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IMMUNOBIOLOGY
From the Division of Pulmonary and Critical Care,
Department of Medicine, the Department of Microbiology, Immunology and
Molecular Genetics, and the Jonsson Comprehensive Cancer Center, UCLA
School of Medicine, Los Angeles; and the West Los Angeles Veterans
Affairs Medical Center, CA.
Dendritic cells (DCs) are rare antigen-presenting cells that
play a central role in stimulating immune responses. The
combination of recombinant granulocyte
macrophage-colony-stimulating factor (rGM-CSF) and
recombinant interleukin-4 (rIL-4) provides an important stimulus for
generating DCs from murine bone marrow precursors in vitro. Using
miniature osmotic pumps, we now demonstrate that continuous infusion of
these cytokines for 7 days had a similar effect in vivo, increasing the
number and function of splenic DCs. Administration of rGM-CSF/rIL-4 (10 µg/d each) increased the concentration of CD11+ DCs by
2.7-fold and the absolute number of splenic DCs by an average of
5.7-fold. DC number also increased in peripheral blood and lymph
nodes. The resultant DCs exhibited a different phenotype and function
than those in control mice or mice treated with rGM-CSF alone.
rGM-CSF/IL-4 increased both the myeloid
(CD11c+/CD11b+) and the lymphoid
(CD11c+/CD8 Dendritic cells (DCs) develop from bone marrow
precursors and are distributed in limited numbers throughout peripheral
tissues and lymphoid organs. Although they represent only a small
percentage of mononuclear leukocytes, they play a sentinel role in
initiating and regulating immune responses.1,2 The use of
cytokines (granulocyte macrophage-colony-stimulating factor
[GM-CSF], interleukin-3 [IL-3], IL-4, tumor necrosis factor- In contrast to results obtained with human cells, murine studies
suggest a more independent role for GM-CSF as a DC growth and
differentiation factor.16-18 Inaba et al16
cultured bone marrow progenitors in GM-CSF and identified budding
clusters of cells expressing dendritic morphology, MHC class I, MHC
class II, CD11c, and DEC-205. These cells exhibited potent
allostimulatory activity consistent with their identification as DCs.
Similarly, the systemic administration of GM-CSF to mice increases DC
number in vivo. Hanada et al19 implanted GM-CSF-secreting
tumors into mice and observed increased numbers of splenic DCs when
compared to controls. The direct administration of GM-CSF alone, in the form of a polyethylene glycol-modified molecule, was also recently shown to increase the number of splenic DCs, but only the myeloid subpopulation expressing CD11c and CD11b.20
Despite these independent effects of GM-CSF, there is evidence that
IL-4 still plays an important synergistic role in generating mouse DCs.
Labeur et al8 compared bone marrow progenitors cultured in
GM-CSF alone to those cultured with the combination of GM-CSF/IL-4. The
presence of IL-4 increased the expression of MHC, CD40, CD80, CD86, and
DEC-205. Cells cultured with GM-CSF/IL-4 produced 3-fold more IL-12
than those cultured in GM-CSF, and they stimulated greater T-cell
proliferation in response to alloantigens or OVA peptide. Others have
reported that DCs grown in low concentrations of GM-CSF alone are
tolerogenic, whereas those generated in response to GM-CSF/IL-4 are
not.20 Gunji et al22,23 failed to observe tumor rejection in mice inoculated with tumor cells producing GM-CSF or
IL-4 alone, but they noted tumor rejection and the development of
tumor-specific immunity in mice injected with a combination of GM-CSF-
and IL-4-producing tumors.
To clarify the role of IL-4 on the differentiation and expansion of
mouse DCs in vivo, we used miniature osmotic pumps to deliver
continuous infusions of GM-CSF, either alone or in combination with
IL-4. Spleens and lymph nodes from cytokine-treated mice were evaluated
for evidence of DC differentiation and function. The combination of
GM-CSF/IL-4 increased the number of myeloid (CD11c+/CD11b+) and lymphoid
(CD11c+/CD8 Mice
Cytokines
In vivo generation of DCs C57BL/6 mice were treated with a 7-day continuous infusion of rGM-CSF, alone or in combination with rIL-4, administered by mini-osmotic pump (model 1007D; Alza, Palo Alto, CA). In brief, osmotic pumps were loaded under sterile conditions with rGM-CSF (2-20 µg/mL), rIL-4 (2-20 µg/mL), or both and were implanted in subcutaneous tissue over the mid-back. Control animals received pumps loaded with diluent alone (0.9% saline). The presence of circulating rGM-CSF and rIL-4 was determined on day 7 serum samples by specific enzyme-linked immunosorbent assay, as described by the manufacturer (BioSource International, Camarillo, CA).Spleen cell preparation and phenotyping Single-cell suspensions from control and experimental spleens and lymph nodes were prepared by cutting the organs into small pieces and then digesting them with 1 mg/mL type II collagenase (Worthington Biochemical, Freehold, NJ) and 0.02 mg/mL bovine pancreatic DNAse (Boerhinger Mannheim, Mannheim, Germany) for 30 minutes at room temperature. Digestion mixtures were then treated with 0.1 M EDTA (Sigma, St Louis, MO) for 5 minutes and were centrifuged to remove tissue debris, and cells were washed in RPMI 1640 (Irvine Scientific, Santa Ana, CA) containing 2% fetal calf serum (FCS; Omega Scientific, Tarazana, CA). Red blood cells were depleted by hypotonic shock. For fluorescence-activated cell sorter (FACS) analysis, cell surface Fc receptors (FcR) were first blocked by incubation with anti-FcRII
monoclonal antibody (mAb) (clone 2.4G2; ATCC, Rockville, MD) for 30 minutes at 4°C. T cells, B cells, and NK cells were identified by
incubation with biotinylated Thy1.2, B220, or NK1.1 mAb (Caltag
Laboratories, Burlingame, CA) for 30 minutes at 4°C and labeling with
fluorescein isothiocyanate (FITC)-conjugated streptavidin (Caltag
Laboratories). DC subsets were identified by incubation with FITC,
allophycocyanin (APC)-, or phycoerythrin (PE)-conjugated mAb directed
against CD11c, CD11b, CD8 , MHC class I, or MHC class II (PharMingen,
San Diego, CA). After washing in phosphate-buffered saline (PBS) with
2% FCS, cells were fixed in 1% paraformaldehyde. The
acquisition of 103 to 105 events was
performed on a FACStar cytometer (Becton Dickinson, San Jose, CA), and
results were analyzed using Cellquest software (Becton Dickinson).
In some experiments, DCs were further enriched by the depletion of T and B cells. Single-cell suspensions of spleen cells were depleted of RBCs and incubated with purified mAbs against Thy-1.2 and B220 for 30 minutes at 4°C (PharMingen). Cells were then incubated for 30 minutes at 37°C in serum-free RPMI 1640 medium containing 10% rabbit complement (Sigma, St Louis, MO). After washing, the remaining cells were used as an enriched population of DCs. Histology and immunohistology Spleens from control, rGM-CSF, and rGM-CSF/rIL-4-treated mice were embedded in Tissue-Tek OCT compound (Miles, Elkhart, IN) by freezing in liquid N2 and stored at 80°C.
Six-micrometer sections were cut on a cryostat (Reichert Jung,
Cambridge Instruments GmbH, Germany) and were mounted on
poly-L-lysine-coated slides. Sections were air dried overnight, fixed
in 10% formalin (Sigma) for 10 minutes, and stained with hematoxylin
(Fisher Scientific) and eosin (Sigma) or for immunohistology as
described for each antibody combination.
Dual detection of CD11c and CD11b was performed by washing slides with PBS and blocking endogenous peroxidase activity with 0.3% H2O2 (10 minutes). Sections were blocked for 20 minutes with PBS containing 5% bovine serum albumin (Fisher Scientific, Springfield, NJ) and 1% goat serum (Jackson Immuno Research, West Grove, PA), rinsed, and stained with anti-CD11c (PharMingen) for 1 hour. After washing, biotinylated goat antihamster antibody (PharMingen) was added for 30 minutes. Specific antibody binding was visualized by treating with a peroxidase substrate for 30 minutes using the Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA). Sections were again treated with H2O2 and were blocked with 1% goat serum before incubation with rat anti-mouse CD11b (PharMingen) for 1 hour. Sections were stained with biotinylated donkey anti-rat antibody (Jackson Immuno Research) and were visualized with an alkaline-phosphatase, fast-blue substrate using the Vectastain ABC-AP kit (Vector Laboratories). Dual detection of DEC-205 (rat antimouse DEC-205; Serotech, Raleigh,
NC) and immunoglobulin Ig Measurement of endocytosis and pinocytosis Enriched DC populations (1 × 106) prepared from spleens of control and cytokine-treated mice were incubated with 1 mg/mL FITC-dextran (70 kd) or Lucifer yellow (both from Molecular Probes, Eugene, OR) for 1 hour at 37°C. Control cells were treated in the same manner but were maintained on ice to block energy-dependent uptake. Antigen uptake was terminated by the addition of ice-cold PBS containing 0.1% azide. Cells were washed 3 times in PBS/1% FCS/0.1% azide, and extracellular antigen was removed by incubation with a 1% trypsin-PBS solution (Sigma) for 3 minutes at 37°C. Cells were counterstained with PE-anti-CD11c and APC-anti-CD11b, and intracellular uptake of FITC-dextran and Lucifer yellow was determined by flow cytometry.Mixed-lymphocyte reaction Splenocytes from control and cytokine-treated C57BL/6 mice were depleted of B and T cells, as described above, to prepare DC-enriched populations. Either they were directly tested as stimulators in mixed-lymphocyte reaction (MLR) assays (fresh) or they were cultured for 36 hours in the presence of 20 ng/mL of rGM-CSF and rIL-4 before testing (cultured). Allogenic T cells were prepared from the lymph nodes of 8- to 12-week-old BALB/c mice. In brief, single-cell suspensions from inguinal and axillary lymph nodes were incubated with mAbs to B220, NK1.1, and Gr-1 for 45 minutes at 4°C. Cells were then incubated at 37°C for 30 minutes in serum-free RPMI 1640 medium containing 10% rabbit complement (Sigma). T cells prepared by this method were at least 90% pure, as determined by FACS analysis. MLR assays were performed in 96-well, round-bottomed culture plates. Allogenic T cells (1 × 105 BALB/c) were incubated with varying numbers of irradiated DC-enriched spleen cells (20 Gy) from control or cytokine-treated mice. Cells were cultured in 0.2 mL RPMI 1640 containing 10% FCS and 10 4 M 2-ME (Sigma) in a
humidified CO2 incubator for 3 days. Culture wells were
pulsed with 1.25 µCi (46.2 kBq) [3H] thymidine
for 12 hours, and the cells were harvested onto glass fiber sheets
using an automated harvester. Proliferation was determined by counting
each sample in a liquid scintillation  -counter. Background counts
for T cells or DCs alone were always less than 200 cpm.
Tumor cell line The murine cell line E-22 (kindly provided by Dr S. Restifo, National Cancer Institute, Bethesda, MD), a clone of the EL4 mouse thymoma line stably expressing the LacZ gene, was used for in vivo tumor studies. Cells were maintained in RPMI 1640, 10% heat-inactivated FCS, 0.03% L-glutamine, 100 µg/mL streptomycin, 100 µg/mL penicillin, and 50 µg/mL gentamicin sulfate (Life Technologies, Rockville, MD) in the presence of 400 µg/mL G418 (Life Technologies).Recombinant adenoviral vectors Recombinant adenovirus Ad5.CMV-LacZ (AdV/-gal) was obtained
from Quantum Biotechnologies (Montreal, Quebec, Canada). Ad5.CMV-LacZ is a first-generation, E-1-deleted adenovirus serotype 5 expressing the bacterial LacZ gene under the control of the CMV-IE
promoter/enhancer. The control adenovirus, AdV/RR5 (kindly provided by
Dr L. Butterfield, University of California Los Angeles) is an
E-1-deleted type 5 vector that carries no reporter gene
construct.24 Viral stocks were amplified on 293 cells.
This was followed by CsCl purification, dialysis, and storage at
80°C. The titer of viral stock was between 109 and
1013 plaque-forming units (PFU)/mL by plaque assay on
293 cells.
In vivo adenoviral immunization and tumor immunotherapy model Eight- to 12-week-old C57BL/6 mice (n = 5/group) were injected subcutaneously with E-22 tumor cells (1 × 105 in 100 µL saline) in the right flank. The following day, mice were implanted with osmotic pumps and were treated with 7-day infusions of saline, rGM-CSF, or the combination of rGM-CSF/rIL-4 (10 µg each per day) to increase the number of DCs in vivo. Fourteen days after tumor inoculation, mice were immunized by intraperitoneal injection with 1 × 108 PFU of either ADV/-gal, expressing the
LacZ transgene, or the control vector, AdV/RR5. Tumor
volumes were measured biweekly in mm3 (maximal length × width × height) with an electronic caliper (Stoelting, Wheat
Lake Wood Dale, IL).
Statistics Data from individual representative experiments were presented as mean values ± SD, and data from multiple experiments were represented as mean group values ± SE. Differences between groups were determined by paired or unpaired Student t test, as applicable to the assay conditions, with P values as indicated.
Systemic rGM-CSF and combined rGM-CSF/IL-4 increased spleen and lymph node sizes and the concentration of CD11c+ DCs Continuous infusions of rGM-CSF and/or rIL-4 were delivered to mice through miniature osmotic pumps, and dosing was initially evaluated by changes in spleen size and the presence of CD11c+ DCs. Cytokine effects reached a plateau by day 7, and serum levels reached 158.3/0 pg/mL (GM-CSF/IL-4) for rGM-CSF-treated mice and 52.8/410 pg/mL (GM-CSF/IL-4) for rGM-CSF/rIL-4-treated mice at the 10 µg/d dosing (data represent pooled serum from 5 mice). When used alone, rGM-CSF increased spleen cellularity and DC percentage in a dose-dependent manner up to 10 µg/d. In contrast, rIL-4 produced minimal effects when infused by itself but induced specific changes in spleen size and composition when given in combination with rGM-CSF (Figure 1). At 10 µg/d, rGM-CSF increased cellularity by 4-fold and the concentration of CD11c+ DC by 2.1-fold compared with control mice (P .05). In
contrast, the combination of rGM-CSF/rIL-4 increased spleen cell number by only 2.1-fold, yet it enriched the percentage of CD11c on average by
2.7-fold to as much as 25% of total spleen cells
(P .05). Although rGM-CSF significantly increased the
percentage of DCs, the response to combined rGM-CSF/rIL-4 was greater
in all experiments (P .05).
A similar increase in total cell number was noted in the lymph nodes of
mice treated with rGM-CSF and rGM-CSF/rIL-4, with the response always
greater in mice receiving rGM-CSF/rIL-4 (1.7-fold vs 2.7-fold increase
compared to control, respectively; Tables 1 and 2).
An increase in the percentage of CD11c cells in lymph nodes also
occurred, though the effects were smaller in magnitude compared with
those observed in spleen (1.3- to 2.3-fold increase compared with
control). The overall effect of cytokine treatment was an increase in
total cell number and CD11c content in spleen and lymph nodes.
rGM-CSF increased all lineage-specific subsets, whereas rGM-CSF/rIL-4 preferentially increased natural killer cells and myeloid cells in spleen Cytokine treatments increased spleen and lymph node cellularity, and flow cytometry was used to determine changes in the B cell (B220+), T cell (Thy1.2+), NK cell (NK1.1+), and myeloid (CD11b+) subsets (Tables 1, 2). In spleen, treatment with rGM-CSF produced a proportional increase in all these subsets. In contrast, rGM-CSF/rIL-4 selectively increased the percentage and number of NK cells and myeloid cells with little effect on the total number of B cells and T cells. These differences suggest a more generalized proliferative response to rGM-CSF and a more targeted effect of rGM-CSF/rIL-4 on spleen cell populations. The response in lymph node was slightly different, with both rGM-CSF and rGM-CSF/rIL-4 resulting in a proportional increase in all cell subsets.rGM-CSF/rIL-4 increased the number of myeloid
(CD11c+/CD11b+) and lymphoid
(CD11c+/CD8
and myeloid DCs delineated by the expression of CD11b. Consistent with
prior reports,17,20,21,25 treatment with 10 µg/d rGM-CSF
for 7 days increased the percentage of myeloid DCs by 3- to 5-fold, making them the predominant DC population in rGM-CSF-treated mice. The
addition of rIL-4 at 10 µg/d increased this response further, boosting the concentration of CD11c+/CD11b+
cells up to 7 times that found in control mice (Figure
2). The effects of cytokine treatment on
the lymphoid subset in spleen were different. rGM-CSF, when used as a
single agent, did not increase the percentage of
CD11c+/CD8+ cells. However, when combined
with rIL-4 (both at 10 µg/d), the concentration of
CD8+ DCs increased by 2- to 3-fold over the
concentration found in control mice (Figure 2). These results were
confirmed by 3-color analysis demonstrating that
CD11c+/CD8+ cells in rGM-CSF/rIL-4-treated
mice also stained for DEC-205, another lymphoid DC marker. The
percentage of cells expressing both DEC-205 and CD11c increased 2- to
3-fold in the rGM-CSF/rIL-4 treatment group (Figure 2). The effects on
DC composition were further magnified by the overall increase in spleen
size. On average, the number of myeloid DCs in the spleens of mice
treated with rGM-CSF increased approximately 8-fold compared with
control mice, averaging 7.1 × 106
CD11c+/CD11b+ cells per spleen (Figure
3). In mice treated with rGM-CSF/rIL-4, the number of myeloid DCs increased by approximately 5-fold, and the
number of lymphoid DCs increased by 3.7-fold. Similar, but not
identical, trends were observed in axillary and inguinal lymph nodes.
Mice treated with rGM-CSF exhibited a 2.2-fold increase in the number
of myeloid DCs in lymph nodes and a 3-fold increase in the number of
lymphoid DC. In rGM-CSF/rIL-4-treated mice, there was a similar 2-fold
increase in myeloid DCs in lymph nodes and a 4- to 6-fold increase in
lymphoid DCs (Figure 3). Dual-staining demonstrated that DEC-205 was
primarily expressed on the CD8+ subset in spleen and
lymph nodes, consistent with its established expression on lymphoid
DCs. In total, the response to rGM-CSF was clearly biased toward
increasing primarily myeloid DCs, whereas the response to rGM-CSF/rIL-4
was more balanced, significantly increasing myeloid and lymphoid
subsets in spleen and lymph node.
Localization of DCs by immunohistology Spleens from control and cytokine-treated mice were stained with hematoxylin and eosin or 2-color immunocytochemistry for evaluation of cytokine-related changes on DC number and distribution (Figure 4). Anti-CD11b was used to identify cells of myeloid origin, anti-DEC-205 to identify lymphoid DCs, anti-CD11c to detect total DCs, and anti-Ig to detect B cells.
Hematoxylin and eosin sections from control mice demonstrated well-organized follicular structures with normal distributions of red and white pulp. In cytokine-treated mice, these structures were disrupted by a diffuse infiltration of mononuclear leukocytes. High-power examination also revealed increased stromal tissue in cytokine-treated mice (not shown). Dual staining for CD11b and CD11c (Figure 4, middle panel) demonstrated
several important features. First, it suggested the presence of
enlarged follicular structures in the spleens of cytokine-treated mice
that were not discerned by simple hematoxylin and eosin stain. This was
confirmed by staining for Ig Combined rGM-CSF/rIL-4 up-regulated MHC expression and antigen uptake by splenic DCs Expression of MHC class I and class II is essential for antigen-presenting activity, and it increases as DCs mature. Flow cytometry was used to determine the mean florescence intensity (MFI) of MHC class I and class II expression on lymphoid (CD11c+/CD8+) and myeloid
(CD11c+/CD11b+) DCs from control and
cytokine-treated mice (Figure 5). The
most dramatic effects occurred with MHC class I, which did not increase after treatment with rGM-CSF alone but increased significantly after
treatment with rGM-CSF/rIL-4. These effects were most prominent on
lymphoid DCs, where MHC class I expression increased by 3- to 4-fold
compared with myeloid DC, where expression increased by only 30% to
75%. The response pattern was different with respect to MHC
class II, which increased moderately in both DC subsets in response to
rGM-CSF and increased further in response to rGM-CSF/rIL-4. The
up-regulation of MHC class I and MHC class II, in response to systemic
rGM-CSF/rIL-4, suggests cytokine-induced maturation in vivo.
Flow cytometry was also used to examine the effects of cytokine therapy
on endocytosis (uptake of FITC-dextran) and macropinocytosis (uptake of
Lucifer yellow). Similar to the effects on MHC class I, the systemic
administration of rGM-CSF did not increase the pinocytotic activity of
either myeloid (CD11c+/CD11b+) or nonmyeloid
(CD11c+/CD11b
Allostimulatory capacity is primed by in vivo exposure to rGM-CSF and rGM-CSF/rIL-4 Purified DCs from the spleens of control and cytokine-treated mice were tested for their ability to stimulate allogenic T cells in a one-way MLR (Figure 7). When tested fresh, without a period of in vitro culture, only DCs from rGM-CSF-treated mice produced significant T-cell proliferation above that of DCs from control spleens. However, when DCs from the different groups were placed in culture for 24 to 36 hours with 20 ng/mL rGM-CSF and rIL-4, there was a dramatic increase in stimulatory activity from those previously exposed to cytokines in vivo. T-cell proliferation increased approximately 8-fold in response to in vivo rGM-CSF and 10-fold in response to in vivo rGM-CSF/rIL-4, with the response consistently higher in the rGM-CSF/rIL-4 group (P .05).
Taken together, these results suggest that rGM-CSF and the combination of rGM-CSF/rIL-4 increased the number of DCs and primed them in vivo
for enhanced T-cell stimulatory activity.
Administration of rGM-CSF/rIL4, but not rGM-CSF alone, enhances the response to an adenoviral-based vaccine and promotes antigen-specific tumor responses in vivo To access the functional impact of cytokine therapy (rGM/CSF vs rGM-CSF/rIL-4) on antigen-presentation and antitumor immunity in vivo, an established tumor immunotherapy model was developed. E-22 tumor cells, expressing-gal as a model tumor antigen, developed into
solid tumors in 100% of mice treated with subcutaneous injection of
1 × 105 cells. Pre-immunization with a single
intraperitoneal administration of 109 PFU of AdV/-gal,
but not the control vector (AdV/RR5), imparted 100% protection from
subsequent tumor challenge and generated tumor-specific immunity (data
not shown). However, in mice with established tumors, vaccination with
109 PFU of AdV/-gal only slowed tumor progression, and
vaccination with 108 PFU had no detectable effect (data not
shown). To evaluate the role of cytokines in enhancing the vaccine
response, mice were injected with tumor and, 1 day later, were treated
with saline, rGM-CSF, or rGM-CSF/rIL-4 by mini-osmotic pump. Subsequent
vaccination with 108 PFU of AdV/-gal significantly
reduced the rate of tumor growth only in mice that had been treated
with rGM-CSF/IL-4 (Figure 8B). The
response in mice pretreated with rGM-CSF was identical to that in
saline-treated controls (not shown), yielding no detectable effect on
tumor growth. The antigen-specific nature of the response was confirmed
in animals immunized with AdV/RR5 (Figure 8A). Vaccination with this
control vector, lacking -gal, produced no effect on the growth of
E-22. In vitro cocultures of cytokines and tumor cells were carried out
to evaluate whether combined rGM-CSF/rIL-4 had a direct cytotoxic or
cytostatic effect on the growth of E-22. No effect on cell growth (as
assessed by cell number and thymidine uptake) was observed in the
presence of cytokines in the range of 0.1 ng/mL to 1µg/mL. These
results suggest that pretreatment with rGM-CSF/rIL-4 enhances the
vaccine response and stimulates antigen-specific, antitumor immunity
in vivo.
Various differentiation and maturation signals have been
used to generate DCs in vitro, including GM-CSF for stimulating murine progenitors from bone marrow or spleen,16-18 GM-CSF in
combination with IL-4 for stimulating human and murine
precursors,7,8,10-12,26,27 or these cytokines in
combination with a variety of accessory stimuli, such as tumor necrosis
factor- An initial challenge in performing these studies was the short half-life of rGM-CSF and rIL-4 when administered to mice in vivo. In contrast to humans, in whom sustained cytokine levels persist for up to 8 to 12 hours after a single subcutaneous injection,14,15,31 the half-lives for rGM-CSF and rIL-4 in the mouse are only 11 minutes20 and 5 minutes,32 respectively. These pharmacokinetics likely explain why an earlier study, in which mice were treated with 10 µg/d GM-CSF and IL-4 as a single daily injection, failed to elicit significant increases in DC.5 Others have approached this problem by implanting gene-modified tumor cells secreting one or both cytokines.19,22,23 These models, though delivering a complex mixture of cytokines and other tumor-derived factors, confirmed that in vivo exposure to GM-CSF alone, or in combination with IL-4, can increase the number of functional DCs. In fact, several studies demonstrated greater induction of antitumor immunity when both cytokines were administered together. Recently, Daro et al20 successfully administered polyethylene-glycol modified GM-CSF (pegGM-CSF) as a mechanism for generating DCs in vivo. To extend this line of investigation and to study the combined effects of GM-CSF/IL-4, we administered unmodified cytokines by miniature osmotic pumps. When implanted subcutaneously, these pumps release cytokine(s) at a continuous rate for up to 1 to 2 weeks.33,34 This technique allowed us to examine the effects of GM-CSF and IL-4 in the absence of other factors, such as tumor burden. Using this approach, systemic rGM-CSF-produced marked splenic
hypertrophy with increases in most cell lineages including DCs, monocytes, granulocytes, B cells, and T cells. Within the DC
population, rGM-CSF expanded only myeloid DCs, identified by their
co-expression of CD11c and CD11b and their lack of expression of
CD8 In contrast to rGM-CSF, the administration of rIL-4 as a single agent did not increase spleen cellularity or DC number (data not shown). This corresponds to in vitro studies in which IL-4 alone failed to induce the proliferation or differentiation of myeloid progenitors.7,37,38 When rIL-4 was administered in combination with rGM-CSF, spleen cellularity increased significantly, but not to the degree observed with rGM-CSF alone. This finding is consistent with prior studies in which IL-4 partially suppressed hematopoietic stem cell proliferation in response to GM-CSF.28,39 However, though limiting the expansion of T cells and B cells, GM-CSF/IL-4 still allowed DCs to increase by approximately 6-fold compared with control spleens. Taken together, these facts suggest a more targeted effect on DC proliferation and differentiation than occurs with GM-CSF alone. In addition to increasing DC number, rGM-CSF/rIL-4 promoted a more balanced expansion of myeloid and lymphoid DCs than observed with rGM-CSF alone. Similar to other reports,20 we found that myeloid DCs predominate de novo in the spleen at a ratio of approximately 1.5:1 compared with lymphoid DCs and that this ratio increased to roughly 7:1 in response to the effects of GM-CSF on the myeloid subset. By comparison, exposure to rGM-CSF/rIL-4 increased the number of myeloid DCs in spleen tissue by approximately 6-fold and the number of lymphoid DCs by approximately 3.7-fold, resulting in a more balanced myeloid-to-lymphoid ratio. A similar pattern was observed in lymph nodes, in which the combination of rGM-CSF/rIL-4 resulted in a greater expansion of the lymphoid DC subset than occurred with rGM-CSF alone. Although treatment with flt3-ligand also increases both DC populations, it favors the expansion of lymphoid DCs in spleen by approximately 2:1 over myeloid DCs.5,6,20,25 In untreated mice, myeloid and lymphoid DCs are present in scant numbers and occupy distinct microenvironments within the spleen.6,40,41 Lymphoid DCs primarily reside in T-cell areas of the white pulp, whereas myeloid DCs are distributed throughout the marginal zones with some extension into the red pulp. In response to rGM-CSF in vivo, a striking splenic enlargement develops with a diffuse increase in CD11c+ DCs throughout all regions, including a significant increase in DEC-205+ cells infiltrating into the T-cell-enriched PALS. These effects are similar to those described for flt3-ligand6 and IL-1242 and suggest a general response pattern to dendropoietic factors. However, in response to rGM-CSF/rIL-4, DCs expressing CD11c and CD11b and those expressing DEC-205 became highly concentrated within enlarged PALS. This localization within T-cell zones and the distinct increase in staining intensity observed in response to rGM-CSF/rIL-4 are similar to the changes that occur in mice treated with lipopolysaccharide in vivo.40 These changes are also reminiscent of the homing that occurs when activated DCs migrate from the periphery to lymphoid tissue.43 In contrast to other dendropoietic factors, these immunohistology results suggest that combined treatment with rGM-CSF/rIL-4 produces the proliferation and mobilization of DCs and some degree of activation and localization to T-cell-enriched areas. Considerable controversy exists regarding the relative origins and
roles of myeloid versus lymphoid DCs. Although it has been reported
that CD11c+/CD8 |
Top
Abstract
Introduction
P 
P 
indicates control; 
, GM; 
,
GM/IL-4.
