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Mario Negri Institute for Pharmacological Research, Bergamo, Italy;
Laboratory of Immunogenetics and Transplantation, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; and
Division of Nephrology and Dialysis, Azienda Ospedaliera, Ospedali Riuniti di Bergamo, Largo Barozzi 1, Bergamo, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) and DCs) from rat thymus expressed
high levels of inducible NO synthase (iNOS) and produced large amounts
of NO in basal conditions whereas iNOS expression and NO production
were very low in thymocytes. Analysis by FACS and by double labeling of
cytocentrifuged preparations showed that DCs and M
both express iNOS
within APC. Analysis of a purified preparation of DCs confirmed that
these cells express high levels of iNOS and produce large amounts of NO
in basal conditions. The capacity of DCs to generate NO was enhanced by
exposure to rat albumin, a self-protein, and required a fully expressed
process of Ag internalization, processing, and presentation. Peptides
derived from portions of class II MHC molecules up-regulate iNOS
expression and NO production by DCs as well, both in self and
allogeneic combinations, suggesting a role of NO in both self and
acquired tolerance. We also found that NO induced apoptosis of rat
double-positive thymocytes, the effect being more evident in
anti-CD3-stimulated cells. Altogether, the present findings might
suggest that DC-derived NO is at least one of the soluble factors
regulating events, in the thymus, that follow recognition of self- and
allo-Ags. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite the growing number of studies in the area, the cellular and molecular mechanisms underlying the induction of intrathymic tolerance are unclear. Some of these data support clonal deletion (10, 11, 12, 13), which physically eliminates reactive T cells by a process of programmed cell death, or apoptosis, as the main mechanism, although clonal anergy, rendering T cells unresponsive to Ags, also plays a part (14). In favor of the clonal/deletion hypothesis are very recent data in transgenic mice expressing a TCR specific for the class I MHC Ag H-2Kb. Intrathymic injection of H-2b spleen leukocytes reduced the CD8-positive thymocyte population by 80% nine days after thymic delivery (15). Double-positive thymocytes were also deleted (15). On the same line, intrathymic injection of OVA in mice transgenic for the TCR specific for peptide 323-339 of OVA led to apoptosis of thymocytes starting as early as 3 h and persisting up to 7 days after injection (16).
Thymic epithelial cells can induce T cell clonal deletion, at least in certain experimental systems (17), but today it seems more likely that dendritic cells (DCs) are the cell population most consistently involved in T cell-negative selection (18, 19). The negative selection potential of DCs implies that the Ag is internalized, processed, and presented to the TCR (20, 21). The Ag retention capacity of thymic DCs is also instrumental to this specialized function (22).
Soluble factors are indispensable to drive either the suppression or
the maturation of a given clone to a functionally mature cell. The
observation that maturing thymocytes, in the earliest stage of T cell
development but not later on, undergo apoptosis when exposed to the
L-arginine derivative NO (23) suggested that
NO released by thymic stromal cells may take part in the cellular
events involved in T cell development. This was supported by the
finding that fetal mouse thymi express mRNA for the inducible type of
NO synthase (iNOS) with levels of iNOS mRNA peaking around gestation
day 18 and declining before birth (24). Moreover, thymic
stromal cells from mouse fetuses spontaneously release NO in certain
culture conditions (24), although it is not known which
cell in the thymus is mainly involved in NO synthesis. Reports that
bone marrow-derived DCs were induced to produce NO by the iNOS inducers
IFN-
and LPS (25) aroused our interest in exploring the
possibility of resident DCs being involved in NO formation in the
thymus.
Overall, our findings indicate that 1) rat thymic DCs express iNOS and generate NO; 2) exposure to either self- or allo-Ags results in up-regulation of iNOS expression and enhances NO production by thymic DCs; 3) the latter phenomenon depends on an intact Ag processing and presenting pathway; and 4) rat immature thymocytes undergo apoptosis when exposed to NO.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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[3H]L-arginine (56.4 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Dowex AG 50 WX-8 was obtained from Bio-Rad (Richmond, CA) and HEPES was from Merck (Darmstadt, Germany). Collagenase type IV, brefeldin A (BfA), chloroquine (ChlQ), amiloride (AML), and all other chemicals were purchased from Sigma (St. Louis, MO). Culture medium was RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with antibiotics, 2 mM glutamine, 50 µM 2-ME, and 10% heat-inactivated FCS.
Rat serum albumin was obtained from Sigma. Synthetic WF polymorphic class II MHC peptides were synthesized in the Protein/Nucleic Acid Laboratory, Department of Medicine, Brigham and Women’s Hospital (Boston, MA). We selected RT1.Bß and RT1.Dß distal domains of RT1u (WF) and synthesized two overlapping peptides of 25 aa (1–25 and 20–44) for each locus using published sequences (26). Rat albumin and class II MHC peptides were tested for endotoxin content by the Limulus amebocyte lysate assay (Sigma) and were found to contain between <15 and 25 endotoxin units (EU) of endotoxin/mg peptides and 200 and 300 EU of endotoxin/mg rat albumin. These extremely low levels do not affect NO production in DCs, as documented by preliminary experiments with endotoxin from two different sources (0111:B4 and 055:B5 serotypes). As many as 10,000 EU endotoxin/ml were needed to significantly stimulate NO production in DCs.
Mouse mAbs specific for rat determinants included Abs specific for CD3
(IF4; Serotec, Oxford, U.K.), TCR-
ß (R73; Serotec), TCR-
(V65; Serotec), CD4 (W3/25; Serotec), CD8ß (341.1; Serotec), CD8
(PE-conjugated OX8; Serotec), Ig 
-chain (OX12; Serotec), CD45RA
(OX33; Serotec), CD45RC (OX22; Serotec), class I MHC (OX18; Serotec),
class II MHC (OX6; Serotec), rat tissue macrophage Ag (ED2; Serotec),
rat macrophage sialoadhesin (ED3; Serotec), and rat DC-restricted Ag
(OX62; Serotec). Rabbit polyclonal Ab against mouse macrophage iNOS was
purchased from Transduction Laboratories (Exeter, U.K.).
FITC-conjugated F(ab')2 goat anti-mouse IgG
(Jackson ImmunoResearch, West Grove, PA), FITC-conjugated
F(ab')2 goat anti-rabbit IgG (Caltag,
Burlingame, CA), Cy-3-conjugated goat anti-rabbit IgG (Jackson
ImmunoResearch) were used as secondary Abs.
Animals
Thymi from Lewis (RT1l, 150–175 g) or WF rats (RT1u, 150–175 g; Charles River Italia, Calco, Italy) were used. Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national (Decreto Legislativo no. 116, Gazzetta Ufficiale, Suppl. 40, February 18, 1992, circolare no. 8, G.U., July 14, 1994) and international laws and policies (European Economic Community Council Directive 86/609, OJL 358, December 12, 1987; Guide for the Care and Use of Laboratory Animals, U.S. National Research Council, 1996).
Cells
Thymi (10–20 for each experiment) were cut into small fragments and digested with collagenase (400 U/ml) for 15 min at 37°C, filtered through a stainless steel screen, and washed in PBS to obtain a total thymocyte suspension (27, 28).
Double-positive CD4+/CD8+ (DP) thymocytes were enriched by removing class I MHC-positive cells since previous studies have shown that most DP thymocytes lack class I MHC expression (29). Briefly, rat anti-mouse IgG-coupled magnetic beads (Dynabeads; Dynal, Oslo, Norway) were coated with anti-polymorphic class I MHC Ab (OX18) and used to deplete class I MHC-positive cells at a 6:1 bead:cell ratio. Class I MHC-positive cells were removed magnetically and the resulting class I MHC-negative cells were then analyzed by flow cytometry. Double labeling with anti-CD8-PE-conjugated and anti-CD4 Abs followed by FITC-conjugated F(ab')2 goat anti-mouse IgG showed that the isolated cells consisted of 95% CD4+/CD8+ thymocytes, on average.
Macrophages (M) and DCs were obtained as previously reported
(23, 28), with some modifications. The total thymocyte
suspension obtained after collagenase digestion and stainless steel
screen passage was washed twice in PBS, resuspended in RPMI 1640
supplemented with 10% FCS, and then cultured in 100-mm petri dishes
(Falcon; Becton Dickinson, Lincoln Park, NJ) for 120 min at 37°C in a
humidified 5% CO2 in air incubator. Nonadherent
cells were removed by extensive washing with warm PBS. Under
phase-contrast microscopy, the adherent cells comprised two
populations: some cells showed the characteristics of DCs in that they
had dendrite-like processes and rapidly changing shape; other cells,
M, were circumferentially spread, ruffled cells with many
vesicles.
The adherent cells were cultured overnight in RPMI 1640 10% FCS at
37°C with 5% CO2. After the overnight culture,
the floating cells were collected. Cells that detached after the
overnight culture consisted of a large cell population that was MHC
I+, MHC II++,
CD4-,
CD8-/+,
CD45RA-, CD3-, and
OX62+, consistent with the expected profile of
DCs (23, 28, 30). However, a considerable number were
ED2+ and ED3+, suggesting
that some M also detached during the overnight culture. Thus, we
called these cells thymic APC. Some (10–15% on average) contaminating
thymocytes (CD3+, CD4+,
CD8+, MHC I-/+, MHC
II-, ED2-,
ED3-, and OX62-) were
also found. Most thymic M remained adherent after the overnight
culture (28). In selected experiments, M were
collected, by treating the petri dishes with EDTA 30 mM, and analyzed
by FACS.
Purified DCs were obtained as described previously (30),
with some modifications. Thymic APC were centrifuged on 55% Percoll
(Pharmacia LKB, Uppsala, Sweden) solution for 20 min at 4°C, and the
low-density fraction was collected and subjected to two rounds of
plastic adherence for 30 min at 37°C. The final enrichment for DCs
was routinely performed by removing T cells and M using magnetic
beads. Briefly, cells were incubated with a mixture of appropriate
dilutions of R73, V65, OX12, OX22, OX33, 341.1, ED2, and ED3 mAbs for
30 min at 4°C, washed three times, and then incubated with rat
anti-mouse IgG-coated magnetic beads for 30 min at 4°C in
agitation. After three rounds of magnetic depletion, the final
population contained 85–95% DCs.
DNA fragmentation
Double-positive CD4+/CD8+ thymocytes were washed once with PBS and resuspended at 2 x 106 cells/ml in RPMI 1640 with 10% FCS with or without the NO-generating agent S-nitrosoacetylpenicillamine (SNAP, 10 µM, 100 µM, and 1 mM; Cayman Chemicals, Ann Arbor, MI) for 8, 15, and 24 h at 37°C. Dexamethasone-treated thymocytes (1 µM) served as positive controls. In negative control experiments, a SNAP solution that had been preincubated overnight at 37°C was used. To evaluate the effect of TCR engagement on NO-induced apoptosis, DP thymocytes were cultured for 15 h in anti-CD3-coated petri dishes and then harvested and recultured in the presence or absence of either 100 µM or 1 mM SNAP for 8 h.
A modified form of the TUNEL assay (31) was used to detect fragmented DNA in apoptotic DP thymocytes (32). Briefly, DP thymocytes were formaldehyde fixed, permeabilized with 0.1% Triton X-100 and 0.1% sodium citrate, and labeled with dUTP-FITC by TUNEL reaction using a cell death detection kit (Boehringer Mannheim, Mannheim, Germany). Labeled cells were visualized by flow cytometry. The apoptosis values were presented after subtracting the percentage of spontaneous apoptosis (with medium alone) which averaged 15–20% at 8 h, 20–25% at 15 h, and 30–37% at 24 h. For the experiment with anti-CD3-coated plates, the apoptosis values were presented after subtracting the percentage of apoptosis in DP thymocytes cultured for 15 h with medium and recultured for 8 h with medium (32–38%).
Flow cytometry
Cell surface immunophenotypic analysis was performed by cytofluorography using the FACSort (Becton Dickinson, Mountain View, CA). Cells were incubated with optimal concentrations of primary Abs for 30 min at 4°C in PBS containing 5% FCS, washed twice with the same buffer, and incubated with FITC-conjugated F(ab')2 goat anti-mouse IgG for unconjugated primary Abs.
For detection of iNOS, cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 and 0.1% sodium citrate. A rabbit polyclonal Ab against mouse macrophage iNOS (that also recognizes the corresponding rat Ag (33)) was used, followed by FITC-conjugated F(ab')2 goat anti-rabbit IgG as secondary Ab. All staining included negative controls from which the primary Abs were omitted.
Light-scattering parameters and propidium iodide staining gates were set to exclude dead cells and debris.
Immunohistochemical examination of rat thymus
Thymic frozen sections (8-µm thick) were cut on a cryostat (HM500-O; Microm, Zeiss Oberkochen, Germany). Sections were air dried, incubated for 1 h with 0.3% H2O2 in methanol to quench endogenous peroxidase, and processed for light microscopy immunohistochemistry using an avidin-biotin HRP complex technique (ABC method, ABC-Elite; Vector Laboratories, Burlingame, CA). Slides were blocked by 30-min incubation with non-immune sera (goat serum for anti-iNOS or horse serum for OX6) and then incubated overnight at 4°C in a moist chamber with the primary Abs (anti-iNOS, 1:150; OX6, 1:150) in PBS/1% BSA (Miles, Milan, Italy), followed by the secondary Abs (biotinylated goat anti-rabbit IgG or biotinylated horse anti-mouse IgG), ABC solution, and finally developed with diaminobenzidine for anti-iNOS and diaminobenzidine-nickel (Vector Laboratories) for OX6, as described elsewhere (33). The sections were counterstained with Harris hematoxylin (Biooptica, Milan, Italy). Negative controls were obtained by omitting the primary Ab on a second section present on all of the slides.
To evaluate whether administration of substances capable of up-regulating iNOS expression in several tissue types can modify iNOS expression and distribution in rat thymus, two animals received 20 mg/kg LPS by i.p. injection. The animals were sacrificed 18 h later and the thymus was removed and treated as above.
Intracellular localization of iNOS
Thymic APC and purified DCs were washed with PBS and cytospun into glass slides (Shandon, Cheshire, U.K.). Cells were fixed and permeabilized with ice-cold methanol for 15 min before staining with anti-iNOS or anti-class II MHC Abs (in PBS/5% FCS) followed by Cy-3-conjugated goat anti-rabbit IgG and FITC-conjugated F(ab')2 goat anti-mouse IgG (in PBS/5% FCS), respectively. Double labeling anti-iNOS and anti-class II MHC was also performed. All staining included negative controls from which the primary Abs were omitted.
To study intracellular iNOS localization, immunoblot analysis was performed on DC whole lysate, cytosol, and particulate fraction. Purified DCs were pelleted, resuspended in 500 µl lysis buffer (50 mM ß-glycerolphosphate, 2 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, 0.5% Nonidet P-40, 1 mM DTT, 1 mM pefabloc, 20 µM pepstatin, 20 µM leupeptin, and 1000 U/ml aprotinin) and sonicated. An aliquot of the whole lysate was stored at -70°C. The remaining lysate was centrifuged at 100,000 x g for 30 min at 4°C to separate cytosol and particulate fraction (34). The cytosol was stored as for the whole lysate and the pellet was resuspended and again centrifuged at 100,000 x g for 30 min at 4°C. The pellet was resuspended and stored at -70°C until assayed. Protein concentration was determined for each sample (whole lysate, cytosol, and particulate fraction) using the Bradford method (Bio-Rad). The proteins (10 µg for each lane) were separated on denaturating a 7.5% SDS-polyacrylamide gel by electrophoresis and then blotted on nitrocellulose membrane by wet electroblotting for 90 min. Blots were blocked overnight at 4°C with 5% nonfat dry milk in TBS-T at pH 7.5 (20 mM Tris base, 137 mM NaCl, and 0.1% Tween 20) and then incubated for 2.5 h with anti-iNOS (1/1000) followed by the secondary Ab (biotinylated goat anti-rabbit IgG), ABC solution, and finally developed with diaminobenzidine (Vector Laboratories).
Conversion of [3H]L-arginine to [3H ]L-citrulline
To evaluate NO production in the different thymic cell populations, total thymocytes, APC, and purified DCs (about 2 x 105/ml) were incubated for 24 h in RPMI 1640/10% FCS in the presence of [3H]L-arginine (0.5 µCi). Total thymocyte incubation was performed with 10 x 106 cells/ml to overcome the assay’s detection limit.
Incubations were stopped by adding one volume of ice-cold 15% TCA. TCA-treated samples were centrifuged at 10,000 x g to precipitate proteins. The supernatant was extracted five times with one volume of water-saturated ether, vacuum lyophilized, and resuspended in 2 ml HEPES (pH 5.5) and applied to 2-ml wet-bed volumes of Dowex AG 50 WX-8 (100–200 mesh, Li+ form), followed by 2 ml of water. [3H]L-citrulline was quantitated by liquid scintillation counting in the 4-ml column effluent and identified by TLC as described previously (35).
For each experiment, aliquots of RPMI1640/10% FCS containing [3H]L-arginine were incubated without cells, as blanks.
Effect of self- and non-self-Ags on iNOS expression and NO production in DCs
To establish the effect of self- or non-self-Ags on iNOS expression and NO production by DCs, in vitro pulsing experiments were performed using 100 µg/ml of either rat serum albumin or sperm whale myoglobin. Since DCs mature in culture in 12–18 h after isolation and lose their Ag-processing capacity (36, 37, 38), either rat albumin or sperm whale myoglobin was added to adherent cells from WF or Lewis thymi during the overnight culture (16 h). In selected experiments, the effect of shorter exposure to a self-Ag was evaluated after 2.5- and 5.5-h incubation with rat albumin. APC floating after the incubation period were collected and DCs were purified and assayed for iNOS expression, by FACS and immunoblot, and NO production (see above). To assess the effect of Ag processing on the induction of NO synthesis in thymic DCs, we used AML (50 µM), which inhibits Ag uptake by blocking macropinocytosis (39, 40), ChlQ (15 µM), which inhibits endocytic function and Ag processing (41, 42), or BfA (1 µg/ml), which blocks the endoplasmic reticulum-Golgi egress of nascent class II MHC molecules (43, 44, 45, 46).
The effect of self- or allopeptides on iNOS expression and NO production by DCs was assessed by in vitro pulsing experiments using the two WF RT.1Bu and the two RT.1Du peptides (25 µg/ml each). The peptide mixture was added to adherent cells from WF (self-pulsing) or Lewis (allopulsing) thymi during the overnight culture. After the overnight culture, DCs were purified and assayed for iNOS expression and NO production. In some experiments, Ag-processing inhibitors (AML, ChlQ, and BfA) were added in combination with peptides.
Statistical analysis
Data are means ± SE. The two-tail Student t test was used for statistical analysis of NO production and iNOS expression in different thymic cell populations and in DCs incubated with self- or allo-Ags. One-way ANOVA was used to analyze NO-induced apoptosis. Statistical level of significance was defined as p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To evaluate the expression and tissue localization of iNOS, the
only NOS isoform identified in the rodent thymus (24),
immunohistochemical analysis was done on cryostat sections of Lewis rat
thymi using anti-iNOS Ab. Results showed iNOS intense staining at
the corticomedullary junction and medulla (Fig. 1
A). The same regions were
strongly stained by anti-class II MHC Ab (Fig. 1, C and
D). At higher magnification, iNOS staining was mainly
localized on large cells surrounded by a number of negative smaller
thymocytes (Fig. 1B). No iNOS expression was detected in the
cortex of normal thymus (Fig. 1A). However, in thymic
sections from rats receiving a single i.p. injection of LPS, some
specific iNOS staining was also found focally in the cortex (data not
shown), suggesting that also cortical cells, maybe M or epithelial
cells, can be induced to express iNOS.
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To determine the cell type(s) within the thymus that express iNOS
and produce NO in basal conditions, rat thymocytes and thymic APC (DCs
and M) were isolated from the thymus by differential adherence, as
described in Materials and Methods. FACS analysis of iNOS in
thymocytes and thymic APC showed specific high staining in the latter
cell population (Fig. 2A),
whereas in thymocytes iNOS staining was very low (Fig. 2A).
As shown in Fig. 2B, ex vivo NO production in unstimulated
total thymocytes, measured as conversion of
[3H]L-arginine to
[3H]L-citrulline after a
24-h incubation, was very low (0.009 ± 0.002
nmol/105 cells, n = 4). APC
produced large amounts of NO in basal conditions (1.39 ± 0.38,
nmol/105 cells, p < 0.01 vs
thymocytes, n = 6), confirming that these cells express
the enzymatic machinery for NO synthesis.
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when viewed on cytospin slides. Double labeling of
cytocentrifuged APC showed two cell populations expressing high iNOS:
the majority of iNOS-positive cells had the exact morphology of DCs
(high nuclear:cytoplasmic ratio and distinct long cytoplasmic processes
or dendrites) and was class II MHC highly positive (Fig. 3, A and B).
However, we also found unequivocal iNOS-positive staining in a second
cell population that was clearly class II MHC negative or had a low MHC
positivity (Fig. 3, A and B). This cell
population was classified as M on the basis of the FACS finding of
low class II MHC, moderate ED2, and high iNOS in a cell preparation
enriched with M from rat thymi (Fig. 4).
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). Nearly 80% of purified DCs were iNOS
positive by FACS when the staining was done on unstimulated cells
(Fig. 5).
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, C and D). To determine iNOS
intracellular distribution in DCs, anti-iNOS immunoblots were
performed on soluble and particulate fractions of DC sonicates. As
shown in Fig. 6, immunoblots revealed two
forms with apparent molecular masses of 135 and 130 kDa, respectively,
both in the soluble and in the particulate fractions. This is
consistent with previous data in primary M showing the existence of
two iNOS variants both found in the cytosolic and in the membrane
compartments of the cell (34). Confocal microscopy
analysis on purified DCs confirmed that iNOS immunoreactivity was
present both in the cytosol and in association with membrane structures
(data not shown).
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Induction of iNOS expression and NO production by thymic DCs after incubation with either self- or non-self-Ags
To establish whether engagement with self-Ags induced iNOS
expression in thymic DCs, rat serum albumin was added to
thymic-adherent cell preparations that were cultured for 2.5, 5.5, and
16 h, respectively. At the end of incubation period, floating
cells were collected and processed as described in Materials and
Methods. FACS analysis revealed that a 2.5-h exposure to rat
albumin did not modify iNOS expression (mean FITC-fluorescence
intensity, rat albumin-pulsed DCs: 375, unpulsed DCs: 325,
n = 2), whereas iNOS expression was almost doubled by a
5.5-h exposure (mean FITC-fluorescence intensity, rat albumin-pulsed
DCs: 472, unpulsed DCs: 287, n = 2). Maximal induction
was observed after a 16-h exposure to albumin (mean FITC-fluorescence
intensity, rat albumin-pulsed DCs: 652 ± 28, unpulsed DCs:
262 ± 52, n = 4, p < 0.01, Fig. 7A). Densitometric analysis of
immunoblots revealed an increased immunoreactivity of protein extracts
from 16-h rat albumin-pulsed DCs over unpulsed cells, either in
cytosolic (150%) or in particulate fraction (200%) (Fig. 6).
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B, 16-h rat albumin pulsing significantly
raised [3H]L-citrulline
release compared with unpulsed cells (2.95 ± 0.55
nmol/105 cells, n = 9,
p < 0.01 vs unpulsed 1.45 ± 0.32
nmol/105 cells, n = 9 (Lewis DCs,
n = 5 experiments; WF DCs, n = 4
experiments)), indicating an increased NO biosynthesis. NO production
in albumin-pulsed DCs corresponded to a concentration of NO of about
10–20 µM which is equivalent to that generated by 100 µM SNAP
(23, 47).
Additional experiments were performed to find whether NO synthesized in
response to DC pulsing with self-Ags reflected an aspecific effect of
albumin itself or implied activation of the process of Ag processing
and presentation. To this purpose AML, which inhibits macropinocytosis
in DCs (39, 40), or ChlQ, which blocks presentation of Ags
by neutralizing intracellular acidic compartments (41, 42), or BfA, which interferes with the egress of newly
synthesized MHC molecules (43, 44, 45, 46), was added before and
maintained throughout the Ag pulsing. As shown in Fig. 7B,
all of the above inhibitors significantly abrogated NO production by Ag
pulsing.
To evaluate whether non-self-Ags were capable as well to induce NO synthesis in DCs, the conversion of [3H]L-arginine to [3H]L-citrulline was evaluated in sperm whale myoglobin-pulsed DCs. Results showed a 2-fold increase of NO production in myoglobin-pulsed vs unpulsed DCs (4.01 vs 1.70 nmol/105 cells).
To establish whether loading with peptides from self-class II MHC
molecules induced NO biosynthesis in DCs, NO production was also
evaluated in WF DCs pulsed in vitro with WF class II MHC peptides. WF
class II MHC peptide loading stimulated NO release, as documented by a
significant increase in
[3H]L-citrulline compared with
unpulsed DCs (4.02 ± 0.84 nmol/105 cells,
p < 0.05 vs unpulsed 2.11 ± 0.41
nmol/105 cells, n = 7, Fig. 8, left panel). To evaluate
whether allogeneic class II molecules could up-regulate iNOS expression
and NO production in DCs as well, DCs from Lewis rats were pulsed with
WF class II MHC peptides. WF class II MHC allopeptides induced
up-regulation of iNOS expression in Lewis DCs (mean FITC-fluorescence
intensity, allopeptide-pulsed DCs: 565, unpulsed DCs: 336; mean of two
experiments) and NO synthesis (4.19 ± 0.95
nmol/105 cells, p < 0.05 vs
unpulsed 1.78 ± 0.29 nmol/105 cells,
n = 7, Fig. 8, right panel) as compared with
unpulsed DCs.
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NO-induced apoptosis in DP thymocytes
The kinetics of apoptosis induced by different concentrations of
the NO donor is shown in Fig. 9. After an
8-h incubation, only the highest (1 mM) SNAP concentration induced
evaluable apoptosis (after subtracting the percentage of spontaneous
apoptosis). After 15 and 24 h, a significant specific apoptosis
was found with all three SNAP concentrations (Fig. 9).
Dexamethasone-treated DP thymocytes, which served as positive control,
showed an average of 55% apoptosis. Negative control experiments in
which DP thymocytes were incubated with SNAP solution (1 mM),
disactivated overnight at 37°C (23), showed a percentage
of apoptosis comparable with untreated cells.
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. As
expected, an 8-h incubation with 100 µM or 1 mM SNAP induced no or
very little specific apoptosis in cells pre-exposed to medium alone. By
contrast, an 8-h reculture of anti-CD3-stimulated thymocytes with
100 µM or 1 mM SNAP dose-dependently increased the percentage of
apoptotic cells as compared with that of DP thymocytes treated with
anti-CD3 and recultured with medium. Taken together, these results
suggest that TCR engagement renders DP thymocytes more sensitive to
NO-induced apoptosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have now provided evidence that thymic DCs possess the enzymatic machinery for synthesizing NO and are actually a major source of NO within the thymus. The capacity of DCs to generate NO was enhanced by exposure to a self-protein and required a fully expressed process of Ag internalization, processing, and presentation. Peptides derived from portions of self-class II MHC molecules up-regulate iNOS expression and NO production by DCs as well. Exactly the same was found with an allogeneic combination of class II MHC molecules and DCs, suggesting a possible role of NO in both self and acquired tolerance. The functional implication of these findings in thymic pathophysiology rests on additional data that exogenous NO induced apoptosis of rat DP thymocytes in a dose- and time-dependent fashion.
NO appears to be an ideal messenger for cell to cell interactions within the thymic parenchyma according to the following arguments: 1) It is synthesized and released upon cell activation (48, 49) and has a very short half-life (50). This would confine the effect of NO in the thymus to cells close enough to the ones initially activated. 2) Induction of apoptosis by NO in DP thymocytes (Refs. 23, 24 and present data) is prompt, whereas single-positive mature T cells are resistant (23). 3) TCR engagement renders DP thymocytes more sensitive to the apoptotic effect of NO (Ref. 24 and present data). Thus, the effect of NO is maximal on cells ready to be clonally deleted in response to self- or allogeneic peptide-TCR-mediated recognition, as a function of its different impact depending on the T cell maturation step and TCR engagement.
Previous data (23, 24, 50) on the cell source of NO within
the thymus are fragmentary and inconclusive. Immunohistochemistry
analysis in this study showed that iNOS was expressed in the normal
adult rat thymus and the signal was mostly localized in the
corticomedullary and medullary regions. Further analysis of different
thymic cell populations showed that APC, largely represented by DCs and
M, express iNOS and generate NO even constitutively soon after
isolation. By contrast, NO production by thymocytes is negligible and
occasionally undetectable. Two different phenotypes were identified
within APC, both iNOS positive. The prevailing phenotype was class II
MHC highly positive and had a high nuclear:cytoplasmic ratio and long
cytoplasmic processes (dendrites), recapitulating the main
characteristics of DCs (30, 51, 52). Less common were
cells with immunophenotype and morphologic characteristics of resident
M (53).
DCs represent a unique cell population in the thymus constitutively
designed to express class I and class II MHC at high levels (28, 54, 55). Functionally, these cells, in contrast to M which
simply capture Ags, process self- and allogeneic Ags and present them
in a self-restricted MHC fashion (18, 56, 57). These
functional properties, unique to DCs in the thymic parenchyma, render
them instrumental to the complex process of negative selection of
maturing T cell. Thus, in fetal thymic reaggregation culture from
C5- TCR transgenic mice, addition of DCs from
C5+ mice drastically reduced
CD4+/CD8+ DP thymocytes,
indicating that self-Ag presented by DCs induced clonal deletion of
TCR-specific thymocytes (53). That thymic DCs actually
induce negative selection events in vivo has been confirmed by data
that targeted expression of class II MHC I-E molecules specific to DCs
do actually negatively select I-E-reactive T cell clones
(19).
Although it is well established that internalization and processing of Ags by thymic DCs are indispensable for negative selection, no studies have investigated whether Ag handling generates messages within the DC itself which eventually trigger cell activation. In this study, we show that thymic DC exposure to a self-circulating Ag, albumin, potently induces iNOS expression and enhances its capacity to release NO in vitro. The ability of Ag-pulsed DCs to enhance their NO synthetic capacity was completely prevented by AML, ChlQ, and BfA, three agents that effectively block presentation of diverse Ags by DCs (38, 39, 40, 45). AML inhibits macropinocytosis, a process that endows DCs with a high capacity of a nonsaturable mechanism for capturing any soluble Ag (39, 40). ChlQ, by neutralizing intracellular acidic compartments, inhibits endocytic function, Ag processing, and invariant chain cleavage from class II MHC molecules following peptide binding (41, 42), whereas BfA interferes with the egress of newly synthesized class II molecules from the endoplasmic reticulum (43, 44, 45, 46). Thus, our data indicate that induction of iNOS and NO release by rat albumin is dependent on processing of the self-Ag by DCs, although it cannot be established which event along the Ag-processing pathway triggers iNOS induction. According to our data, iNOS up-regulation occurs at a late stage during Ag processing; indeed, maximal iNOS expression in DCs was found after a 16-h exposure to albumin, when Ag processing has been completed and Ag peptides are being presented on class II MHC molecules (58). Unpulsed thymic DCs express iNOS although at a lesser degree than Ag-pulsed DCs and immunohistochemistry findings showed iNOS-positive cells, with dendritic morphology, in the normal rat thymus. This finding can be taken as to suggest that iNOS expression in DCs is induced in vivo by endogenous thymic Ags.
When thymic DCs were exposed to either self- or allogeneic class II MHC peptides, up-regulation of iNOS and NO release was observed to an extent comparable to that elicited by albumin pulsing. Again, ChlQ and BfA completely blocked NO synthesis up-regulation by self- and allogeneic peptides. Recent work with mouse, rat, and human peptides representing portions of the polymorphic regions of class I and II MHC molecules indicate that exogenous self-peptides and allopeptides are taken up by APC and presented in a self-MHC binding site for recognition after endogenous pinocytosis, processing in the Golgi, and transport to the cell surface (59, 60, 61, 62, 63). In an earlier study, we found that thymic recognition of class II MHC allopeptides is sufficient for induction of tolerance in the rat renal allograft model (7). Further studies on the mechanisms of acquired thymic tolerance by class II MHC allopeptides showed that the induction phase depended on the presence of an intact thymus and at least partially on a process of T cell anergy, whereas the maintenance phase implied clonal deletion of specific alloreactive T cell clones (64).
TCR-mediated recognition of self-MHC-peptide complexes is instrumental to negative selection so that thymocytes that express TCR with high avidity for MHC/peptide undergo apoptosis (65). However, it is also clear that additional signals, other than the TCR-mediated ones, are required to determine whether thymocytes will undergo clonal deletion or maturation and that such signals are provided by APC (65). That NO produced by thymic DCs upon Ag processing and presentation may function as a molecule that delivers such an additional signal is supported by the finding that administration of exogenous NO greatly increased apoptosis in anti-TCR Ab-stimulated DP thymocytes, whereas DP thymocytes that did not have their TCR engaged were less sensitive. If this were true, it is possible to hypothesize that thymocytes, that express TCR with high avidity for a given presented Ag, respond to NO and undergo negative selection whereas those with low affinity are spared.
NO-mediated apoptosis was reported in several cell types including macrophages (66), lymphocyte cell lines (67, 68), pancreatic ß cells (69), and mouse thymocytes (23). Although mechanisms of NO-mediated apoptosis are still controversial, activation of tumor suppressor protein p53 (70), inhibition of the proteasome (70), impairment of mithocondrial function and energy depletion (71), direct DNA damage by deamination and cross-linking of DNA (72), and activation of caspase cascade (67, 68), all have been proposed. Preliminary experiments in our laboratory showing that exposure of DP thymocytes to exogenous NO activates caspases 3 and 6 and particularly caspase 8 (S. Aiello, M. Noris, and G. Remuzzi, unpublished observations) support a role for caspase cascade activation in NO-induced DP thymocyte apoptosis. Consistently, treatment of DP thymocytes with z-VAD-fmk (a broad-spectrum caspase inhibitor) partially prevented NO-induced apoptosis (Aiello et al. unpublished observations). On the other hand, other studies in rat hepatocytes (73) and human mature lymphocytes (74) have shown that NO can even suppress apoptosis via a direct inhibition of caspases activation (75, 76), indicating that not all cell types are equally sensitive to the apoptotic effect of NO.
A recent study showed that thymocyte apoptosis and animal survival after induction of polymicrobial abdominal sepsis is significantly less in animals lacking iNOS as compared with wild-type mice, providing in vivo evidence that NO has indeed a role in mediating thymocyte apoptosis and selection (77).
In summary, the information from the present study and from few other studies (23, 24) can be used to support the possibility that, upon self- and alloantigen processing, DCs up-regulate iNOS and enhance NO synthesis. Engagement of TCR with specific Ags sensitizes reactive T cell clones to the apoptotic properties of paracrinally released NO.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Giuseppe Remuzzi, "Mario Negri," Institute for Pharmacological Research, Via Gavazzeni 11, 24125 Bergamo, Italy.
3 Abbreviations used in this paper: WF, Wistar-Furth; DC, dendritic cell; iNOS, inducible NO synthase; DP, double positive; M, macrophage; AML, amiloride; ChlQ, chloroquine; BfA, brefeldin A; SNAP, S-nitrosoacetylpencillamine; ABC, avidin-biotin complex; EU, endotoxin unit.
Received for publication April 19, 1999. Accepted for publication February 16, 2000.
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, p < 0.01 vs 15-h medium + 8-h SNAP (100 µM
and 1 mM).