Blood ASH 95/7 - Blood Journal

From www.bloodjournal.org by guest on January 13, 2017. For personal use only.
HEMATOPOIESIS
Pharmacologic doses of granulocyte colony-stimulating factor affect cytokine
production by lymphocytes in vitro and in vivo
Elaine M. Sloand, Sonnie Kim, Jaroslaw P. Maciejewski, Fritz Van Rhee, Aniruddho Chaudhuri, John Barrett, and Neal S. Young
Peripheral blood stem cell (PBSC) transplantation is successful in improving
engraftment without increasing acute
graft-versus-host disease (GVHD), despite much larger numbers of T cells in
unmanipulated PBSCs than in bone marrow grafts. In mouse models and retrospective human studies, granulocyte
colony-stimulating factor (G-CSF) therapy
has been associated with less acute
GVHD. We studied the effect of G-CSF on
interferon (IFN)-␥ and IL-4 expression in
CD4ⴙ lymphocytes. CD4ⴙ cells co-cultivated with G-CSF and stimulated with
PHA or CD3 monoclonal antibodies
showed significant decreases in IFN-␥
and increases in IL-4 expression (n ⴝ 13;
P F .01). G-CSF appeared to have a direct
effect on CD4ⴙ cells independent of
monocytes present in the culture because purified CD4ⴙ cells exposed to
G-CSF, washed, and cocultivated with
untreated monocytes demonstrated similar changes in IFN-␥ and IL-4 expression,
whereas untreated CD4ⴙ cells cocultured
with G-CSF–stimulated monocytes behaved as controls. We then studied
peripheral blood mononuclear cells
(PBMCs) from G-CSF–mobilized PBSC
donors. When their PBMCs were cultured
with PHA or CD3 monoclonal antibody,
the percent of IFN-␥–expressing cells de-
creased by a mean of 55% and 42%,
respectively, whereas the percent of IL-4–
containing cells increased by a mean of 39%
and 58%, respectively, following G-CSF
stimulation. Increased apoptosis of IFN-␥–
producing CD4ⴙ cells was not responsible
for the shift in TH1/TH2 subsets. G-CSF-R
mRNA was present in both CD4ⴙ and CD8ⴙ
cells. These results suggest that G-CSF
decreases IFN-␥ and increases IL-4 production in vitro and in vivo and likely modulates
a balance between TH1 and TH2 cells, an
effect that may be important in PBSC
transplantation. (Blood. 2000;95:2269-2274)
r 2000 by The American Society of Hematology
Introduction
Use of peripheral blood stem cells (PBSC) for allogeneic bone
marrow (BM) transplantation has a number of advantages over
conventional BM grafting, including an improved yield of progenitor cells, ease of harvest, and shortened time to achieve engraftment.1,2 Despite a 10-fold increase in the number of CD3⫹ cells in
the apheresis product used for the transplantation of stem cells,
patients undergoing PBSC transplantation do not demonstrate an
apparent increased incidence or severity of acute graft-versus-host
disease (GVHD).3,4 This observation may be accounted for by a
number of differences observed in BM and PB stem cells. PBSC
obtained from granulocyte colony-stimulating factor (G-CSF)mobilized donors contain more CD3⫹CD8⫺CD4⫺ cells than BM
cells5; these cells decrease the severity of acute GVHD in the
murine model and are associated with decreased cytotoxic lymphocyte activity in vitro.6 Similarly, monocytes obtained from G-CSF
mobilized donors suppress cytotoxic responses and T-cell proliferation when they are cocultivated with T cells.7,8 Recently, G-CSF
was shown to polarize the balance within the murine T-lymphocyte
subpopulations toward the TH2 cytokine production in vitro.9
CD4⫹ lymphocytes can be functionally subdivided into TH1
and TH2 cells based on their pattern of cytokine expression.10 TH1
cells can secrete tumor necrosis factor (TNF)-␣ and IFN-␥,
whereas TH2 cells are capable of producing IL-2, IL-4, and IL-10.
The balance between these 2 subsets is altered in a number of
pathophysiologic conditions and is associated with progression
of or protection against some infectious and autoimmune conditions.11-13 Recent results have suggested that acute GVHD is
mediated by TH1 lymphocytes and that TH2 lymphocytes may
exert a protective effect but may have a role in mediating chronic
GHVD.14 In a murine model of GVHD with TH1 and TH2
knock-out mice, TH1 cells mediated the symptoms of severe
diarrhea and wasting, ultimately leading to death from acute
GVHD15; TH2 cells caused skin disease and were partially
effective in antagonizing the effect of TH1 cells. Experiments using
the IFN-␥ knockout mouse showed significant modification of
acute GVHD, confirming the importance of the role of IFN-␥.16
We hypothesized that G-CSF, in addition to its trophic effects on
hematopoiesis, may have regulatory effects on T lymphocytes and
may modulate immune responsiveness in unmanipulated PBSC
grafts and autoimmune hematologic diseases in which it is used.
Therefore, in this study we investigated the effects of G-CSF on
TH1 and TH2 cell development in mitogen-stimulated human CD4⫹
cells and studied the mechanisms of G-CSF–mediated changes within
the lymphocyte subpopulations. The in vitro findings were correlated
with the effects of G-CSF on TH1 and TH2 lymphocyte distribution in PBSC donors receiving G-CSF for stem cell mobilization.
From the National Heart, Lung and Blood Institute, National Institutes of Health
(NIH), Bethesda, MD.
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.
Submitted March 17, 1999; accepted December 2, 1999.
Reprints: Elaine M. Sloand, NIH, National Heart, Lung, and Blood Institute, 31
Center Dr, MSC 2490, Bldg 31, Room 4A11, Bethesda, MD 20892-2490;
e-mail: [email protected].
BLOOD, 1 APRIL 2000 • VOLUME 95, NUMBER 7
Materials and methods
Stem cell donor selection and studies
Peripheral blood for in vitro studies was obtained from normal volunteers.
For studies involving in vivo G-CSF stimulation of normal donors, samples
were obtained from normal stem cell donors before and after 5 days of
r 2000 by The American Society of Hematology
2269
From www.bloodjournal.org by guest on January 13, 2017. For personal use only.
2270
BLOOD, 1 APRIL 2000 • VOLUME 95, NUMBER 7
SLOAND et al
G-CSF treatment (10 µg/kg subcutaneously daily) before apheresis donation. Heparinized blood for cytokine expression studies was obtained on
day 1, before the administration of G-CSF, and on day 6. In all cases,
informed consent was obtained according to protocols approved by the
Institutional Review Board of the National Heart, Lung, and Blood
Institute. PBMC from G-CSF–stimulated donors were separated as described below, and cells were cultured for 48 hours with PHA (1 µg/mL) or
anti-CD3 monoclonal antibody (mAb) (0.1 µg/mL). Cells were then surface
stained with anti-CD4–phycoerythrin (PE) and intracellularly stained with
either anti–IFN-␥-fluorescein isothiocyanate (FITC) or anti–IL-4 FITC as
described below.
d(T)16 primer (RNA CORE KIT; Perkins-Elmer Cetus, Foster City, CA). After
reverse transcription, the cDNA was amplified using 58 and 38 primer
pairs, 58-AGTACAGTCCTCACCCTGATG-38, 58-AAAGTATGCAGATCGCCTGGG-38 specific for G-CSF receptor.18 The following reverse
transcription and amplification conditions were used: 42°C for 30 minutes,
99°C for 2 minutes, at 95°C for 1 minute, 55°C for 1 minute, and 72°C for 2
minutes for 40 cycles. PCR products were electrophoresed in 1% agarose
gel, and the bands were visualized after staining with ethidium bromide and
ultraviolet light exposure.
Cocultivation of G-CSF with T lymphocytes
Results
Peripheral blood mononuclear cells (PBMCs) from normal unstimulated
donors were separated using density gradient centrifugation with lymphocyte separation media (Organon, Durham, NC). Cells were washed twice
with phosphate-buffered saline (PBS) and resuspended in Iscove’s modified
medium supplemented with fetal calf serum (FCS; both Life Technologies,
Gaithersburg, MD). Cultures of normal unstimulated donor cells were
performed at a cell density of 0.5 ⫻ 106 cells/mL. When appropriate,
natural lymphocyte-derived IL-2 (Boehringer Mannheim, Indianapolis, IN)
or phytohemagglutinin (PHA; Boehringer Mannheim) were used for
stimulation at concentrations of 20 U/mL or 8 µg/mL, respectively. G-CSF
was used at a concentration of 100 ng/mL (Amgen, Thousand Oaks, CA).
GCSF was added to cultures; either PHA or CD3 mAb were added to
cultures 24 hours later to stimulate cytokine expression. Cells were stained
and enzyme-linked immunosorbent assay (ELISA) was performed on
supernatants 2 days later.
Isolation of CD4⫹ cells, CD8⫹ cells, and monocytes. For lymphocyte
subset separation, after washing with PBS supplemented with 2% human
albumin, cells were applied to either a CD4⫹ or a CD8⫹ affinity column
(R&D Systems, Mineapolis, MN), and the cell fraction was eluted with PBS
according to manufacturer’s instructions. An aliquot of eluted cells was
stained with PE-conjugated anti-CD4 or CD8 HPCA-12 mAb (Becton
Dickinson, Mountain View, CA) to assess purity. Monocytes were separated
by adhesion to 250-mL polystyrene flasks in the presence of 20% FCS for 2
hours; this was followed by the removal of nonadherent cells and by
PBS/2% FCS washes. Adherent cells were detached and resuspended by
agitation at 4°C in the presence of 1X Versene (BRL; Life Technologies).
The usual purity of the adhesion-separated monocytes ranged between 75%
and 85% as determined by the expression of CD14⫹ antigens by flow
cytometry. Cell viability was measured using a standard trypan blue
exclusion assay (Life Technologies).
Intracellular staining. Intracellular staining was performed on density
gradient-separated PBMC. Intracellular staining for ICE expression was
performed using the Pharmingen Intracellular Staining Kit (Pharmingen).
PBMC were stained with FITC-conjugated CD4 mAb and fixed. After
membrane permeabilization, cells were stained with PE-conjugated antiIFN-␥, anti–IL-4 mAb, or appropriate isotypic control mAb (Pharmingen).
Permeabilization of cells was controlled using PermaSure reagents (Biosource). Samples were analyzed using an Epics ELITE flow cytometer
(Coulter).
Apoptosis assay. Cultured PBMC were prepared as described above,
washed with PBS, and stained with an annexin apoptosis kit (Pharmingen)
according to the manufacturer’s17 specifications and as previously described. Samples were analyzed using flow cytometry.
G-CSF stimulated PBSC donors demonstrate decreases
in IFN-␥ and increases IL-4 expression in CD4ⴙ cells
Cytokine ELISA
Concentrations of IL-4 and IFN-␥ in tissue culture supernatants were
measured using commercially available ELISA systems (R&D Systems).
All determinations were made in duplicate.
Reverse transcriptase–polymerase chain reaction for detection
of G-CSF receptor expression
Peripheral blood mononuclear cells were sorted for CD4⫹ and CD8⫹ by
flow cytometry. Total RNA was extracted using RNA STAT-60 (Tel-Test,
Friendswood, TX). Reverse transcription was performed using an oligo
To determine whether G-CSF modulated the cytokine expression
pattern of T lymphocytes, we used flow cytometry to examine
intracellular cytokine expression in mononuclear cells derived
from PBSC of donors receiving G-CSF. Normal donors receiving
10 µg/kg subcutaneously daily for 5 days were studied immediately
before and after G-CSF administration for PBSC donation by
apheresis. After blood sampling, PBMCs were stimulated for 48
hours with either PHA or CD3 mAb and were subsequently stained
for intracellular IL-4 and IFN-␥. Donor CD4⫹ cells stimulated with
G-CSF demonstrated significant changes in cytokine expression
(Figures 1 and 2). In comparison to pretreatment values, G-CSF
increased IL-4 and decreased IFN-␥ expression, resulting in a
decreased TH1/TH2 ratio (determined by dividing the percentage
of cells expressing IL-4 by the percentage of cells expressing
IFN-␥). In addition to a decreased proportion of cells expressing
IFN-␥ and an increased proportion of IL-4–expressing cells, there
were characteristic changes in the mean channel fluorescence
(MCF) indicating changes in the intracellular cytokine content.
IFN-␥ and IL-4 MCF was measured on a relative scale (PHAstimulated normal lymphocytes showed MCF of 5 to 6, whereas
isotype controls registered at less than 0.10). CD3 mAb-stimulated
CD4⫹ cells from G-CSF–treated patients showed a mean decrease
in MCF of ⫺4.2 for IFN-␥, with a decrease in the percent of cells
staining by 42% with a concomitant increase in the number of cells
staining for IL-4 by 58% with ⌬MCF ⫽ ⫹6.0 (n ⫽ 10; P ⬍ .01).
Determination of IL-4 and IFN-␥ concentrations in culture supernatants by ELISA resulted in similar changes (Table 1A).
G-CSF decreased the proportion of IL-4 and IFN-␥ producing
lymphocytes in vitro
In a subsequent set of experiments, we asked whether the effects of
G-CSF observed in PBSC donors could be replicated in vitro.
Normal PBMCs were incubated with varying concentrations of
G-CSF, then stimulated with IL-2 and CD3 mAb and stained 2 days
later with CD4-PE and (after permeabilization) with either IL-4 or
IFN-␥. Flow cytometric analysis showed that the number of CD4⫹
cells expressing IFN-␥ decreased, whereas the number of IL-4–
producing cells increased in cultures performed in the presence of
G-CSF (Figure 3). The effect was less pronounced in cells that were
not preincubated with G-CSF before stimulation with either PHA
or CD3 mAb (data not shown). The culture supernatants were also
tested for IFN-␥ and IL-4 by ELISA. G-CSF resulted in changes in
the cytokine production pattern that paralleled those detected by
flow cytometry (Table 1B). The effects of G-CSF were dose
dependent (data not shown).
From www.bloodjournal.org by guest on January 13, 2017. For personal use only.
BLOOD, 1 APRIL 2000 • VOLUME 95, NUMBER 7
Figure 1. Effects of G-CSF administration on IFN-␥ and IL-4 production by
peripheral blood lymphocytes. Eleven normal stem cell donors received G-CSF
(10 µg/kg per day) for 5 days. Blood was obtained before and after the administration
of G-CSF. PBMC cells were purified by density-gradient centrifugation and were
cultured with PHA for 48 hours. After permeabilization, cells were stained for IFN-␥
and IL-4 as described in ‘‘Materials and methods.’’ Scattergrams represent log PE
(CD4 or CD8) fluorescence activity versus log FITC (IFN-␥ and IL-4) fluorescence
activity for representative donors (A, B).
G-CSF has a direct effect on CD4ⴙ cells
To determine whether the effects of G-CSF on cytokine production
by T lymphocytes were directly mediated, we studied the growth
factor’s effects on purified cell populations contained in PBMC
preparations. We compared IFN-␥ expression in purified control
CD4⫹ and those treated by G-CSF after PHA stimulation. In the
absence of accessory cells, IFN-␥ production was not detected in
PHA-stimulated CD4⫹ cells (data not shown).
When monocyte preparations, purified CD4⫹ cells, or CD8⫹ cells
(98% pure by flow cytometry) were cultured in the presence of G-CSF,
washed, and then mixed—G-CSF–exposed CD4⫹ cells with untreated
monocytes; G-CSF–exposed monocytes with untreated CD4⫹ cells not
treated with G-CSF; G-CSF–exposed CD4⫹ cells with G-CSF–exposed
monocytes—and stimulated for 48 hours with either CD3 mAb or PHA,
only CD4⫹ cells directly exposed to G-CSF showed changes in IFN-␥
expression (Figure 4 and Table 2). Neither CD4⫹ cells cultured with
G-CSF–exposed monocytes nor CD8⫹ cells exposed directly or cultured with G-CSF–treated monocytes had altered IFN-␥ production.
G-CSF AND CYTOKINE PRODUCTION
2271
Figure 2. Effects of G-CSF on the percentages of CD4ⴙ cells secreting IFN-␥
and IL-4. Summary of the results obtained from 10 PBSC donors, including those
exemplified in Figure 1. Bars represent percentages (mean ⫾ SEM) in the IFN-␥ and
IL-4 expressing CD4⫹ cells before (black) and after (gray) treatment. Corresponding
shifts in the mean fluorescence intensity for IFN-␥ and IL-4 are described in the text.
Statistical analysis (nonparametric Kruskal–Wallis test) for PHA: IL-4, P ⬍ .05; IFN-␥,
P ⬍ .01. Statistical analysis for CD3 ⫹ IL-2: IL-4 P ⬍ .01; IFN-␥ P ⬍ .01.
Table 1. Production of IFN-␥ and IL-4 in supernatants from cultures of
peripheral blood mononuclear cells stimulated with G-CSF in vivo and in vitro
Before G-CSF
After G-CSF
A
IL-4
1.75 ⫾ 0.25
7.5 ⫾ 4.54
IFN-␥
400 ⫾ 100
175 ⫾ 75
B
IL-4
1.8 ⫾ 0.5
4 ⫾ 0.4
IFN-␥
350 ⫾ 87
110 ⫾ 35
In part A, samples of lymphocytes were obtained before and after the administration of G-CSF. IL-4 and IFN-␥ concentrations were measured after 24-hour PHA
stimulation. In part B, samples of lymphocytes obtained from normal donors were
cocultured with G-CSF for 24 hours and subsequently stimulated with anti-CD3 mAb
for 48 hours. Concentrations (mean ⫾ SEM of IL-4 and IFN-␥ concentration in
supernatants) were determined using ELISA and expressed as pg/mL. Results
represent the summary of 2 experiments for part A and 5 experiments for part B.
From www.bloodjournal.org by guest on January 13, 2017. For personal use only.
2272
BLOOD, 1 APRIL 2000 • VOLUME 95, NUMBER 7
SLOAND et al
Figure 4. Direct effects of G-CSF on IFN-␥ and IL-4 production by CD4ⴙ T
lymphocytes. Monocytes and CD4⫹ cells from normal donors were purified to obtain
preparations with more than 98% purity as determined by flow cytometry using CD4
and CD14 mAb. Monocytes and CD4⫹ cells were separately exposed to G-CSF for
24 hours, washed, and mixed together (ratio 1:1). The expression of IFN-␥ and IL-4
was measured using an intracellular staining technique and flow cytometry. Bars
represent the changes (mean ⫾ SEM) in the percentage of cells expressing IFN-␥
(gray) and IL-4 (black) over a control mixture of monocytes and monocytes not
exposed to G-CSF (summary of 2 independent experiments).
Figure 3. In vitro effects of G-CSF on IFN-␥ and IL-4 production. After exposure to
G-CSF and polyclonal stimulation with PHA, IL-4 and IFN-␥ expression were
determined using flow cytometry as previously described. The columns represent the
percentages (mean ⫾ SEM) of cells expressing IFN-␥ and IL-4. Summary of 13
experiments performed. Statistical analysis (nonparametric Kruskal-Wallis test) for
IL-4: P ⬍ .01. Statistical analysis for IFN-␥: P ⬍ .01.
G-CSF does not decrease TH1/TH2 ratio by increasing apoptosis
The G-CSF-induced modulation pattern of cytokine expression in
CD4⫹ cells could potentially be mediated by the deletion of TH1
cells secreting IFN-␥, as for example by selective apoptosis. Using
the annexin technique, we investigated the effects of G-CSF on
apoptosis of CD4⫹ cells. The presence of G-CSF in culture did not
affect the numbers of apoptotic cells, as determined by flow
cytometry. After 48 hours of culture, a mean of 48% ⫾ 10 TH1
cells and 54% ⫾ TH2 cells stained with annexin (n ⫽ 3).
RT-PCR detects GCSF-R m-RNA expression in CD4
and CD8 cells
Peripheral blood mononuclear cells were sorted for CD4⫹ and
CD8⫹ by FACS, resulting in a preparation that was 99.6% and 97%
pure, respectively. Total RNA was extracted using RNA STAT-60,
and RT-PCR was performed using an oligo d(T)16 primer. After
reverse transcription, the cDNA was amplified using primer pairs
58-AGTACAGTCCTCACCCTGATG-38 and 58-AAAGTATGCAGATCGCCTGGG-38 specific for G-CSF receptor. A band of 900 kbp
was present for CD4 and CD8 cells, indicating the molecular presence
of G-CSF-R (Figure 5).
Discussion
In this study, we demonstrated that the pretreatment of T cells with
G-CSF resulted in diminished IFN-␥ and increased IL-4 production
when these cells were subsequently subjected to polyclonal stimulation with IL-2, PHA, or CD3 mAb in vitro. In the presence of
G-CSF, resting CD4⫹ cells that expressed low levels of IL-4
produced significant amounts of this cytokine after polyclonal
activation. In agreement with in vitro results, we found that when
Table 2. Direct effects of G-CSF on IFN-␥ and IL-4 production by CD4ⴙ T
lymphocytes
Mixture
G-CSF
Exposure
IFN-␥
Concentration
IL-4
Concentration
Lymphocytes
No
300
2.4
Monocytes
No
Lymphocytes
Yes
100
4
Monocytes
No
320
2
Lymphocytes
No
Monocytes
Yes
Monocytes and CD4⫹ cells from normal donors were purified to obtain preparations with greater than 98% purity as determined by flow cytometry using CD4 and
CD14 mAb. Monocytes and CD4⫹ cells were separately exposed to G-CSF for 24
hours, washed, and mixed together (ratio 1:1). IFN-␥ and IL-4 were measured using
ELISA and were expressed as pg/mL.
From www.bloodjournal.org by guest on January 13, 2017. For personal use only.
BLOOD, 1 APRIL 2000 • VOLUME 95, NUMBER 7
G-CSF AND CYTOKINE PRODUCTION
Figure 5. Expression of G-CSF mRNA in PB CD4ⴙ and CD8ⴙ cells. Purified CD4⫹
and CD8⫹ cells were derived by sorting PBMCs obtained from a normal healthy
control. mRNA was extracted from the cells, and RT-PCR was performed using
G-CSFR–specific primers. RT-PCR products were electrophoresed in agarose gel
containing ethidium bromide and visualized under ultraviolet light. Unseparated
normal bone marrow (nbm) from a normal control was used as a positive control.
PBSC donors were stimulated with G-CSF for 5 days, their T cells
produced less IFN-␥ and more IL-4 in comparison to pretreatment
levels. This result implies that G-CSF decreased the TH1/TH2 ratio
within the CD4⫹ T-cell population. Because monocytes constitute a
major population in PBSC harvests, they could in principle mediate
the immunomodulatory effects of G-CSF on T lymphocytes. Using
cocultivation experiments, we demonstrated that the effects of
G-CSF on CD4⫹ T cells were direct and that monocytes were not
required for G-CF–mediated modulation. Although previous results have shown that monocytes undergo apoptosis as a result of
exposure to IFN-␥, survival of monocytes in our cultures was
improved by their coculture with G-CSF–treated CD4⫹ cells. This
effect may have been caused by the diminished quantities of IFN-␥
in G-CSF–supplemented cultures or the survival-promoting effects
of G-CSF. G-CF–mediated modulation of the cytokine pattern
produced by T cells could be related to apoptosis of a particular
T-cell subset (either TH1 or TH2) or their preferential survival.
From the lack of changes in the apoptosis rate of lymphocytes
stimulated with G-CSF, we concluded that this cytokine exerts its
effect on IFN-␥ and IL-4 production through modulation rather
than the preferential elimination of TH1 cells.
Other investigators7,8,19 have demonstrated that G-CSF–
mobilized monocytes exert inhibitory effects on T-cell proliferation
and responsiveness. G-CSF–mobilized CD14⫹ cells express significantly lower levels of the costimulatatory molecules B7-2, imply-
2273
ing that low levels of B7-2 expression may contribute to the
decreasing T-cell responsiveness by providing suboptimal amounts
of stimulatory signals.8 The observation that the blockade of B7
with soluble CD28 fusion protein leads to subresponsiveness of the
T cells also supports the theory that G-CSF down-modulates the
GVHD reactivity of PBSC grafts. Our results are not in contrast
with this thesis, and both mechanisms may simultaneously operate
in PBSC donors, resulting in an unexpected low rate and intensity of
GVHD in recipients of PBSC allografts. Another group20 demonstrated
the increased production of both IL-4 and IFN-␥ by PBMCs obtained
from patients with non-Hodgkin’s lymphoma who undergo autologous transplantation compared to normal controls. However, the
contributions of the underlying disease, previous chemotherapy,
and GCSF in changing cytokine responsiveness cannot be ascertained. In this study we showed that both CD4⫹ and CD8⫹ cells
contain G-CSF receptor mRNA, suggesting a mechanism by which
G-CSF directly interacts with T cells.
Clinically, the use of unmanipulated PBSC transplants derived
from donors treated with G-CSF is associated with an unexpected
low rate of GVHD2,3 given the relatively large number of T cells
infused. If G-CSF can mediate a shift in the balance between TH1
and TH2 cells, G-CSF administration to the PBSC donors might
influence the course of GVHD. Acute GVHD appears to be
mediated by TH1 cells and their proinflammatory cytokines IL-2,
TNF, and IFN-␥.21 Although IFN-␥ is a marker for TH1 cells, IFN-␥
secreted by CD4⫹ and CD8⫹ donor T cells is one of the earliest events
occurring in acute GVHD.22 IFN-␥ is thought responsible for the
delayed hematopoietic recovery and development of cytopenias.22,23
IFN-␥ is also present in the intestinal lesions of acute GVHD, and
anti–IFN-␥ antibodies prevented bowel damage in a murine GVHD
model. In contrast, TH2 cells appeared to favorably modify acute
GVHD. IL-4, a product of TH2 cells, decreases the expression of
other proinflammatory cytokines, which may lead to immunologic
tolerance.24 TH2 cells, however, may mediate chronic GVHD.
G-CSF also has been used to treat aplastic anemia. Especially in
combination with SCF25 or with antithymocyte globulin and
cyclosporine,26 G-CSF may have therapeutic activity apart from its
stimulatory effects on hematopoiesis. Both GVHD and aplastic
anemia have been reported to be related to the overexpression of
IFN-␥.27,28 For both, the beneficial effects of G-CSF may be
secondary to changes in IFN-␥ production and the balance in the
TH1 and TH2 lymphocyte subsets. The effects of G-CSF on the
cytokine secretion pattern of lymphocytes derived from G-CSF–
treated normal subjects were comparable to those observed in
aplastic anemia. In studies to be reported elsewhere, we found that
when patients with aplastic anemia and abnormally decreased
TH1/TH2 ratios were treated with G-CSF, significant changes in
the TH1/TH2 subsets were observed (unpublished data).
In summary, our results suggest that G-CSF, in addition to its
stimulatory effects on hematopoiesis, can exert immunomodulatory
effects on T lymphocytes, and these may be therapeutically
exploited in conditions associated with TH1/TH2 imbalance such
as immune-mediated bone marrow failure syndromes and GVHD.
References
1. Pan L, Bressler S, Cooke KR, Krenger W, Karandikar M, Ferrara JLM. Long-term engraftment,
graft-vs.-host disease, and immunologic reconstitution after experimental transplantation of alloge-
neic peripheral blood cells from G-CSF-treated
donors. Biol Blood Marrow Transplant. 1996;2:
126-133.
2. Mielcarek M, Torok-Storb B. Phenotype and en-
graftment potential of cytokine-mobilized peripheral blood mononuclear cells. Curr Opin Hematol.
1997;4:176-182.
3. Bensinger WI, Clift RA, Anasetti C, et al. Trans-
From www.bloodjournal.org by guest on January 13, 2017. For personal use only.
2274
BLOOD, 1 APRIL 2000 • VOLUME 95, NUMBER 7
SLOAND et al
plantation of allogeneic peripheral blood stem
cells mobilized by recombinant human granulocyte colony stimulating factor. Stem Cells. 1996;
14:90-105.
4. Schmidtz N, Bacigalupo A, Hasenclever D, et al.
Allogeneic bone marrow transplantation vs filgrastim-mobilised peripheral blood progenitor cell
transplantation in patients with early leukaemia:
first results of a randomised multicentre trial of
the European Group for Blood and Marrow Transplantation. Bone Marrow Transplant. 1998;21;
995-1003.
5. Kusnierz-Glaz CR, Still BJ, Amano M, et al.
Granulocyte colony-stimulating factor-induced
comobilization of CD4- CD8- T cells and hematopoietic progenitor cells (CD34⫹) in the blood of
normal donors. Blood. 1997;89;2586-2595.
6. Strober S, Cheng L, Zeng D, et al. Double negative (CD4-CD8-alpha beta⫹) T cells which promote tolerance induction and regulate autoimmunity. Immunol Rev. 1996;149;217-230.
7. Mielcarek M, Graf L, Johnson G, Torok-Storb B.
Production of interleukin-10 by granulocyte
colony-stimulating factor-mobilized blood products: a mechanism for monocyte-mediated suppression of T-cell proliferation. Blood. 1998;91:
215-222.
8. Mielcarek M, Martin PJ, Torok-Storb B. Suppression of alloantigen-induced T-cell proliferation by
CD14⫹ cells derived from granulocyte colonystimulating factor-mobilized peripheral blood
mononuclear cells. Blood. 1997;89:1629-1634.
9. Pan L, Delmonte J Jr, Jalonen CK, Ferrara JLM.
Pretreatment of donor mice with granulocyte
colony-stimulating factor polarizes donor T-lymphocytes toward type-2 cytokine production and
reduces severity of experimental graft-versushost disease. Blood. 1995;86:4422-4429.
10. Krenger W, Ferrara JLM. Graft-versus-host disease and the Th1/Th2 paradigm. Immunol Res.
1996;15:50-73.
11. Lafaille JJ. The role of helper T cell subsets in
autoimmune diseases. Cytokine Growth Factor
Rev. 1998;9:139-151.
12. Rocken M, Biedermann T, Ogilvie A. The role of
TH1 and Th2 dichotomy: implications for autoimmunity. Rev Rhum Engl Ed. 1997;64:131S-137S.
13. Gately MK, Renzetti LM, Magram J, et al. The
interleukin-12-receptor system: role in normal and
pathologic immune responses. Annu Rev Immunol. 1998;16:495-521.
14. Krenger W, Snyder KM, Byon JCH, Falzarano G,
Ferrara JLM. Polarized type 2 alloreactive CD4⫹
and CD8⫹ donor T cells fail to induce experimental acute graft-versus-host disease. J Immunol.
1995;155:585-593.
15. Nikolic B, Grusby MJ, Sykes M. Th1 and Th2 responses causes distinct clinical GHVD syndromes after fully MHC-mismatched STAT4 and
STAT6 KO bone marrow transplantation. Blood.
1997;90(suppl):395a. Abstract 1756.
16. Ellison CA, Fischer JM, HayGlass KT, Gartner
JG. Murine graft-versus-host disease in an F1hybrid model using IFN-gamma gene knockout
donors. J Immunol. 1998;161:631-640.
17. Verhoven B, Schlegel RA, Williamson P. Mechanisms of phosphatidylserine exposure, a phagocyte recognition signal, on apoptotic T lymphocytes. J Exp Med. 1995;182:1597-1601.
18. White SM, Ball ED, Ehmann WC, Rao AS,
Tweardy DJ. Increased expression of the differentiation-defective granulocyte colony-stimulating
factor receptor mRNA isoform in acute myelogenous leukemia. Leukemia. 1998;12:899-906.
19. Ageitos AG, Varney ML, Bierman PJ, Vose JM,
Warkentin PI, Talmadge JE. Comparison of
monocyte-dependent T cell inhibitory activity in
GM-CSF vs G-CSF mobilized PSC products.
Bone Marrow Transplant. 1999;23:63-69.
20. Singh RK, Ino K, Varney ML, Heimann DG, Tal-
madge JE. Immunoregulatory cytokines in bone
marrow and peripheral blood stem cell products.
Bone Marrow Transplant. 1999;23:53-62.
21. Rus V, Svetic A, Nguyen P, Gause WC, Via CS.
Kinetics of TH1 and TH2 cytokine production during the early course of acute and chronic murine
graft-versus-host disease: regulatory role of donor CD8⫹ T cells. J Immunol. 1995;155:23962406.
22. Ferrara JL. Cytokine inhibitors and graft-versushost disease. Ann N Y Acad Sci. 1995;770:227236.
23. Selleri C, Sato T, Anderson S, Young NS, Maciejewski JP. Interferon-gamma and tumor necrosis
factor-alpha suppress both early and late stages
of hematopoiesis and induce programmed cell
death. J Cell Physiol. 1995;165:538-546.
24. Lee JD, Rhoades K, Economou JS. Interleukin-4
inhibits the expression of tumor necrosis factors
alpha and beta, interleukins-1 beta and -6 and
interferon-gamma. Immunol Cell Biol. 1995;73:
57-61.
25. Kumar M, Alter BP. Hematopoietic growth factors
for the treatment of aplastic anemia. Curr Opin
Hematol. 1998;5:226-234.
26. Bacigalupo A, Piaggio G, Podesta M, et al. Early
hemopoietic progenitors in the peripheral blood of
patients with severe aplastic anemia (SAA) after
treatment with antilymphocyte globulin (ALG),
cyclosporin-A and G-CSF. Haematologica. 1997;
82:133-137.
27. Nistico A, Young N. Interferon-␥ gene expression
in the bone marrow of patients with aplastic anemia. Ann Intern Med. 1994;120:463-469.
28. Dickinson AM, Sviland L, Dunn J, Carey P, Proctor JS. Demonstration of direct involvement of
cytokines in graft-versus-host reactions using an
in vitro human skin explant model. Bone Marrow
Transplant. 1991;7:209-216.
From www.bloodjournal.org by guest on January 13, 2017. For personal use only.
2000 95: 2269-2274
Pharmacologic doses of granulocyte colony-stimulating factor affect
cytokine production by lymphocytes in vitro and in vivo
Elaine M. Sloand, Sonnie Kim, Jaroslaw P. Maciejewski, Fritz Van Rhee, Aniruddho Chaudhuri, John
Barrett and Neal S. Young
Updated information and services can be found at:
http://www.bloodjournal.org/content/95/7/2269.full.html
Articles on similar topics can be found in the following Blood collections
Hematopoiesis and Stem Cells (3386 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.