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 Previous Article | Table of Contents
Blood, 1 January 2001, Vol. 97, No. 1, pp. 333-335
CORRESPONDENCE
To the editor:
Modulation of Th1/Th2 subsets by granulocyte-colony stimulating
factor
The recent article published in Blood by Sloand
et al1 suggests that pharmacologic doses of
granulocyte-colony stimulating factor (G-CSF) affect cytokine
production by lymphocytes in vitro and in vivo. Sloand et al
demonstrated that G-CSF decreases interferon (IFN)- and increases
interleukin-4 (IL-4) production and modulates a balance between T
helper 1 (Th1) and T helper 2 (Th2) cells in favor of Th2
cells. Interestingly, a subsequent article by Arpinati et
al2 published in Blood reports that G-CSF
mobilizes Th2-inducing dendritic cells (the so-called DC2 cells). Both
reports propose that this immunomodulatory effect of G-CSF may be
exploited in clinical situations. Sloand et al suggest the use of this
growth factor in conditions associated with Th1/Th2 imbalance such as immune-mediated bone marrow failure syndromes and graft-versus-host disease (GVHD), whereas Arpinati et al advocate the use of
G-CSF-mobilized DC2 cells to induce immune deviation after
transplantation of hematopoietic stem cells or organ transplants. As
indicated by Liu and Blom3 in their commentary, the finding
of G-CSF-induced mobilization of DC2 cells (and Th2 cells) is a major
advancement with several potential clinical applications. However, I
would like to point out that none of these reports addresses the
relevance and the implications of the above immunomodulatory effect of
G-CSF in other clinical situations where this growth factor is
widely used. G-CSF is commonly used to accelerate neutrophil recovery
following high dose chemotherapy for various hematogenous and
nonhematogenous malignant diseases. The available scientific
evidence indicates that the cytokine profile of the T helper cell plays
an important role in "cancer immunity."4 The Th1 cells
that produce IFN- and IL-2 have been shown to exert a powerful
antitumor effect, whereas a Th2 profile (IL-4 and IL-10) may have an
opposite effect, that is, down-regulation of innate and acquired
antitumor immunity. The corollary of these observations is that a Th1
profile is "protective" and therefore beneficial, but a Th2 profile
may be deleterious because it may promote tumor growth and
dissemination. For this reason, if the findings of Sloand et al and
Arpinati et al are confirmed, we may have to ask (1) whether the use of
G-CSF in malignant conditions is indeed harmful because of the
potential risk of tumor growth and dissemination mediated by the Th2
cytokines and, therefore, should be avoided in these situations, and
(2) whether an alternative growth factor that predominantly induces a
Th1 profile (in addition to the effect on myelopoiesis) such as
granulocyte-macrophage colony-stimulating factor (GM-CSF) should be
preferred for this purpose. I suppose it may be scientifically logical
to suggest that the use of G-CSF should be limited to stem cell
mobilization of allogeneic donors (to reduce the risk of graft
rejection and acute GVHD), but GM-CSF should be preferred in the
treatment of chemotherapy-induced neutropenia in malignant and
premalignant diseases (to avoid depression of antitumor activity). Induction of a Th2 cytokine profile may also be detrimental in certain
infections. For example, neutropenia is a relatively common
manifestation of acquired immunodeficiency syndrome (AIDS) either as a
result of bone marrow damage or due to drug therapy, and G-CSF is used
in the management of neutropenic sepsis in these patients. It has been
reported that a Th1 to Th2 switch is associated with progression of HIV
disease.5 Hence, this growth factor may adversely
influence the outcome of this condition by inducing a Th2 cytokine profile. Should, therefore, the recent findings that G-CSF induces or enhances a
Th2 cytokine profile prompt a discussion as to the rational use of
colony stimulating growth factors in various clinical situations?
Muttuswamy Sivakumaran
Department of Haematology, Peterborough District Hospital
Peterborough, United Kingdom
References
1.
Sloand EM, Kim S, Maciejewski P, et al.
Pharmacologic doses of granulocyte colony-stimulating factor affect cytokine production by lymphocytes in vitro and in vivo.
Blood.
2000;95:2269-2274[Abstract/Free Full Text].
2.
Arpinati M, Green CL, Heimfeld S, Heuser JE, Anasetti C.
Granulocyte-colony stimulating factor mobilizes T helper 2-inducing dendritic cells.
Blood.
2000;95:2484-2490[Abstract/Free Full Text].
3.
Liu Y-J, Blom B.
TH2-inducing DC2 for immunotherapy.
Blood.
2000;95:2482-2483[Free Full Text].
4.
Shurin MR, Lu L, Kalinski P, Stewart-Akers AM, Lotze MT.
Th1/Th2 balance in cancer, transplantation and pregnancy.
Springer Seminars in Immunopathology.
1999;21:339-359[Medline]
[Order article via Infotrieve].
5.
Clerici M, Shearer M.
A Th1 Th2 switch is a critical step in the etiology of HIV infection.
Immunology Today.
1993;14:107-111[CrossRef][Medline]
[Order article via Infotrieve].
To the editor:
Th1/Th2 lymphokine profile of T cells present in the blood of
granulocyte-colony stimulating factor-treated stem-cell donors: up or
down modulation?
The article by Sloand et al1 that was published in
the April 1 issue of Blood suggests a direct regulatory role
of granulocyte-colony stimulating factor (G-CSF) over T cells.
The authors show a shift towards Th2, or interleukin 4 (IL-4)
production, of CD4+ T lymphocytes from peripheral blood
mononuclear cells (PBMCs) obtained from stem-cell donors
treated with G-CSF (G-PBMC) on the basis of intracellular detection of
IL-4 and interferon (IFN)- by flow cytometry. Detection of
intracellular lymphokines by flow cytometry requires controls usually
not required for detection of surface molecules2 such as
competition of the specific staining with the unlabeled antibody, since
isotype control monoclonal antibody (mAb) does not perform well
as a negative control for intracellular staining as it does for surface
staining.2 To address the same question raised by our colleagues, we recently
performed studies on G-PBMC T cells. Using the same approach as the one
used by Sloand et al,1 our results were initially very
similar to those published by Sloand's group (Figure 1A, left
panels). However, we were surprised by
the fact that the frequency of IL-4+ cells plus
IFN-+ cells added up to almost 100%, a result very
different from that obtained using healthy volunteers.2-5
In fact, data in the literature estimate the mean relative number of
IL-4-producing cells to be about 4% to 5% and IFN-- producing
cells to be about 25% to 30% of T cells. In addition, the
staining pattern we obtained was not repetitive and did not allow us to
set, with confidence, quadrants for statistical analysis.
View larger version (24K):
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| Figure 1.
Cytokine profile of T cells obtained with the same
reagents but different fixation time and temperature.
(A) PBMCs were fixed with PP for 30 minutes at 4°C (left panels) or 5 minutes at 37°C (right panels) and then intracellular stained.
Quadrant settings were based on unstained negative controls. From top
to bottom: unstained cells; cells stained with anti-IL-4 FITC,
anti-IFN- PE (Pharmingen, Palo Alto, CA) and anti-CD3 PercP (B&D,
San Jose, CA); cells incubated with 25-fold excess of unlabeled
anti-IFN- and stained as above; cells incubated with 25-fold excess
of unlabeled anti-IL-4 and stained as above. The data was collected in
a FACScan and analyzed using CellQuest software (B&D, San Jose, CA).
Data gated on the CD3+ population are shown. (B)
Comparative analysis of the 2 techniques used on PBMCs obtained before
(PBMC) and after (G-PBMC) G-CSF treatment. The data represent mean ± SD obtained from 7 (30 minutes at 4°C ) or 5 (5 minutes at 37°C)
different stem cell donors for each technique shown.
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In order to understand the reasons for these discrepancies, we
used 2 different techniques to stain PBMCs. When the cells are fixed
with paraformaldehyde (PF) for 30 minutes at
4°C,6 the whole cell population can be
considered positive for either lymphokine, and the staining is only
marginally inhibited by a 25-fold excess of unlabeled antibody (Figure
1A, left panels). On the other hand, if the cells are fixed with PF for
5 minutes at 37°C, the percentage of positive cells agrees with the
literature (Figure 1A, right panels), and the staining for both IL-4
and IFN- can be specifically inhibited by the matched unlabeled
antibodies (Figure 1A, right panels). This apparently minor technical
difference generates data that can mislead the interpretation of the
regulatory effects of G-CSF over the Th1/Th2 phenotype. As shown in Figure 1B, a long fixation time (30 minutes) at 4°C
suggests an augmentation of both IL-4- and IFN--producing cells
after in vivo G-CSF treatment, whereas a short fixation time (5 minutes) at 37°C suggests exactly the opposite; that is, any
inhibition on both cell types. Since the former technique cannot be
confirmed with the specific controls (Figure 1A), we conclude that only
the latter should be used. In the present situation, if an
interpretation can be drawn from our preliminary data, it would be that
G-CSF plays an inhibitory role over the frequency of IL-4- and
IFN--producing cells. More importantly, these results alert
us to the significance of showing the specificity controls when
performing intracellular staining for cytokines, in order to avoid
misinterpretations based on technical artifacts.
Zilton F. M. Vasconcelos
Centro Nacional de Transplante de Medula
Óssea, Instituto Nacional do Câncer Divisão de
Medicina Experimental, Coordenação de
Pesquisa, Instituto Nacional do Câncer
Hilda R. Diamond, and Daniel G. Tabak
Centro Nacional de Transplante de Medula
Óssea, Instituto Nacional do Câncer
Marcello A. Barcinski
Divisão de Medicina Experimental, Coordenação
de Pesquisa, Instituto Nacional do Câncer Departamento de
Parasitologia, Universidade de São Paulo
Adriana Bonomo
Divisão de Medicina Experimental, Coordenação
de Pesquisa, Instituto Nacional do Câncer Departamento de
Imunologia, Centro de Ciências da Saúde, Universidade
Federal do Rio de Janeiro Supported by the Brazilian National
Cancer Institute (INCA) and CNPq.
References
1.
Sloand EM, Kim S, Maciejewski JP, et al.
Pharmacologic doses of granulocyte colony-stimulating factor affect cytokine production by lymphocytes in vitro and in vivo.
Blood.
2000;95:2269-2274.
2.
Prussin C.
Cytokine flow cytometry: understanding cytokine biology at the single-cell level.
J Clin Immunol.
1997;17:195-204.
3.
Carrock Sewell WA, North M, Webster D, Farrant J.
Determination of intracellular cytokines by flow cytometry following whole blood culture.
J Immunol Methods.
1997;209:67-74[CrossRef][Medline]
[Order article via Infotrieve].
4.
Jung T, Schauer U, Heusser C, Neumann C, Rieger C.
Detection of intracellular cytokines by flow cytometry.
J Immunol Methods.
1993;159:197-207[CrossRef][Medline]
[Order article via Infotrieve].
5.
Prussin C, Metcalf DD.
Detection of intracytoplasmic cytokines using flow cytometry and directly conjugated anti-cytokine antibodies.
J Immunol Methods.
1995;188:117-128[CrossRef][Medline]
[Order article via Infotrieve].
6. Schimid I, Giorgi JV, Holmes K, Fowlkes BJ. Preparation of cells and
reagents for flowcytometry. In: Coligan JE, Kruisbeek AM, Margulies DH,
Shevach EM, Strober W, eds. Current Protocols in Immunology. New York:
John Wiley & Sons.
1994;5.3.1-5.3.23.
Response:
Pharmacologic concentrations of granulocyte-colony stimulating
factor affect cytokine expression by lymphocytes
In a manuscript recently published in
Blood,1 we demonstrated that lymphocytes
stimulated with either CD3 monoclonal antibody (mAb) or
phytohemagglutinin (PHA) in the presence of granulocyte-colony stimulating factor (G-CSF) altered the cytokine expression, resulting in a shift in cytokine response from a Th1 to a Th2 pattern.
Lymphocytes obtained from healthy individuals following G-CSF
mobilization for the purpose of stem cell donation also showed shifted
patterns of cytokine expression following coculture with either CD3 mAb or PHA. We certainly agree with Vasconelos et al that appropriate controls are
of paramount importance when performing intracellular staining. In
order to ensure that our staining methods were sensitive and specific,
we (1) optimized the amount of antibody that was added so as to
decrease nonspecific staining, (2) performed neutralizing studies with
unlabeled interferon- (IFN-) and IFN- mAb, (3) checked
concordance between intracellular staining using antibodies from
different manufacturers and by enzyme-linked immunosorbent assay
(ELISA) measurements on duplicate samples, (4) measured IFN- and
interleukin 4 (IL-4) using 2-color staining to ensure that the cell
populations did not overlap extensively, and (5) used appropriate
isotypic controls. We found that the addition of excessive anticytokine antibody to the
lymphocytes resulted in significant nonspecific staining, particularly
when using IL-4 mAb. Results from titration experiments were used to
optimize antibody concentrations. In addition, we performed multiple
experiments using stimulated and unstimulated lymphocytes from healthy
donors: PHA stimulation of cultured cells resulted in a mean of 35% of
cells staining with IFN- with IFN--fluorescein isothiocyanate
(FITC) with a mean channel fluorescence (MCF) of 5.6, whereas
isotype controls resulted in an MCF of less than 0.10. Lymphocytes
cultured in the presence of IFN- or IL-10 stimuli expected to favor
development of the Th1 phenotypestained primarily with IFN-, and
lymphocytes cocultivated with IL-4 or IL-12stimuli that favor
development of Th2 cellsstained primarily with IL-4. Preincubation of
anti-IFN- mAb with unlabeled IFN- competitively eliminated the
staining, as did preincubation of cells with unlabeled IFN- mAb;
unlabeled irrelevant mAb had no effect. The failure of Vasconelos et al
to observe neutralization with the permeabilization protocol
recommended by the manufacturer of the IFN- mAb may be related to
use of a cold IFN- mAb directed at a different epitope than the
labeled IFN- mAb, or to excess labeled IFN- mAb in the sample. To
assess the reproducibility of intracellular staining, we also tested
another antibody from a different manufacturer (BioSource, Camarillo,
CA; Pharmingen, San Diego, CA). Results obtained from comparison of 26 sets of samples showed a highly significant correlation (Pearson
correlation coefficient = 0.96). In addition, IL-4 and IFN-
ELISA measurements of lysed cell samples correlated with the results of
intracellular cytokine staining. Measurement of IL-4 and IFN- on CD4
cells using 3-color staining showed no overlap between CD4 cells
staining with each antibody. We performed appropriate isotypic controls using labeled
antibodies provided by the manufacturer of the cytokine mAbs and as
described in Current Protocols in Immunology.2
Although there are inherent problems in using isotypic controls, they
still provide great advantages over unstained cells for negative gating cells. In none of the papers referenced by Vasconelos et al were unstained cells used as controls, and more recent articles by Gurunathan et al3 utilize the same isotypic controls as we report in our study. When we applied the staining method suggested by Vasconcelos et al (a
short fixation period at 37°C), we found significant nonspecific
uptake of stain in the isotypic control as well as in unstimulated
normal cells. When appropriately gated using an isotypic control also
prepared in this fashion, there appeared to be no difference in the
amount of uptake of stain by the sample when compared to the other
fixation method. We believe that the comparison of stained to
unstained cells (Figure 1 of the letter by Vasconcelos et al) may
therefore not be valid. With respect to the comments made by Sivakumaran et al, extrapolation
of in vitro data for clinical purposes must always be made cautiously.
There is no evidence that administration of G-CSF is detrimental in
immunosuppressed patientsin fact, quite to the contrary, as multiple
trials involving neutropenic HIV-infected patients show that G-CSF
consistently decreased the frequency of serious
infection.4,5 Short courses of G-CSF are especially unlikely to influence tumor progression or chronic infections. Nevertheless, as we describe in the "Discussion" segment of our paper, long-term G-CSF therapy may affect immune processes like graft-versus-host disease or autoimmune marrow destruction.
Elaine Sloand, Sonnie Kim, Jaroslaw Maciejewski, and Neal Young
National Heart, Lung, and Blood Institute National
Institutes of Health, Bethesda, MD
References
[CrossRef][Medline]
[Order article via Infotrieve]
1.
Sloand EM, Kim S, Maciejewski J, et al.
Pharmacologic doses of granulocyte colony-stimulating factor affect cytokine production by lymphocytes in vitro and in vivo.
Blood.
2000;95:2269-2274.
2. Holmes K, Fowlkes B. Immunofluorescence and cell sorting. In:
Coligan J, Kruisbeek A, Margulies D, Shevach
E, Strober W, eds. Current Protocols In Immunology. New York: John Wiley & Sons. 2000;5.3.9.
3.
Gurunathan S, Stobie L, Prussin C, et al.
Requirements for the maintenance of Th1 immunity in vivo following DNA vaccination: a potential immunoregulatory role for CD8+ T cells.
J Immunol.
2000;165:915-924.
4. Ambati BK, Peerlman DC, Salomon N. Outcomes of granulocyte
colony-stimulating factor or granulocyte-macrophage
colony-stimulating factor use in neutropenic patients infected with
human immunodeficiency virus. Int J Infect Dis. 1998-99 Winter;3(2):70-75.[Abstract/Free Full Text]
5.
Kuritzkes DR, Parenti D, Ward DJ, et al.
Filgrastim prevents severe neutropenia and reduces infective morbidity in patients with advanced HIV infection: results of a randomized, multicenter, controlled trial. G-CSF 930101 Study Group.
AIDS.
1998;12:65-74.
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