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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 428-435, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.4824

Granulocyte Macrophage Colony–Stimulating Factor–Driven Respiratory Mucosal Sensitization Induces Th2 Differentiation and Function Independently of Interleukin-4

Stacey A. Ritz, Meghan J. Cundall, Beata U. Gajewska, David Alvarez, José-Carlos Gutierrez-Ramos, Anthony J. Coyle, Andrew N. J. McKenzie, Martin R. Stämpfli and Manel Jordana

Department of Pathology and Molecular Medicine and Centre for Gene Therapeutics, Division of Respiratory Diseases and Allergy, McMaster University, Hamilton, Ontario, Canada; Millennium Pharmaceuticals Incorporated, Cambridge, Massachusetts; and MRC Laboratory of Molecular Biology, Cambridge, United Kingdom

Address correspondence to: Manel Jordana, M.D., Ph.D., HSC-4H21, McMaster University, 1200 Main Street West, Hamilton, ON, L8S 3Z5 Canada. E-mail: jordanam@mcmasterca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The development of T helper (Th)2 responses is a key step in the pathogenesis of asthma. Interleukin (IL)-4 is thought to be important, although not strictly necessary, for Th2 differentiation, although triggers of IL-4–independent Th2 polarization have not been identified. We examined whether IL-4 is necessary for Th2-polarized responses during granulocyte macrophage colony–stimulating factor (GM-CSF)–driven respiratory mucosal sensitization. Balb/c wild type (WT) or IL-4 knockout (4KO) mice were exposed to aerosolized ovalbumin (OVA) in the context of airway GM-CSF expression. We examined the extent of Th2 polarization using real-time quantitative polymerase chain reaction on lymph node mRNA, flow cytometric analysis of lung Th cells, and measurement of cells, cytokines, and immunoglobulins in bronchoalveolar lavage (BAL) and serum. GATA-3 and CCR3, -4, and -8 were expressed in the lymph nodes of WT and 4KO mice at similar levels, as were IL-5 and IL-13 levels in the BAL, T1/ST2 on lung Th cells, and BAL eosinophils after recall challenge. With the exception of immunoglobulin production, expression of GATA-3, CCR-3, -4, -8, IL-5, and T1/ST2, and the generation of blood eosinophilia, were intact in mice doubly deficient in both IL-4 and IL-13. We conclude that IL-4 is not required for the generation of Th2-polarized responses in the presence of GM-CSF.

Abbreviations: analysis of variance, ANOVA • bronchoalveolar lavage, BAL • double knockout, DKO • IL-4 knockout, 4KO • interferon, IFN • interleukin, IL • granulocyte macrophage colony–stimulating factor, GM-CSF • monocyte chemotactic protein, MCP • ovalbumin, OVA • phosphate-buffered saline, PBS • polymerase chain reaction, PCR • room temperature, rt • wild-type, WT


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In spite of their innocuous character, aeroallergens induce asthma in a growing proportion of the population (1). Asthma is a syndrome distinguished by a distinct cytokine and immunoglobulin profile, eosinophilic airway inflammation, and bronchial hyperreactivity. Studies in murine models of asthma have established a critical role for T helper (Th) cells (2, 3), and it is well established that interleukin (IL)-4, IL-5, and IL-13 are the preeminent effector molecules (4–10). Insofar as Th2 cells are the primary producers of these cytokines, the differentiation of precursor Th cells into Th2 cells is of paramount importance to the generation of asthmatic inflammation.

It is frequently asserted that IL-4 is necessary for the development of Th2-polarized responses, in spite of evidence that these responses can sometimes be generated in the absence of IL-4. In murine models of allergic inflammation and infectious disease models, some studies show that Th2 responses are apparently hampered in the absence of IL-4 signaling (11–22), whereas others demonstrate that they are not impaired (17, 23–27). However, none of these latter studies have established a mechanism by which Th2 responses are elicited in the absence of IL-4.

Until recently, Th2 responses were defined entirely by the cytokine profile observed, and previous documentation of Th2 responses in the absence of IL-4 have relied almost exclusively on these cytokine readouts. However, more recently a number of noncytokine markers of Th2 responses have been described, including the transcription factor GATA-3, the chemokine receptors CCR3, -4, and -8, and the cell surface protein T1/ST2. We have used these markers to more comprehensively assess the presence of Th2 responses in mice in which the prototypical Th2 cytokine, IL-4, is absent.

Murine models have been very fruitful for investigating mechanisms of human allergic inflammation, but not sensitization, because sensitization in these models is typically accomplished by the introduction of antigen into the intraperitoneal cavity in conjunction with alum as an adjuvant, which is very dissimilar to the conditions under which allergic sensitization occurs in humans. In contrast, this study is unique in that we examined the differentiation and activity of Th2 cells in vivo in a model of allergic airways inflammation in which mice are exposed to an innocuous antigen, exclusively via the respiratory tract, and in the absence of adjuvants. Such a system recapitulates the route of allergen sensitization in humans. Granulocyte macrophage colony–stimulating factor (GM-CSF) is employed because exposure to antigen only has been shown to induce inhalation tolerance (28, 29), and we have previously shown that expression of GM-CSF in the airways during antigen exposure overrides the tendency to elicit tolerance, and promotes the generation of an immune-inflammatory response reminiscent of asthma (30).

To examine the role of IL-4 in the generation of Th2-polarized responses via the respiratory tract, IL-4 knockout (4KO) mice were compared with their wild type (WT) controls. In addition, IL-4 and IL-13 double-knockout (DKO) mice were used to investigate the possibility that IL-13 could drive Th2 responses in the absence of IL-4 (31). We found that indices of Th2 differentiation and function were intact in 4KO and DKO mice. These studies suggest a novel role for GM-CSF in the generation of Th2 responses in the mucosae, independently of IL-4 and IL-13.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects
Female Balb/c mice (6–8 wk old) were obtained from Charles River Laboratories (Ottawa, ON, Canada). Female 4KO mice on a Balb/c background were obtained from Jackson Laboratories (Bar Harbor, ME). IL-4 and IL-13 DKO mice on the Balb/c background (31) were bred in the McMaster facility (Hamilton, ON, Canada). Mice were kept under specific pathogen–free conditions, with a 12-h light/dark cycle, and food and water ad libitum. These experiments were approved by the Animal Research Ethics Board of McMaster University.

Respiratory Mucosal Sensitization and Challenge
Airway expression of GM-CSF was achieved by delivery of a replication-deficient human type 5 adenovirus (Ad) with the gene for GM-CSF in the E1 region of the genome (30). A quantity of 3 x 107 pfu of Ad/GM-CSF in 30 µl phosphate-buffered saline (PBS) was delivered intranasally to isoflurane-anaesthetized animals, 1 d before ovalbumin (OVA) exposure (Day -1). We have previously demonstrated that this dose of the Ad/GM-CSF vector results in airway expression of GM-CSF for 10 d, with bronchoalveolar lavage (BAL) expression peaking at ~ 80–100 pg/ml on Day 7 (30); this level of expression is well within physiologically achievable concentrations.

For sensitization, mice were exposed to OVA for 20 min daily from Day 0 to Day 9. For rechallenge, OVA was delivered for 1 h daily for 3 d. Mice were placed in a Plexiglas chamber, and OVA aerosol (1% wt/vol in 0.9% saline, Grade V; Sigma-Aldrich, Oakville, ON, Canada) delivered using compressed medical air at 10 liters/min through a Bennet/Twin nebulizer (Bennet/Twin, Kansas City, MO). We have previously reported an extensive characterization of this model (30) in which we observed that neither exposure to OVA only, nor exposure to OVA in the presence of an empty Ad vector, results in immunologic sensitization to OVA. Similarly, 4KO mice exposed to OVA in the context of a control adenoviral vector did not exhibit any indication of antigen-specific immune-inflammatory responses to OVA; total BAL inflammation in these mice was comparable to that seen in naive 4KO controls (total cells: 6.8 ± 0.7 versus 4.3 ± 0.8 x 105 cells/ml, respectively), and no eosinophilia was observed.

Isolation of mRNA and Analysis of mRNA Expression by Real-Time Quantitative Polymerase Chain Reaction
Thoracic lymph nodes were pooled from 4–6 mice and placed in RNAlater (Ambion Inc., Austin, TX), and RNA extracted with TriPure (Roche, Indianapolis, IN). Genomic DNA was removed using the Qiagen RNeasy kit (Qiagen Inc., Mississauga, ON, Canada). RNA was reverse transcribed using the Qiagen OMNIscript kit (Qiagen) using random hexamers (Gibco, Rockville, MD) and oligo-dT (Gibco) as primers. Primers and FAM-labeled probes were designed with PrimerExpress v1.5 software (Applied Biosystems, Foster City, CA). For GATA-3, the forward primer was 5'-CTACCGGGTTCGGATGTAAGTC; the reverse was 5'-GTTCACACACTCCCTGCCTTCT; and the probe was 5'-AGGCCCAAGGCACGATCCAGC. For t-bet the forward primer was 5'-ACCAGAACGGACAGATCACTCA; the reverse was 5'-CAAAGTTCTCCCGGAATCCTT; and the probe was 5'-CTGAAAATCGACAACAACCCTTTGCC. CCR3, -4, and -8, primers and FAM-labeled probes, and GAPDH primers and VIC-labeled probe were obtained from Applied Biosystems. Polymerase chain reaction was done in the ABI Prism 6700 Sequence Detection operated by Sequence Detector v1.7 software (Applied Biosystems), using TaqMan Universal polymerase chain reaction Master Mix (Applied Biosystems). Gene expression was quantitated relative to the expression of glyceraldehyde-3-phosphate dehydrogenase. The value of the relative expression for the WT naive samples are defined as 100%, and all other values are plotted compared with them.

Collection and Measurement of Specimens
Mice were anaesthetized by isoflurane and killed for sample collection. Peripheral blood was collected, total cell counts determined using a hemocytometer, and leukocyte smears prepared. Blood samples were incubated at 37°C for 30 min, and the serum supernatant stored at -20°C.

Lungs were dissected and BAL performed. Two aliquots of PBS (250 µl and 200 µl) were delivered and recovered through a polyethylene tube (Becton Dickinson, Sparks, MD) inserted in the trachea; 300 µl were consistently recovered. Total cell counts were determined with a hemocytometer. The BAL was centrifuged and the supernatant stored at -20°C for cytokine analysis. The cell pellet was resuspended in PBS and smears were prepared by cytocentrifugation (Shandon Inc., Pittsburgh, PA) at 300 rpm for 2 min.

Peripheral blood and BAL smears were stained with the Protocol Hema 3 Stain Set (Fisher Scientific, Toronto, ON, Canada). Differential cell counts were made from 300 leukocytes.

Lung tissue was fixed in 10% formalin and embedded in paraffin. Sections 3 µm thick were stained with hematoxylin and eosin (H&E), or with periodic acid-Schiff (PAS).

Flow Cytometric Analysis of Lung Cells
Lungs were perfused with 10 ml warmed Hanks' balanced salt solution, cut into 2-mm pieces, and agitated for 1 h at 37°C in 150 U/ml collagenase III (Life Technologies, Rockville, MD). Pieces were ground through a metal screen into HBSS, filtered through fine-gauge nylon mesh, washed and resuspended in HBSS, and layered over 30% and 60% Percoll (Amersham Pharmacia Biotech, Uppsala, Sweden) for density gradient centrifugation at 2,000 rpm (25 min at room temperature [rt]). Cells at the 30/60% interface were collected and washed with PBS. A quantity of 106 cells in 50 µl PBS was incubated with 1 µg Fc block (anti-CD16/CD32; PharMingen, Mississauga, ON, Canada) on ice for 15 min, and then stained with fluorescently-conjugated Ab: anti-CD3{varepsilon}-phycoerythrin (clone 2C11; PharMingen) (2 µg/106 cells); anti-CD4-cychrome C (clone L3T4; PharMingen) (0.05 µg/106 cells); anti- T1/ST2-FITC (clone 3E10) (1.25 µg/106 cells); or its isotype control. Flow cytometry was performed on a FACScan (Becton Dickinson, Sunnyvale, CA) and analyzed using WinMDI version 2.8 (Scripps Research Institute, La Jolla, CA).

Enzyme-Linked Immunosorbent Assay Measurement of Cytokines and Immunoglobulins
Cytokine levels in BAL were measured by enzyme-linked immunosorbent assay (ELISA) using commercially available kits for IL-13, eotaxin, monocyte chemotactic protein (MCP)-1, and interferon (IFN)-{gamma} (R&D, Minneapolis, MN), and IL-5 (Amersham, Buckinghamshire, UK). The level of sensitivity for these assays was ~ 5 pg/ml.

Sandwich ELISA was used to measure OVA-specific IgG1 and IgG2a in serum. One hundred microliters per well of 5 mg/ml OVA in a borate buffer was applied to 96-well plates and incubated for 1 h at 37°C, 3 h at rt, and overnight at 4°C. Plates were blocked with 1% bovine serum albumin (Sigma) in PBS (150 µl/well) for 2 h at rt. Fifty µl of each sample was added and left overnight at 4°C. After washing, 50 µl/well of 0.25 mg/ml biotinylated anti-mouse IgG1 or IgG2a Ab (Southern Biotechnology Associates, Birmingham, AL) was added. After 2 h at rt, plates were washed, and alkaline phosphate/streptavidin was added for 1 h at rt (diluted to 1:1,000, 50 µl/well). p-Nitrophenyl phosphate in diethanolamine buffer was used for color development. OVA-specific IgE levels were determined using an antigen-capture ELISA method, which has been previously described (30). Briefly, anti-mouse IgE Ab was in the solid phase, serum samples were added, and OVA-specific IgE detected using biotinylated OVA. Units of OVA-specific Ig were determined relative to standard sera. Units of the standard serum are defined as the largest dilution factor giving an optical density reading greater than background plus 2 SD; therefore, by definition, the sensitivity of the assay is 1 U/ml.

Data Analysis
Data are expressed as mean ± SEM. Statistical analysis was performed using SigmaStat version 2.03. Differences were considered statistically significant when P <= 0.05 by analysis of variance (ANOVA), with Fisher's PLSD where applicable.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of Transcription Factor and Chemokine Receptor mRNA Expression
We used real-time quantitative PCR to measure expression of mRNA for Th2-associated transcription factors and chemokine receptors in the thoracic lymph nodes at various time points during sensitization.

Compared with naive, WT mice had increased expression of the Th2-associated transcription factor GATA-3 (32, 33) by Day 4 of the sensitization phase, returning to near-naive levels by Day 7 (Figure 1A). The kinetic and magnitude of GATA-3 expression in 4KO mice was similar to that observed in the WT. Although the fold increases in GATA-3 expression are not dramatic, they were consistently reproducible in repeated experiments. Neither WT nor 4KO mice significantly upregulated the Th1-associated transcription factor t-bet (34) at any time point (Figure 1B).



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  		Figure 1. Expression of GATA-3 and t-bet in the thoracic lymph nodes of WT and 4KO mice. WT (black bars) and 4KO mice (gray bars) were exposed to OVA in the context of GM-CSF. At Days 4, 7, and 11, RNA was isolated from thoracic lymph nodes and GATA-3 and t-bet were analyzed by real-time quantitative PCR. A shows the relative expression of GATA-3, and B shows the relative expression of t-bet. Data shown is mean ± SD of triplicate measurements; * indicates a statistically significant (P <= 0.05) difference from the expression levels of the naive control, as determined by ANOVA.

 
The Th2-associated chemokine receptors CCR3, -4, and -8 (32, 35) were also examined. In WT mice, CCR3 expression was increased on Day 4, and peaked on Day 7 (Figure 2A); although basal expression of CCR3 was lower in 4KO mice, expression was equivalent to that seen in WT mice on Day 4 and Day 7. CCR4 expression peaked on Day 4 in WT mice (Figure 2B), and was identical to the expression seen in 4KO mice. Expression of CCR8 also peaked on Day 4 in WT mice (Figure 2C); the pattern of expression was the same in 4KO mice, although CCR8 expression was higher in 4KO mice at all time points examined.



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  		Figure 2. Expression of CCR3, CCR4, and CCR8 in the thoracic lymph nodes of WT and 4KO mice. RNA was isolated from pooled thoracic lymph nodes of WT (black bars) or 4KO mice (gray bars) at Day 4 and Day 7 of exposure to OVA in the context of GM-CSF expression. Real-time quantitative PCR was performed to assess CCR3, CCR4, and CCR8 expression. Relative expression of CCR3 is shown in A, of CCR4 in B, and of CCR8 in C. Data shown is mean ± SD of triplicate measurements; * indicates a statistically significant (P <= 0.05) difference from the expression levels of the naive control, as determined by ANOVA.

 
Measurement of Cytokines and Chemokines in the BAL
BAL cytokine and chemokine levels were measured on Day 7 of the sensitization protocol, as we previously determined that this is the peak of cytokine expression (30).

BAL from naive WT and 4KO mice had no detectable levels of IL-5 or IL-13 as measured by ELISA (data not shown). On Day 7, both WT and 4KO mice had upregulated IL-5 protein expression to the same extent (Figure 3A). On Day 7, IL-13 expression was lower in 4KO mice than in WT mice, although this difference did not reach statistical significance; furthermore, this amount, although reduced compared with WT, was still significantly greater than that seen in naive mice. IFN-{gamma} was not detected in the BAL of WT or 4KO mice at any time point examined (data not shown); similarly, real-time quantitative PCR analysis of lymph node RNA did not demonstrate any upregulation of mRNA for IFN-{gamma} above naive levels in WT or 4KO mice at any time point examined (data not shown).



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  		Figure 3. Levels of IL-5, IL-13, eotaxin, and MCP-1 in BAL fluid of WT and 4KO mice. BAL samples were obtained on Day 7 of exposure to OVA in the context of GM-CSF expression, and cytokine levels measured by ELISA. IL-5 levels are depicted in A, IL-13 in B, eotaxin in C, and MCP-1 in D. Data shown is mean ± SEM, n = 4–9.

 
No eotaxin was detectable in the BAL of naive mice of either strain and MCP-1 was not detectable in naïve WT mice (data not shown), but was detectable at low levels in 4KO mice (12.9 ± 2.6 pg/ml). Eotaxin (Figure 3C) and MCP-1 (Figure 3D) levels in the BAL on Day 7 were significantly increased above naive levels, and were not significantly different between WT and 4KO mice.

T1/ST2 Expression on Lung Th Cells
On Day 9, T1/ST2 expression was analyzed on lung Th cells by flow cytometry to identify effector Th2 cells (36–38). Th cells were defined as CD3+ CD4+ cells in the lymphocyte gate (as defined by forward and side scatter characteristics). Approximately 2% of Th cells from the lungs of naive WT and 4KO mice were T1/ST2+ (data not shown). On Day 9, 10.7% of lung Th cells were T1/ST2+ in WT mice, compared with 10.4% in 4KO mice (Table 1). These data were reproducible in three separate experiments.


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  		TABLE 1 Flow cytometric and immunoglobulin analysis in WT and 4KO mice

 
Inflammation and Eosinophilia
As a hallmark of allergic asthma, we assessed eosinophils in blood and BAL during sensitization, in BAL after rechallenge, and in histologic sections.

WT and 4KO mice had similar numbers of total blood leukocytes on Day 11 (Figure 4A). 4KO mice had a slight reduction in peripheral blood eosinophilia compared with WT mice (Figure 4B), but this was not statistically significant.



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  		Figure 4. Eosinophilia in the peripheral blood and BAL of WT and 4KO mice. WT and 4KO mice were exposed to OVA aerosol in the context of GM-CSF expression. On Day 11, peripheral blood (A and B) and BAL (C and D) were collected from both mouse strains; another group of WT and 4KO mice were rechallenged on Days 40–42, and BAL collected on Day 47 (E and F). Data show total leukocytes (A, C, and E) and eosinophils (B, D, and F) in the blood or BAL. Data shown is mean ± SEM, n = 4–14; * indicates a statistically significant (P <= 0.05) difference from WT mice as determined by ANOVA.

 
Both WT and 4KO naive mice had similar total cell numbers in the BAL (6.9 ± 0.6 versus 4.3 ± 0.8 x 105 cells/ml), and no eosinophils. On Day 11, WT and 4KO mice had statistically significant increases in total BAL leukocytes (Figure 4C) and eosinophils (Figure 4D) compared with naive, although 4KO mice had 40% fewer total cells and 65% fewer eosinophils than WT. Histologic sections of lung tissue at this time point revealed peribronchial and perivascular inflammatory leukocytes in both strains, and eosinophils were apparent among the infiltrating cells in H&E-stained sections (Figures 5A and 5B). PAS stain revealed mucous-containing goblet cells in the bronchial lining of both WT and 4KO mice (Figures 5C and 5D).



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  		Figure 5. Light photomicrograph of lung tissues from WT and 4KO mice. Lung tissues from WT (A and B) and 4KO mice (C and D) exposed to OVA aerosol in the context of GM-CSF were obtained on Day 11, fixed in formalin, and embedded in paraffin. Sections were stained with haematoxylin and eosin (A and C) to reveal general morphology, or periodic acid-Schiff (B and D) to stain for mucous. Note that peribronchial and perivascular inflammation are present both in WT (A) and 4KO mice (C), and that eosinophils are present in both strains (arrows). Also, magenta-stained mucous is apparent in both WT (B) and 4KO mice (D). "A" indicates airways, and "V" indicates blood vessels. Magnification: A and C, x250; B and D, x100.

 
In some experiments, the inflammation caused by sensitization was allowed to resolve for 1 mo, and the mice were rechallenged with OVA to examine antigen-specific memory responses. Inflammation in the BAL was assessed at the peak of the inflammatory response, 72 h after the third OVA rechallenge. In WT mice the eosinophilic inflammation was recapitulated (Figures 4E and 4F). In contrast with the observations at Day 11, after long term rechallenge 4KO mice had eosinophilic inflammatory responses indistinguishable from that seen in WT mice.

Immunoglobulin Profile
After sensitization, OVA-specific Ig were measured in the serum of WT and 4KO mice by ELISA. As expected, no OVA-specific Ig were detected in naive WT or 4KO mice (data not shown). OVA-specific IgE, IgG1, and IgG2a were all present in WT mice after sensitization to OVA (Table 1). In 4KO mice, no OVA-specific IgE was detected and IgG1 was significantly decreased, whereas IgG2a levels were significantly increased.

Evidence of Th2-Polarized Responses in DKO Mice
We used DKO mice to determine whether IL-13 can substitute for IL-4 in the generation of Th2-polarized responses. None of GATA-3 (Figure 6A), CCR-3, -4, or -8 (Figure 6B), IL-5 (Figure 6C), or peripheral blood eosinophilia (Figure 6D) were significantly different between WT, 4KO, and DKO mice. Furthermore, T1/ST2 expression on lung Th cells from DKO mice was, at 9.2%, upregulated to an extent comparable to that seen in WT and 4KO mice (Table 1). In contrast, BAL eosinophilia (Figure 6E) was completely absent in DKO mice at Day 11. Whereas eotaxin expression was expressed to a comparable degree in the BAL of WT and 4KO mice, it was reduced by ~ 85% in DKO mice (Figure 6F).



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  		Figure 6. Indices of Th2 polarization and eosinophilic inflammation in IL-4/IL-13 DKO mice. WT, 4KO, and DKO mice were exposed to OVA aerosol in the context of GM-CSF expression. mRNA expression of GATA-3 (A) and CCR3, -4, and -8 (B) was analyzed in the thoracic lymph nodes by real-time quantitative PCR on Day 7. IL-5 (C) and eotaxin (F) were measured by ELISA on Day 7. Peripheral blood (D) and BAL (E) eosinophilia was assessed on Day 11. In C, black bars indicate WT mice, light gray bars are 4KO mice, and dark gray bars are DKO mice. Data in A and B are mean ± SD of triplicate measurements; data in (C–F) are mean ± SEM, n = 4–13; * indicates a statistically significant (P <= 0.05) difference as determined by ANOVA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Molecular signals delivered to a Th cell during primary activation critically influence its differentiation pathway, and ultimately determine the nature of the immune-inflammatory response that will ensue. The primary objective of this study was to investigate the role of IL-4 in the development of Th2-polarized responses during respiratory exposure to aerosolized OVA in the context of a GM-CSF–enriched airway microenvironment. This model mimics the natural route of exposure to antigen while providing a signal (GM-CSF) which overrides the tendency of the lung to induce inhalation tolerance when exposed to harmless antigens (28, 29).

The Th2-associated transcription factor GATA-3 and Th2-affiliated CCRs were analyzed to evaluate whether Th2 differentiation had taken place. Our data show that GATA-3 is upregulated in the lymph nodes of WT mice; importantly, our data also show that GATA-3 is upregulated in 4KO mice. Furthermore, analysis of chemokine receptors associated with Th2 cells demonstrated the upregulation of CCR3, -4, and -8 in both WT and 4KO mice. These findings have several implications. First, insofar as these molecules are markers of Th2 cells, these data confirm that Th2 cells are present. Second, the upregulation of GATA-3, CCR3, -4, and -8 in 4KO mice indicates that their expression can occur independently of IL-4 in vivo.

Expression of the prototypical Th2 cytokines in the BAL was analyzed to examine whether these Th2 cells were functional. Previous studies using 4KO mice have generally shown a marked decrease in IL-5 and/or IL-13 production in in vitro recall assays (11, 13, 16, 19–22, 24, 25). However, the findings reported here are consistent with reports (17, 23, 27) in which these cytokines are present at equivalent levels in WT and 4KO mice. We did not directly measure the T cell contribution to cytokine levels, and hence cannot rule out the possibility that cells other than T cells may have supplemented the overall cytokine levels; however, given that GATA-3 was equivalently expressed in WT and 4KO mice, and because GATA-3 expression is sufficient for the transcription of these cytokines (39), we infer that Th2 cells are most likely responsible for the cytokine levels measured in the BAL.

With respect to eosinophilia, a number of groups have reported a drastic reduction in BAL eosinophils when IL-4 was blocked or absent during allergen sensitization (11, 13–16), whereas others have not demonstrated such a change (16). Our data show that the ability of 4KO mice to generate peripheral blood eosinophilia, and BAL eosinophilia after recall challenge, is intact, consistent with the fact that IL-5 production is intact in 4KO mice. The extent of the BAL eosinophilic response in 4KO mice observed on Day 11 was considerably greater than that observed by some others (11, 14–16), but still significantly less than in WT controls; this transient inhibition of eosinophilia remains unexplained. However, because this reduction occurred while the adenoviral vector was present, we suspect that this was due to an unknown process induced in response to the virus. This suspicion was confirmed when we used recombinant GM-CSF to induce sensitization instead of the Ad/GM-CSF; in this case, the number of eosinophils was statistically indistinguishable between WT and 4KO mice (8.0 ± 2.0 versus 6.0 ± 1.9 x 105 eosinophils/ml; P = 0.496, n = 4). One possibility is that the vector induced a concurrent Th1 response, antagonizing the Th2 effector response in the lung (40). However, our data suggest that is unlikely, because neither the Th1-associated transcription factor t-bet nor IFN-{gamma} were upregulated. Thus, the mechanism by which the viral vector reduced BAL eosinophilia at Day 11 is not obvious at present.

Immunoglobulin profiles are often used as indices of the nature of Th responses, because Th1 and Th2 cytokines signal for distinct patterns of isotype switching in B cells. As shown in Table 1, we observed no production of antigen-specific IgE in 4KO mice, as expected, given the requirement for IL-4 for {varepsilon} isotype switching (4). In addition, OVA-specific IgG1 production was lower in 4KO mice than in WT mice, whereas IgG2a production was increased. Although IgG2a was increased in 4KO mice, it is not likely that this is indicative of a Th1-polarized response because we were unable to detect t-bet or IFN-{gamma} using very sensitive methods (real-time quantitative PCR) in the lymph nodes of WT or 4KO mice; more probably, in the absence of IL-4, isotype switching to IgG2a was not inhibited in 4KO mice. This skewed pattern of Ig production, taken by itself, is suggestive of an absence of Th2 differentiation in 4KO mice. However, in the context of our more direct markers indicating that Th2 polarization indeed occurred, we conclude that Ig profiles do not fully reflect the nature of Th responses.

IL-4 and IL-13 have a number of redundant biologic functions, and it is possible that IL-13 may have been responsible for our observation of Th2 differentiation in the absence of IL-4 (31). We used DKO mice to establish whether indices of Th2 polarization in the absence of IL-4 were attributable to IL-13. It is clear that they were not; the equivalent expression of GATA-3, CCR-3, -4, -8, T1/ST2, IL-5, and blood eosinophils in DKO mice are compelling evidence that neither IL-4 nor IL-13 are required for Th2 polarization per se, although a downstream effect of Th2 responses, BAL eosinophilia, was not fully realized in their absence. IL-13 is known to upregulate eotaxin production (41–43); therefore, in this case, it would seem that eosinophils were not recruited from the blood into the lung tissues because of the absence of eotaxin, which in turn was likely due to the congenital absence of IL-13.

GM-CSF has been documented to be present in a variety of allergic airway diseases (44–47); polymorphisms in the GM-CSF gene have been identified in allergic populations (48); and airway expression of GM-CSF appears to be differentially regulated in atopic and nonatopic individuals (49–51). Furthermore, the known functions of GM-CSF (such as the expansion of antigen-presenting cells and upregulation of antigen-presenting functions and costimulatory molecules [52–54], and downregulation of the immunosuppressive functions of alveolar macrophages [55, 56]) are events conducive to the initiation of adaptive immune responses through the ability of such antigen-presenting cells to activate naive T cells. Others have previously shown that GM-CSF expands a population of myeloid dendritic cells which are able to preferentially induce Th2 responses (57), independently of IL-4 (58), and we speculate that such a mechanism could be at work in this model.

Collectively, our data provide compelling evidence that Th2 differentiation occurred in vivo in the absence of IL-4. We demonstrate that respiratory exposure to antigen in the context of GM-CSF expression overrides the requirement for IL-4 to induce Th2-polarized immune-inflammatory responses. We further propose that the ability of GM-CSF to elicit Th2 responses suggests an important and novel role for this cytokine in the initiation of allergic responses.


   Acknowledgments
 
The authors thank Monika Cwiartka, Susanna Goncharova, Duncan Chong, Xueya Feng, and Steve Manning for technical assistance, and Mary Kiriakopolous for secretarial support. S.A.R., B.U.G., and D.A. hold doctoral fellowships from the Canadian Institutes for Health Research (CIHR). M.R.S. holds a Parker B. Francis Research Fellowship. This research was funded by the CIHR, the Hamilton Health Sciences Corporation, and the St. Joseph's Hospital Foundation.

Received in original form January 25, 2002

Received in final form May 14, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Beasley, R., J. Crane, C. K. Lai, and N. Pearce. 2000. Prevalence and etiology of asthma. J. Allergy Clin. Immunol. 105:S466–S472.[Medline]
  2. Garlisi, C. G., A. Falcone, T. T. Kung, D. Stelts, K. J. Pennline, A. J. Beabis, S. R. Smith, R. W. Egan, and S. P. Umland. 1995. T cells are necessary for Th2 cytokine production and eosinophil accumulation in airways of antigen-challenged allergic mice. Clin. Immunol. Immunopathol. 75:75–83.[Medline]
  3. Gonzalo, J. A., C. M. Lloyd, L. Kremer, E. Finger, A.-C. Martinez, M. H. Siegelman, M. Cybulsky, and J. C. Gutierrez-Ramos. 1996. Eosinophil recruitment to the lung in a murine model of allergic inflammation: the role of T cells, chemokines, and adhesion receptors. J. Clin. Invest. 98:2332–2345.[Medline]
  4. Finkelman, F. D., I. M. Katona, J. F. Urban, J. Holmes, J. Ohara, A. S. Tung, J. V. Sample, and W. E. Paul. 1988. IL-4 is required to generate and sustain in vivo IgE responses. J. Immunol. 141:2335–2341.[Abstract]
  5. Hansel, T. T., R. K. Braun, I. J. De Vries, C. Boer, L. Boer, S. Rihs, and C. Walker. 1993. Eosinophils and cytokines. Agents Actions Suppl. 43:197–208.[Medline]
  6. Ohnishi, T., S. Sur, D. S. Collins, J. E. Fish, G. J. Gleich, and S. P. Peters. 1993. Eosinophil survival activity identified as interleukin-5 is associated with eosinophil recruitment and degranulation and lung injury twenty- four hours after segmental antigen lung challenge. J. Allergy Clin. Immunol. 92:607–615.[Medline]
  7. Corry, D. B., H. G. Folkesson, M. L. Warnock, D. J. Erle, M. A. Matthay, J. P. Wiener-Kronish, and R. M. Locksley. 1996. Interleukin 4, but not interleukin 5 or eosinophils, is required in a murine model of acute airway hyperreactivity. J. Exp. Med. 183:109–117.[Abstract/Free Full Text]
  8. Hogan, S. P., A. Koskinen, and P. S. Foster. 1997. Interleukin-5 and eosinophils induce airway damage and bronchial hyperreactivity during allergic airway inflammation in BALB/c mice. Immunol. Cell Biol. 75:284–288.[Medline]
  9. Grunig, G., M. Warnock, A. E. Wakil, R. Venkayya, F. Brombacher, D. M. Rennick, D. Sheppard, M. Mohrs, D. D. Donaldson, R. M. Locksley, and D. B. Corry. 1998. Requirement for IL-13 independently of IL-4 in experimental asthma. Science 282:2261–2263.[Abstract/Free Full Text]
  10. Wills-Karp, M., J. Luyimbazi, X. Xu, B. Schofield, T. Y. Neben, C. L. Karp, and D. D. Donaldson. 1998. Interleukin-13: central mediator of allergic asthma. Science 282:2258–2261.[Abstract/Free Full Text]
  11. Hogan, S. P., K. I. Matthei, J. M. Young, A. Kosikinen, I. G. Young, and P. S. Foster. 1998. A novel T cell-regulated mechanism modulating allergen-induced airways hyperreactivity in Balb/c mice independently of IL-4 and IL-5. J. Immunol. 161:1501–1509.[Abstract/Free Full Text]
  12. Kuperman, D., B. Schofield, M. Wills-Karp, and M. J. Grusby. 1998. Signal transducer and activator of transcription factor 6 (Stat6)-deficient mice are protected from antigen-induced airway hyperrepsonsiveness and mucus production. J. Exp. Med. 187:939–948.[Abstract/Free Full Text]
  13. Hogan, S. P., A. Mould, H. Kikutani, A. J. Ramsay, and P. S. Foster. 1997. Aeroallergen-induced eosinophilic inflammation, lung damage, and airways hyperreactivity in mice can occur independently of IL-4 and allergen-specific immunoglobulins. J. Clin. Invest. 99:1329–1339.[Medline]
  14. Kips, J. C., G. G. Brusselle, G. F. Joos, R. A. Peleman, R. R. Devos, J. H. Tavernier, and R. A. Pauwels. 1995. Importance of interleukin-4 and interleukin-12 in allergen-induced airway changes in mice. Int. Arch. Allergy Immunol. 107:115–118.[Medline]
  15. Brusselle, C. G., J. C. Kips, J. H. Tavernier, G. H. van der Heyden, C. A. Cuvelier, R. A. Pauwels, and H. Bluethmann. 1994. Attenuation of allergic airway inflammation in IL-4 deficient mice. Clin. Exp. Allergy 24:73–80.[Medline]
  16. Coyle, A. J., G. Le Gros, C. Bertrand, S. Tsuyuki, C. H. Heusser, M. Kopf, and G. P. Anderson. 1995. Interleukin-4 is required for the induction of lung Th2 mucosal immunity. Am. J. Respir. Cell Mol. Biol. 13:54–59.[Abstract]
  17. Herrick, C. A., H. MacLeod, E. Glusac, R. E. Tigellar, and K. Bottomly. 2000. Th2 responses induced by epicutaneous or inhalational protein exposure are differentially dependent on IL-4. J. Clin. Invest. 105:765–775.[Medline]
  18. Akimoto, T., F. Numata, M. Tamura, Y. Takata, N. Higashida, T. Takashi, K. Takeda, and S. Akira. 1998. Abrogation of bronchial eosinophilic inflammation and airway hyperreactivity in signal transducers and activators of transcription (STAT)6-deficient mice. J. Exp. Med. 187:1537–1542.[Abstract/Free Full Text]
  19. Kopf, M., G. G. Le Gros, M. Bachmann, M. C. Lamers, H. Bluethmann, and G. Koehler. 1993. Disruption of the murine IL-4 gene blocks Th2 cytokine responses. Nature 362:245–248.[Medline]
  20. Noben-Trauth, N., L. D. Shultz, F. Brombacher, J. F. Urban, H. Gu, and W. E. Paul. 1997. An interleukin 4 (IL-4) -independent pathway for CD4+ T cell IL-4 production is revealed in IL-4 receptor-deficient mice. Proc. Natl. Acad. Sci. USA 94:10838–10843.[Abstract/Free Full Text]
  21. Hogarth, P. J., M. J. Taylor, and A. E. Bianco. 1998. IL-5-dependent immunity to microfilariae is independent of IL-4 in a mouse model of onchocerciasis. J. Immunol. 160:5436–5440.[Abstract/Free Full Text]
  22. Kopf, M., F. Brombacher, G. Kohler, G. Kienzle, K. H. Widmann, K. Lefrang, C. Humborg, B. Ledermann, and W. Solbach. 1996. IL-4-deficient Balb/c mice resist infection with Leishmania major. J. Exp. Med. 184:1127–1136.[Abstract/Free Full Text]
  23. Brewer, J. M., M. Conacher, C. A. Hunter, M. Mohrs, F. Brombacher, and J. Alexander. 1999. Aluminum hydroxide adjuvant initiates strong antigen-specific Th2 responses in the absence of IL-4- or IL-13-mediated signaling. J. Immunol. 163:6448–6454.[Abstract/Free Full Text]
  24. Chensue, S. W., K. Warmington, J. H. Ruth, N. Lukacs, and S. L. Kunkel. 1997. Mycobacterial and schistosomal antigen-elicited granuloma formation in IFN-{gamma} and IL-4 knockout mice. J. Immunol. 159:3565–3573.[Abstract]
  25. Noben-Trauth, N., P. Kropf, and I. Muller. 1996. Susceptibility to Leishmania major infection in interleukin-4-deficient mice. Science 271:987–990.[Abstract]
  26. Mohrs, M., C. Holscher, and F. Brombacher. 2000. Interleukin-4 receptor alpha-deficient BALB/c mice show an unimpaired T helper 2 polarization in response to Leishmania major infection. Infect. Immun. 68:1773–1780.[Abstract/Free Full Text]
  27. Kropf, P., L. R. Schopf, C. L. Chung, D. Xu, F. Y. Liew, J. P. Sypek, and I. Muller. 1999. Expression of Th2 cytokines and the stable Th2 marker ST2L in the absence of IL-4 during Leishmania major infection. Eur. J. Immunol. 29:3621–3628.[Medline]
  28. Holt, P. G., and C. McMenamin. 1989. Defence against allergic sensitization in the healthy lung: the role of inhalation tolerance. Clin. Exp. Allergy 19:255–262.[Medline]
  29. Swirski, F. K., B. U. Gajewska, D. Alvarez, S. A. Ritz, M. J. Cundall, E. C. Cates, A. J. Coyle, J. C. Gutierrez-Ramos, M. D. Inman, M. Jordana, and M. R. Stämpfli. 2002. Inhalation of a harmless antigen (ovalbumin) elicits immune activation but divergent immunoglobulin and cytokine activities in mice. Clin. Exp. Allergy 32:411–421.[Medline]
  30. Stämpfli, M. R., R. E. Wiley, G. S. Neigh, B. U. Gajewska, X. F. Lei, D. P. Snider, Z. Xing, and M. Jordana. 1998. GM-CSF transgene expression in the airway allows aerosolized ovalbumin to induce allergic sensitization in mice. J. Clin. Invest. 102:1704–1714.[Medline]
  31. McKenzie, G. J., P. G. Fallon, C. L. Emson, R. K. Grencis, and A. N. McKenzie. 1999. Simultaneous disruption of interleukin (IL)-4 and IL-13 defines individual roles in T helper cell type 2-mediated responses. J. Exp. Med. 189:1565–1572.[Abstract/Free Full Text]
  32. Glimcher, L. H., and K. M. Murphy. 2000. Lineage commitment in the immune system: the T helper lymphocyte grows up. Genes Dev. 14:1693–1711.[Free Full Text]
  33. Zhang, D. H., L. Cohn, P. Ray, K. Bottomly, and A. Ray. 1997. Transcription factor GATA-3 is differentially expressed in murine Th1 and Th2 cells and controls Th2-specific expression of the interleukin-5 gene. J. Biol. Chem. 272:21597–21603.[Abstract/Free Full Text]
  34. Szabo, S. J., S. T. Kim, G. L. Costa, X. Zhang, C. G. Fathman, and L. H. Glimcher. 2000. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 100:655–669.[Medline]
  35. Bonecchi, R., G. Bianchi, P. P. Bordignon, D. D'Ambrosio, R. Lang, A. Borsatti, S. Sozzani, P. Allavena, P. A. Gray, A. Mantovani, and F. Sinigaglia. 1998. Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s. J. Exp. Med. 187:129–134.[Abstract/Free Full Text]
  36. Lohning, M., A. Stroehmann, A. J. Coyle, J. L. Grogan, S. Lin, J. C. Gutierrez-Ramos, D. Levinson, A. Radbruch, and T. Kamradt. 1998. T1/ST2 is preferentially expressed on murine Th2 cells, independent of interleukin 4, interleukin 5, and interleukin 10, and important for Th2 effector function. Proc. Natl. Acad. Sci. USA 95:6930–6935.[Abstract/Free Full Text]
  37. Meisel, C., K. Bonhagen, M. Lohning, A. J. Coyle, J. C. Gutierrez-Ramos, A. Radbruch, and T. Kamradt. 2001. Regulation and function of T1/ST2 expression on CD4+ T cells: induction of type 2 cytokine production by T1/ST2 cross-linking. J. Immunol. 166:3143–3150.[Abstract/Free Full Text]
  38. Townsend, M. J., P. G. Fallon, D. J. Matthews, H. E. Jolin, and A. N. McKenzie. 2000. T1/ST2-deficient mice demonstrate the importance of T1/ST2 in developing primary T helper cell type 2 responses. J. Exp. Med. 191:1069–1076.[Abstract/Free Full Text]
  39. Zheng, W., and R. A. Flavell. 1997. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89:587–596.[Medline]
  40. Stampfli, M. R., S. A. Ritz, G. S. Neigh, P. J. Sime, X. F. Lei, Z. Xing, K. Croitoru, and M. Jordana. 1998. Adenoviral infection inhibits allergic airways inflammation in mice. Clin. Exp. Allergy 28:1581–1590.[Medline]
  41. Matsukura, S., C. Stellato, S. N. Georas, V. Casolaro, J. R. Plitt, K. Miura, S. Kurosawa, U. Schindler, and R. P. Schleimer. 2001. Interleukin-13 upregulates eotaxin expression in airway epithelial cells by a STAT6-dependent mechanism. Am. J. Respir. Cell Mol. Biol. 24:755–761.[Abstract/Free Full Text]
  42. Pope, S. M., E. B. Brandt, A. Mishra, S. P. Hogan, N. Zimmerman, K. I. Matthaei, P. S. Foster, and M. E. Rothenberg. 2001. IL-13 induces eosinophil recruitment into the lung by an IL-5- and eotaxin-dependent mechanism. J. Allergy Clin. Immunol. 108:594–601.[Medline]
  43. Li, L., Y. Xia, A. Nguyen, Y. H. Lai, L. Feng, T. R. Mosmann, and D. Lo. 1999. Effects of Th2 cytokines on chemokine expression in the lung: IL-13 potently induces eotaxin expression by airway epithelial cells. J. Immunol. 162:2477–2487.[Abstract/Free Full Text]
  44. Nonaka, M., R. Nonaka, M. Jordana, and J. Dolovich. 1996. GM-CSF, IL-8, IL-1R, TNF-{alpha} R, and HLA-DR in nasal epithelial cells in allergic rhinitis. Am. J. Respir. Crit. Care Med. 153:1675–1681.[Abstract]
  45. Ohno, I., R. Lea, S. Finotto, J. Marshall, J. Denburg, J. Dolovich, J. Gauldie, and M. Jordana. 1991. Granulocyte/macrophage colony-stimulating factor (GM-CSF) gene expression in eosinophils in nasal polyposis. Am. J. Respir. Cell Mol. Biol. 5:505–510.
  46. Sousa, A. R., R. N. Poston, S. J. Lane, J. A. Nakhosteen, and T. H. Lee. 1993. Detection of GM-CSF in asthmatic bronchial epithelium and decrease by inhaled corticosteroids. Am. Rev. Respir. Dis. 147:1557–1561.[Medline]
  47. Marini, M., E. Vittori, J. Hollemborg, and S. Mattoli. 1992. Expression of the potent inflammatory cytokines, granulocyte-macrophage-colony-stimulating factor and interleukin-6 and interleukin-8, in bronchial epithelial cells of patients with asthma. J. Allergy Clin. Immunol. 89:1001–1009.[Medline]
  48. Rohrbach, M., U. Frey, R. Kraemer, and S. Liechti-Gallati. 1999. A variant in the gene for GM-CSF, I117T, is associated with atopic asthma in a population of asthmatic children. J. Allergy Clin. Immunol. 104:247–248.[Medline]
  49. Devalia, J. L., H. Bayram, M. M. Abdelaziz, R. J. Sapsford, and R. J. Davies. 1999. Differences between cytokine release from bronchial epithelial cells of asthmatic patients and non-asthmatic subjects: effect of exposure to diesel exhaust particles. Int. Arch. Allergy Immunol. 118:437–439.[Medline]
  50. Gauvreau, G. M., P. M. O'Byrne, R. Moqbel, J. Velazquez, R. M. Watson, K. J. Howie, and J. A. Denburg. 1998. Enhanced expression of GM-CSF in differentiating eosinophils of atopic and atopic asthmatic subjects. Am. J. Respir. Cell Mol. Biol. 19:55–62.[Abstract/Free Full Text]
  51. Nakamura, Y., T. Ozaki, T. Kamei, K. Kawaji, K. Banno, S. Miki, K. Fujisawa, S. Yasuoka, and T. Ogura. 1993. Increased granulocyte/macrophage colony-stimulating factor production by mononuclear cells from peripheral blood of patients with bronchial asthma. Am. Rev. Respir. Dis. 147:87–91.[Medline]
  52. Christensen, P. J., L. R. Armstrong, J. J. Fak, G. H. Chen, R. A. McDonald, G. B. Toews, and R. Paine III. 1995. Regulation of rat pulmonary dendritic cell immunostimulatory activity by alveolar epithelial cell-derived granulocyte macrophage colony-stimulating factor. Am. J. Respir. Cell Mol. Biol. 13:426–433.[Abstract]
  53. Suda, T., K. McCarthy, Q. Vu, J. McCormack, and E. E. Schneeberger. 1998. Dendritic cell precursors are enriched in the vascular compartment of the lung. Am. J. Respir. Cell Mol. Biol. 19:728–737.[Abstract/Free Full Text]
  54. Wang, J., D. P. Snider, B. R. Hewlett, N. W. Lukacs, J. Gauldie, H. Liang, and Z. Xing. 2000. Transgenic expression of granulocyte macrophage colony-stimulating factor induces the differentiation and activation of a novel dendritic cell population in the lung. Blood 95:2337–2345.[Abstract/Free Full Text]
  55. Bilyk, N., and P. G. Holt. 1993. Inhibition of the immunosuppressive activity of resident pulmonary alveolar macrophages by granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 177:1773–1777.[Abstract/Free Full Text]
  56. Bilyk, N., and P. G. Holt. 1995. Cytokine modulation of the immunosuppressive phenotype of pulmonary macrophage populations. Immunology 86:231–237.[Medline]
  57. Pulendran, B., J. L. Smith, G. Caspary, K. Brasel, D. Pettit, E. Maraskovsky, and C. R. Maliszewski. 1999. Distinct dendritic cell subset differentially regulate the class of immune response in vivo. Proc. Natl. Acad. Sci. USA 96:1036–1041.[Abstract/Free Full Text]
  58. Maldonado-Lopez, R., C. Maliszewski, J. Urbain, and M. Moser. 2001. Cytokines regulate the capacity of CD8{alpha}+ and CD8{alpha}- dendritic cells to prime Th1/Th2 cells in vivo. J. Immunol. 167:4345–4350.[Abstract/Free Full Text]



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