TauCl, at noncytotoxic concentrations (< 300 μM) inhibited the cytokine and nitric oxide production by macrophages. H ₂O₂ completely abolished the biological activities of TauBr but not those of TauCl. Nitrites did not affect any activity of TauBr or TauCl while they diminished the HOCl^- mediated bacterial killing. Conclusion: TauBr, despite very low concentration of Br^- in body fluids, may support TauCl and HOCl in the regulation of inflammatory response and in killing of bacteria by neutrophils. However, TauBr activity in vivo will depend on the presence of H₂O₂ and possible other mediators of inflammation which can compete with target molecules for TauBr. Birkhaeuser Verlag, Basel, 2005.

Full text of DOI:10.1007/s00011-004-1322-9

© Birkhäuser Verlag, Basel, 2005
Inflamm. res. 54 (2005) 42–49
1023-3830/05/010042-08
DOI 10.1007/s00011-004-1322-9
Inflammation Research
Is there a role of taurine bromamine in inflammation?
Interactive effects with nitrite and hydrogen peroxide
J. Marcinkiewicz1, M. Mak1, M. Bobek1, R. Biedron´ ,A. Białecka2, M. Koprowski1, E. Kontny3 and W. Mas´lin´ ski3
1
Department of Immunology, Jagiellonian University Medical College, 18 Czysta St., 31-121 Craców, Poland, Fax: ++4812 633 94 31,
e-mail: mmmarcin@cyf-kr.edu.pl
Center of Microbiological Research and Autovaccines Ltd., Cracow, Poland,
Department of Pathophysiology and Immunology, Institute of Rheumatology,Warsaw, Poland
2
3
Received 16 August 2004; returned for revision 16 September 2004; accepted by A. Falus 13 October 2004
Abstract. Objective and Design: The myeloperoxidase sys-
tem of neutrophils generates chlorinating and brominating
oxidants in vivo. The major haloamines of the system are tau-
rine chloramine (TauCl) and taurine bromamine (TauBr). It
has been demonstrated in vitro that TauCl exerts both anti-
inflammatory and anti-bacterial properties. Much less is
known about TauBr. The present study was conducted to
compare bactericidal and immunoregulatory capacity of
TauBr with that of the major chlorinating oxidants: HOCl
andTauCl. Moreover, the effect of nitrites and H2O2 onTauBr
activity was investigated.
presence of H2O2 and possible other mediators of inflamma-
tion which can compete with target molecules for TauBr.
Key words: Inflammation – Neutrophils – Macrophages –
MPO-halide system – Taurine bromamine – Taurine
chloramine – Hydrogen peroxide – Bactericidal activity –
Cytokine secretion
Introduction
Materials: TauBr was prepared by reaction of HOBr with tau-
rine. The reaction was monitored by UV absorption spectra.
Methods: Bactericidal activity of TauBr, TauCl and HOCl
was tested by incubation of E. coli with the compounds and
determined by the pour-plate method. To test the anti-inflam-
matory activity the compounds were incubated with LPS-
and IFN-g stimulated murine peritoneal macrophages. The
production of following mediators was measured: nitrites by
Griess reaction; TNF-a, IL-6, IL-10, IL-12p40 using capture
ELISA. In some experiments the compounds were incubated
with either nitrites or H2O2.
Activated neutrophils and eosinophils generate a variety of
reactive oxygen intermediates (ROI) including hypohalous
acids (HOCl, HOBr), the products of peroxidase-halide sys-
tem [1–4]. Myeloperoxidase (MPO), the heme enzyme syn-
thesized/secreted by neutrophils and monocytes, uses H2O2
and Cl– to produce hypochlorous acid (HOCl), its major ini-
tial product [5, 6].
Cl– + H2O2+ H+ Æ HOCl + H2O
Results: In our experimental set-up TauBr and HOCl exert-
ed strong bactericidal effects on E. coli (MBC = 110 mM
and 8 mM, respectively), while TauCl (< 1000 mM) did not
kill test bacteria. However, both, TauBr and TauCl, at non-
cytotoxic concentrations (< 300 mM) inhibited the cytokine
and nitric oxide production by macrophages. H2O2 com-
pletely abolished the biological activities of TauBr but not
those of TauCl. Nitrites did not affect any activity of TauBr
or TauCl while they diminished the HOCl– mediated bacter-
ial killing.
HOCl is an unstable, highly reactive oxidant with strong bac-
tericidal activity. MPO-halide system generates also sec-
ondary oxidants such as mono-chloramines, di-chloramines
and amino acid-derived aldehydes [7, 8]. As taurine is the
most abundant free amino acid in leukocyte cytosol [9], the
major chloramine generated by activated neutrophils is tau-
rine chloramine (N-chlorotaurine, TauCl) [8]. TauCl is less
toxic than HOCl, long lived oxidant with well documented in
vitro microbicidal activity [10–12]. Concerning biological
functions, it is assumed that the formation of TauCl protects
body cells from damage by HOCl [8, 13]. Moreover, data
from several laboratories demonstrate that TauCl is a power-
ful regulator of inflammation [14–17]. TauCl exerts anti-
inflammatory properties by suppressing the production of
such mediators as nitric oxide, PGE2, TNF-a, IL-6, IL-8, IL-
12 and chemokines in both rodent and human leukocytes
[14–18]. Studies investigating the mechanisms of action of
Conclusion: TauBr, despite very low concentration of Br– in
body fluids, may support TauCl and HOCl in the regulation
of inflammatory response and in killing of bacteria by neu-
trophils. However, TauBr activity in vivo will depend on the
Correspondence to: J. Marcinkiewicz

Vol. 54, 2005
N-bromotaurine and inflammation
43
TauCl have shown that it inhibits the activation of NF-kB, a
potent signal transducer for inflammatory cytokines, by oxi-
dation of IkB-a at Met45 [19].
of TauCl and/or HOCl, the major products of MPO-halide
system. Moreover, the effect of nitrite (NO–2) and hydrogen
peroxide (H2O2) on the activity of TauBr has been tested.
Eosinophil peroxidase (EPO), a heme protein structural-
ly related to MPO, is released by activated eosinophils, which
helps to kill invading parasites [4]. At plasma concentrations
of halide (100 mM chloride; 20–100 mM bromide; < 1 mM
iodide) eosinophil peroxidase preferentially oxidizes bro-
mide (Br–) to produce hypobromous acid, HOBr [4].
Materials and methods
Preparation of taurine bromamine (TauBr) and taurine
chloramine (TauCl)
Br– + H2O2 + H+ Æ HOBr + H2O
TauCl was prepared by dropwise addition of 5 ml of 20 mM NaOCl
(Aldrich, Steinham, Germany) solution in 0.05 M phosphate buffer (pH
7.4), with vigorous stirring, to 5 ml of 24 mM taurine (Tau) (Sigma, St.
Louis MO). Each preparation of TauCl was monitored by UV absorp-
tion spectra (l = 200 to 400 nm) to assure the authenticity of mono-
chloramine (TauCl) (lmax = 252 nm) and the absence of dichloramine
(TauCl2) (lmax = 300 nm) and unreacted HOCl/OCl– (lmax = 292 nm).
The concentration of synthesizedTauCl was determined using the molar
extinction coefficient 429 M–1 cm–1 at A252 [23].
Only recently it has been published that HOBr may be also
generated by the myeloperoxidase system of neutrophils [4, 7].
HOCl + Br– Æ HOBr + Cl–
HOBr is a weaker oxidant than HOCl, but exerts even
stronger microbicidal and cytotoxic activity [3, 4, 21, 22]. At
physiological pH, both HOCl and HOBr react readily with
amines to form haloamines (chloramines, bromamines) and
with the unsaturated bonds of fatty acids to form halohydrins
[7, 10, 23]. In studies with isolated leukocytes, evidence for
the formation of brominating agents was obtained, but long
lived bromamines were not detected in the medium. Reduc-
tion of HOBr– and bromamines by H2O2 may account for
these results [20]. Nevertheless HOBr reacts with amino
acids to form bromoamines, and Taurine bromamine (N-bro-
motaurine, TauBr) is considered to be the major product of
these reactions [3, 4]. In contrast to large body of evidence
demonstrating the role of TauCl in inflammation [13–19,
24], much less is known about biological properties of TauBr
[25, 26].
TauBr preparation: HOBr/OBr– was generated by mixing equimo-
lar amounts of HOCl– and NaBr–. Than, the product of this reaction was
mixed with taurine. The mono-bromamine was obtained with a 10-fold
excess of amine over the amount of HOBr/OBr–. Each preparation of
TauBr was monitored by UV absorption spectra (l = 200 to 400 nm) to
assure the authenticity of monobromamine (TauBr). Taurine bro-
mamines (mono-, di-bromamine) have absorption spectra similar to
those of chloramines, but shifted 36 nm towards longer wavelengths.
TauBr and TauBr2 give UV spectra with lmax = 288 nm and lmax = 241 nm,
respectively [4].
The concentration of synthesized TauBr was determined using the
molar extinction coefficient 415 M–1 cm–1 at A288.
Stock solutions of TauCl and TauBr were kept at 4 °C for maximum
5 days before use.
Preparation of bacteria
Activated eosinophils as well as neutrophils can simulta-
neously generate a variety of reactive oxygen species (ROS)
and nitric oxide (NO) [1, 27]. It has been demonstrated that
leukocyte derived oxidants such as nitric oxide (NO/NO2–)
and hydrogen peroxide (H2O2) differentially react with
HOCl, HOBr and their derivatives [28, 29]. For example,
HOCl, HOBr and bromamines are reduced by H2O2 while
chloramines are resistant [4, 20].
E. coli (EC) (ATCC 25922) was grown in Trypticase soy broth (Difco,
Detroid, MI) at 37 °C for 24 h. Bacteria were centrifuged at 1800 ¥ g,
washed twice in 0.9% NaCl, and diluted in saline to a concentration of
1 ¥ 108 CFU/ml before use.
Bactericidal activity of TauBr, TauCl and HOCl
Basing on the kinetics studies of bacterial killing by neutrophils [12]
and our preliminary experiments demonstrating time- and dose-depen-
dent bactericidal and cytotoxic activity of TauBr, we have established
experimental conditions for this study. In our experimental set-up, at
concentrations up to 1 mM, neither H2O2, NO2– nor taurine affected via-
bility of bacteria. Briefly, bacteria were suspended and further diluted
in 0.2 M phosphate buffer (pH-7.4) to achieve a final concentration of
1 ¥ 105 CFU/ml and then were incubated with different concentrations
of the components (1 – 1000 mM). Immediately after the incubation
(30 min.), aliquots were removed and the viable cell count was deter-
mined by the pour-plate method [28]. Control samples (bacteria dilut-
ed in the buffers only), were treated the same way. Bactericidal activi-
ty of TauBr, TauCl and HOCl are expressed either as a log CFU/ml of
bacteria survived at the indicated concentration of the component or as
MBC (minimal bactericidal concentration), the lowest concentration of
the component at which 100% of bacteria were killed.
HOBr + H2O2 Æ H2O + H+ + Br– + O2
NO and its more stable metabolites, nitrite (NO2–) and nitrate
(NO3–) may also react with hypohalous acids to form novel
biologically active species [30]. We have previously shown
that nitrite, the product of NO degradation, inhibits bacteri-
cidal activity of HOCl [24]. On the contrary, nitric oxide
potentiates hydrogen peroxide-induced killing of Escheri-
chia coli, as described by others [31]. Thus, the interactions
between ROS, NO and MPO-halide system products may
results in both the enhancement and the inhibition of neu-
trophil bactericidal – cytotoxic activities determining the bal-
ance between immune defence and host injury.
The bactericidal and immunoregulatory activities of
TauBr in the presence of ROS have never been investigated
systemically.
The present study was conducted to evaluate some bio-
logical functions of TauBr. We have compared bactericidal,
cytotoxic and anti-inflammatory potency of TauBr with that
Mice
Inbred Balb/c male mice from the breeding unit, Department of
Immunology, Jagiellonian University Medical College, Cracow, Poland
were used between 6 and 8 weeks of age. The authors were granted per-
mission by the Local Ethics Committee to use mice in this study.

44
J. Marcinkiewicz et al.
Inflamm. res.
Mingen, San Diego, CA, USA) were used as detecting antibodies.
Recombinant mouse TNF-a (Sigma, Steiham, Germany) was used as a
standard. The limit of detection was 30 pg TNF-a/ml.
Macrophages
Peritoneal mouse macrophages (Mf) were induced by intraperitoneal
injection of 1.0 ml of paraffin oil (Sigma, St. Luis, MO, USA). Cells
were collected 48 h later by washing out the peritoneal cavity with
5 ml of PBS (phosphate buffer solution) containing 5U heparin/ml
(Polfa, Warsaw, Poland). Cells were centrifuged and red blood cells
were lysed by osmotic shock using distilled water; osmolarity was
restored by addition of 2¥ concentrated PBS. The presence of
macrophages (85–90%) was judged by cytochemical demonstration of
non-specific esterase-positive mononuclear cells, using a-naphtyl
acetate (Sigma).
Determination of H2O2 production
H2O2 detection was based on the oxidation of phenol red by H2O2 in the
presence of horseradish peroxidase (HRPO) [32]. Briefly, samples were
suspended in an assay solution containing 0.56 mM of phenol red and
20 U/ml HRPO (both Sigma, Steiham, Germany) in phenol red-free
HBSS medium and seeded in tissue culture plates in a final volume
of 100 ml per well. After incubation for 2 h the reaction was stopped
by addition of 10 ml of 1N NaOH per well and absorbance was read at
600 nm. Wells with NaOH added at the beginning of testing were used
as blanks. H2O2 concentration was calculated from H2O2 (Sigma, Stei-
ham, Germany) standard curve.
Cell culture. Activation of cells for production
of inflammatory mediators
Mf were cultured in 24-well flat-bottom cell culture plates at 5 ¥ 105/
well in RPMI 1640 medium (JR Scientific Inc., Woodland, CA) sup-
plemented with 5% FCS, at 37 °C in an atmosphere of 5% CO2. Cells
were activated with 20 U/ml of IFN-g (Sigma, Steiham, Germany) and
100 ng/ml of LPS (E. coli 0111 B:4, Sigma Steiham, Germany) and
cultured in the presence of test agents. After 24 h culture supernatants
were collected and frozen at –80 °C until used.
Statistical analysis
If not otherwise stated the statistical significance of differences between
groups was analysed using a factorial ANOVA (Microsoft Excel) fol-
lowed by Student’s test, if appropriate. Non-parametric statistical analy-
ses (Mann-Whitney U test) were done using Statistica PLTM v. 6.0 (Stat-
Soft, Poland). Results are expressed as mean SE. A p-value less than
0.05 was considered statistically significant.
Measurement of cell viability
Viability of the cells was routinely monitored by cellular exclusion of
trypan blue. In some experiments cell respiration, an indicator of cell
viability, was assessed by mitochondrial-dependent reduction of MTT
to formazan. Cells in 96-well plates were incubated at 37°C with MTT
(0.2 mg ml–1 for 60 min). Then, culture medium was removed by aspi-
ration and cells were solubilized in DMSO (200 ml). The extent of
reduction of MTT to formazan within cells was quantitated by mea-
surement of absorbance at 550 nm.
Results
Stability of TauBr
To determine the stability of TauBr in our experimental set-
up, UV spectrum of TauBr was analysed. TauCl was used as
a reference agent. Time course analysis has shown similar
stability of TauBr and TauCl at room temperature for 24 h
when incubated alone in a phosphate buffer. At higher tem-
perature (37 °C) the concentration of TauBr decreased by
about 20%, while TauCl remained unchanged (Fig. 1).
Cytokines determination
Cytokine concentrations in culture supernatants were measured using
capture ELISA. For IL-6, IL-10, IL-12 determination 96-well plates
(Corning, NY, USA) were coated overnight with rat mAb against a
mouse cytokine (capture antibody). After blocking the plates with 4%
albumin (2 h) or 3% milk standards and tested supernatants were added
and incubated overnight. Finally, plates were coated with biotinylated
antibodies against the same cytokine detecting antibody for 1h. The
ELISA was developed with horseradish peroxidase streptavidin (Vector,
Burlingame, CA, USA), followed by o-phenylenediamine and H2O2
(both Sigma, Steiham, Germany) as substrates 30 min. The reaction was
stopped with 3 M H2SO4 and the optical density of each well at 492 nm
was measured in a plate reader.
IL-6: Rat anti-IL-6 mAbs, clone: MP5-20F3 (2 mg/ml) and biotiny-
lated rat anti-IL-6, mABs clone: MP5-32C11 (0.5 mg/ml) (both Phar
Mingen, San Diego, CA, USA) were used as detecting antibodies.
Recombinant mouse IL-6 (PeproTech Rocky Hill, NewYork, USA) was
used as a standard. The detection limit was about 15 pg IL-6/ml.
IL-10: Rat anti-mouse IL-10 mAbs, clone: JES5-2A5 (5 mg/ml)
and biotinylated rat anti-mouse IL-10 mAbs, clone: JES5-16E3 (2 mg/
ml) (PharMingen, San Diego, CA, USA) were used as detecting anti-
bodies. Recombinant mouse IL-10 (Genzyme, Cambridge, UK) was
used as a standard. The limit of detection was 30 pg IL-10/ml.
IL-12 p40: Rat anti-mouse IL-12 (p40p70) mAbs, clone C15.6
(2 mg/ml) (PharMingen, San Diego, CA, USA) and biotinylated rat anti-
mouse IL-12 mAbs, clone: C17.8 (1 mg/ml) (Endogen, Woburn, MA,
USA) were used detecting antibodies. Recombinant mouse IL-12 (Gen-
zyme, Cambridge, UK) was used as a standard. The detection limit was
about 30 pg IL-12 p40/ml.
Fig. 1. Stability of TauBr and TauCl in phosphate buffer (pH 7.4) at dif-
ferent temperatures. TauBr and TauCl were monitored by UV absorp-
tion spectra and the concentrations of taurine monobromamine and tau-
rine monochloramine were estimated as described in Methods. The
results are the means SE of 3 separate experiments.
TNF-a: Rat anti-mouse TNF-a mAbs, clone: TN3-19.12 (2 mg/ml)
and biotinylated rabit anti-mouse/rat TNF-a pAbs (1.6 mg/ml) (Phar

Vol. 54, 2005
N-bromotaurine and inflammation
45
Table 1. Interaction of TauBr with nitrite and hydrogen peroxide.
(A) Nitrite
(B) Hydrogen peroxide
100 mM of H2O2 incubated with:
None (control)
100 mM of NO–2 incubated with:
Recovery of NO–2 (%)
100
Recovery of H2O2 (%)
100
None (control)
TauBr 1000
TauBr 500
TauBr 250
TauBr 100
TauBr 50
84
94
97
100
100
3
3
1
1
1
TauBr 1000
TauBr 500
TauBr 250
TauBr 100
TauBr 50
0
0
3
1
3
8
58 5**
TauBr 25
77 3*
HOCl 100
TauCl 100
Tau 1000
10 2**
HOCl 100
TauCl 100
Tau 1000
< 2**
95
2
99
3
101
4
97
3
The reaction mixture contained: 100 mM NO–2 (A); or 100 mM H2O2 (B) and TauBr, TauCl, Tau or HOCl at the concentration indicated (mM). Sam-
ples were incubated in 0.2 M phosphate buffer (pH 7.4). After 1 h, the level of NO–2(A) or H2O2 (B) was measured as described in Methods. The results
are the mean SE of five separate experiments. *p < 0.05; **p < 0.001 vs. control.
Fig. 3. Bactericidal activity of TauBr, TauCl and HOCl against E. coli
(EC). Bacteria (1¥105/ml) were incubated with different concentrations
of either TauBr, TauCl or HOCl at phosphate buffer pH.7.4 for 30 min.
The reduction of bacteria growth was estimated as described in Meth-
ods. Each point on the killing curves represents the mean SE calcu-
lated from 12 separate experiments.
contrast to TauCl, HOCl consumed H2O2 completely when
both reagents were used at the equimolar concentrations.
Interactions of TauBr with nitrite (NO2–)
Figure 2 shows that at neutral pH, the absorption spectrum of
TauBr was not changed by nitrite when both reagents were
used at the equimolar concentrations. We also tested a con-
sumption of nitrites by TauBr, HOCl or TauCl by measuring
NO2– levels in the reaction mixture. As shown in Table 1 (the
bolded lines), neitherTauBr norTauCl, when used at equimo-
lar concentrations, decreased the level of NO2–. By contrast,
nitrites were consumed by HOCl.
Fig. 2. The UV absorption spectra of TauBr incubated with equimolar
concentrations of either NO2– or H2O2 in 0.2 M phosphate buffer at pH 7.4.
Samples of controlTauBr were incubated in the buffer alone.After 30 min
at 23 °C, absorption spectra were measured against blanks without TauBr.
Interactions of TauBr with hydrogen peroxide (H2O2)
Figure 2 indicates that at pH 7.4, TauBr reacts directly with
H2O2 to produce new compounds with no absorption in the
UV spectrum between 210–400 nm. The analysis of H2O2
concentrations at the reaction mixture shows the consump-
tion of H2O2 by TauBr (Table 1). The reaction is well known:
TauBr + H2O2 Æ Tau + H+ + Br– + O2.
Bactericidal activity of TauBr, TauCl and HOCl
TauBr bactericidal activity was tested at pH 7.4, at concen-
trations ranging from 1 to 1000 mM. In our experimental
set-up TauBr exerted strong bactericidal activity to E. coli
(MBC ~ 100 mM) (Fig. 3). The bactericidal activity of HOCl
was significantly higher as compared toTauBr (MBC ~8 mM).
Unlike TauBr, TauCl is not reduced by H2O2 [4]. As
shown in Table 1, TauCl did not affect the level of H2O2. In

46
J. Marcinkiewicz et al.
Inflamm. res.
Table 2. The role of MPO-halide system products in inflammation.
Agent
Biological activity
Immune defense
Host injury
Micro-
bicidal
Immuno-
regulatory
Cytotoxic [Ref.]
HOCl
TauCl
HOBr
TauBr
H2O2
(+++)
( )
(+++)
(++)
(–)
(–)
(+)
(?)
(+)
(+)
(++)
(–)
(+++)
(–)
[1, 5, 33]
[11, 14, 15]
[7, 10, 22]
[4, 25, 26]
[1, 5, 31]
A
( )
The hypothetic role of MPO-halide system products at a site of inflam-
mation as suggested by the data from in vitro studies in which MPO-
halide system products were tested at physiological concentrations
(< 300 mM).
TauCl, in contrast to TauBr and HOCl, at these experimental
conditions (< 1000 mM) did not kill E. coli bacteria. So, in
further experiments bactericidal activity of TauBr was com-
pared only to that of HOCl.
B
Fig. 4. Influence of nitrite (A) and H2O2 (B) on the bactericidal activi-
ty of TauBr and HOCl against EC. TauBr and HOCl were pre-incubated
with equimolar concentrations of either NO2– or H2O2. After 30 min of
the incubation at pH 7.4, bacteria (EC) were added (1¥105/ml) and bac-
tericidal activity of the test samples was estimated as described in the
Methods. Nitrites (A) and H2O2 (B) were examined in the separate sets
of experiments. The results are shown as MBC values (the concentration
that give 100% killing of bacteria) taken from 3 independent experi-
ments for each panel. Mann-Whitney U test was used to test the differ-
ences in numerical data between two independent groups. Medians
along with interquartile ranges depicted all variables. *p < 0.05
Nitrites and H2O2 differentially affect the bactericidal
activities of TauBr and HOCl
Under the conditions used, neither nitrite nor hydrogen per-
oxide exerted bactericidal activity when incubated alone with
the test bacteria (data not shown). Interestingly, nitrite and
H2O2 differentially affected bactericidal activity ofTauBr and
HOCl.
Fig. 5. Effect of TauBr and TauCl on cytokine production by macrophages. Macrophages, stimulated with LPS and IFN-g were incubated with differ-
ent concentrations of either TauCl or TauBr. The production of TNF-a (A), IL-6 (B), IL-10 (C) and IL-12p40 (D) was estimated as described in Meth-
ods. Results are expressed as percentage of the cytokine production by control macrophages. Data were calculated from 11 separate experiments. The
production of cytokines by macrophages stimulated with LPS and IFN-g (control group): TNF-a = 977 449 pg/ml; IL-6 =16.7 5.7 ng/ml; IL-10
= 320 166 pg/ml; IL-12p40 = 7.2 3.7 ng/ml. Taurine alone did not affect the production of cytokines (data not shown). The statistical significance
of differences between groups was analysed using a factorial ANOVA. *p < 0.05; **p < 0.01 (Experimental groups vs. control)

Vol. 54, 2005
N-bromotaurine and inflammation
47
A
Fig. 6. The effect of H2O2 interactions with TauBr on nitrite generation
by macrophages. TauBr was pre-incubated with equimolar concentra-
tions of H2O2. After 30 min of the incubation at pH 7.4, the test agents,
at indicated concentrations, were added to macrophages stimulated with
LPS and IFN-g. After 24 h, supernatants were collected and nitrites
were measured using Griess reagents (see Materials and Methods).
Results are the mean SE of five separate experiments. The statistical
significance of differences between groups was analysed using a fac-
torial ANOVA. *p < 0.05; **p < 0.01 (TauBr vs. TauBr + H2O2)
As shown in Fig. 4B, at neutral pH bactericidal activity of
both HOCl and TauBr to EC was completely neutralized by
H2O2 used at equimolar concentrations.
Nitrite also affected bactericidal activity of the test
agents, however the effect was different from that evoked by
addition of H2O2. The bactericidal activity of HOCl was sig-
nificantly inhibited by nitrite. On the contrary, the effect of
nitrite on the bactericidal activity of TauBr was negligible
(Fig. 4A).
B
Fig. 7. The effect of nitrite and H2O2 interaction with TauBr on IL-10
production by activated macrophages. TauBr was pre-incubated with
equimolar concentrations of either NO2– or H2O2. After 30 min of the
incubation at pH 7.4, the test agents, at indicated concentrations (A –
100 mM; B – 300 mM), were added to the culture of macrophages stim-
ulated with LPS. After 24 h supernatants were collected and the level
Effect of TauBr and TauCl on the release of cytokines
by stimulated macrophages
of IL-10 was measured by ELISA. Results are the mean
three separate experiments. *TauBr vs TauBr + H2O2 (A) p < 0.05;
(B) p < 0.001
SE of
To determine TauBr cytotoxic activities against cells
involved in inflammation, viability of macrophages incubat-
ed with TauBr (1–1000 mM) was tested. In our experimental
set-up, TauBr, at concentrations up to 300 mM did not affect
viability of macrophages. In further experiments macropha-
ges were cultured with TauBr and TauCl used at non-cyto-
toxic concentrations (50–300 mM). At these concentrations,
HOCl shows cytotoxic effect on macrophages [24].
As shown in Fig. 5, TauBr andTauCl exerted similar, dose
dependent, inhibitory effects on cytokine production by peri-
toneal macrophages stimulated with LPS and IFN-g. No sta-
tistically significant differences were found in the inhibitory
activities of 100 mM of TauBr against TNF-a , IL-6, IL-10
and IL-12 p40. At the highest, non-cytotoxic concentration
TauBr differentially affected the cytokine production. TauBr
showed the strongest inhibitory activity against IL-10, while
TNF-a production was inhibited the least.
influence of the test agents on generation of selected inflam-
matory mediators by activated macrophages was studied. As
shown in Fig. 6 TauBr and H2O2 exerted similar, dose-depen-
dent inhibitory effect on nitrite production by macrophages.
Interestingly, the mixture of TauBr and H2O2, used at the con-
centrations in which both agents separately exerted suppres-
sive activity, did not inhibit the production of NO2–. TauCl has
not been used as a reference agent in these experiments since
TauCl does not react with H2O2 .
Figure 7 shows the effect of NO2– and H2O2 interactions
with TauBr on IL-10 production. As expected, NO2– did not
affect the suppressive activity of TauBr on IL-10 release. On
the contrary, such suppressive activity of TauBr was com-
pletely neutralised by H2O2 when these agents were added
simultaneously at equimolar concentrations. The same effect
was observed when the production of TNF-a, IL-6 and IL-12
was tested (data not shown).
Nitrites and H2O2 differentially affect the immunoregulatory
potency of TauBr
To determine whether NO2– and H2O2 affect bactericidal and
immunoregulatory activities of TauBr in a similar way, the

48
J. Marcinkiewicz et al.
Inflamm. res.
that physiologically plausible variations in Br– concentration
will support neutrophil dependent defence system
(HOCl/TauCl) by formation of HOBr and TauBr, very
strong bactericidal agents. Importantly, TauBr, in contrast to
HOCl, at bactericidal concentrations (< 200 mM) does not
exert cytotoxic activity. The contribution of chlorinating and
brominating oxidants in bacterial killing, in the regulation of
inflammatory cells function and in tissue injury will also
depend on the interactions with other biologically active
agents present at a site of inflammation. For example, activ-
ity of HOCl may be neutralised by both, nitrites and H2O2
[28, 33], while activity of TauBr is affected only by the pres-
ence of H2O2 [4, 7].
In the present study we have also examined immunoreg-
ulatory potential of TauBr. The ability of TauBr to suppress
the production of inflammatory mediators by activated
macrophages was compared with that of TauCl. Both taurine
haloamines inhibited the generation of nitrites and cytokines
(TNF-, IL-6, IL-12, IL-10) in a similar, dose-dependent man-
ner. These results are in agreement with our recent findings
[26]. We have shown that TauBr and TauCl can induce
expression of heme oxygenase-1 (HO-1), a stress inducible
protein, in both non-activated and LPS-activated macro-
phages. Importantly, dose-dependent induction of HO-1 was
associated with a concomitant fall in NOS-2 protein level.
Thus, at a site of inflammation, TauCl andTauBr may provide
a link between taurine-dependent and HO-1-dependent cyto-
protective mechanisms.
In conclusion, the present study, together with previous
reports, suggests that TauBr may be a part of neutrophil
defence system [7]. TauBr is generated by neutrophil MPO-
halide system and at low non-toxic concentrations exerts
in vitro bactericidal activity, even stronger than TauCl.
Now, TauBr contribution to pathogen killing by phagocytes
in vivo remains to be established. TauBr shows similar to
TauCl capacity to modulate inflammation by induction of
heme oxygenase expression and by inhibition of inflamma-
tory mediators generation by activated macrophages. Thus,
bromide may be a previously unexpected, but important
component of neutrophil activity at a site of inflammation.
On the other hand, TauBr may be easily neutralised by
hydrogen peroxide generated by both bacteria and phago-
cytes at a site of inflammation. Nevertheless, further studies
are necessary to compare the effect of TauBr and TauCl on
the balance between pro-(TNF-a) and anti-(IL-10) inflam-
matory cytokines production by the cells engaged in inflam-
mation.
Discussion
Although the biological activities of both chlorinating and
brominating oxidants in vitro are in general well charac-
terised, the role of the latter in acute inflammatory response
is still the matter of discussion. For years bromide has attract-
ed little attention because its extracellular concentration is at
least 1,000-times lower than that of Cl– [1, 6]. Thus, it could
imply that in vivo chlorinating oxidants will strongly com-
pete with brominating oxidants limiting their physiological
role to minimum. Meanwhile, it has been reported that neu-
trophil myeloperoxidase generates chlorinating and bromi-
nating oxidants; the production of the latter was previously
ascribed solely to eosinophil peroxidase [4]. Moreover, it has
been shown that the bromination pathways operate when Cl-
concentrations are 1,000-times to 10,000-times higher than
Br– concentrations [7, 20]. Therefore, the bromination path-
ways may be physiologically relevant.
In this study we have compared some biological activi-
ties of micromolar concentrations of TauBr with these of
HOCl and TauCl, the major products of MPO-halide system.
These oxidants, generated by monocytes and neutrophils at
sites of inflammation, can be involved both in a host defence
mechanism and as a means of tissue injury [2, 5, 22]. How-
ever, their activity in vivo will depend not only on the local
concentrations, but also on their reactions with other reac-
tive species which may result in the formation of products
with new biological activities [24, 28, 30]. Indeed, our study
confirmed previous reports [4] that at physiological pH (pH
7.4) TauBr, but not TauCl, is decomposed by H2O2 to form
non active products. On the other hand, nitrite (NO2–), a
major end-product of nitric oxide metabolism, does not react
with TauBr and TauCl. Nitrite, however, competes with tau-
rine for reaction with HOCl to form nitryl chloride (NO2Cl)
[24, 30]. Thus, it is reasonable to speculate that the balance
between H2O2 and nitrite at sites of inflammation will favour
either chlorinating or brominating oxidants (e.g TauCl vs
TauBr). The concentration of MPO-halide system products
which can be achieved in body fluids is difficult to estimate
due to their extremely high reactivity [8]. The chemical basis
of these reactions can be reduced to: oxidation of thiols;
halide substitution of activated C-H compounds; transhalo-
gation and hydrolytic degradation [5, 12, 23]. This may rise
doubts whether in vitro demonstrated activity of chlorinat-
ing and brominating oxidants is relevant to the situation in
vivo. Nevertheless, in our and others’ opinion [8], the phys-
iologically relevant parameter is not the concentration, but
rather the amount of HOCl, TauCl or TauBr to which a bio-
logical target is exposed. Stimulated neutrophils produce
200 nmol HOCl per 107 cells over 30 min [8]. Therefore, it
is, possible that the amounts of oxidants used in this study
could be achieved in vivo at sites of inflammation. Whether
TauBr at the physiological concentrations plays a role in
inflammation, is an additional question. Our study has
shown that TauBr and HOCl, at similar micromolar concen-
trations, exert strong microbicidal activity. At these condi-
tions TauCl did not kill bacteria. These results are in agree-
ment with the studies of Gaut et al. [7]. They have shown
that addition of low concentration (1 mM) of Br– markedly
increased bactericidal activity of the complete myeloperoxi-
dase-H2O2–Cl– system in vitro. Therefore, one may suggest
Acknowledgements. This study was supported by the grant from “Pol-
ish Pharmacy and Medicine development” and partly by Center of
Microbiological Research and Autovaccines Ltd., Krakow, Poland.
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