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* Department of Experimental Medicine and Biochemical Science, University of Perugia, Perugia, Italy;
Department of Environmental Sciences, Swiss Federal Institute of Technology, Zurich, Switzerland; and
Immunology Research Division, Department of Pathology, Brigham and Women’s Hospital, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing CD4+ T cells
and to induce apoptosis through a CD80/CD28-dependent mechanism. A
population of CD80+Gr-1+ myeloid cells was
found to be expanded in conventional as well as in bone
marrow-transplanted mice with disseminated candidiasis, but its
depletion increased the IFN--mediated antifungal resistance. These
data indicate that alternatively activated PMN expressing CD80 may
adversely affect Th1-dependent resistance in fungal
infections. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is known that cells expressing Gr-1 or CD11b myeloid lineage cell
markers are capable of activating or suppressing the function of T
cells, depending on cytokine-dependent maturation pathways
(16, 17, 18). Myeloid suppressor cells (MSC), found in adult
bone marrow (BM) (19), in sites of intense hematopoiesis
(20), and in tumor-bearing hosts (21), are
capable of inhibiting the T cell proliferative response induced by
alloantigens (22), CD3/CD28 ligation (23),
and various mitogens (24) through contact-dependent
(25) and -independent (23, 26, 27)
mechanisms. Two distinct subpopulations of MSC have been characterized.
Classically activated (CA) MSC are NO and IFN- dependent (23, 24), whereas alternatively activated MSC are IL-4 dependent
(16, 17, 28, 29).
We were interested in finding out whether PMN, abundantly recruited in
candidiasis (10), could directly affect the activation of
Th cells. Because signal(s) delivered through costimulatory molecules
are required for proper T cell activation (30, 31), and
considering that human peripheral blood and inflammatory PMN express a
functional B7-1-like molecule (32), in the present study
we addressed the issue of whether murine PMN express costimulatory
molecules and the functional consequence of this expression in
candidiasis. To this purpose, we assessed 1) the expression of CD80
(B7-1) and CD86 (B7-2) molecules on peripheral and inflammatory murine
Gr-1+ PMN, 2) its modulation upon interaction
with C. albicans in vitro, in vivo, and in human PMN, 3) the
effect of Candida exposure on the ability of
Gr-1+ PMN to affect CD4+
Th1 cell proliferation, and 4) the mechanisms underlying this
effect. Like the human counterparts, murine Gr-1+
PMN constitutively express CD80 molecules; however, in murine and
human PMN, CD80 expression was differentially modulated upon
interaction with Candida yeasts or hyphae in vitro, as well
as in infected mice. A population of
CD80+Gr-1+ myeloid cells
was found to be expanded in mice with disseminated infection, but its
depletion increased IFN--mediated antifungal resistance. In vitro,
Gr-1+ PMN were found to inhibit the activation of
IFN--producing CD4+ T cells and to induce
apoptosis through a CD80/CD28-dependent mechanism.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c and C3H/HeJ mice, 8–10 wk old, were purchased
from Charles River (Calco, Italy). Homozygous IFN--, IL-4-, CD28-,
CD80-, and CD86-deficient mice on a BALB/c background and homozygous
IL-10-deficient on a C57BL/6 background were bred under specific
pathogen-free conditions in the Animal Facility of Perugia University
(Perugia, Italy). Procedures involving animals and their care were
conducted in conformity with national and international laws and
policies.
C. albicans strains, culture conditions, and infection
The origin and characteristic of the C. albicans virulent strain and the live vaccine strain used in this study have already been described in detail (6, 7). Because the virulent strain is capable of undergoing yeast to hyphal transition in vivo and in vitro, whereas the live vaccine strain is not, the two strains were used as sources of hyphae and yeasts, respectively (33). For disseminated infections in vivo we resorted to two well-characterized models of infection, in which mice are i.v. injected with either the virulent strain (5 x 105 cells/0.5 ml; thereafter referred to as hypha-infected mice) or the low-virulence strain (106/0.5 ml; yeast-infected mice) (primary disseminated infection). Hypha-infected mice succumb to the infection with signs of yeast and hyphal overgrowth in the target organs, such as the kidneys and brains (11). In contrast, yeast-infected mice survive the infection, with limited yeast growth in the kidneys (11). In BM-transplanted mice, mice were i.v. infected with the low-virulence Candida strain followed 14 days later by reinfection with the virulent strain (106/0.5 ml) (secondary disseminated infection). Treatments with Gr-1- or CD80-neutralizing mAbs (a total of 500 µg of mAb, given i.p.) were done on days 3, 5, and 7 after the infection. Control mice were injected with an unrelated, isotype-matched Ab. Quantification of fungal growth in the organs of infected mice (four to six mice per group) was performed by plating serial dilutions of homogenized organs in Sabouraud dextrose agar, and results (mean ± SEM) were expressed as CFU per organ. Mice succumbing to fungal challenge were routinely necropsied for histopathological confirmation of candidiasis.
Irradiation
C3H/HeJ mice were exposed to a single, lethal dose of 9 Gy from a gamma beam 150A, 60Co source (Clinac 600/C Varian; Cernusco, Milan, Italy) with focus to skin distance of 75 cm and a 0.7 Gy/min dose rate. Unless BM transplanted, mice died within 14 days (34).
Infusion of T cell-depleted BM cells
Donor BM cells were prepared by differential agglutination with
soybean agglutinin, as described (34). T cell-depleted
soybean agglutinin-positive cells (containing <1% of contaminating T
cells on FACS analysis) were injected at the concentration of 
4
x 106/ml into recipient mice i.v. According to
previous studies (35), >95% of the mice survived showing
stable, donor-type hematopoietic chimerism, as revealed by donor-type
MHC class I Ag expression on cells from spleens. Total and differential
white blood cell counts were done by hemocytometry and by staining
blood smears from transplanted mice with May-Grünwald Giemsa
reagents (Sigma-Aldrich, St. Louis, MO) before analysis.
Isolation of murine and human PMN
Murine PMN were isolated from blood, spleens, and the peritoneal cavity of mice as described (7, 8). Peritoneal PMN were obtained 18 h after the i.p. injection of 1 ml endotoxin-free 10% thioglycolate solution (Difco, Detroit, MI), as described (7, 8). Endotoxin was depleted from all solutions with Detoxi-gel (Pierce, Rockford, IL). To purify Gr-1+ PMN, 107 cells were incubated with biotin-conjugated anti-mouse Gr-1 mAbs (clone RB6-8C5; BD PharMingen, Palo Alto, CA) for 30 min at 4°C and then with avidin-conjugated magnetic MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 min at 6°C, and were magnetically separated with a positive selection column (Miltenyi Biotec) according to the manufacturer’s instructions. On FACS analysis, Gr-1+ PMN were >98% pure and stained positive for the CD11b myeloid marker. Cytospin analysis confirmed that the population consisted of polymorphonuclear cells (>98%). Human PMN were obtained from the heparinized whole blood of healthy donors after lysis of hypotonic shock with ammonium chloride and fractionation by Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden) centrifugation. The purity of PMN preparations was >97%, as determined by Giemsa morphology.
Exposure of murine or human PMN to Candida in vitro
Murine Gr-1+ PMN or human PMN (106/ml) were cultured with 2 x 106 C. albicans hyphae or yeasts or both in RPMI containing 10% FCS, 50 mM 2-ME, 1 mM sodium pyruvate, and 10 mM HEPES (complete medium) in polypropylene tubes (Falcon; BD Labware, Franklin Lakes, NJ) for 2 h in a 5% CO2 incubator. Cells were collected and resuspended in complete medium plus 2.5 µg/ml amphotericin B (Fungizone; Bristol-Myers Squibb, Sermoneta, Italy) to prevent overgrowth of residual fungal cells. Control experiments indicated that amphotericin B did not modify the functional properties of PMN.
Immunoprecipitation and Western blotting
Murine Gr-1+ PMN were isolated from blood and exposed to hyphae, as described above. B cells, purified from spleens by magnetic cell separation with CD45R(B220)-conjugated magnetic MicroBeads (Miltenyi Biotec), were stimulated with 10 µg/ml LPS for 72 h. The P1.HTR tumor variant transfected with murine B7-1 (P1.HTR.B7-1), generated as described (36), was kindly provided by Dr. F. Fallarino (University of Perugia, Perugia, Italy). Cells were lysed in 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 1% (w/v) Triton X-100, 5 mM EGTA, 2 mM MgCl2, 1 mM PMSF, 50 µM leupeptin, 50 mM E-64, 10 µM pepstatin A, and 10 µg/ml chymostatin (lysis buffer) on ice for 45 min, and then centrifuged at 13,000 x g for 15 min. The resulting supernatants were removed and the immunoprecipitation was done as described (37), by the addition of the 16-10A1 mAb (final dilution 1/100, v/v; BD PharMingen) followed by the incubation at 4°C for 2–4 h with constant mixing. Protein G-Sepharose (Pierce) was then added (250 µl of a 50% suspension in lysis buffer per milliliter of lysate) and the resulting suspension was incubated for a further 12–16 h. The immunoabsorbent was then collected by centrifugation (2 min at 2,000 x g) and washed three to five times with 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl and 0.5% Triton X-100; bound proteins were eluted by incubation for 30 min at 40°C in SDS sample buffer. After incubation, the samples were loaded onto 12% polyacrylamide slab gels under denaturing conditions as described (37). Electrophoresis was conducted for 14 h at 180 V, and the gels were then transferred electrophoretically to polyvinylidene difluoride transfer membranes (Immobilon-P; Millipore, Bedford, MA) for 3 h at 4 mA/cm2 using an electroblotter (Trans-blot cell; Bio-Rad, Richmond, CA), as described (38). Membranes were incubated with primary Abs (1/100 diluted) followed by peroxidase-conjugated secondary Abs and developed with H2O2 and diaminobenzidine tetrahydrochloride in Tris-HCl 0.1 M (pH 7.6).
Flow cytometry
For analysis of costimulatory molecule surface expression, murine Gr-1+ PMN, freshly purified or after exposure to Candida hyphae or yeasts, were sequentially reacted with PE-conjugated anti-Gr-1 (rat IgG2b, clone RB6-8C5) and with FITC-conjugated anti-CD80 (hamster IgG2a, clone 16-10A1) or anti-CD86 (rat IgG2a, clone GL1) mAbs (BD PharMingen). Human PMN were similarly exposed to Candida and reacted with FITC-conjugated anti-human CD80 (mouse IgM, clone BB1) from Ancell (Bayport, MN). For intracytoplasmic staining, cells were labeled with Cytofix/Cytoperm Plus containing brefeldin A to inhibit protein transport (39), as per the manufacturer’s instructions (BD PharMingen). For double surface and cytokine intracellular staining, Candida-exposed Gr-1+ PMN were reacted with FITC-conjugated anti-CD80 mAb and with PE-conjugated anti-IL-10 (rat IgG2b, clone JES5-16E3) or anti-IL-12 p40 (rat IgG1, clone C15.6) mAbs, by using the Cytofix/Cytoperm Plus kit as above. Cells were analyzed with a FACScan flow cytofluorometer (BD Biosciences, Mountain View, CA). Nonviable cells were excluded from analysis by accepted procedures involving propidium iodide (PI) and narrow forward-angle light scatter gating. Control staining of cells with irrelevant mAb was used to obtain background fluorescence values. Data are expressed as a percentage of positive cells over total cells analyzed. Aliquots of cells were spun down on slides on a cytocentrifuge and mounted in buffered glycerol to be examined by fluorescent microscopy. Photographs were taken using a Zeiss Axiophot equipment (Carl Zeiss, Milan, Italy) and a Kodak Tmax 400 film (Kodak, Rochester, NY).
Lymphoproliferation assay
CD4+ T cells were purified from spleens of
mice by means anti-CD4 magnetic MicroBeads (Miltenyi Biotec) as
described elsewhere (6, 7, 33). Separation of
CD4+CD45RBhigh and
CD4+CD45RBlow cells was
done by magnetic separation of CD4+ cells reacted
with R-PE anti-mouse CD45RB (BD PharMingen) with anti-PE
magnetic MicroBeads (Miltenyi Biotec). A total of 5 x
105 CD4+ T splenocytes were
stimulated with plate-immobilized (20 µl/ml) anti-CD3
Abs
(clone 145-2C11; BD PharMingen) in the presence of
105 Gr-1+ cells, either
unexposed or exposed to Candida yeasts or hyphae, in 200
µl complete medium in round-bottom 96-well plates (Falcon; BD
Labware). Anti-CD80-, anti-CD86-, and anti-CTLA-4
(9H10)-neutralizing mAbs (BD PharMingen) were used at final
concentrations of 10 µg/ml. In selected experiments, 0.5 mM of the
competitive inhibitor of NO-synthase
NG-monomethyl-L-arginine
(L-NMMA; Calbiochem, San Diego, CA) was added to
the cultures. Cells were cultured for 3 days at 37°C, 5%
CO2. Eight hours before harvesting, cells were
pulsed with 0.5 µCi of [3H]thymidine per
well. Incorporation into cellular DNA was measured by liquid
scintillation counting. The results are expressed as mean cpm ±
SEM of triplicate cultures. In parallel experiments, culture
supernatants were collected for cytokine determination.
Cytokine assays
The levels of IFN- and IL-2 in culture supernatants were
determined by means of specific ELISA, as previously described. The
capture/biotinylated detection mAbs were as follows: IFN-,
R4-6A2/XMG1.2, IL-2, JES6-1A12/JES6–5H4. Cytokine titers were
calculated by reference to standard curve constructed with known
amounts of recombinant cytokines. All reagents were from BD
PharMingen.
Apoptosis assay
After a 24-h coculture, cells (105) were
surface stained with anti-CD4-PE, washed, and then stained with
FITC-labeled annexin V and PI (Sigma-Aldrich) as described
(40). At least 10,000 CD4+ events
were collected for annexin/PI analysis (see Fig. 7
;
CD4+ cells in early apoptosis
(annexin+PI-) are in the
lower right quadrant; live cells
(annexin-PI-) are in the
lower left quadrant; dead cells
(annexin+PI+) are in the
upper right quadrant).
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For TEM, 106 Candida-exposed Gr-1+ PMN were pelleted at 1200 rpm for 5 min, washed twice with PBS, and fixed in cold 2.5% glutaraldehyde in 0.1 M sodium cacodylate 1% osmium tetroxide (50 min), encapsulated in 1% agar, stained with uranyl acetate and phosphotungstic acid, and dehydrated in a series of graded ethanolic solutions, finishing with propylene oxide before finally being embedded in Epon 812-Araldite mixture. Ultrathin sections (50 nm) were cut on an ultramicrotome (LKB Wallac, Uppsala, Sweden) and placed under 200-mesh standard copper grids, contrasted with uranyl acetate and lead citrate, and examined with a Philips TEM 400 transmission electron microscope (Phillips, Eindhoven, The Netherlands).
RNA preparation and RT-PCR
Gr-1+ PMN were subjected to RNA extraction and amplification of synthesized cDNA from PMN were done as previously described (6, 7). Briefly, 5 µg of total RNA was reverse transcribed into cDNA using Moloney murine leukemia virus reverse transcriptase (Life Technologies, Gaithersburg, MD). The cDNA was then amplified using specific primers for B7 Ig V-like exon, as described (41). Amplifications were performed in 2 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 0.2 mM of each deoxynucleotide triphosphate, 1 µM of each primer, and 2.5 U AmpliTaq polymerase (PerkinElmer/Cetus, Norwalk, CT). The cDNA was amplified in an automated thermal cycler (PerkinElmer/Cetus) as described elsewhere (6, 7). Amplification was stopped at 35 cycles. The hypoxanthine phosphoribosyltransferase primers were used as a control for both reverse transcription and the PCR, and also for comparing the amount of products from samples obtained with the same primer. The PCR fragments were analyzed by 1.5% agarose gel electrophoresis, stained with 0.5 mg/ml ethidium bromide, and visualized using a UV transilluminator.
Statistical analysis
Student’s t test was used to determine significance of values among experimental groups. Significance was defined as p < 0.05. The data reported are either from one representative experiment of five with similar results (FACS analysis) or were pooled from three to five experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine PMN were isolated from blood, spleen, and the peritoneal
cavity, double stained with anti-Gr-1 and anti-CD80 or
anti-CD86 mAbs, and analyzed for surface molecule expression by
flow cytometry and fluorescence microscopy. The results show that a
significant fraction of peripheral cells (14.2% in blood and 8.4% in
spleens) constitutively expressed CD80 molecules, as evidenced by dot
plot FACS analysis (Fig. 1A)
and immunofluorescence microscopy (Fig. 1B). CD80 molecule
expression was also observed, and actually increased, in elicited
peritoneal PMN (16.6%), a finding suggesting that CD80 expression is
maintained upon inflammation. No expression of CD80 molecules could be
detected in peripheral PMN from CD80-deficient mice (Fig. 1A). RT-PCR analysis confirmed the above results by showing
the presence of CD80 RNA message in the cells (Fig. 1C). In
contrast, but in line with the results obtained with human PMN
(32), the expression of the CD86 molecules was not
constitutive but was induced upon inflammation (Fig. 1, A
and C). Similar to that observed with peritoneal exudate
cells (37), immunoprecipitation experiments performed with
the mAb 16-10A1 reactive with murine CD80 molecules (37)
revealed the presence of a band of an apparent molecular mass of 60 kDa
from the P1HTR.B7-1 cell line as well as from peripheral
Gr-1+ PMN and B cells (Fig. 1D). The
60-kDa band was not immunoprecipitated by a control hamster mAb of
irrelevant specificity. Altogether, these experiments demonstrate the
presence of CD80 molecules reacting with the 16-10A1 mAb on murine
Gr-1+ PMN.
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To assess whether exposure to C. albicans would affect
the PMN expression of CD80 molecules, peripheral murine or human PMN
were exposed to either yeasts or hyphae of the fungus, because we have
shown that PMN are capable of discriminating between the two forms of
the fungus in terms of functional responses (6, 7, 10).
TEM revealed that the two forms of the fungus were differently handled
by murine PMN. Yeasts cells were rapidly internalized through a coiling
mechanism of phagocytosis, whereas hyphae could not be internalized and
were found to be surrounded by multiple cells (Fig. 2). On assaying costimulatory Ag
expression, we found that the exposure to Candida yeasts
significantly reduced the surface expression of CD80, although to a
variable degree among the different experiments, whereas the exposure
to hyphae significantly increased this expression (Fig. 3A). No effect was observed on
the expression of the CD86 molecule (data not shown). Because the
viability of PMN, as assessed by the dye exclusion test and apoptosis
assay, was not affected upon exposure to either type of the fungus
(42% of necrotic cells upon exposure to yeasts or hyphae vs 38% of
unexposed PMN; 22% of apoptotic PMN upon exposure to either type of
the fungus vs 31% of unexposed cells), these results suggest that
surface expression of CD80 on PMN is differently modulated upon contact
with yeasts or hyphae of the fungus. Interestingly, the expression of
CD80 also increased upon exposure of cells to yeasts and hyphae
simultaneously (Fig. 3A), a finding suggesting that the
effect of hyphae is predominant. Intracytoplasmic staining revealed the
presence of CD80 molecule inside the cells (Fig. 3B), a
finding confirming the finding obtained with human PMN, whose B7-1-like
molecule is localized to the cytoplasmic granules (32).
Similar results were obtained with human PMN, as increased expression
of CD80 was observed on the surface of cells exposed to hyphae and not
to yeasts (Fig. 3C). The cytoplasmic localization of the
molecule was also confirmed (data not shown). In vivo, in mice with
primary disseminated candidiasis, the number of
Gr-1+ PMN greatly increased in mice infected with
Candida hyphae, but not Candida yeasts, as
observed in the kidneys as well as in the spleens (Fig. 3D).
Because both yeasts and hyphae are present in the kidneys of mice
infected with virulent Candida (10, 11), these
data confirm the in vitro finding that, when simultaneously present,
the effect of hyphae predominates over that of yeasts.
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Because the interaction of PMN with Candida yeasts or
hyphae resulted in the production of IL-12 or IL-10, respectively
(6, 7, 10), we assessed the possible association of CD80
expression with cytokine production in murine PMN exposed to the
fungus. To this purpose, peripheral Gr-1+ PMN,
exposed to Candida yeasts or hyphae as above, were double
stained for surface CD80 and intracellular IL-10 and IL-12 and assessed
by FACS analysis. The results showed that the expression of CD80
correlated with IL-10 production; i.e., the number of IL-10-producing
Gr-1+ cells decreased or increased after yeast
phagocytosis or hypha exposure, respectively (Fig. 4A). In contrast, the number
of IL-12-producing Gr-1+ cells did not vary upon
exposure to either type of the fungal form. Therefore, signaling for
CD80 Ag expression may also involve signaling for cytokine (IL-10)
production in PMN to C. albicans. Although IL-10 is a
negative regulator of costimulatory Ag expression (42), we
have shown that IL-10 is nevertheless required for the proper
expression of costimulatory molecules in mice with candidiasis
(43). In addition, IL-4 is known to be one most important
regulator of CD80 expression on myeloid cells (26). To
assess whether these cytokines could be involved in the modulation of
the CD80 expression, PMN from IL-10- or IL-4-deficient mice were
exposed to hyphae and assessed for CD80 expression. CD80 expression did
not increase in PMN from IL-4-deficient mice upon exposure to hyphae,
as it did in PMN from IL-10-deficient mice (Fig. 4B). In
addition, IL-10 neutralization did not affect CD80 expression on
wild-type PMN upon exposure to hyphae (data not shown). Therefore,
IL-4, more than IL-10, appears to be involved in the regulation of CD80
expression on PMN after exposure to Candida.
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To investigate whether the modulation of CD80 Ag expression
observed after exposure to C. albicans yeasts or hyphae
could impact differently on the activation of naive or activated
CD4+ T lymphocytes, murine peritoneal PMN, either
unexposed or after exposure to Candida yeasts or hyphae,
were cocultured with CD4+ T lymphocytes in the
presence of anti-CD3 mAb, in the absence of APCs. Lymphocyte
activation was measured in terms of proliferation and cytokine
production in culture supernatants. The results show that unexposed PMN
greatly inhibited the proliferative activity and the IL-2 and IFN-
production of CD4+ T lymphocytes, of both the
naive and activated phenotypes (Fig. 5).
Similarly, PMN exposed to hyphae, but not to yeasts, inhibited
lymphoproliferation and IL-2 and IFN- production. Therefore, PMN
behave as functional MSC, capable of inhibiting the CD3-mediated
activation of CD4+ Th1 lymphocytes, an activity
preserved upon exposure to hyphae, but not to yeasts.
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/NO-independent mechanism of apoptosis
To understand the possible mechanisms through which
Gr-1+ PMN suppress T cell activation, the
lymphoproliferation was assessed in conditions of selective
blockade of costimulatory-dependent pathways by means of
neutralizing mAbs. In addition, CD80- or CD86-deficient mice were used
as a source of PMN and CD28-deficient mice were used as a source of
CD4+ T cells. The functional blockade of CD80,
obtained either with the relevant mAb or by gene deficiency, restored
the CD3-mediated activation of CD4+ T cells,
although to a slightly different extent (Fig. 6A). As expected, no such an
effect was observed upon functional blockade of CD86 molecules by
either the relevant mAb or with the use of CD86-deficient mice (Fig. 6A). The suppressive activity of PMN could not be observed
with CD4+ T cells from CD28-deficient mice (Fig. 6B). Because blockade of CTLA-4 by the anti-CD152
neutralizing mAb did not restore the responsiveness of
CD4+ T cells from either wild-type or
CD28-deficient mice (Fig. 6, A and B), these
results would suggest that a CD80/CD28-dependent pathway exists that
negatively regulates the activation of T lymphocyte upon contact
with PMN.
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-dependent production of
peroxynitrite (23). To evaluate whether this pathway of
lymphocyte inhibition could be at work in our system, the ability of
PMN to inhibit CD4+ T cells activation was
assessed in the presence of the NO synthase inhibitor,
L-NMMA, or in a system devoid of IFN-. The results show
that the addition of L-NMMA partially restored
lymphoproliferation (Fig. 6
C). However, when
CD4+ T cells and PMN from IFN--deficient mice
were assessed for CD3-dependent proliferation in the presence of
L-NMMA or the anti-CD80 mAb, the cell
proliferation was still inhibited through a CD80-dependent pathway and
was not restored in the presence of L-NMMA (Fig. 6C). Therefore, it appears that the CD80/CD28-mediated
inhibition pathway of CD4+ T cell activation
mainly occurs through an IFN-/NO-independent mechanism. Because the
addition of IL-10-neutralizing mAb or the addition of exogenous IL-12
did not restore lymphoproliferation (data not shown), the suppressive
activity of MSC does not involve IL-10 or IL-12 production either. To
assess whether responding cells undergo apoptosis, we measured
apoptosis of CD4+ T cells upon contact with
Gr-1+ cells in vitro with a quantitative flow
cytometry assay that uses annexin-V-FITC (40). Apoptosis
of CD4+ cells was significantly higher (14.7 vs
3.1%) in the population cultured with Gr-1+
cells as compared with cells cultured alone (Fig. 7). Apoptosis was also confirmed by DNA
ladder analysis on agarose gel (data not shown). Evidence for the occurrence of CD80+ MSC in mice with candidiasis
To find out whether CD80+ MSC are generated
in vivo in mice with candidiasis, we resorted to the primary or
secondary disseminated infection in immunocompetent or allo-BM
transplanted mice, respectively, both models being characterized by a
sustained neutrophilia (6, 7, 10, 34). In the acute
disseminated infection, we have already observed that early PMN
depletion accelerates mortality (6, 7, 10). In this study
we show that, while the number of
CD80+Gr-1+ cells
progressively increased in the course of the infection (from 12 to 55%
at 6 days after the infection), depletion of
Gr-1+ or CD80+ cells late
in the infection improves the outcome, as revealed by the increased
survival and the reduced fungal growth in the kidneys (Fig. 8A). In parallel, increased
production of IFN- was also observed (data not shown). Although the
anti-Gr-1 treatment may also deplete cells other than PMN
(44), the finding that the treatment had an opposite
effect if done earlier or later in the course of the infection suggests
a possible correlation between the treatment and the appearance of
suppressive Gr-1+CD80+
cells. In transplanted mice we found that the temporally regulated
expression of specific antifungal Th1 activity correlated with the
number of peripheral
CD80+Gr-1+ myeloid cells
(Fig. 8B). At the earlier weeks after transplantation, at
the time when minimal antifungal resistance was observed, both the
absolute number (at wk 2) of PMN and the percentage (at both wk 1 and
2) of CD80+Gr-1+ PMN were
elevated. However, at the time when functional Th1 reactivity to the
fungus was restored (Fig. 8 and Ref. 34), both the
absolute and the relative numbers of
CD80+Gr-1+ PMN had greatly
dropped out. Together, these data point to the existence of cells,
sensitive to either PMN or CD80 ablation, that negatively regulate the
protective, IFN--dependent immunity in candidiasis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In particular, the number of
CD80+Gr-1+ myeloid cells
progressively increased during the acute lethal infection, as well as
during the early engraftment period after the allo-BM transplantation.
Interestingly, depletion of
CD80+Gr-1+ cells late in
the infection increased host resistance to the acute infection as well
as IFN- production. Thus, it is possible that the early expansion of
myeloid CD80+Gr-1+ cells in
the transplanted mice could adversely affect the development of
antifungal Th1 immunity. In this regard, it has already been reported
that a population of myeloid progenitor cells inhibiting T cell
responsiveness is increased in mice upon immunosuppression
(45).
There has been considerable recent interest in MSC, expressing the Gr-1
and CD11b myeloid markers and capable of inhibiting T and B cell
proliferation (reviewed in Ref. 19). Studies have shown
that MSC are responsible for the immunosuppression observed in
pathologies as dissimilar as tumor growth, immunosuppression,
overwhelming infections (17, 46, 47, 48, 49, 50), graft-vs-host
disease (51), and pregnancy (19). Two
distinct subpopulations of myeloid suppressors have been characterized.
CA MSC are NO and IFN- dependent (24, 28), whereas
alternatively activated MSC are IL-4 dependent (17, 18, 26, 29, 30, 52). An immature myeloid cell line strongly up-regulated its
suppressive functions after exposure to IL-4 (26) and,
interestingly, the exposure to IL-4 also up-regulated CD80 expression
on these cells (26), whereas exposure to IFN- did not
up-regulate CD80 expression on human PMN (32). It
has been suggested that alternative differentiation of myeloid
precursors might represent a main characteristic of immunocompromised
hosts (19).
The MSC population we describe in this study does not fall in the CA
category. Myeloid cells, IFN-, and a combination of NO and reactive
oxygen intermediates all contributed to a common pathway of T cell
death that targets activated CD4+ T cells
(24, 27, 28). We found that the inhibitory activity of
Gr-1+ myeloid cells on CD4+
T cells of either naive or activated phenotype was still retained in
the absence of IFN- and was only partially restored in the presence
of the NO inhibitor L-NMMA. Thus, the
Gr-1+ suppressor cells described in this work are
different from those found to be expanded in response to a
schistosome-expressed immunomodulatory glycoconjugate and to suppress
the proliferative activity of naive CD4+ T cells
via an IFN- and NO dependent mechanism (24). A
population of alternatively activated macrophages that suppress
CD4+ T cells reactivity by an as-yet-unknown
mechanism, but in an IFN-/NO-independent manner, has been reported
(53).
One interesting observation of the present study is the detection of CD80 molecules on murine Gr-1+ PMN. The molecule was detected both on the surface and intracellularly, similarly to what observed in human PMN (32). PMN from healthy donors did not express MHC class II Ags or CD80 or CD86 molecules; however, expression of these molecules was seen in patients with chronic inflammatory diseases (32). The B7-1-like molecule was located in the cytoplasmic granules and translocated to the cell surface after LPS stimulation (32). It was suggested that the expression of CD80 molecules on PMN may be of biological significance in conditions of prolonged stimulation in vivo, such as at sites of inflammation (32). We found that an increased expression of CD80 occurred in both murine and human PMN after exposure to hyphae but not yeasts of Candida in vitro and in vivo. Whether this is due to a prolonged stimulation by hyphae is not known at the moment. However, because different receptors appear to be involved in the recognition of either form of the fungus by murine PMN (our unpublished observation), it is also possible that the engagement of different receptors may be responsible for the differential up-regulation observed not only between yeasts and hyphae but also between CD80 and CD86. In this regard, differential up-regulation of the CD80 and CD86 costimulatory molecules was observed after Ag receptor engagement (54, 55).
Together with the up-regulated expression of CD80 molecule, murine PMN also increased the production of IL-10, a finding that corroborates previous data showing that PMN discriminate between yeasts and hyphae of the fungus in terms of IL-12 or IL-10 production, respectively (6, 7). Preliminary evidence suggest that human PMN also produced higher levels of IL-10 upon exposure to hyphae than yeasts (data not shown). Therefore, it seems that a signaling pathway leading to both IL-10 and CD80 up-regulation takes place when hyphae are sensed by PMN. However, because CD80 was up-regulated in IL-10-deficient mice, but not in IL-4-deficient mice, it appears that IL-4, more than IL-10, is autocrinally involved in the regulation of CD80 expression on myeloid cells, as suggested (26).
IL-10 did not contribute to the impairment of T cell reactivity either, as blocking IL-10 with neutralizing mAb did not reverse the inhibitory effect of Gr-1+ cells. A similar result was obtained by adding exogenous IL-12, a finding suggesting that the differential production of these cytokines may not account for the suppressive activity of Gr-1+ cells. Instead, the suppressive activity was dependent upon the CD80/CD28 interaction, as it was not observed in the presence of CD80 blocking mAbs or in the absence of CD28. The finding that blocking the CTLA-4R did not abrogate the suppressive activity of Gr-1+ PMN ruled out the possibility of an inhibitory effect mediated by the CD80/CTLA-4 interaction (56). Altogether, these result point to the existence of a costimulatory molecule on murine Gr-1+ PMN, whose engagement deliver a negative, rather than positive, stimulatory signal to T lymphocytes.
In an attempt to elucidate mechanisms underlying this phenomenon, we looked for apoptosis in CD4+ T cells upon contact with Gr-1+ PMN and found that the number of apoptotic cells was 4-fold increased in the presence of PMN. Although signaling through costimulatory molecules has been shown to inhibit apoptosis of lymphocytes (57, 58), it has recently been reported that cosignaling through the CD80/CD28 pathway may account for the programmed cell death occurring in HIV-seropositive patients (59). It would appear that CD80 molecule expressed on Gr-1+ PMN are able to deliver an apoptotic signal to CD4+ T cells. Whether the apoptotic signal would be sufficient to mediate the suppressive function of myeloid cells or other mechanisms are at work is not presently known. In this regards, studies are ongoing to assess whether the NO synthase 2- and arginase-1-dependent pathways, known to play distinct roles on macrophage activation (60), also are critical determinants in the disparate activation and functional activity of PMN in response to the different forms of fungi. Whatever the case would be, the inhibitory signal delivered by CD80 would be in line with the negative regulatory function of the B7-1 molecule observed in B7-1 transgenic mice, where the temporally regulated expression of CD80 was suggested to contribute to either initiation or down-regulation of T-dependent immunity (61). In addition, further studies will help clarify the contribution of the different murine CD80 isoforms (62) to the contrasting roles this molecule may have in the regulation of T cell functional activities.
Overall, the data of the present study point to an important immunomodulatory role of Gr-1+ PMN in mice with candidiasis. It appears that a population of suppressive Gr-1+ cells expressing the CD80 molecule are expanded in mice with an overwhelming fungal infection as well as in transplanted mice.
Other studies have already shown the superior activity of CD86 over that of CD80 in the induction of antifungal Th1 resistance to candidiasis (63, 64), as well as in experimental aspergillosis (65) and histoplasmosis (66). Studies in CD80- or CD86-deficient mice confirm that CD80 deficiency, but not CD86 deficiency, is associated with increased resistance to candidiasis and aspergillosis (C. Montagnoli, manuscript in preparation). Because a similar population of suppressive Gr-1+ PMN was isolated from the lungs of mice with invasive aspergillosis (our unpublished observation), it is tempting to speculate that alternatively activated Gr-1+ PMN expressing CD80 may act as MSC in immunocompromised hosts with fungal infections, eventually vanishing the therapeutic efficacy of the CSFs in fungal infections (67, 68).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Luigina Romani, Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Via del Giochetto, 06122 Perugia, Italy. E-mail address: lromani{at}unipg.it
3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; BM, bone marrow; CA, classically activated; MSC, myeloid suppressor cell; L-NMMA, NG-monomethyl-L-arginine; TEM, transmission electron microscopy; PI, propidium iodide.
Received for publication February 4, 2002. Accepted for publication July 8, 2002.
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double-d
