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Departments of
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Biochemistry,
Rheumatology,
Pathology, and
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Medicine, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; and Unit of Immunohematology and Immunopathology, Pasteur Institute, Paris, France
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A stock solution of aminoguanidine was prepared from a bicarbonate salt (Sigma, St. Louis, MO) by suspending it in a minimum quantity of distilled water. Concentrated phosphoric acid was then added until no more CO2 evolution was visualized. The pH was adjusted to 7.4 with saturated NaOH, and the final volume was adjusted with PBS to the desired stock concentration. Mn-TBAP (manganese [III] tetrakis [4-benzoic acid] porphyrin) was purchased from Alexis (San Diego, CA). PHA, PMA, L-tyrosine, 3-amino-L-tyrosine, o-phospho-L-tyrosine, 3-nitro-L-tyrosine, and monoclonal anti-phosphotyrosine (mouse IgG1, clone PT-66) were purchased from Sigma.
Cell preparation and culture conditions
PBMC were isolated from peripheral venous blood obtained from healthy volunteers by centrifugation over a Ficoll gradient. T lymphocytes were purified from nonadherent cells obtained after adherence of PBMC to plastic for 2 h at 37°C. Nonadherent cells were then incubated with saturating concentrations of anti-CD14, anti-CD19, anti-CD16, and anti-CD56 murine mAbs (Dakopatts, Glostrup, Denmark) for 1 h at 4°C to completely eliminate contaminating monocytes, B lymphocytes, and NK cells. After washing, cells were incubated in the presence of magnetic beads precoated with sheep anti-mouse Abs (Dynabeads, Dynal, Oslo, Norway) to achieve a 5:1 bead to cell ratio. After incubation (1 h at 4°C) cell fractions were separated using a magnet applied to the outside of the tube. Unbound T lymphocytes were carefully aspirated, and a second round of immunomagnetic separation was performed with a bead to cell ratio of 10:1; this fraction was referred to as purified lymphocytes. Purified monocytes were isolated by adherence to plastic for 2 h at 37°C from a fraction of untreated whole PBMC that were not exposed to the Abs used in lymphocyte preparation. The purity of the subsets was evaluated by either flow cytometry or immunofluorescence, and the purity of the cell preparations used was >95%. The possibility of minor contaminant cells includes plasma cells, which, in contrast to B cells, do not react with CD19. Cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS (LPS free; Life Technologies, Grand Island, NY), 4 mM glutamine, 1 mM HEPES, 1 mM sodium pyruvate, and penicillin (200 IU/ml)-streptomycin (50 µg/ml; Life Technologies).
Synthesis of peroxynitrite
Peroxynitrite was synthesized in a quenched flow reactor as
previously reported 17, 18 . Briefly, solutions of 1) 0.6 M
NaNO2 and 2) 0.6 M H2O2 were pumped
into a T junction and mixed in a glass tube. The reaction was quenched
by pumping 1.5 M NaOH at the same rate into a second T junction at the
end of glass tube. Excess H2O2 was removed by
addition of MnO2 powder. The solution was then frozen at
-80°C, at which temperature peroxynitrite forms a yellow top layer
due to freeze fractionation. The concentration of this layer was
usually in the range of 250–400 mM as determined
spectrophotometrically at 302 nm (
302 = 1670
M-1·cm-1). Working dilutions were made in 1
N NaOH.
Agarose gel analysis of DNA fragmentation
The presence of apoptotic cells was assessed by visualization of a characteristic banding pattern of internucleosomal DNA fragmentation. For this, 8 x 106 cells were pelleted for 10 min at 200 x g and resuspended in 0.5 ml of TTE buffer (10 mM Tris-HCl (pH 7.6) and 1 mM EDTA containing 0.2% Triton X-100). Fragmented DNA was separated from intact chromatin by microcentrifugation for 10 min at 13,000 x g at 4°C. Supernatants (0.5 ml) were mixed with 0.1 ml of ice-cold 5 M NaCl and vortexed vigorously. After addition of 0.7 ml of ice-cold isopropanol, DNA was precipitated overnight at -20°C. Samples were centrifuged for 10 min at 13,000 x g at 4°C and washed in ice-cold 70% ethanol. Dried pellets were solubilized in 20 µl of TE buffer (10 mM Tris-HCl (pH 7.6) and 1 mM EDTA). Samples were mixed with loading buffer, heated for 10 min at 65°C, and electrophoresed on 1% agarose gels with TBE buffer until bromophenol blue dye migrated 3–4 cm. Fragmented DNA was visualized by staining with ethidium bromide.
Flow cytometric analysis of apoptotic cells
Flow cytometric analysis was performed on an EPICS 752 FACS (Coulter, Hialeah, FL). The forward and right angle scatter signals were used to gate out cellular debris, damaged cells, and aggregates. The fluorescence emission was displayed on a log scale to yield histograms of log fluorescence intensity (x-axis) vs cell number (y-axis). The fluorescent dye merocyanin 540 (MC-540, Fluka, Ronkonkona, NY) has been successfully used as a fluorescent probe in cytometric analysis by its ability to bind symmetric membranes with exposed phosphatidylserine on apoptotic cells 39 . Staining of cells with MC-540 was performed according to the method described by McEvoy et al. 40 with minor modifications. In brief, a stock solution of MC-540 was prepared in 50% ethanol at 1 mg/ml and stored in the dark at -20°C. A working solution was freshly prepared by diluting merocyanine in HEPES-buffered salt solution with 0.1% BSA to achieve a final concentration of 2 µg/ml. Cells (106) were resuspended in 100 µl of working solution and incubated for 5 min in the dark at room temperature, then washed once with HEPES-buffered salt solution without BSA and immediately analyzed by flow cytometry.
Production of rabbit anti-nitrotyrosine polyclonal Abs
New Zealand rabbits were immunized with 500 µg of peroxynitrite-modified keyhole limpet hemocyanin (NO2-KLH) emulsioned with an equal volume of CFA according to the procedure of Ye et al. 41 . KLH (Sigma) diluted in 100 mM PBS was nitrated in the presence of 0.5 mM peroxynitrite. Boosters were performed every 3 mo after the first injection with NO2-KLH emulsioned in IFA. Rabbits were bled 15 days after the last booster according to serum anti-nitrotyrosine titration as assessed by ELISAs against native KLH and NO2-KLH. Pooled sera were fractionated in ammonium sulfate, and after dialysis overnight unwanted Abs against native KLH were eliminated using a Sepharose-4B column coupled to native KLH. Specific anti-nitrotyrosine Abs were affinity purified using a Sepharose-4B column coupled to 3-nitro-L-tyrosine.
ELISAs for specificity and competition analysis of anti-nitrotyrosine Abs
Polyclonal anti-nitrotyrosine Abs were screened for specificity and unwanted cross-reactions by Western blot and ELISA. Briefly, 96-well polystyrene ELISA plates were coated with either native or peroxynitrite-nitrated proteins diluted at 30 µg/ml in carbonate buffer (50 mM; pH 9.5) and incubated overnight at 4°C. After washing, plates were blocked with 5% gelatin in PBS plus 0.05% Tween-20 (v/v) for 1 h at 37°C. After washing, the anti-nitrotyrosine polyclonal Ab was added at a 1/2000 dilution (0.12 µg/ml) in PBS-0.05% Tween for 2 h at 37°C. The plates were washed and then incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (Dakopatts) for 2 h at 37°C. The plates were developed with o-phenylenediamine dihydrochloride, and absorbance was read at 492 using a microplate reader. SDS-PAGE and Western blotting were performed as described below. When competition analysis were performed, the anti-nitrotyrosine Ab was preincubated for 30 min at room temperature with several concentrations of L-tyrosine, 3-amino-L-tyrosine, o-phospho-L-tyrosine, and 3-nitro-L-tyrosine as competitors for Ab binding.
Nitrotyrosine and phosphotyrosine immunodetection by Western blot assays
For Western blot analysis, cell suspensions were washed twice in PBS and resuspended in hot lysis buffer (10 mM Tris-HCl (pH 6.8), 1% SDS, and 5% glycerol) followed by immediate boiling for 5 min, sonication, and determination of protein concentration by the bicinchoninic acid method (Pierce, Rockford, IL). Samples were stored at -70°C until analysis on 10% SDS-PAGE (30 µg of protein/lane). Next, proteins were electrophoretically transferred to 0.45-µm pore size nitrocellulose membranes, and nonspecific binding sites were blocked overnight in blocking in TBS-T blocking buffer (5% BSA and 0.3% Tween in Tris-buffered saline, pH 7.5). For nitrotyrosine immunodetection, nitrocellulose filters were probed with anti-nitrotyrosine polyclonal Abs diluted 1/1000 in blocking buffer for 1 h at room temperature. After extensive washings in TBS-Tween 0.3%, blots were further incubated for 45 min with a horseradish peroxidase-linked anti-rabbit IgG polyclonal Ab raised in donkeys and developed using the enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL). For phosphotyrosine immunodetection, nitrocellulose filters were blocked as described above and then probed with an anti-phosphotyrosine mAb diluted in TBS-T with 0.1% BSA. After washing in TBS-T, blots were incubated for 45 min with a horseradish peroxidase-linked anti-mouse IgG polyclonal Ab and developed as described above.
Proliferation assays
For [3H]thymidine uptake, cells were cultured in 96-well culture plates at 5 x 104 cells/well in a total volume of 0.2 ml. Stimulation was conducted in the presence of immobilized anti-CD3 mAbs, PHA, and soluble (sCD3) anti-CD3 mAbs (at 5 and 10 µg/ml, respectively), or PMA (100 ng/ml) plus calcium ionophore A-23187 (0.2 µg/ml). After 3 days, cultures were pulsed with 1 µCi/well of [3H]thymidine and further cultured for 18 h. Cells were then harvested, and radioactivity was measured in a beta scintillation counter. All samples were incubated in quadruplicate, and for each experimental condition a control with culture medium alone was included.
Immunohistochemical studies for nitrotyrosine detection
Immunohistochemistry to detect anti-nitrotyrosine was performed on human lymph nodes obtained from routine surgical resections for lung and colonic cancers obtained at the Hospital de Clínicas (Facultad de Medicina, Universidad de la República, Montevideo, Uruguay). Lymph nodes were fixed in 10% formalin and paraffin embedded, and 5-µm sections were mounted in silanized microscope slides. Tissue sections were probed with anti-nitrotyrosine polyclonal Abs (working dilution, 1/50) and developed with a secondary Ab coupled to biotin using a streptavidin-peroxidase kit (Sigma) and either aminoethylcarbazol or diaminobenzidine as chromogens. Histological sections were counterstained with hemalum. Controls were performed with an irrelevant polyclonal rabbit serum as the first Ab. The technical control was omission of the primary Ab. The specificity of immunostaining was always assessed by competition with soluble 3-nitrotyrosine at 5 mM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fig. 1
depicts the range of nontoxic
concentrations of ONOO-. It is shown that doses up to 1 mM
ONOO- result in no significant loss of lymphocyte
viability after 2 h of in vitro culture as assessed by trypan blue
exclusion tests. Thus, we selected a working range from 0.01 to 1 mM
ONOO- for further in vitro experiments. Pretreatment of
purified T lymphocytes with ONOO- induced a dose-dependent
impairment in their proliferative activity (Fig. 2) upon activation with immobilized
anti-CD3 mAbs (iCD3), PHA plus anti-sCD3 mAb, and PMA plus
calcium ionophore. Compared with either iCD3 or PHA-sCD3, impairment of
proliferative responses to PMA plus calcium ionophore was only observed
when ONOO- doses were higher than 0.25 mM. Proliferative
responses to iCD3 and sCD3-PHA were significantly reduced at the lowest
doses of ONOO- used in this work (<0.1 mM).
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To better define the mechanisms of peroxynitrite-induced
impairment in lymphocyte proliferative responses we investigated the
eventual existence of apoptotic cell death. Normal purified lymphocytes
were pretreated with increasing concentrations of ONOO-
(ranging from 0.01 to 1 mM), and after 30 min cells were either
nonactivated or activated in the presence of PMA-ionophore, iCD3,
and PHA plus sCD3. Apoptotic cell death was assessed by DNA
fragmentation on agarose gels or by flow cytometry using the MC-540
fluorochrome at different culture times. Fig. 3 (left panel) shows a
clear pattern of DNA fragmentation in resting purified lymphocytes
after 6 h of culture only at doses of ONOO- higher
than 0.25 mM. When peroxynitrite-treated lymphocytes were activated by
anti-iCD3, DNA fragmentation started at very low concentrations
(
0.05 mM) of ONOO- (Fig. 3, middle panel).
Almost identical results were observed by flow cytometric analysis
(Fig. 4, A and B).
In contrast to CD3-mediated activation, when purified lymphocytes were
stimulated by PMA plus calcium ionophore, DNA fragmentation was
undetectable in the range of ONOO- doses used here. The
absence of apoptotic death in the case of PMA and calcium ionophores
for all doses of peroxynitrite was verified by both DNA fragmentation
(Fig. 3) and flow cytometry (data not shown). These results indicated
that low doses of ONOO- pretreatment primed purified
lymphocytes to undergo apoptotic cell death upon CD3-mediated
activation. The absence of DNA fragmentation after calcium ionophore
plus phorbol esters in purified lymphocytes pretreated with
ONOO- suggested that CD3-mediated induction of apoptosis
after treatment with ONOO- could reflect an impairment in
early protein tyrosine phosphorylation. As depicted in Fig. 4B the ratio of apoptotic cells decreased 48 h after
ONOO- treatment, suggesting that the apoptotic process was
stopped, and a relative increase in the ratio of nonapoptotic cells was
occurring at this time. Because ONOO- is also a strong
oxidant, a set of experiments was performed to exclude the effects of
ONOO-mediated changes in the redox capacity of these cells. For this,
cells were treated with peroxynitrite at the indicated doses and
incubated for 2 h with either N-acetylcysteine (10 mM)
or 2-ME (50 µM) before CD3-mediated activation to restore the
redox capacity of ONOO-treated cells. This treatment (data not shown)
was not able to either inhibit apoptotic death or restore impaired
proliferative responses.
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To examine the effects of ONOO- on the
activation-induced tyrosine phosphorylation, normal purified
lymphocytes were pretreated with increasing doses of ONOO-
and activated after 15 min with immobilized anti-CD3 mAbs. As shown
in Fig. 5, ONOO- induced a
progressive inhibition of early protein tyrosine phosphorylation in a
dose-dependent manner. Of note, compared with peroxynitrite-untreated
lymphocytes there was a relative increase in tyrosine phosphorylation
over the basal values and the level after the first 5 min of
activation at the lowest doses of ONOO- pretreatment.
Overall, these results showed that ONOO- induced
dose-dependent impairment of early tyrosine phosphorylation in normal
lymphocytes, with almost complete inhibition at doses higher than 0.1
mM.
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The presence of protein tyrosine nitration during immune
activation in vitro was assessed by Western blot assays using specific
polyclonal anti-nitrotyrosine Abs raised in rabbits. The
specificity of Ab binding was assessed with either nitrated or native
BSA by Western blot assays as illustrated in Fig. 6A. These results showed that
anti-nitrotyrosine polyclonal Abs specifically recognized nitrated
BSA but not their native form and did not cross-react with either
L-tyrosine or
o-phospho-L-tyrosine. In contrast,
3-nitro-L-tyrosine successfully blocked Ab
immunoreactivity. Fig. 6B depicts results from competition
analysis performed by ELISA using peroxynitrite-modified KLH
(KLH-NO2) and BSA (BSA-NO2). Nitrotyrosine can
completely block Ab binding to nitrated BSA and KLH, with a 50%
inhibitory concentration (IC50) of about 30 micromolar. Up
to 20-mM concentrations of aminotyrosine, tyrosine, or phosphotyrosine
had no effect on Ab binding to nitrated KLH or nitrated BSA (data not
shown). In ELISA assays anti-nitrotyrosine Abs did not recognize
either native KLH or native BSA (data not shown). To assess the
putative role of PBMCs in producing nitrating species in vitro,
additional experiments were conducted on normal purified lymphocytes
cultured alone (Fig. 7B;
purified T lymphocytes) or in the presence of autologous monocytes
(Fig. 7A; purified T lymphocytes from whole PBMC cultures).
Both cell preparations were cultured in either the presence or the
absence of immobilized anti-CD3 mAbs (nonactivated and CD3-mediated
activation, respectively, in Fig. 7). In all cases, lymphocytes were
isolated from cultures at different times and were lysed to study the
time course of the appearance of protein tyrosine nitration (30 min
after activation up to 96 h in culture).
Nitrotyrosine-immunoreactive bands greater than basal levels
appeared after a minimum of 6–12 h following activation or after
24 h for nonactivated lymphocytes. As depicted in Fig. 7A a clear pattern of protein tyrosine nitration was
observed in purified lymphocytes 12 h after activation (Fig. 7A, right panel) when isolated from whole PBMC
cultures and activated by immobilized anti-CD3 mAb. Analysis of
lysates from nonactivated lymphocytes isolated from whole PBMC cultures
(Fig. 7A, middle panel) revealed nitrotyrosine
formation but to a lesser extent than activated lymphocytes cocultured
with autologous monocytes. In contrast, cell lysates from cultures of
purified lymphocytes (Fig. 7B) revealed a slight pattern of
nitrotyrosine-immunoreactive bands only in the case of CD3-mediated
activation and no significant increase in their intensity after the
observation period (Fig. 7B, right panel). No
significant increase in nitrotyrosine-immunoreactive bands was observed
for purified nonactivated T lymphocytes compared with their respective
basal levels (Fig. 7B, left panel). These results
clearly demonstrated that nitrating species were produced in vitro
after immune activation as revealed by nitrotyrosine-immunoreactive
bands observed in cell lysates upon CD3-mediated activation of PBMC. In
addition, these experiments suggested that endogenously produced
nitrating species in PBMC cultures depended on the presence of
monocytes during lymphocyte activation. Further evidence of this
phenomenon is depicted in Fig. 8. Cell
lysates from whole PBMC cultures showed a clear pattern of protein
nitration when activated by immobilized anti-CD3 mAb, PMA plus
calcium ionophore, or PHA plus sCD3 mAb (Fig. 8A). Fig. 8B showed that when purified monocytes and lymphocytes were
cultured alone in the absence (nil, lines 1 for each) or the
presence of immobilized anti-CD3 mAb (iCD3, lines 3 for
each), there was not a significant rise in nitrotyrosine over the
respective basal levels. After whole PBMC cultures for 24 h in the
absence or the presence of immobilized anti-CD3 mAb, monocytes and
lymphocytes were isolated and analyzed separately for nitrotyrosine
immunodetection. These experiments revealed that protein nitration
after CD3-mediated activation was observed in monocytes or lymphocytes
only when they were cocultured (Fig. 8B, line 4
for each). When cocultured monocytes and lymphocytes were analyzed
separately in the absence of CD3-mediated activation there was not a
significant increase in nitrotyrosine levels over their respective
basal levels (Fig. 8B, line 2 for each). These
results suggested that activated lymphocytes through soluble mediators
as cytokines or cell to cell interactions were able to induce monocytes
to produce nitrating species that, in turn, nitrated their own cellular
proteins and also cellular proteins of the neighboring lymphocytes.
Fig. 9 shows the pattern of
nitrotyrosine-immunoreactive bands induced on lymphocytes by
exogenously added ONOO-. Of note, nitrated cellular
proteins induced by authentic peroxynitrite were more homogeneously
distributed than those observed by CD3-induced tyrosine nitration,
which were concentrated at lower relative m.w. These differences might
be attributed to several factors, including bolus addition of authentic
ONOO- vs continuous production at lower levels of
endogenous ONOO- and protein degradation and turnover in
the case of prolonged in vitro cultures.
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Fig. 10 depicts the results of
inhibition experiments conducted by using the cell-permeable superoxide
dismutase mimetic 42 Mn-TBAP and aminoguanidine, an inhibitor of the
inducible isoform of nitric oxide synthase 43, 44 . Aminoguanidine at
doses ranging from 1–20 mM was able to almost completely inhibit
nitrotyrosine formation in whole PBMC cultures after CD3-mediated
activation. Mn-TBAP was also effective in inhibiting nitrotyrosine
formation in a dose-dependent manner. The combination of these two
inhibitors of ONOO- generation completely abrogated
nitrotyrosine immunoreactivity of cellular proteins. These results
strongly indicated that ONOO- was at least the main
nitrating agent in our experimental conditions. If we assumed that
ONOO- impairs lymphocyte activation and prime them to
undergo activation-dependent cell death, it can be speculated that
either aminoguanidine or Mn-TBAP, through inhibition of
ONOO- production, should enhance lymphocyte proliferation.
Thus, we have studied the effects of these pharmacological treatments
on lymphocyte proliferation after CD3-mediated activation. As expected,
both aminoguanidine and Mn-TBAP significantly enhanced CD3-induced
thymidine uptake of normal PBMCs (Fig. 11). Of note, at the higher doses of
both inhibitors used here (>15 mM and 150 µM for aminoguanidine and
Mn-TBAP, respectively), there was a significant decrease in
proliferative response compared with that of control cells.
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To evaluate the in vivo production of nitrating species during
immune responses, we studied human lymph nodes obtained from radical
surgical resections for lung and colonic cancers from six different
patients. Lymph nodes included in this study were free of metastatic
invasion and showed mild to strong reactive follicular hyperplasia and
sinusoidal histiocytosis. In all tissue specimens a diffuse ganglionar
immunostaining for protein nitrotyrosine was observed (Fig. 12, A–D), including
interstitium, lymphocytes, macrophages, and sinuses. A weak reactivity
was seen at the interstitial level between lymphocytes, drawing a ring
around some of them. However, the strongest nitrotyrosine
immunoreactivity was observed principally in macrophage-rich zones,
such as sinuses, perifollicular areas, and germinal centers (Fig. 12, B–D). Macrophages frequently depicted a strong
intracytoplasmic immunoreactivity with a clear granular pattern (Fig. 13). In all cases controls were
performed to substantiate the specificity of immunostaining, including
coincubation of the first Ab with 5 mM nitrotyrosine, which completely
blocked the reactivity, and using duplicated specimens incubated with
irrelevant isotype-matched rabbit Abs as negative controls (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present work we have demonstrated that authentic peroxynitrite primes normal T lymphocytes to undergo peroxynitrite-driven apoptotic death in a dose- and time-dependent manner. Recently, it has been demonstrated that ONOO- induces apoptotic death in several transformed cell lines, including the promyelocytic leukemic HL-60 cell line, the human monocytic tumor cell line U-937 46 , the neuronal PC-12 cell line 47 , and the murine macrophage RAW 264.7 cell line 48 . With respect to nontransformed cells, Lin and co-workers 46 failed to demonstrate peroxynitrite-induced apoptotic changes in normal PBMC from healthy volunteers exposed to peroxynitrite concentrations up to 0.1 mM and analyzed 5–6 h after this treatment. In agreement with this report we failed to demonstrate significant apoptotic changes in resting peripheral lymphocytes treated with ONOO- concentrations <0.1 mM and analyzed up to 5–6 h. However, we extended these results, showing that ONOO- treatment is able to induce apoptotic death in resting human lymphocytes both when the observation period was extended beyond 6 h after exposure to ONOO- and when ONOO- concentrations were higher than 0.1 mM. Additionally, our results demonstrate that ONOO- primes resting lymphocytes to undergo peroxynitrite-driven apoptotic cell death at low ONOO- concentrations (<0.1 mM). Interestingly, there was more than a fourfold increase in the ratio of apoptotic cells when peroxynitrite-treated resting lymphocytes were further activated through the TCR/CD3 pathway but not by phorbol esters plus calcium ionophores. Phorbol esters in association with calcium ionophores activate purified lymphocytes and effectively by-pass the early protein tyrosine phosphorylation signals associated to CD3-mediated activation.
These results suggested that peroxynitrite-induced apoptotic changes in CD3-activated lymphocytes could be related to an impairment in the tyrosine phosphorylation cascade proximal to the TCR/CD3 complex. Thus, resting normal lymphocytes appear more resistant than transformed cell lines and activated lymphocytes to peroxynitrite-mediated apoptotic death because either high doses of ONOO- or extended culture times are needed to produce apoptotic death. These assumptions suggest that the activated phenotype of transformed cell lines or the activated state of normal lymphocytes could play a permissive role in the susceptibility to peroxynitrite-induced apoptotic cell death. Although the precise mechanism involved in peroxynitrite-induced apoptosis is still unclear, recent data demonstrated that peroxynitrite is a potent initiator of DNA strand breakage 49, 50 , which is an obligatory stimulus for the activation of the nuclear enzyme poly(A)DP ribosyl synthetase (PARS). Thus, the cellular response to peroxynitrite exposure can be influenced by the activation of PARS as a DNA nick sensor enzyme. Although the role of PARS activation in apoptosis is still controversial, it has been claimed that it can lead to the depletion of cellular energy pools and to rapid cell death by necrosis, which interferes with the execution of the apoptotic program 50, 51, 52, 53, 54 . In addition, it has been recently reported that Bcl-2 appears to block peroxynitrite-induced apoptosis in Bcl-2-overexpressing HL-60 cells 55 . Further work is needed to substantiate the putative role of either the PARS or the Bcl-2 pathways in the development of peroxynitrite-induced apoptosis.
Our results suggest that nitration of tyrosine residues could play a
major role in the impairment of tyrosine phosphorylation and the
enhancement of programmed cell death induced by peroxynitrite 33, 56, 57 . In agreement with previous reports 32, 58 , our results show that
peroxynitrite at low concentrations induces a transient rise in
tyrosine phosphorylation compared with that in nontreated lymphocytes
both basally and 5 min after CD3-mediated activation. In addition to
the nitrating abilities of ONOO-, a large body of
published data showed that peroxynitrite can induce oxidative
modifications of several biomolecules, including lipids and proteins
and protein fragmentation 19, 22, 59 . Thus, reaction of peroxynitrite
with glutathione depletes glutathione pools 18 , which may sensitize
cells to oxidant stress. Studies of the redox state of T lymphocytes
have suggested that T cell responses and survival are affected by
oxidative stress through inhibition of tyrosine phosphorylation events,
calcium mobilization, and proliferative responses 60, 61, 62, 63 . However, it
has been demonstrated that oxidants (when oxidant insult is not beyond
the capacity of the cellular redox buffer) can elicit positive or
protective responses in cells through a tyrosine
phosphorylation-dependent activation of several transcription factors,
including NF-
B and activating protein-1 64, 65 . In human B and T
lymphocytes these responses were achieved through combined inhibition
of thiol-dependent phosphotyrosine phosphatases and activation of
tyrosine kinases, and the use of tyrosine kinase inhibitors provided
evidence that activation of these signaling pathways are, in fact,
protective responses against oxidant-induced apoptotic death 38, 61, 66, 67 . The transient increase in tyrosine phosphorylation described
here could be a response to a moderate oxidative insult mediated by
peroxynitrite; thus, apoptotic cell death appears as a common
consequence when these protective responses against oxidative stress
are blocked by tyrosine nitration 68, 69 . When peroxynitrite-treated
lymphocytes were further activated by PMA plus calcium ionophore, we
could not observe any apoptotic changes regardless of the method
employed, the dose of peroxynitrite, and/or the incubation time. It can
be suggested that the absence of apoptotic death could be explained
because PMA and calcium ionophores effectively by-pass the early events
of tyrosine phosphorylation and, in turn, trigger protective responses
against oxidative stress, which could able to inhibit apoptotic death.
In contrast, apoptotic changes observed in resting lymphocytes exposed
to high doses of ONOO- could be explained by the inability
to trigger these tyrosine phosphorylation-dependent protective
responses as a consequence of tyrosine nitration. Decreased
proliferative responses to PMA and calcium ionophores in lymphocytes
pretreated with high doses of peroxynitrite were not associated with
either apoptotic changes or cellular loss due to a necrotic process,
which could suggest a peroxynitrite-induced anergic state.
Given the reactive nature of ONOO- in biological systems 70 , one could speculate that peroxynitrite-mediated oxidative modifications could explain the impairment in lymphocyte activation and survival described here. Despite the strong oxidizing potential of ONOO-, kinetic factors determine that a limited number of reactions are relevant in vivo. In particular, the reactions with thiols, metal centers, and CO2 account for the most of peroxynitrite consumption. Since nitration is strongly favored by metals and CO2, nitration constitutes a key cellular event after peroxynitrite formation in vivo. It is unlikely that depletion of endogenous antioxidants by ONOO- could have a relevant place in explaining our results because we cannot prevent apoptotic cell death after restoration of cellular redox capacity with either N-acetylcysteine or 2-ME. In addition, the absence of apoptotic cell death after PMA-mediated activation supports the idea that oxidizing ability of ONOO- is not the main factor explaining our results. Nevertheless, further work is necessary to definitively confirm this idea.
Although other factors could play a role in peroxynitritemediated impairment of lymphocyte signaling proliferation and survival, our results suggest that nitration-mediated impairment of protein tyrosine phosphorylation plays a major role in this process through a defective coupling of the TCR-CD3 complex to proximal intracellular events such as tyrosine phosphorylation 67, 70, 71, 72, 73 . In addition, we provide strong experimental evidence for the endogenous production of nitrating species by cells of the monocyte-macrophage lineage in cultures of PBMC activated either by the CD3 pathway or in the presence of PMA plus ionophores. Generation of reactive nitrogen intermediates by human monocytes/macrophages requires the production of nitric oxide as one essential precursor for the generation of nitrating species either in vivo or in vitro. Indeed, evidence does exist for the identification of the endothelial constitutive and inducible isoforms of nitric oxide synthase mRNA, nitric oxide synthase protein, and nitric oxide production in human monocytes and macrophages 71, 72, 73 . On the other hand, recent work demonstrated that nitrotyrosine is not the unique biomarker of peroxynitrite production and other NO-derived nitrating species could be formed in vivo 45 . In this respect it has been recently reported that nitrite (NO2-), a major end product derived from nitric oxide metabolism, readily promotes tyrosine nitration through formation of nitryl chloride (NO2Cl) and nitrogen dioxide (NO2) through reaction with the inflammatory mediator hypochlorous acid (HOCl) or myeloperoxidase 74, 75, 76 .
Thus, it has been proposed that the nitrite oxidation mechanisms are probably relevant during inflammatory processes, where neutrophils are recruited and activated, providing an additional pathway for the generation of reactive nitrating intermediates. Although with the present evidence, it cannot be definitively stated that the formation of nitrotyrosine in vitro is due to peroxynitrite, the absence of neutrophils in our culture conditions and the results of experiments performed with Mn-TBAP associated or not associated with aminoguanidine favor the view that under our experimental conditions formation of nitrotyrosine was dependent on the presence of superoxide and NOS activity. These arguments support the idea that peroxynitrite is the main nitrating species. Our results also showed that monocyte-derived nitrating species were able to induce long-lasting protein tyrosine nitration of their own cellular proteins and neighboring lymphocytes present in whole cultures. The persistence of nitrated proteins over time suggests that lymphocytes lack the enzymatic ability to remove nitro groups from tyrosine residues as has been demonstrated for tyrosine kinases/phosphatases.
It is presently unclear whether activated lymphocytes are able to
induce the production of nitrating species by monocytes either through
membrane-associated interactions or through soluble mediators as
cytokines, including TNF-
77 . The pattern of nitrated proteins
after in vitro activation showed that the proteins more heavily
nitrated were principally concentrated <50 kDa. However, the pattern
of tyrosine nitration induced by authentic peroxynitrite (Fig. 9
)
evolves over time to acquire a profile indistinguishable from that
induced by in vitro activation (data not shown). In this respect it has
been demonstrated that peroxynitrite-induced protein modifications are
associated with increased degradation, which could explain our results
33 . Since our results suggested that the pattern of nitrated proteins
induced during immune activation might be the consequence of
endogenously produced ONOO-, we have showed in vivo that
lymph nodes from surgical specimens with histological evidence of
hyperplasia and nonspecific immune activation displayed a strong
pattern of reactivity with anti-nitrotyrosine polyclonal Abs.
Although we cannot elucidate which species nitrated tyrosines, the
results show the generation of nitrating species in vivo.
Overall, our results suggest a role for nitrating species such as
peroxynitrite as major modulators of immune responses. This assumption
is reinforced by the enhancement of lymphocyte proliferative responses
in conditions of low to moderate inhibition of either ·NO
or O2 (Fig. 11), which provides experimental evidence for the
physiological relevance of peroxynitrite-mediated tyrosine nitration.
Our results also lead to the idea that this effect is at least in part
mediated through nitration of tyrosine residues, which result in
inhibition of tyrosine phosphorylation. In this respect, one can
speculate that upon activation only heavily tyrosine-phosphorylated
lymphocytes will proceed to acquire adequate effector and regulator
functions. Besides the potential of peroxynitrite as a down-modulator
of normal immune responses, nitration of cellular proteins may be
injurious via multiple mechanisms, including altered protein function,
increased turnover due to enhanced proteolysis, and formation of
neoantigens with eventual autoimmune reactions. Thus, peroxynitrite
generation in vivo may represent a major mediator in
inflammation-mediated protein modification and tissue injury at sites
of chronic or intense immune activation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. A. Cayota, Departmento de Bioquímica-Facultad de Medicina, Av. Gral Flores 2125 CP 11800, Montevideo, Uruguay.
3 Abbreviations used in this paper: ·NO, nitric oxide; ONOO-, peroxynitrite; O2, superoxide; SOD, superoxide dismutase; Mn-TBAP, manganese [III] tetrakis [4-benzoic acid] porphyrin; MC-540, merocyanin 540; KLH, keyhole limpet hemocyanin; sCD3, soluble CD3; iCD3, immobilized CD3; PARS, poly(A)DP ribosyl synthetase.
Received for publication July 29, 1998. Accepted for publication December 2, 1998.
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