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Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 1028-1037
Nitric Oxide Mediation of Active Immunosuppression Associated With
Graft-Versus-Host Reaction
By
Pierre Bobé,
Karim Benihoud,
Danièle Grandjon,
Paule Opolon,
Linda Louise Pritchard, and
Roger Huchet
From INSERM Unité 267 "Immunogénétique des
Allogreffes", Hôpital Paul Brousse, Villejuif Cedex, France;
Université Paris XI, Orsay Cedex, France; Service de Pathologie
Expérimentale, Institut Gustave Roussy, Villejuif Cedex, France;
and CNRS UPR 9079, IFR1221, Villejuif Cedex, France.
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ABSTRACT |
In the immunosuppression accompanying the lethal systemic
graft-versus-host reaction (GVHR) directed against minor
histocompatibility antigens in irradiated adult mice, we previously
determined that non-T, non-B, L-leucine methyl ester (LME)-sensitive
cells were implicated via two different mechanisms: one, which is
interferon- (IFN-)-dependent and affects both T-cell
proliferative responses and thymus-independent antibody production by
CD5+ B cells; and a second, which is IFN--independent
and affects B-cell proliferative responses. Because IFN- induces the
production of nitric oxide (NO), a potent
immunosuppressive molecule, we investigated the involvement of NO in
the suppression mediated by the LME-sensitive cells. Inducible NO
synthase (iNOS) mRNA, iNOS protein, and the stable end products of iNOS
pathway, L-citrulline and nitrite, were detected early in GVHR in
LME-sensitive spleen cells taken ex vivo and could be amplified in
vitro by T and B mitogens. Inhibition of NO production with arginine
analogs (aminoguanidine, NG-monomethyl-L-arginine
[LMMA]), like anti-IFN- antibodies, reversed suppression of both
T-cell responses to concanavalin A and CD5+ B-cell
responses, but not of B-cell response to lipopolysaccharides (LPS). The
GVHR-associated, IFN--dependent immunosuppression of T-cell
proliferation and of antibody synthesis by CD5+ B cells
is the consequence of NO production by LME-sensitive cells.
Immunohistochemical analyses indicate that these cells belong to the
macrophage lineage.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE GRAFT-VERSUS-HOST reaction (GVHR) is
a major complication of bone marrow transplantation. GVHR is initiated
by mature alloreactive T cells of donor origin, specific for antigenic differences that can be encoded by genes located outside the major histocompatibility complex (MHC). This may occur both in humans, when
donor and recipient are HLA compatible,1,2 and in mice, when donor and recipient bear the same H-2 haplotype.3-8
The development of the GVHR is associated with an active
immunosuppression inducing a severe immunodeficiency syndrome
responsible for much of the mortality.9,10
In a previous study,11 we analyzed GVHR immunosuppression
in response to non-H-2 histocompatibility antigens in a model of
murine lethal GVHR induced by injecting B10.D2 (H-2d
Mlsb) bone marrow and spleen cells into lethally irradiated
(DBA/2 × B10.D2)F1 (H-2d/d Mlsa/b)
recipients. We found that GVHR spleen cells exhibit a suppressive action that alters T- and B-cell functions such as the concanavalin A
(Con A) and lipopolysaccharride (LPS) mitogenic responses of normal
spleen cells, as well as the response of CD5+ B cells
against a cryptic determinant on mouse erythrocytes revealed by
proteolytic treatment with the enzyme bromelain (anti-Br-MRBC response). CD5+ B cells form the predominant B-cell subset
(B-1) in the peritoneal and pleural cavities, and their specificity is
predominantly against bacterial polysaccharides and
self-antigens.12 A study of the mechanisms responsible for
the GVHR suppression showed that: (1) elimination of T lymphocytes by a
treatment with anti-Thy-1 monoclonal antibody (MoAb) did not modify
the suppressive activity of GVHR spleen cells, indicating that T cells
were not responsible for the active, nonspecific suppression; (2) an
anti-interferon- (IFN-) MoAb greatly attenuates the suppression
of the anti-Br-MRBC antibody response and of the mitogenic response to
Con A, but not to LPS, suggesting the existence of both
IFN--dependent and IFN--independent immunosuppression; (3)
treatment of GVHR spleen cells by L-leucine methyl esther (LME), a
lysosomotropic agent,13 considerably diminishes both types
of suppressive activity. From these and other observations, we
determined that the GVHR-associated active immunosuppression is linked
to the interactions of at least two populations: (1) a T-cell
population synthesizing IFN-, which is devoid of direct inhibitory
activity; (2) an LME-sensitive, non-T-cell population, which mediates
suppression of all responses studied through an effector molecule (or
molecules) not identified in that study.
In the present work, we investigated whether nitric oxide (NO), whose
synthesis is mainly induced by IFN-, is one of the effectors of the
GVHR-associated immunosuppression. NO has been shown to mediate the
suppressive effect of activated macrophages on lymphocyte responses to
a T-cell mitogen in rats,14 and enhanced NO production has
been observed in several experimental models of immunosuppression
associated with GVHR.15-17 We have analyzed: inhibition of
iNOS (inducible NO synthase) induction by anti-IFN- MoAb;
inhibition of NO production by NG-monomethyl-L-arginine
(LMMA) and by aminoguanidine, both competitive inhibitors of iNOS; the
selective destruction of macrophages by LME treatment. The effects of
these treatments on the ability of GVHR spleen cells to mediate the
GVHR-associated immunosuppression were explored. We found that NO,
synthesized during GVHR by an iNOS whose transcription is induced by
IFN-, mediates the IFN--dependent, but not the
IFN--independent immunosuppression by LME-sensitive cells.
Immunohistochemical analyses using anti-iNOS and anti-CD11b/Mac-1 antibodies showed that the LME-sensitive cells synthesizing NO are macrophages.
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MATERIALS AND METHODS |
Animals.
DBA/2 (H-2d, Mlsa), B10.D2
(H-2d, Mlsb) and (DBA/2 × B10.D2)F1 (H-2d/d, Mlsa/b) mice
were raised in our own animal facility from stock purchased from
Jackson Laboratories (Bar Harbor, ME) or Harlan OLAC (Gannat, France).
Preparation of cell suspensions.
Single-cell suspensions of spleen or femoral bone-marrow cells were
prepared in RPMI 1640 medium; cells from three to four donors were
pooled. Viable cell numbers were determined using Trypan blue dye
exclusion as criterion of viability.
Induction of GVHR.
(DBA/2 × B10.D2)F1 adult recipient mice were given a lethal dose
(9 Gy) of whole-body irradiation using a 137Cs source at a
dose rate of approximately 0.8 Gy/min. Twenty-four hours after
irradiation, they were grafted intravenously with 107 bone
marrow and 8 × 106 spleen cells from sex-matched
normal B10.D2 donors; the control groups consisted of either irradiated
B10.D2 recipients reconstituted with 107 bone marrow and 8 × 106 spleen cells from syngeneic donors (isografted
mice), or else normal B10.D2 mice, as indicated. Grafted mice were
killed at various intervals from day 5 to day 21 after transplantation
for immunologic testing.
Antibodies and reagents.
Hamster antimouse IFN- MoAb and normal hamster IgG were purchased
from Genzyme (Cambridge, MA) and Immunotech (Marseille, France)
respectively. Rat anti-Thy-1 MoAb and rat antimouse CD11b/Mac-1 MoAb
were obtained from PharMingen (San Diego, CA). Rabbit anti-iNOS affinity-purified polyclonal Ab were acquired from Transduction Laboratories (Lexington, KY). Rabbit anti-transforming growth factor 1 (TGF- 1) polyclonal Ab were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Biotinylated goat antirabbit immunoglobulin (Ig) was
purchased from BIOSYS (Compiègne, France). Rabbit antirat Ig
antibodies and rat anti-alkaline phosphatase MoAb (DAKO Rat APAAP) were
obtained from DAKO (Glostrup, Denmark). Alkaline phosphatase substrate
kit (Vector Blue substrate kit), Horseradish Peroxidase Streptavidin,
and AEC chromogen kit were acquired from Vector Laboratories
(Burlingame, CA) and Sigma (St Louis, MO), respectively. LMMA and
NG-monomethyl-D-arginine (DMMA) were purchased from
Calbiochem (San Diego, CA). Aminoguanidine, LME, and bromelain were
obtained from Sigma. Aquatex (aqueous mounting media for microscopy)
was obtained from MERCK (Darmstadt, Germany).
Cell treatments.
Where required, spleen cells were treated twice with anti-Thy-1 plus
complement (C) under conditions that abrogate the response of normal
spleen cells to Con A. Briefly, spleen cells (2 × 107/mL) in RPMI 1640 were first incubated at 4°C with
anti-Thy-1 MoAb at 1/200 final dilution for 30 minutes. Guinea pig C
was then added at a 1/10 final dilution, and the cells were incubated at 37°C for 45 minutes. At the end of this period, the cells were washed three times in cold RPMI. The entire procedure was then repeated.
Treatment of the GVHR spleen cell population with LME was performed
according to the method of Thiele et al.13 Spleen cells (5 × 106/mL) were incubated for 40 minutes at room
temperature in RPMI containing 5 mmol/L LME, and were then washed three
times in RPMI 1640. This treatment causes lysosomal disruption and
selective death of cells with a high lysosomal content, such as
macrophages, natural killer (NK) cells, and precytotoxic T
lymphocytes.18 Between days 5 and 21 after grafting, the
proportion of LME-sensitive cells in GVHR spleen were found to range
from approximately 40% to 75%; observed percentages varied from one
experiment to another, but within a given experiment did not seem to be
time-dependent during the period studied.
Mitogen stimulation assays.
Mitogens used were Con A (Pharmacia Fine Chemicals, Uppsala, Sweden) at
a final concentration of 2.5 µg/mL and LPS (Escherichia coli
055: B5; Difco, Detroit, MI) at a final concentration of 100 µg/mL.
Proliferation assays were performed in RPMI 1640 medium containing 1 mmol/L L-arginine, -mercaptoethanol (5 × 10 5 mol/L), and gentalline (Boehringer, Mannheim,
Germany) and supplemented with 0.6% normal mouse serum. Experiments
were performed in 96-well flat-bottomed plates (Microtest III;
Falcon; Becton Dickinson, Franklin Lakes, NJ). Spleen
cells taken from normal DBA/2 mice were cultured (2 × 106 cells/mL, 0.2 mL/well) either without treatment, for
the response to Con A, or after treatment with anti-Thy-1 plus C, for
the response to LPS. After a 48-hour period of incubation at 37°C
in a 5% CO2 atmosphere, cells counts were determined and
cultures were pulsed with 37 kBq of
[3H]thymidine and cultured for an additional period of 18 hours. Cells were then harvested onto glass fiber filters with a
Skatron unit (Skatron Instruments, Lier, Norway), and
incorporated [3H]thymidine measured by liquid
scintillation counting (LKB Rackbeta 1218; Wallac OY,
Turku, Finland). In experiments where the suppressive activity of GVHR
spleen cells was studied, normal DBA/2 cells at the same concentrations
as given above were cocultured with either GVHR or B10.D2 (control)
spleen cells (106 cells/mL, 0.1 mL/well). The total volume
was maintained at 0.2 mL/well. Cultures were incubated and
pulse-labeled as described above. In all proliferation assays, each
test group was assayed in quadruplicate and, where tested, cell counts
paralleled [3H]thymidine uptake.
Antibody plaque-forming cells (PFC) against bromelain-treated
syngeneic erythrocytes (Br-MRBC).
One percent to 2% of B cells in normal murine spleen express the CD5
antigen and secrete, without T-cell help, antibodies against a cryptic
determinant (phosphatidyl choline) on mouse erythrocytes (MRBC), which
is unmasked by proteolytic treatment with the enzyme
bromelain12,19; such bromelain-treated MRBC are designated
herein as, Br-MRBC. LPS treatment increases the anti-Br-MRBC response
of CD5+ B cells in vitro. Normal DBA/2 responding spleen
cells were pretreated with anti-Thy-1 plus C, and then resuspended in
RPMI 1640 medium containing -mercaptoethanol, gentalline, and
0.6% normal mouse serum before seeding into 24-well tissue culture
plates (Falcon 3008; Becton Dickinson). A total of 5 × 106 responding cells were cultured for 3 days with LPS (10 µg/mL), in the presence of either 106 GVHR or
106 control spleen cells in a total volume of 1 mL/well.
Br-MRBC were prepared according to the method of
Cunningham.19 The blood of B10.D2 mice was obtained by
retroorbital venous puncture and was mixed with heparin. Immediately
thereafter, the red blood cells were washed three times in
phosphate-buffered saline (PBS), incubated for 30 minutes at 37°C
as a 50% suspension with bromelain at a final concentration of 10 mg/mL in PBS, and then washed three more times. Direct PFC specific for
Br-MRBC were detected by the method of Cunningham and
Szenberg.20 Results are expressed as the mean number of PFC
obtained with LPS minus the mean number of PFC in the absence of LPS.
Determination of NO production.
Nitrite present in cell culture supernatants was measured by a
microplate assay method21: 100-µL aliquots were removed
from conditioned medium and incubated with 100 µL of the Griess
reagents: N(1-naphtyl) ethylene diamine dihydrochloride (0.15% in
H2O), and sulfanilamide (1.5% in 1 N HCl) at room
temperature for 10 minutes. Nitrites form a chromophore with the Griess
reagents, absorbing at 543 nm. The absorbance was determined with a MR
700 microplate reader (Dynatech, Chantilly, VA). Nitrite
concentration was calculated from an NaNO2 standard curve.
Measuring NO synthase (NOS) activity by monitoring the accumulation of
nitrite, a stable oxidative end product of NO, is currently the
standard assay for NOS activity. However, the NOS activity assay based
on the biochemical conversion of L-arginine to L-citrulline by NOS is
an incomparably specific and sensitive method compared with nitrite
dosage. To analyze the whole set of radiolabeled L-arginine-derived
metabolites produced by GVHR spleen cells, a fast radioactive high
performance liquid chromatography (HPLC) method was used. This method,
extensively described elsewhere,22 is based on the fact
that L-arginine is converted either to ornithine and urea via the
arginase pathway, or to L-citrulline and the toxic split product NO via
the NOS pathway. In brief, GVHR spleen cells (3 × 106/mL) were incubated in RPMI 1640 medium with 37 MBq of
L-[2, 3-3H]arginine (NEN, Dupont de Nemours, Le Blanc
Mesnil, France) for 48 hours. The cell supernatant was
then collected, and the L-[3H]arginine,
L-[3H]citrulline, and [3H]ornithine it
contained were measured by HPLC. Concentrations are expressed in
relative terms, with (arginine) + (citrulline) + (ornithine) = 100% in
each case.
iNOS mRNA analysis.
Spleen cell RNAs extracted with RNA B (Bioprobe, Montreuil,
France) were fractionated by electrophoresis on 1%
agarose/2.2 mol/L formaldehyde gel in MOPS running buffer
(Sigma), blotted onto Hybond-N membranes (Amersham, les Ulis,
France), and probed with 15 ng of random-primed
32P-labeled cDNA fragments synthesized with a random
priming kit as directed by the manufacturer (Appligene, Illkirch,
France). Hybridizations were performed at 42°C in
50% formamide solution with the cDNA probe (950 bp 5'-end
fragment) specific for murine macrophage iNOS23 and with an
actin cDNA fragment.24 After two washes at 65°C in high
(2X SSC [Bioprobe], 0.1% sodium dodecyl sulfate
[SDS]) and low concentration (0.1X SSC, 0.1% SDS) salt solutions,
the blots were exposed to x-ray films at  80°C in the presence of intensifying screens. Quantitative determination of iNOS
mRNA present in each of the various samples was normalized with respect
to the concentration of actin mRNA detected in the same sample.
Cell lines.
CTLL2 cells were maintained in RPMI 1640 medium
supplemented with 5% heat-inactivated fetal calf serum (FCS) and
recombinant interleukin-2 (IL-2). The EMT6 and RAW264.7 (American Type
Culture Collection [ATCC], Rockville, MD, #TIB71) cell
lines were cultured and expanded by serial transplantation in RPMI 1640 containing 5% FCS. EMT6 is a mammary adenocarcinoma from BALB/c mice,
and RAW264.7 is a mouse macrophage tumor; both express iNOS activity after incubation with LPS and IFN-.
Dosage of IFN- and IL-2 in the supernatant of GVHR
spleen cells.
A total of 50 µL of supernatants was tested for the presence of
IFN- using a commercial enzyme-linked immunosorbent assay (ELISA)
kit (R&D Systems, Abingdon, UK).
A total of 50 µL of supernatant was added to 5 × 103 CTLL2 (previously starved of IL-2 for 4 hours), and proliferation was measured 24 hours later by a colorimetric
method.25 IL-2 concentration is given as international
units per milliliter calculated from a standard curve using human
recombinant IL-2, which was tested in parallel under the same conditions.
Immunohistochemistry.
GVHR or isografted control spleen cells were spotted on SuperFrost/plus
slides (Menzel-Glaser, Braunschweig, Germany) after cell
treatment, mitogen stimulation, and coculture with normal DBA/2 cells.
Additional controls consisted of RAW264.7 cells, either uninduced
(negative control) or treated (positive control) with 100 U/mL IFN-
and 1 mg/mL LPS for 12 hours. Cytospin preparations (5 × 104 spleen cells/spot) were air dried and kept at
80°C before immunostaining. Briefly, cytospins were
incubated with anti-iNOS, anti-CD11b/Mac-1, and anti-TGF-1 primary
Ab at 1/100 final dilution. iNOS is detected in the cytoplasm of
NO-producing cells, CD11b/Mac-1 molecules are expressed on cell surface
membranes of macrophages and granulocytes, and TGF-1 is shown in the
cytoplasm of a broad spectrum of cells. The binding of anti-iNOS and
anti-TGF-1 Ab was detected by a biotinylated goat antirabbit Ig
second Ab followed by the Horseradish Peroxidase Streptavidin reagent.
The binding of anti-CD11b MoAb was evidenced by a rabbit antirat Ig
second Ab, which also reacts with the complex formed by a rat
monoclonal anti-alkaline phosphatase antibody and alkaline phosphatase
enzyme. All incubations were performed at room temperature for 1 hour
with the primary antibody and 30 minutes with each of the conjugates,
followed by 5-minute washes in Tris-buffered saline plus 0.3% bovine
serum albumin (BSA). Endogenous alkaline phosphatase activity was
blocked using Levamisole. Slides were developed at room temperature by
two successive incubations in AEC chromogen (6 minutes) and in alkaline
phosphatase substrate (15 minutes). Finally, slides were mounted in Aquatex.
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RESULTS |
Effect of arginine analogs on GVHR-associated active
immunosuppression.
To determine whether NO might be responsible for the GVHR-associated
immunosuppression, experiments were designed to assess the effects of
GVHR spleen cells, either untreated or treated with LME or arginine
analogs (LMMA, aminoguanidine), on the proliferative response of normal
DBA/2 spleen cells to the mitogens Con A and LPS, as well as on the
CD5+ B-cell response to Br-MRBC antigen. DBA/2 cells were
chosen as responders for reasons of convenience and not because they
express parent-strain antigens, which are targets of the GVHR: indeed, our previous results clearly show that the GVHR-associated
immunosuppression is nonspecific and not restricted by H-2
antigens.11 Results (Table 1)
show that day 7 GVHR spleen cells cocultured with normal DBA/2 spleen
cells abrogate the mitogenic response of the latter to both Con A and
LPS, as well as the anti-Br-MRBC antibody response. Treatment of GVHR
spleen cells with LME abrogated their suppressive activity; this
treatment eliminates a population of non-T, non-B cells.11
LMMA, a competitive inhibitor of iNOS (the enzyme that cleaves
L-arginine into L-citrulline and NO) partially reverses the inhibition
by GVHR spleen cells of both the mitogenic response to Con A and the
anti-Br-MRBC antibody response (35% and 32% of control,
respectively). Under the same experimental conditions, LMMA fails to
reverse the suppression of the mitogenic response to LPS and has no
effect on the Con A and LPS responses of normal DBA/2 spleen cells
cocultivated with B10.D2 control spleen cells. It should be mentioned
that qualitatively similar results were obtained at time points ranging
from 5 to 21 days after grafting, that GVHR spleen cells cultured alone
exhibit no mitogenic responses to either Con A or LPS, and that LMMA
does not modify this nonresponsiveness (not shown). Aminoguanidine,
another competitive inhibitor of iNOS, was used in some experiments in
parallel with LMMA. LMMA and aminoguanidine have strictly the same
effect on the inhibition of mitogenic responses mediated by GVHR cells
(not shown). The relatively moderate concentration of LMMA or
aminoguanidine (1.5 to 2 mmol/L) used in our experiments never altered
the proliferation of normal B10.D2 spleen cells stimulated by mitogens,
as shown in Table 1. But, as we also observed, it was recently
reported26 that arginine analogs, and in particular
aminoguanidine at high concentrations (5 mmol/L), suppressed both the
antigen-specific and nonspecific proliferation of T lymphocytes.
In conclusion, these results suggest that NO is implicated in the
GVHR-mediated suppression of the mitogenic response of T cells to Con A
and of the secretion of anti-Br-MBRC autoantibody by CD5+
B-cell subset, but do not support the hypothesis that NO is involved in
suppression of the mitogenic response of B cells to LPS. It should be
emphasized that this pattern of activity of LMMA on GVHR-associated
immunosuppression is the same as that of anti-IFN- MoAb, which we
described previously.11
Time course of iNOS mRNA expression in GVHR spleen cells.
To determine the time course of iNOS transcription in GVHR spleen
cells, Northern blot analyses were performed from days 5 to 14 after
grafting (Fig 1). EMT6 cells stimulated
with LPS and IFN- were included as a positive control for detection
of iNOS mRNA (not shown). iNOS mRNA of 4.7-kb size became detectable in untreated GVHR spleen cells taken ex vivo in most experiments beginning
on day 5 after grafting; the level of transcripts decreased on day 9 and disappeared on day 12, but was restored by in vitro stimulation
with Con A or LPS (Fig 1B). Densitometric analyses of iNOS mRNA
expression relative to actin are represented below the blot. Spleen
cells from control mice grafted with syngeneic bone marrow (either
untreated or treated by mitogens) were negative for iNOS
transcription (Fig 1A).
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| Fig 1.
Analysis by Northern blot of iNOS mRNA expression. Total
RNA was extracted, and 10 µg of each sample was electrophoresed. The
Northern blot transfer was hybridized with the specific murine
macrophage iNOS cDNA probe (950 bp 5'-end fragment), and an actin
probe was used as control for RNA expression. (A) iNOS RNA expression
in spleen cells from control mice on day 7 after grafting with
syngeneic bone marrow (either untreated or stimulated by Con A or LPS)
and in mitogen-stimulated GVHR spleen cells. (B) Time course of iNOS
RNA expression in GVHR spleen cells (either untreated or stimulated by
Con A or LPS). Densitometric analysis of iNOS mRNA expression relative
to actin is represented below the blot.
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When LMMA was added at the beginning of culture to day 7 GVHR spleen
cells stimulated by either Con A or LPS, the 48-hour mitogen-induced
iNOS mRNA levels were not decreased compared with those seen in the
absence of LMMA, consistent with the role of the latter as a specific
antagonist for the enzyme (not shown). A slight increase in iNOS mRNA
was observed in the presence of LMMA (not shown), possibly indicative
of a feedback regulatory loop influencing transcription of the iNOS
gene, as suggested by others.27
Effect of LMMA and anti-IFN- MoAb on GVHR-mediated
suppression and on nitrite and L-citrulline production.
The effects of LMMA, LME, and anti-IFN- MoAb on the GVHR spleen
cell-mediated suppression and on the synthesis of L-citrulline and
nitrite, the stable end products of the NO pathway, were studied. LMMA
and anti-IFN- MoAb (IgG; 1.5 µg/mL), but not hamster IgG control
(1.75 µg/mL), are both endowed with the ability to reverse the
suppression of the mitogenic response to Con A and to reduce by 50%
the level of nitrite produced by day 9 GVHR spleen cells. ELISA
measurements of IFN- concentrations confirmed the efficacy of the
anti-IFN- MoAb for complete inactivation of the IFN- present in
the cultures. However, their failure to reverse the suppression of the
mitogenic response to LPS contrasts with their ability to reduce by
50% the nitrite synthesis in the culture. It should be pointed out
that when normal DBA/2 spleen cells are cocultured with normal B10.D2
spleen cells (controls) for 48 hours with Con A or LPS, no nitrite
synthesis is observed (Table 2).
To unequivocally confirm that the observed nitrite production by
LME-sensitive GVHR spleen cells was due to the iNOS pathway, we
measured NOS activity by monitoring the conversion of L-arginine to
L-citrulline. L-citrulline and NO are produced simultaneously by the
action of iNOS on L-arginine, whereas accumulation of ornithine reflects the activity of arginase, the other enzyme that uses L-arginine as substrate in activated murine macrophages. IFN- exclusively upregulates iNOS, whereas IL-4 and IL-10 induce arginase activity.28 However, little information about possible
functions of murine macrophage arginase is available. On day 7, GVHR
spleen cells, either untreated or treated by mitogens, anti-IFN-,
or LME were cultured with L-[3H]arginine in the presence
or absence of LMMA. Forty-eight hours later,
L-[3H]citrulline, [3H]ornithine, and
L-[3H]arginine concentrations were quantified in the
supernatants by HPLC. As observed in Fig 2,
L-citrulline, synthesized from L-arginine (which reflects the amount of
NO synthesized) is produced even by untreated GVHR spleen cells taken
ex vivo, as well as after stimulation of these cells by Con A or LPS.
An arginase activity is also present in GVHR spleen cells taken ex vivo
and is eliminated, as expected, after LME treatment, which removes macrophages. The presence of products from both metabolic routes is
indicative of the production during GVHR of Th1 and Th2 cytokines. L-citrulline production is reduced by LMMA and abrogated by
anti-IFN- MoAb and LME treatment. As expected, the negative
control, DMMA, an inactive enantiomer of LMMA, had no effect on the
L-citrulline synthesis (not shown). This shows that the observed NO
synthesis by GVHR spleen cells is linked to iNOS activity in
LME-sensitive cells. In parallel with the HPLC analyses, GVHR
immunosuppression was studied (Fig 2). As already mentioned (Tables 1
and 2), the inhibition of NO production by LMMA, confirmed by the
corresponding reduction of L-citrulline synthesis, abolished the
suppressive effect of GVHR spleen cells on the T-cell response of
normal cells to Con A. In contrast, their suppressive effect on the
B-cell response of normal cells to LPS was not affected by a complete blockade of NO production, confirmed by the absence of L-citrulline in
the presence of anti-IFN- MoAb.
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| Fig 2.
Analysis by radio-HPLC of the conversion of
L-[3H]arginine into L-[3H]citrulline and
[3H]ornithine by GVHR spleen cells (either untreated or
treated by Con A or LPS). The effects of LMMA (1.5 mmol/L),
neutralizing anti-IFN- MoAb (1.5 µg/mL), and LME treatment on the
production of L-arginine-derived metabolites is also presented (see
Materials and Methods for details of experimental procedures). In
parallel with HPLC analyses, the GVHR-associated immunosuppression was
tested: (+) indicates that suppression was observed, () that it
was not.
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LME-sensitive GVHR spleen cells producing NO belong to the macrophage
lineage.
We next asked which GVHR spleen cell subpopulation expressed the iNOS
and was responsible for nitrite synthesis. In these experiments, GVHR
spleen cells were cultured either in medium alone or else in the
presence of Con A or LPS. The nitrite concentrations were measured in
the supernatants simultaneously with determination of the presence of
iNOS protein by immunohistochemistry (see below). Results presented in
Table 3 indicate that day 7 GVHR spleen cells cultured in the absence of mitogen exhibit a spontaneous nitrite
synthesis (consistent with the already mentioned presence of iNOS mRNA
and of L-citrulline production in GVHR spleen cells taken directly ex
vivo), and that the level of this nitrite synthesis is strongly
increased when GVHR cells are stimulated by Con A or LPS. Treatment of
GVHR spleen cells with anti-Thy-1 + C does not modify their ability to
produce nitrite after stimulation by Con A or with LPS, whereas such
treatment leads to a nearly sixfold reduction of Con A-induced IL-2
synthesis. On the other hand, treatment of GVHR spleen cells with LME
leads to an abrogation of nitrite synthesis after stimulation by Con A
and LPS without altering the level of IL-2 synthesis. Likewise, the
level of IFN- was not affected by LME treatment: 852 versus 827 ng/mL when stimulated by Con A and 22.5 versus 19 ng/mL when stimulated
by LPS. No nitrite production could be detected in the spleens of mice
grafted with syngeneic bone marrow (not shown). Taken together, these
results confirm that the nitrite is synthesized in GVHR spleens by
non-T, LME-sensitive cells.
To determine the phenotype of the GVHR spleen cell subpopulation
responsible for synthesizing nitrite, immunohistochemical analyses were
performed at days 7, 14, and 21 after grafting, using anti-CD11b/Mac-1
antibodies, which recognize the cell surface membranes of macrophages
and granulocytes, and anti-iNOS antibodies (Fig 3). As expected, RAW264.7 mouse
macrophage tumor cells (positive control) stained positively with
anti-CD11b/Mac-1 MoAb and with anti-iNOS Ab after stimulation with
IFN- and LPS (not shown). Spleen cells from control mice grafted
with syngeneic bone marrow were negative when stained by anti-iNOS Ab
(not shown). On the other hand, in nontreated GVHR spleen cells taken
ex vivo on day 7, numerous cells were intracytoplasmically stained
(red) with the anti-iNOS Ab; the cell number as well as the intensity
of the staining was increased when GVHR spleen cells were stimulated by
Con A or LPS. In parallel, the suppressive activity of these GVHR
spleen cells was tested and found to suppress at greater than 99% the
mitogenic responses of normal DBA/2 cells to Con A and LPS. On day 14, immunohistochemical analyses performed on nontreated GVHR spleen cells
taken ex vivo and on GVHR spleen cells stimulated by Con A or LPS
showed, in both groups, a strong decrease in both the number of iNOS
positive cells and the intensity of staining. Despite this pronounced
reduction in staining by anti-iNOS Ab, the GVHR-associated suppression
remained strong (81% and 95% for Con A and LPS, respectively). On day
21, only a few cells appeared intracytoplasmically stained; however, in this case, too, the suppression remained strong (56% and 62%). As
expected, spleen cells from days 7 to 21 of the GVHR that were treated
by LME lost their capacity to suppress the mitogenic responses of
normal DBA/2 spleen cells to Con A and LPS; and immunostaining by
anti-iNOS run in parallel on these cells was totally negative (data not
shown). In addition, May-Grünwald-Giemsa staining showed that a
large percentage of GVHR spleen cells, either untreated or treated by
mitogens, have a monocyte/macrophage morphology (not shown). Our
previous data showed that 40% to 75% of GVHR cells were LME
sensitive.11 LME selectively removes macrophages, NK cells,
and pre-cytotoxic T lymphocyte (CTL)13,18;
and, as shown in Table 3, NO is synthesized by non-T, LME-sensitive
cells. Thus, to confirm that the NO-producing cell belongs to the
macrophage lineage, GVHR spleen cells were double-stained by the
anti-iNOS Ab and an anti-CD11b/Mac-1 MoAb. All iNOS-positive cells were found to be stained by the CD11b/Mac-1 MoAb (data not shown). Hence,
based on both immunohistochemical and cell morphological analyses, we
conclude that the NO-producing cells are of the monocyte/macrophage lineage.
View larger version (113K):
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| Fig 3.
Immunohistochemical detection of NO-producing cells in
GVHR spleen cell cytospin preparations: (GVH) untreated GVHR cells;
(GVH Con A) and (GVH LPS) GVHR cells treated by Con A and LPS,
respectively. Spleen cells were taken on days 7, 14, and 21 of the
GVHR. Anti-iNOS antibodies stain in red the cytoplasm of NO-producing
cells. Original magnification × 400.
|
|
Features of TGF-1 expression by GVHR spleen cells.
In our previous report,11 we found that TGF-1
synthesized by T cells was one of the cytokines mediating the active
immunosuppression associated with GVHR, especially the anti-Br-MRBC
response and, to a lesser extent, the mitogenic response to LPS. To
evaluate the part played by NO and TGF-1 in the GVHR-associated
suppression, we determined by immunohistochemistry the expression of
TGF-1, using anti-TGF-1 Ab in parallel with that of iNOS (see
above). Only a few scattered TGF-1-positive cells were observed in
both groups analyzed (untreated GVHR spleen cells taken ex vivo and GVHR spleen cells stimulated by Con A or LPS), and they were not markedly diminished by LME treatment (data not shown).
| |
DISCUSSION |
We previously showed that a nonspecific immunosuppression mediated by
IFN- and TGF-1 accompanies the lethal GVHR provoked by minor
histocompatibility differences in irradiated adult (DBA/2 × B10.D2)F1 mice receiving bone marrow and spleen cells from
semiallogeneic, H-2d identical, B10.D2
donors.11 However, IFN- and the latent form of TGF-
secreted by GVHR T cells have no direct suppressive effects; and only
LME treatment of GVHR spleen cells, which remove macrophages, NK cells,
and pre-CTL,13,18 completely abrogated their ability to
suppress the responses of normal T and B cells to Con A and LPS,
respectively, as well as the thymus-independent antibody response of
CD5+ B cells against Br-MBRC. Antibodies produced by
CD5+ B cells have a broad specificity for polysaccharides,
lipids, and proteins of bacterial components. CD5+ B cells,
which represent a distinct B-cell population (B-1 cells) endowed with a
strong capacity for self-renewal,12,19 could therefore be
important in the control of GVHR infections. Indeed, the cryptic
determinant recognized on mouse erythrocytes, Br-MRBC, was identified
as the phospholipid phosphatidyl choline, expressed in the outer
leaflet of the plasma membrane of organisms by enzymatic cleavage of
coated glycoprotein.29
The experiments reported here were designed to determine whether NO
synthesized by LME-sensitive GVHR spleen cells was the effector
molecule of the IFN--mediated immunosuppression. NO is an important
biologic mediator produced either by a constitutive enzyme in the
cardiovascular and nervous system30 or by an inducible NOS
in hepatocytes31 and macrophages.32 In rats, NO
production by Bacillus of Calmette and Guerin (BCG)-,
LPS- or IFN--activated macrophages mediates their cytostatic and
cytotoxic activities against microbial pathogens and tumor
cells,32 as well as their inhibition of lymphocyte
responses to Con A and alloantigens.14 By analogy, in our
GHVR model, the involvement of IFN- in the nonspecific suppression
of lymphocyte proliferative responses by spleen cells suggested that NO
might be the effector molecule. This is in agreement with the
LME-sensitivity of GVHR-associated suppressor cells, which are Thy-1
negative and µm negative, have no NK activity, and
could therefore belong to the macrophage lineage. NO is endowed with
multiple inhibitory activities including the ability to provoke
intracellular iron loss, to inhibit the enzymes required for
mitochondrial respiration, the citric aconitase
enzyme,33,34 and the ribonucleotidyl
transferase,35,36 and finally to inhibit protein
synthesis.31,37 These activities are sufficient to account
for the inhibition of the mitogenic responses and that of the
anti-Br-MRBC antibody response observed with GVHR spleen cells.
The results presented here show that, in GVHR, NO synthesized by GVHR
spleen cells mediates, in part, their suppressive activities. In this
way LMMA or aminoguanidine, competitive inhibitors of iNOS (which
cleaves L-arginine into NO and L-citrulline), partially reverse the
suppression by GVHR spleen cells of both the mitogenic response to Con
A and the anti-Br-MRBC antibody response by DBA/2 spleen cells.
Indicators of NO release are therefore observed: GVHR spleen cells
taken ex vivo exhibit a spontaneous synthesis of nitrite and
L-citrulline; and the production of nitrite (which is moderate) can be
amplified by triggering with Con A or LPS. These syntheses were reduced
in the presence of LMMA or aminoguanidine. Both mRNA iNOS and iNOS
protein were detected early in the GVHR, and their abundance was
greatly increased by Con A and LPS stimulation. In contrast,
L-citrulline, nitrite production, iNOS mRNA, and iNOS protein could
never be detected in the spleens of mice grafted with syngeneic bone
marrow. Treatment of GVHR spleen cells by LME removes the cell
subpopulation involved in NO synthesis (as shown by the dosage of
L-citrulline and of nitrite, and by the presence of iNOS enzyme in the
cytoplasm), and abrogates their suppressive activity without altering
the production of IFN- and the synthesis of IL-2 by T cells. NO
synthesis is regulated by IFN-, as anti-IFN- MoAb, like LMMA,
reduces the NO synthesis. The effector cells belong to the
monocyte/macrophage lineage, as shown by immunohistochemistry using
anti-iNOS and anti-CD11b/Mac-1 Ab.
It is noteworthy that LMMA, anti-IFN-, and LME treatment reduce NO
synthesis by GVHR spleen cells and affect their capacity to suppress
T-cell responses to Con A and CD5+ B-cell responses to
Br-MRBC, but not their suppressive effect on B-cell responses to LPS.
However, LME treatment abrogates the ability of GVHR spleen cells to
inhibit the B-cell response to LPS. Taken together, these results show
that IFN-, via NO production by an LME-sensitive population,
mediates the suppressive effect of GVHR spleen cells on proliferation
of normal T cells and on antibody production by normal CD5+
B cells, whereas inhibition of the proliferation of normal B cells to
LPS involves an LME-sensitive, IFN--independent population, which
does not act via the NO pathway. The implication of NO in GVHR
immunosuppression has also been described by others, but mainly in
experimental models involving H-2 disparities, a situation, which does
not reflect that encountered in bone marrow transplantation in man. For
instance, in a model of GVHR induced across an H-2 barrier (MHC class I
and class II disparity) in semiallogeneic nonirradiated recipients
(parent B6  B6D2F1),15 NO production of
undetermined cellular origin was reported to be responsible for a low
mitogenic responsiveness of GVHR spleen cells. In that model, addition
of LMMA partially restored Con A, but not LPS responsiveness; no
attempt was made to measure the suppressive effect of GVHR cells on the
activity of a normal responding-cell population. Consequently, the
investigators postulated that a low frequency of B cells in GVHR
spleens might account for the ineffectiveness of LMMA in restoring the
LPS response, despite its effectiveness in decreasing nitrite levels in
the same experiments. While these results are in agreement with our
data, our results concerning the suppression by GVHR spleen cells of
the LPS-induced proliferative response of normal spleen cells lead us
to propose an alternative explanation: because LME treatment restores
proliferation, but LMMA or anti-IFN- MoAb do not, inhibition of the
LPS response during GVHR may proceed via an NO-independent mechanism.
In contrast, in a fully allogeneic, but H-2 compatible model (ie,
involving only minor histocompatibility differences: B10.BR irradiated CBA/J), LMMA treatment totally restored LPS
responsiveness,17 indicating that for some other minor
histocompatibility differences, the NO pathway may be sufficient to
account for the GVHR-induced decrease in B-cell proliferation. In a
third model implicating NO in GVHR immunosuppression, GVHR was induced
by donor T cells in response to an isolated MHC class II difference
(bm12 B6).16 In those studies, the investigators
demonstrated the existence of a NO-dependent pathway involved in the
impairment of mitogen-induced T-cell proliferation; the downregulation
of IFN- production decreased NO production 10-fold and virtually
restored splenocyte responses to Con A. We show here that the
LME-sensitive cells responsible for the NO-mediated effects of IFN-
in our model belong to the monocyte/macrophage lineage. However,
whereas all NO-producing cells were found CD11b/Mac-1-positive, the
converse does not hold. Thus, among the population of LME-sensitive
cells, those not secreting NO might, a priori, be suspected of playing
a role in suppression of the mitogenic response to LPS, a suppression
that was relatively insensitive to LMMA, but completely abolished by
LME treatment. In our previous report,11 we showed that
TGF- plays a role in the suppression of the mitogenic response to
LPS, although synthesized in a biologically inactive (latent) form. T
cells were the source of TGF-1 in our model of GVHR-associated
immunosuppression, whereas the principle source of TGF- during
inflammation is the macrophage. The present study confirmed through
immunohistochemical staining that the TGF-1-producing cells are
distinct from the LME-sensitive population. Interestingly, conversion
of the latent form of TGF- to active TGF- by macrophages has been
reported.38 Hence, we hypothesized a cooperation between a
T-cell population producing TGF-1 and an LME-sensitive GVHR cell
population as activator of the TGF- latent form. However, other
studies have provided evidence that IFN- suppresses the production
of TGF-1 by both macrophages and T lymphocytes.39,40
This regulation of TGF- by IFN- could explain our finding of a
total absence of TGF- protein in inflammatory macrophages during
GVHR. In addition, other cytokines may intervene in GVHR-associated immunosuppression.
At present, the full range of effects of NO during GVHR is unknown.
While NO production is critical for many aspects of host defense
mechanisms, its production is responsible for a defective T-lymphocyte
response to mitogen as well as the impairment of antibody synthesis by
CD5+ B cells whose expansion and activation are induced by
bacterial infections.41 NO has been shown to mediate
tumoricidal activity by macrophages.32 Also, NO could be
important for the graft-versus-leukemia effect associated with GVHR,
although its inhibition of cytotoxic T-lymphocyte responses might be
expected to counteract this potential beneficial effect. In addition to
its suppressive effects on immune responses, NO has been shown to
mediate the development of inflammation in the course of acute cardiac
rejection in rats42 and of autoimmune MRL/lpr
disease.43 It remains to be determined whether NO also plays a role in the pathogenesis of the inflammatory lesions associated with GVHR, such as the severe panniculitis observed in our experimental model.5 Despite detrimental effects on hematopoietic
reconstitution44 and, possibly, on host reactivity to
opportunistic infections, blockade of NO production holds some promise
as a therapeutic strategy to suppress GVHR-induced immunosuppression.
| |
ACKNOWLEDGMENT |
The authors are indebted to Alexandre Yapo (CNRS, ERS 571, Université Paris-Sud, Orsay) who performed HPLC analysis and to Mohamed Oukka for the dosage of IL-2.
| |
FOOTNOTES |
Deceased.
Submitted November 20, 1998; accepted April 5, 1999.
Supported by INSERM, by the Association contre le Cancer du Val de
Seine, and by Association pour la Recherche contre le Cancer (Grant No.
ARC 6178).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Pierre Bobé, INSERM Unité 267, 14, avenue Paul Vaillant-Couturier 94807 Villejuif Cedex, France;
e-mail: bobe{at}infobiogen.fr.
| |
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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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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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D. I. Gabrilovich, M. P. Velders, E. M. Sotomayor, and W. M. Kast
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B. Almand, J. I. Clark, E. Nikitina, J. van Beynen, N. R. English, S. C. Knight, D. P. Carbone, and D. I. Gabrilovich
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F. P. Nestel, R. N. Greene, K. Kichian, P. Ponka, and W. S. Lapp
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ABSTRACT
INTRODUCTION