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Blood, Vol. 95 No. 1 (January 1), 2000:
pp. 212-220
IMMUNOBIOLOGY
Nitric oxide-producing
CD11b+Ly-6G(Gr-1)+CD31(ER-MP12)+
cells in the spleen of cyclophosphamide-treated mice:
implications for T-cell responses in immunosuppressed mice
Iñigo Angulo,
Federico Gómez de las Heras,
José F. García-Bustos,
Domingo Gargallo,
M. Angeles Muñoz-Fernández, and
Manuel Fresno
From the Centro de Biología Molecular, CSIC-Universidad
Autónoma de Madrid, Spain; GlaxoWellcome S.A., Tres Cantos,
Spain; and the Department of Immunology, Hospital Gregorio
Marañón, Madrid, Spain.
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Abstract |
During recovery from intensive chemotherapy with cyclophosphamide
(CTX), mice suffer a severe but transitory impairment in spleen cell
proliferation to T-cell mitogens (Con A or anti-CD3 plus IL-2).
Although CTX treatment reduced spleen T-cell cellularity, this cannot
fully account for T-cell unresponsiveness. The results showed that CTX
induces the colonization of spleen by an immature myeloid
CD11b+Ly-6G+CD31+ population.
Its presence closely correlated with the maximum inhibition of T-cell
proliferation. Moreover, this suppressive activity was dependent on
nitric oxide (NO) production in cultures since (1) higher amounts of
nitric oxide and inducible nitric oxide synthase (iNOS) mRNA were
produced in CTX spleen cells (CTX-SC) than in control splenocyte
cultures and (2) NOS inhibitors greatly improved the proliferation of T
lymphocytes. Nitric oxide production and suppressive activity were also
dependent on endogenous interferon- (IFN-) production since
anti-IFN- abrogated both effects. Finally, iNOS protein expression
was restricted to a heterogeneous population of CD31+
cells in which CD11b+Ly-6G+ cells were
required to suppress T-cell proliferation. These results indicated that
CTX might also cause immunosuppression by a mechanism involving the
presence of immature myeloid cells with suppressor activity. This may
have implications in clinical praxis since inappropriate
immunotherapies in patients treated with intensive chemotherapy could
lead to deleterious T-cell responses. (Blood. 2000;95:212-220)
© 2000 by The American Society of Hematology.
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Introduction |
Recovery from immunodeficiency caused by high-dose
chemotherapy or radiotherapy remains as a major clinical problem. In
medical praxis, restoration of neutrophil and platelet counts is often seen as the hallmark of hematological reconstitution. This view somewhat neglects the important role of cellular interactions in
lymphoid organs, which are needed for functional recovery of the immune
system. This issue may have important implications in order to design
therapeutic approaches to improve the anti-infectious or antitumoral
resistance of patients undergoing bone marrow transplantation and/or
anti-cancer treatment.1,2
Cyclophosphamide (CTX) is a widely used antineoplasic drug, employed
alone or in combination with other products.3 Used as an
anticancer drug or in bone marrow transplantation conditioning regimes,
treatment with CTX severely injures hematopoietic and lymphoid tissues,
thereby leading to a profound leukopenia. However, when CTX is used as
a single agent, hematopoietic recovery always occurs, even at the
highest doses of the drug. This probably reflects the relative
resistance of stem cells to cytotoxic effects of CTX.3,4,5
Still, during the repopulation period, a transient unresponsiveness in
T and B lymphocytes is observed.1,2 In mice, those
anomalies have been correlated with a population of cells in the spleen
that are capable of nonspecifically suppressing, in an
major histocompatibility complex-unrestricted
fashion,9,10 primary and secondary in vitro antibody
responses6,7,8 as well as the in vitro proliferative
response of normal T lymphocytes. These cells have been poorly defined,
and in some cases, they have been characterized as a heterogeneous
population of cells that mainly belong to the monocyte/macrophage
lineage.9,11,12
On the other hand, the role of NO in immune response has received much
attention since its discovery as an activated-macrophage secretory
product.13 Nitric oxide is produced by the NO synthase (NOS, EC 1.14.13.39), of which 3 different isoforms have been identified in mammals.14 They are designated as neuronal or type 1 NOS (nNOS), inducible or type 2 NOS (iNOS), and
endothelial or type 3 NOS (eNOS). Both nNOS and eNOS isoforms are
constitutively expressed in neurons and endothelium as well as in other
tissues, and these isoforms produce low amounts of NO upon transient
raise in intracellular Ca++ concentration. In contrast,
iNOS is induced in macrophages and several other cell types by
cytokines and/or products derived from microorganisms. iNOS produces
high quantities of NO for extended periods of time,14,15
and it has become increasingly evident that iNOS-derived NO is involved
in suppression of several immune responses.14,16-20
In this study, we show that immature myeloid cells are the main agents
responsible for defective in vitro responses to mitogens in splenic T
cells obtained from CTX-treated mice by a NO-dependent mechanism. NO
production is dependent on endogenously synthesized interferon-
(IFN-) and is restricted to a heterogeneous population of
CD31+ cells.
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Materials and methods |
Mice
Specific pathogen-free female syngeneic Balb/c mice
(8-12 weeks of age) were used throughout the study. The mice (Harlan; Gannat, France) were maintained in the GlaxoWellcome Centro de Investigación Farmacológica (C.I.F.) animal facilities
(Tres Cantos, Spain). The animal research described in this paper
complied with national and European union legislation and with related codes of practice.
Reagents
We used the following reagents: CTX (Genoxal; Prasfarma, Barcelona,
Spain); streptavidin-R-phycoerythrin, lipopolysaccharide from
Salmonella typhi, concanavalin A,
L-N6-(1-iminoethyl)lysine (L-NIL), aminoguanidine
hemisulfate (AMG), and RPMI-1640 (Sigma, St. Louis, MO); horseradish
peroxidase-conjugated streptavidin (Zymed; San Francisco, CA);
penicillin/streptomycin mixture, L-glutamine, and fetal calf serum
(BioWhittaker, Walkersville, MD); mouse recombinant interleukin-2
(IL-2), IFN-, and tumor necrosis factor- (TNF-) (Genzyme;
Cambridge, MA); and NG-monomethyl-L-arginine (Dr José
L. Subiza, Madrid, Spain).
Antibodies
We used monoclonal antibodies (mAbs) against the
following cell-surface antigens (CSA) (Pharmingen, San Diego, CA):
CD3 (purified and biotin-conjugated hamster IgG, clone 145-2C11);
CD16/CD32 (FcBlock, rat IgG2b, clone 2.4G2); CD19 (fluorescein
isothyocynate-rat [FITC-rat] IgG2a, clone 1D3); CD45 (FITC-rat
IgG2b, clone 30-F11); CD45R/B220 (FITC-rat IgG2a, clone RA3-6B2); Ly-6G
(biotin-rat IgG2b, clone RB6-8C5); CD11b (biotin-rat IgG2b, clone
M1/70); CD90.2 (Thy-1.2) (FITC-conjugated rat IgG2a, clone 53-2.1); and erythroid lineage-specific TER-119 mAb (phycoerythrin-rat [PE-rat] IgG2b, clone TER-119).
Additional CSA were used: CD31 (biotin-rat IgG2a, ER-MP12; BMA, Augst,
Switzerland) and  TcR and CD116 (FITC-hamster IgG, clone H57-597, and
FITC-rat IgG2b, clone M1/70.15; Caltag, Burlingame, CA). We also used
the following isotype control mAbs (Pharmigen): biotin-, FITC-, and
PE-conjugated rat IgG2b (clone R35-38); FITC- and PE-conjugated rat
IgG2a (clone R35-95); and anti-TNP biotin-, FITC-, and PE-conjugated
hamster IgG (clone G235-2356).
We used anti-iNOS mAb (FITC-mouse IgG2a, clone 6; Transduction
Laboratories, Lexington, KY) and mouse IgG2a FITC-conjugated isotypic
control (Dr J Rullas, Hospital Clínico San Carlos, Madrid, Spain). Neutralizing rat IgG1 antimouse IFN- mAb and TNF- mAb (clones XMG1.2 and XT3; Endogen, Woburn, MA) were used at 10 µg/mL. Their specificity and activity were assayed by inhibiting NO production using resident peritoneal macrophages stimulated with IFN- (25 ng/mL) plus TNF- (50 ng/mL).
The characterization of T lymphocytes was assessed by the expression of
CD3, TcR, or CD90.2 (Thy-1.2). The characterization of B
lymphocytes was assessed by analyzing the cells expressing CD19 or
CD45/B220, both expressed from pro-B lymphocytes to mature B cells. The
cells of myeloid/monocytic lineage were identified on the basis of
CD11b (Mac-1) or Ly-6G (Gr-1). The expression of leukocyte common
antigen CD45 was used to distinguish the cells of hematopoietic origin
but of nonerythrocytic lineage. Cells of erythrocytic
lineage express an antigen recognized by TER-119 mAb.
Cell cultures
SC or immunomagnetically sorted cells (0.4 × 106
cells/well) were cultured in 96-well flat-bottom culture plates
(Costar, Cambridge, MA) in 250 µL of culture medium (RPMI 1640, 10%
FCS, 2 mmol/L L-glutamine, 5 × 10-5 mol/L
2-mercaptoethanol, 100 units/mL penicillin, 0.1 µg/mL streptomycin) in an atmosphere of 5% CO2 at 37°C. Proliferation
assays were done in triplicate, culturing the cells for 72 hours. For
the last 18 hours of the culture period, proliferative activity was estimated measuring thymidine deoxyribose
([3H]TdR) incorporation after a pulse with 0.5 µCi/well
(1450 MicroBeta Liquid Scintillation Counter; Wallac, Turku, Finland).
Immunomagnetic depletion of spleen cells
In some experiments, populations of SC were selectively depleted by
immunomagnetic beads (Dynabeads; Dynal, Oslo, Norway). Depletion of CD3
cells or CD31-positive cells was carried out by streptavidin-conjugated
beads, following the manufacturer's instructions. On the other hand,
CD11b+ (Mac-1) cells were depleted by sheep antirat
IgG-coated immunomagnetic beads. Briefly, CTX-SC were incubated for 20 minutes in HBSS 1% FCS 20% normal mouse serum at
4°C. Then the cells (30 × 106 cells/mL) were
stained with CD11b-FITC-conjugated (0.5 µg/106)
cells for 30 minutes, washed, and incubated at the same cell
concentration with antirat IgG-coated immunomagnetic beads (4:1
bead/cell ratio) for 1 hour at 4°C in a rotor. The efficacy of the
process was assessed by flow cytometry. In all cases contamination with
remaining positive cells was below 5%.
Flow cytometry
A known number of cells (0.2 × 106 cells) were
incubated with CD16/CD32 mAbs at 10 µg/mL in 50 µL of staining
buffer (HBSS, 1% FCS, 0.05% azide) for 10 minutes at 4°C.
Subsequently, appropriate biotin-conjugated mAbs were added at 10 µg/mL and incubated for 30 minutes at 4°C. After washing with
staining buffer, samples were stained with FITC-conjugated or
PE-conjugated mAbs (10 µg/mL) plus PE- or FITC-streptavidin
(20µg/mL), respectively, for 30 minutes at 4°C in the dark. After
final washing steps, the cells were fixed with 250 µL of
phosphate-buffered saline (PBS) 1% paraformaldehyde. Finally, 5000 viable cells were acquired on the basis of forward/side light
scattering and analyzed (Coulter EPICS-XL; Coulter Electronics, Miami, FL).
iNOS protein expression was assessed by flow cytometry. Briefly, after
24-30 hours of stimulation, the cells were recovered from culture wells
(about 0.8 × 106 cells per sample) and stained for
surface markers as above. After washing with cold HBSS, the samples
were fixed with PBS-paraformaldehyde 4% at 4°C for 30 minutes in
the dark. Then the cells were washed twice with cold HBSS, twice with
permeabilization buffer (PEB, saponin 0.1% in staining buffer),
pelleted, and resuspended in 0.05 mL PEB. Subsequently, the cells were
stained with mouse IgG-FITC-conjugated antimouse iNOS or isotypic
control at 10 µg/mL for 1 hour, after which they were carefully
washed 3 times with PEB and twice with staining buffer.
Measurement of cytokines
Cytokine contents (IL-4 and IFN-) were measured in 24-hour
supernatants of the aforementioned cell cultures using a specific enzyme-linked immunosorbent assay (ELISA) for mice (MiniKit; Endogen), according to manufacturer's instructions.
Reverse transcriptase-polymerase chain reaction
We cultured SC (0.4 × 106 cells/well) in medium
alone and in medium with Con A or with anti-CD3 plus IL-2 for 24 hours,
as described above. Then total mRNA was isolated (PolyATtract Series 9600 mRNA isolation and cDNA synthesis system; Promega, Madison, WI) as
recommended. Subsequently, the cDNA obtained was employed as a template
for PCR amplification of mouse glyceraldehyde-3-phosphate dehydrogenase
(G3PDH) and mouse iNOS using 5' and 3' available primers
(Clontech Lab, Palo Alto, CA). PCR reactions and analysis were
conducted according to previously described conditions.21
Nitric oxide production
Nitric oxide production was assessed by measuring nitrite
accumulation in 72-hour culture supernatants. This was carried out using the Griess reaction.22 Briefly, 100 µL of
0.5% sulfanilamide and 0.05%
N-naphtyl-ethylenediamine hydrocloride in 2.5%
H3PO4 (Griess reagent) were added to 100 µL
of supernatants and incubated for 5 minutes at room temperature in the
dark. The absorbance was then measured at 550 nm, and nitrite
concentrations were extrapolated from a sodium nitrite standard curve.
Statistical analysis
Experimental differences over the controls were analyzed by the
Student's t test. Probability values P > 0.05 were
considered nonsignificant. All the experiments described were performed
at least twice in order to warrant the reproducibility of the results.
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Results |
The effect of CTX treatment on spleen T-lymphocyte proliferative
activity
Female BALB/c mice were treated with 3 intraperitoneal (i.p.)
injections of CTX at 100 mg/kg of body weight every 72 hours, whereas
control mice received PBS. At days 3, 6, 10, and 20 after the last dose
of drug was administered, SC were obtained and assayed for T-cell
proliferation induced with Con A or anti-CD3 plus IL-2. A strong
impairment of T-cell proliferation in response to both stimuli was
observed at day 6 (more than 98% suppression) and day 10 (between 50%
and 90% suppression, depending on the experiment) after CTX
administration (Figure 1A). No relevant
suppression was noted at days 3 and 20.
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| Fig 1.
Kinetics of splenic T-cell responsiveness and the major
presence of cell populations in the spleen after CTX treatment.
Female Balb/c mice were injected i.p. with CTX (100 mg/kg of body
weight) or PBS 3 times every 72 hours. At days 3, 6, 10, and 20 after
CTX delivery, SC were aseptically obtained and assayed for T-cell
proliferation with Con A (1µg/mL) or anti-CD3 (2 µg/mL) plus IL-2
(50 units/mL) as previously described. Data are the percentage of
proliferation over mitogen-stimulated SC control cultures (A) to
account for differences in proliferative response of these cells in
different days of the experiment. From the very same spleens,
CD19+ B cells, TcR+ T cells, and
CD11b+ macrophage/myeloid cells were analyzed by flow
cytometry as described (B). In this figure, the absolute number of each
population is represented by the height of each stacked bar. The data
are the means of positive cells of PBS-injected control (2 mice/d) and
CTX-injected animals (3 mice/d). SD deviations were <10% in all
cases.
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Theoretically, the impaired response to mitogens could be attributed to
the cytotoxic effect of CTX on SC. To address this point, we used flow
cytometry to analyze the major cell populations present in the spleen.
The results obtained are summarized in Figure 1B. CTX treatment induced
a sharp decrease in splenic cellularity at day 3, after the last dose
(approximately 80% reduction was noted, compared with control mice).
Most of the remaining cells were TcR+ T lymphocytes
(comprising about 70% in CTX-SC versus an average of 35% in control
SC). This represented a drop of around 60% in total T-cell numbers
with respect to those found in control SC. On the other hand,
CD19+ B lymphocytes, which represented 54% in control SC,
were <12% in CTX-SC, which is a drop of more than 20 times in total
number of B cells. However, the percentage of CD11b+ cells
remained unchanged (between 6% and 15% both in SC and CTX-SC).
Overall, the absolute numbers of T and B lymphocytes and myeloid cells
markedly decreased in CTX-SC, but T cells seemed to be more resistant
to the cytotoxic effects of CTX than B lymphocytes. Conversely, at day
6 after CTX treatment, there was a great increase in CD11b+
cells, both in percentage and absolute numbers, that persisted at day 10. At day 20 we found that CD11b+
cells were only slightly elevated (about 20%) compared with
percentages found in SC. Once more, nearly normal percentages and
absolute numbers of T and B lymphocytes were reached at day 20. Therefore, it seems that the decrease in T-cell numbers could not fully
explain the strong impairment of T-cell proliferation seen at days 6 or 10. The same number of residual T cells found at day 3 was also found at days 6 or 10, and they could, theoretically,
proliferate as well.
Involvement of NO in suppression of proliferative responses of T
lymphocytes
The results shown above suggested the existence of an active
suppression phenomenon. Thus, we assessed the involvement of NO or
other reactive nitrogen intermediates (RNI) in the reduced proliferative activity of spleen T cells from CTX-treated mice at days
6 or 10 after treatment. Unstimulated cultures from both SC and CTX-SC
did not release detectable amounts of NO. Interestingly, supernatants
of CTX-SC cultures stimulated with ConA or anti-CD3 plus IL-2 produced
much larger amounts of NO or other RNI (between 10 and 30 µmol/L), as
assessed by nitrite accumulation in 72-hour culture supernatants, than
SC from control mice (<3 µmol/L in all cases).
We also analyzed iNOS mRNA expression in cultures of SC or CTX-SC.
There was a marked correlation between the increase of iNOS mRNA
expression in CTX-SC and the production of high amounts of nitrite in
culture supernatants (Figure 2A). These
results indicate that there is a strong relationship between NO
production and the suppression of proliferation. More interestingly, in
Con A- or anti-CD3 plus IL-2-stimulated cultures, the addition of NOS
inhibitors markedly improved the proliferation of T lymphocytes (Figure
2B). Moreover, there was no significant difference in improving
proliferation between the structurally different NOS inhibitors used,
such as NG-monomethyl-L-arginine (LMMA), a nonspecific NOS
inhibitor, and L-N6-(1-iminoethyl)lysine (LNIL) or AMG,
more specific inhibitors of iNOS.23-25 Thus, it seems
unlikely that their effect in spleen cultures was unspecific. Taken
together, the former results indicate that iNOS-dependent NO production
substantially contributes to the inhibition of proliferation of T cells
from the spleen of CTX-treated mice.
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| Fig 2.
NO involvement in CTX-induced inhibition of T-cell
proliferation.
(A) iNOS mRNA analysis by RT-PCR. 0.4 × 106
cells/well were cultured for 24 hours in the presence of Con A
(1µg/mL) or anti-CD3 (2µg/mL) plus IL-2 (50 units/mL) in the same
conditions used for proliferative assays, and iNOS or control G3PDH
mRNA was analyzed by RT-PCR as previously. The figure also shows
PCR-positive and -negative controls. (B) The effect of NOS inhibitors
on T-cell proliferation. Day 6 or 10 after the last dose of CTX, T-cell
proliferative activity induced with Con A (1µg/mL) or anti-CD3 (2 µg/mL) plus IL-2 (50 units/mL) was assessed in the absence or the
presence of LMMA (0.5 mmol/L), L-NIL (0.5 mmol/L), or AMG (0.5 mmol/L)
added at culture initiation. The cells were cultured for 72 hours, and
proliferation was estimated by [3H]TdR
incorporation in the last 18 hours of culture. Data are the mean
±SEM of triplicate cultures from 3 mice injected i.p. with CTX. At
the concentrations used, iNOS inhibitors did not have any significant
effect over proliferation of control SC cultures that ranged between
100 × 103 and 150 × 103 cpm
(not shown). (C) Nitrite accumulation in culture supernatants from SC
cultures. Nitrite was measured in the very same cultures used for
proliferation assays after 72 hours of culture. As above, data are
expressed as the mean ±SEM. Nitrite production by control SC
cultures was always <3 µmol/L.
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Endogenous IFN- is required for NO production by CTX-SC
In most cell types, iNOS induction relies on cytokine signaling,
particularly by IFN-.14,26 Hence, we studied the role of
IFN- in the production of NO in mitogen-stimulated cultures. First,
we assessed IFN- production by CTX-SC cultures. As expected, control
SC challenged with T-cell mitogens produced large amounts of IFN-
after 24 hours, being higher in anti-CD3 plus IL-2-stimulated cultures
than Con A-stimulated cultures (Table 1).
Surprisingly, Con A-stimulated CTX-SC produced as much IFN-
as control SC, despite the almost complete block in proliferation
and the lower number of T cells found in CTX-SC (Figure 1). When
stimulated with anti-CD3 plus IL-2, about 50% of IFN- production,
with respect to SC controls, was detected in CTX-SC cultures.
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Table 1.
The effect of CTX treatment on IFN- or IL-4
production and proliferative activity of spleen cells stimulated with
T-Cell mitogens
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Next we examined the effect of a neutralizing mAb to IFN- in CTX-SC
or SC cultures treated with Con A or anti-CD3 plus IL-2. The addition
of anti-IFN- mAb did not affect proliferation by SC (Figure
3A). However, it enhanced the proliferation
of CTX-SC to Con A or anti-CD3 plus IL-2 in a similar way as NOS
inhibitors did. Again, this correlated with a total inhibition of the
NO produced in these cultures (Figure 3B). We also determined whether IFN- was sufficient to induce high amounts of NO. To assess this, we
added increasing concentrations of IFN- alone or with other co-stimuli to CTX-SC cultures. Data obtained in a representative experiment are shown in Figure 4.
Recombinant IFN- (up to 40 ng/mL) was unable to induce the
production of large amounts of NO. However, the addition of recombinant
TNF- to IFN--treated cultures triggered a massive NO production.
Similar results were obtained with LPS (10 µg/mL) or an agonistic mAb
anti-CD40 (1C10)27 employed as costimulus,
although this mAb by itself was able to induce NO production in the
absence of exogenous IFN- in some experiments (data not shown).
Therefore, IFN- production is necessary, but not sufficient, to
achieve high NO levels in cultures of CTX-SC, and IFN-
neutralization markedly improved the proliferative response of T cells
from CTX-SC.
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| Fig 3.
The effect of IFN- neutralization in SC cultures.
SC (0.4 × 106 cells/well) were stimulated with Con
A (1 µg/mL) or anti-CD3 (2 µg/mL) plus IL-2 (50 units/mL) and
cultured for 72 hours with or without antimurine IFN- neutralizing
mAb (10 µg/mL) added at culture initiation.
[3H]TdR incorporation (A) and nitrite
production (B) were assessed at 72 hours after the starting point. Data
are the mean ±SD of triplicate cultures of pooled cells from PBS-
or CTX-injected mice.
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| Fig 4.
IFN--dependent NO production by CTX-SC.
CTX-SC (0.4 × 106 cells/well) were cultured in the
absence or presence of increasing amounts of IFN- (5-20 ng/mL) plus
TNF- (4-40 ng/mL). Nitrite accumulation was measured after 72 hours
of culture. Data are the mean ±SD of triplicate cultures from
pooled spleens.
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Characterization of NO-producing suppressive cells
To characterize the phenotype of NO-producing cells, we used flow
cytometry to analyze the cell populations found in CTX-SC. In all
cases, at least 2 markers per cell population were studied in order to
control for any effect of CTX on the expression of critical surface
molecules.28 B lymphocytes, assessed by CD19 (already shown
in Figure 1B) or B220 expression, were severely depleted at day 6 and
particularly day 10, after the last CTX dose, which in most cases was
<7% CTX-SC (Figure 5). Moreover, the
percentage of CD3+ (Figure 5), CD90.2
(Thy-1.2+), or TcR+ T cells (not shown) was
reduced from 50% to 35% of positive cells found in control SC,
depending on the experiment (10%-20% of total cells in CTX-SC versus
35%-42% of total cells in control SC). However, a prominent
population of large and granular cells
CD45+CD11b+Ly-6G+CD31
(ER-MP12)+ accounted for about 50%-75% of splenocytes
from CTX-treated mice, whereas PBS-treated control SC contained <7%
of that population. Accordingly, Giemsa-stained cytospin preparations
of CTX-SC showed the predominance of blasts, immature granulocytic, and
monocytic cells (Figure 6).
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| Fig 5.
Major cell populations in CTX-SC.
Fresh SC from PBS- and CTX-injected mice were obtained 6 days after the
last injection of PBS/CTX and stained with anti-CD45, CD11b, or B220
FITC-labeled mAbs or biotinylated CD31, CD3, or anti-Ly-6G followed by
streptavidin R-phycoerythrin conjugate (AvPE). Isotypic controls showed
<0.5% in selected regions.
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| Fig 6.
Presence of immature myeloid cell in CTX-SC.
Giemsa staining was performed in cytospin preparations of concentrated
fresh SC of PBS-injected (top, SC) or CTX-injected (bottom, CTX-SC)
mice 6 days after the last dose of CTX. Samples were analyzed using an
Olympus Vanox AHBT3 microscope, original magnification ×1000.
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Subsequently, we used flow cytometry to verify the expression of iNOS
in CD11b+ CD31+ cells of CTX-SC after
stimulation with Con A or anti-CD3 plus IL-2. CTX-SC were stimulated
with mitogens for 24 or 30 hours. The cells were then recovered,
stained for suitable surface markers, and assayed for intracellular
expression of iNOS. The results depicted in Figure
7 show that intracellular expression of
iNOS protein is restricted to CD31 (ER-MP12)+ cells or
between 30%-60% of cells in mitogen-treated cultures. In addition, it
was clearly seen that iNOS protein was induced in
CD11b+CD31 (ER-MP12)+ cells, although a
substantial percentage of iNOS+ cells did not express
CD11b. We concluded that the NO-producing ability was induced by
mitogens in a heterogeneous population, such as CTX-SC, and in
particular in CD11b+Ly-6G+CD31+
cells.
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| Fig 7.
iNOS expression in CTX-SC.
CTX-SC (0.4 × 106 cells/well) were cultured for
24-36 hours in the absence of or with Con A (1µg/mL) or anti-CD3 (2 µg/mL) plus IL-2 (50 units/mL). The cells were then recovered and
stained with biotinylated anti-CD11b, anti-CD31, or PE-conjugated
control TER-119 mAbs followed by AvPE, if required, for surface markers
and FITC-conjugated antimouse iNOS. Isotypic controls showed <0.8%
background in the selected regions. The results shown were obtained in
3 separate experiments.
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To confirm the involvement of
CD11b+Ly-6G+CD31+ cells in the
suppression of T-cell proliferation, we performed a series of
experiments by selective cell depletion. Thus, we depleted
CD3+ or CD11b+ cells from CTX-SC. Data depicted
in Figure 8A show that depletion of
CD3+ cells abolished the potential of CTX-SC to release NO
promoted by T-cell mitogens (Con A or anti-CD3 plus IL-2)
but not that promoted by the exogenous addition of
IFN- plus TNF-. As expected, there was a lack of proliferation in
CD3 cells to both mitogens (Figure 8B). Interestingly,
depletion of CD11b+ cells reduced NO production by about
50% but fully restored T-cell responsiveness. This suggests that
CD11b+Ly-6G+CD31 (ER-MP12)+ cells
are the main NO producers in mitogen- or cytokine-stimulated CTX-SC and
are the cells responsible for suppressing the proliferation of splenic
CTX-SC T lymphocytes.
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| Fig 8.
The effect of selective cell depletion in T-cell
proliferation of CTX-SC.
SC and CTX-SC were obtained at day 6 (CD11b+ cell
depletion) or day 10 (CD3+ cell depletion) after the last
dose of PBS/CTX; 0.4 × 106 cells/well were
stimulated with Con A (1µg/mL), anti-CD3 (2µg/mL) plus IL-2 (50 U/mL), or IFN- (5 ng/mL) plus TNF- (40 ng/mL) as indicated. The
cells were assayed for (A) NO production after 72 hours of culture and
(B) T-cell proliferation. Data are the mean ±SD of triplicate
cultures.
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Discussion |
In this work, we have investigated the mechanisms
responsible for T-cell deficiency observed after a course of intensive
chemotherapy with CTX. Our results show that, besides its well-known
cytotoxic effect, the immunosuppressive activity of CTX also results
from the presence of a heterogeneous splenic cell population induced by
CTX treatment. Those cells have been characterized as mostly immature
myeloid/monocytic cells,
CD11b+Ly-6G+CD31+, and their
inhibitory activity is dependent on iNOS-derived NO production. They
can account for the profound impairment in the proliferative activity
displayed by splenic T cells during recovery from chemotherapy. Nitric
oxide-producing suppressor cells are CD11b+, although
there is also a population of CD11b cells that express
iNOS. In this regard, preliminary data from our laboratory indicate
that immature CD11bCD117
(c-kit)+ cells are also able to express iNOS.
Our results are compatible with a mechanism in which activated T
lymphocytes release cytokines (especially IFN-), which together with
other yet undefined signals, induce iNOS expression by immature myeloid
cells. Nitric oxide or other RNI generated in this way, acting alone or
together with other suppressive signals, can suppress further T-cell
activation and proliferation. Similar mechanisms involving
IFN--induced NO-mediated suppression have been proposed in order to
account for the suppression observed in a number of experimental
systems including hematopoietic tissues,21,29,30 infectious
diseases,14,17,18,31 graft-versus-host disease (GVHD),19,32 tumor bearers,33,34 and exposure
to toxic compounds.35 The following evidence supports the
aforementioned model.
First, NO production is required to suppress the proliferation of T
lymphocytes. The fact that all 3 NOS inhibitors tested (LMMA, AMG,
L-NIL) greatly improved the proliferative activity of T lymphocytes
from CTX-SC in response to Con A or anti-CD3 plus IL-2 suggests that
the suppressive activity relies on iNOS induction. Moreover, given
their different structures and mechanism of
inhibition,23-25 it is highly improbable that their effects were due to unspecific activities and not to iNOS inhibition. In
addition, much higher levels of nitrite and iNOS mRNA expression were
found in CTX-SC cultures stimulated with mitogens than in SC control
cultures. This evidence indicates that NO or other RNI contribute
substantially to the profound inhibition of T-cell proliferation in
CTX-SC cultures. However, although it has been shown that NO itself is
able to inhibit proliferation of T lymphocytes36 and other
cell types,37 we cannot discard the fact that other signals
could also be involved in the mechanism of suppression, as it has been
described in the Fas-mediated destruction of pancreatic -cells in
insulin-dependent diabetes mellitus.38 In this work we have
not addressed the molecular mechanism of NO-mediated immunosuppression. Preliminary results suggest that most of the suppressive activity is
due to NO-dependent apoptosis of T cells in mitogen-stimulated CTX-SC
cultures. T-cell apoptosis is clearly reduced by iNOS inhibitors (data
not shown). In this regard, it is worth mentioning that NO has been
shown to induce apoptosis in several other experimental systems.39,40
Second, T lymphocytes are absolutely required for mitogen-induced NO
production as deduced from cell-depletion experiments. This points to
signals derived from activated T lymphocytes as the main inducers of NO
production. Nevertheless, depletion of T cells did not impair the
NO-releasing ability of CTX-SC promoted by exogenous IFN- plus
TNF-. In this regard, IFN- has been shown to be an essential
signal for NO induction in most experimental systems.14,16-19,21,26,32 Upon suitable activation, IFN-
is produced in large amounts by T lymphocytes and NK
cells26 and some macrophages.41,42 In our
experimental system, IFN- also seems to be a key molecule, both in
NO production and in the inhibition of T-cell proliferation. However,
our results suggest that additional signals are also required to
achieve a significant NO production, in accordance with previous
reports from a variety of cell systems.16,18-21,26,32 Specifically, TNF- could contribute to NO production.14
Nevertheless, although a neutralizing anti-TNF- mAb strongly
inhibited NO production in our cultures, it did not recover T-cell
proliferation (data not shown). This effect appeared related to the
fact that this mAb markedly diminished the normal mitogen-induced
proliferation of control SC (data not shown). So, it is likely that the
dominant activity of anti-TNF- in our cultures was the impairment
of early T-cell activation events rather than the signaling
interference of NO-producing cells. Similar inhibition of cell
proliferation by neutralizing anti-TNF- mAb has been previously
reported in human T cells.43 Although other cytokines and
colony stimulating factors are putative candidates to cooperate with
IFN-, it seems likely that cognate cell interactions can play a
decisive role both in NO production and in T lymphocyte suppression. In
support of this possibility we have found that the agonist
CD40 mAb (1C10)27 acted synergistically with
IFN- in inducing high amounts of NO by CTX-SC. This is not
surprising because CD40-CD40L(CD154) interactions have been shown to
play a role in NO production in allograft rejection44 and
in macrophages,45 dendritic cells,46 and bone
marrow-derived immature myeloid cells with natural suppressor (NS)
activity (Angulo et al, submitted manuscript).
Third, the dramatic drop in proliferative activity of T lymphocytes
from CTX-SC is associated with the colonization of spleen by large and
granular immature myelo-monocytic
CD45+CD11b+Ly-6G+CD31+
cells and not with depletion of T cells. Kinetic analysis showed that
the absolute number of T lymphocytes remained relatively unchanged
between days 3 and 10 after the last dose of CTX. The recovery of
cellularity seen at days 6 and 10 was due to the colonization of the
spleen mainly by immature cells expressing myeloid markers 47-49 and not due to T or B lymphocytes. This fact agrees
with previous data showing that after chemotherapy, progenitor cells are mobilized from the bone marrow to the periphery, and the spleen becomes a major site of extramedullary hematopoiesis.50,51 Moreover, NO production by hematopoietic progenitors has been demonstrated by a number of authors.52 Indeed,
CD11b+CD31+ cells are able to express iNOS in
cultures stimulated with T-cell mitogens. Thus, those cells have a high
potential to produce NO in CTX-SC cultures upon induction by
T-cell-derived signals. This was further supported by depletion of
selected cell populations. Depletion of CD11b+ cells, which
included almost all Ly-6G+ and expressed CD31 cells,
reduced NO production and restored T-cell responsiveness, indicating
that this population is mainly responsible for inhibition of
T-lymphocyte proliferation.
Consistent with these results were those obtained by depletion of
CD31+ cells from CTX-SC. Unfortunately, interpretation of
data obtained in these latter experiments has been hampered by 2 facts:
(1) CD31 cells seemed to be directly or indirectly up-regulated by Con
A or anti-CD3 plus IL-2, and (2) depletion of CD31+ cells
abolished proliferation of T lymphocytes from CTX-SC despite the high
percentage of remaining TcR+ cells. Thus, although
CD31+ depletion nearly eliminated NO production in response
to mitogens, it also caused a severe impairment of T-cell activation,
possibly due to depletion of accessory cells. This view is further
supported by the strong inhibition of NO production observed when
CTX-SC cells are stimulated with IFN- plus TNF-. Overall, the
evidence points to
CD11b+Ly-6G+CD31+ immature myeloid
cells as the main actors responsible for T-cell inhibition in CTX-SC cultures.
It is also noteworthy that, as given in previous reports, the
generation of suppressor cells in the spleen of mice treated with CTX
is able to inhibit in vitro responses of T and B lymphocytes obtained
from normal lymphoid organs.6-12 In some cases, this inhibition occurs in an IFN--dependent fashion.10,12 In
the best characterized system, the suppressor activity was assayed in
mixed lymphocyte reaction (MLR), and the suppressive cell population was heterogeneous, comprising null cells or B220+,
TcR+, or Mac-1+ subpopulations, this latter
being the most potent in suppressing lymphocyte
proliferation.12 Although these results are basically in
agreement with ours, the authors discarded NO involvement in the
mechanism of suppression. Still, suppression appeared to be dependent
on IFN- production and was attributed in part to prostaglandin production and/or blocking of IL-2/IL-4 utilization.53 The
discrepancy with our results could be explained by differences in the
experimental models; Brooks et al12 used a
single injection of 200 mg/kg CTX and assayed suppressive activity by
MLR. Importantly, Krenger et al32 found that
inhibition of proliferative responses of spleen cells from mice
undergoing acute GVHD was due to NO production if the cells were
stimulated by Con A. This did not occur in MRL assays. Therefore, the
nature of the mitogenic stimulus and the assay system used could have
decisive importance in the mechanisms of suppression. In fact, in our
experimental system, which lacks allogeneic cells, nitrite levels were
quite similar in Con A-stimulated and anti-CD3 plus IL-2-stimulated
CTX-SC cultures. Microscopic examination, however, showed an extensive
cellular death in the latter, whereas in Con A-stimulated cultures,
this phenomenon was not so evident. Therefore, it seems likely that
different signals are involved in the mechanism of suppression, as a
function of the T-cell mitogen employed.38,54
Among the problems associated with intensive chemotherapy, restoration
of immunocompetence is a critical issue in patient management.1,2 Here we have shown that CTX treatment could induce an state of immunodepression by at least 2 mechanisms: (1)
direct cytotoxic effects that severely reduce the number of T
lymphocytes and (2) the presence in the spleen of a heterogeneous population of immature myeloid cells able to suppress T-cell
proliferation upon activation. So, it is conceivable that activation of
splenic T cells in an altered context could lead to their NO-dependent deletion or functional inactivation effected by those immature CD11b+Ly-6G+CD31+ cells. This
hypothesis is in close agreement with some recent data from other
authors. Thus, it has been described that vaccination-induced splenic
immunosuppressive Mac-1+/Gr-1+, comprising
monocytes and a population of immature myeloid cells, can suppress
specific CTL responses in vivo and in vitro in mice immunized with powerful immunogens.55 Moreover, from our
point of view, this phenomenon is likely related to the NO-dependent suppressive effects caused by IL-12 in different systems of
vaccination.56-58 Accordingly, in both Trypanosoma
cruzi and Histoplasma capsulatum infectious models, it
has been suggested that splenic T cells can be deleted by NO-dependent
apoptosis.59,60
In conclusion, our results may have clinical importance in implementing
immunotherapies in immunocompromised patients due to the risk of
undesired activation of T lymphocytes in the presence of suppressive
cells able to delete or inactivate T lymphocytes in lymphoid tissues.
| |
Acknowledgments |
We are grateful to Drs J. L. Subiza and J. Rullas (Hospital
Clínico, Madrid, Spain) for their help and expertise in flow cytometry experiments. We are also indebted to Dr F. J. Gamo (Glaxo Wellcome, Tres Cantos, Spain) for his help in PCR analysis, Dr Andrew
W. Heath (University of Sheffield, Sheffield, England) for the generous gift of purified 1C10, and Lucía Horrillo for secretarial assistance.
| |
Footnotes |
Submitted May 17, 1999; accepted August 17, 1999.
Supported by grants from Glaxo Wellcome S.A., Dirección General
de Investigación Científico y Técnica, and the
Fundación Ramón Areces.
Reprints: Manuel Fresno, Centro de Biología Molecular
Severo Ochoa, C.S.I.C.-U.A.M., Cantoblanco, 28049 Madrid, Spain.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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C. Sanchez-Valdepenas, A. G. Martin, P. Ramakrishnan, D. Wallach, and M. Fresno
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S. Noda, S. A. Aguirre, A. Bitmansour, J. M. Brown, T. E. Sparer, J. Huang, and E. S. Mocarski
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J. Honeychurch, M. J. Glennie, and T. M. Illidge
Cyclophosphamide Inhibition of Anti-CD40 Monoclonal Antibody-Based Therapy of B Cell Lymphoma Is Dependent on CD11b+ Cells
Cancer Res.,
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L. Brys, A. Beschin, G. Raes, G. H. Ghassabeh, W. Noel, J. Brandt, F. Brombacher, and P. D. Baetselier
Reactive Oxygen Species and 12/15-Lipoxygenase Contribute to the Antiproliferative Capacity of Alternatively Activated Myeloid Cells Elicited during Helminth Infection
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N. Deruytter, O. Boulard, and H.-J. Garchon
Mapping Non-Class II H2-Linked Loci for Type 1 Diabetes in Nonobese Diabetic Mice
Diabetes,
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[Abstract]
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K. M. Heinonen, F. P. Nestel, E. W. Newell, G. Charette, T. A. Seemayer, M. L. Tremblay, and W. S. Lapp
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M. Terabe, S. Matsui, J.-M. Park, M. Mamura, N. Noben-Trauth, D. D. Donaldson, W. Chen, S. M. Wahl, S. Ledbetter, B. Pratt, et al.
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C. Melani, C. Chiodoni, G. Forni, and M. P. Colombo
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A. D. Billiau, S. Fevery, O. Rutgeerts, W. Landuyt, and M. Waer
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Y. Liu, J. A. Van Ginderachter, L. Brys, P. De Baetselier, G. Raes, and A. B. Geldhof
Nitric Oxide-Independent CTL Suppression during Tumor Progression: Association with Arginase-Producing (M2) Myeloid Cells
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H. F. Ismail, P. Fick, J. Zhang, R. G. Lynch, and D. J. Berg
Depletion of Neutrophils in IL-10-/- Mice Delays Clearance of Gastric Helicobacter Infection and Decreases the Th1 Immune Response to Helicobacter
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O. Goni, P. Alcaide, and M. Fresno
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A. Mencacci, C. Montagnoli, A. Bacci, E. Cenci, L. Pitzurra, A. Spreca, M. Kopf, A. H. Sharpe, and L. Romani
CD80+Gr-1+ Myeloid Cells Inhibit Development of Antifungal Th1 Immunity in Mice with Candidiasis
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September 15, 2002;
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A. Mazzoni, V. Bronte, A. Visintin, J. H. Spitzer, E. Apolloni, P. Serafini, P. Zanovello, and D. M. Segal
Myeloid Suppressor Lines Inhibit T Cell Responses by an NO-Dependent Mechanism
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January 15, 2002;
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E. A. Patton, A. C. La Flamme, J. A. Pedras-Vasoncelos, and E. J. Pearce
Central Role for Interleukin-4 in Regulating Nitric Oxide-Mediated Inhibition of T-Cell Proliferation and Gamma Interferon Production in Schistosomiasis
Infect. Immun.,
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O. Atochina, T. Daly-Engel, D. Piskorska, E. McGuire, and D. A. Harn
A Schistosome-Expressed Immunomodulatory Glycoconjugate Expands Peritoneal Gr1+ Macrophages That Suppress Naive CD4+ T Cell Proliferation Via an IFN-{gamma} and Nitric Oxide-Dependent Mechanism
J. Immunol.,
October 15, 2001;
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B. Pelaez, J. A. Campillo, J. A. Lopez-Asenjo, and J. L. Subiza
Cyclophosphamide Induces the Development of Early Myeloid Cells Suppressing Tumor Cell Growth by a Nitric Oxide-Dependent Mechanism
J. Immunol.,
June 1, 2001;
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E. Apolloni, V. Bronte, A. Mazzoni, P. Serafini, A. Cabrelle, D. M. Segal, H. A. Young, and P. Zanovello
Immortalized Myeloid Suppressor Cells Trigger Apoptosis in Antigen-Activated T Lymphocytes
J. Immunol.,
December 15, 2000;
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V. Bronte, E. Apolloni, A. Cabrelle, R. Ronca, P. Serafini, P. Zamboni, N. P. Restifo, and P. Zanovello
Identification of a CD11b+/Gr-1+/CD31+ myeloid progenitor capable of activating or suppressing CD8+ T cells
Blood,
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Top
Abstract
Introduction