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The Journal of Immunology, 2003, 171: 2374-2383.
Copyright © 2003 by The American Association of Immunologists

Regulation of the Class II MHC Pathway in Primary Human Monocytes by Granulocyte-Macrophage Colony-Stimulating Factor 1

Tara M. C. Hornell2,*, Guy W. Beresford{dagger}, Alyssa Bushey{dagger}, Jeremy M. Boss{dagger} and Elizabeth D. Mellins*

* Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305; and {dagger} Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GM-CSF stimulates the growth and differentiation of hematopoietic progenitors and also affects mature cell function. These effects have led to the use of GM-CSF as a vaccine adjuvant with promising results; however, the mechanisms underlying GM-CSF-mediated immune potentiation are incompletely understood. In this study, we investigated the hypothesis that the immune stimulatory role of GM-CSF is in part due to effects on class II MHC Ag presentation. We find that, in primary human monocytes treated for 24–48 h, GM-CSF increases surface class II MHC expression and decreases the relative level of the invariant chain-derived peptide, CLIP, bound to surface class II molecules. GM-CSF also increases expression of the costimulatory molecules CD86 and CD40, but not the differentiation marker CD1a or CD16. Furthermore, GM-CSF-treated monocytes are better stimulators in a mixed leukocyte reaction. Additional analyses of the class II pathway revealed that GM-CSF increases total protein and RNA levels of HLA-DR, DM, and DO{alpha}. Expression of class II transactivator (CIITA) types I and III, but not IV, transcripts increases in response to GM-CSF. Furthermore, GM-CSF increases the amount of CIITA associated with the DR promoter. Thus, our data argue that the proinflammatory role of GM-CSF is mediated in part through increased expression of key molecules involved in the class II MHC pathway via induction of CIITA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Monocytes/macrophages play an important role in host defense, acting as APCs to activate T cells and producing a variety of inflammatory mediators to influence immune responses. Monocytes/Macrophages are important not only in lymphoid tissues, but are also critical for activation of effector and memory T cells at the site of infection or inflammation. Such activation can be beneficial in the case of a response to pathogens or tumors, but deleterious in autoimmune responses. Thus, an understanding of the regulation of monocyte/macrophage Ag-presenting capability is critical to devising ways to manipulate immune responses. During inflammation or a local infection, chemokines and/or inflammatory mediators are generated locally which stimulate monocytes to migrate into the site (1). In response to these environmental signals, monocytes, along with tissue macrophages already present, develop mature Ag presentation function, enabling efficient presentation to T cells. Before activation, however, these cells are not efficient stimulators of T cells. This is in part due to low cell surface expression of MHC molecules and costimulatory molecules such as CD40, CD80, and CD86 (2, 3). Although the action of some mediators, e.g., IFN-{gamma}, of monocyte/macrophage APC function has been well characterized, other mediators important for their activation have been poorly studied.

Critical for the development of mature APC capability is the expression of surface class II MHC molecules stably loaded with peptide. Expression of several molecules in addition to class II MHC molecules is important for this to occur. These include invariant chain (Ii), 3 cathepsins, and, in particular, the nonclassical class II molecule HLA-DM. DM acts intracellularly on class II molecules loaded with class II-associated Ii-derived peptides (CLIP) to catalyze peptide exchange and stabilize empty class II molecules. Cell lines deficient in DM exhibit increased CLIP levels and, in some MHC alleles, decreased surface class II expression (4). Another nonclassical class II molecule, HLA-DO, associates tightly with DM and may modulate DM function (5, 6, 7). Although DO expression has been reported in B cells and thymic epithelial cells, the available data suggest that DO is not expressed by monocytes/macrophages (8, 9, 10, 11, 12). An understanding of the regulation of class II MHC as well as molecules involved in class II peptide loading is critical for efforts to manipulate Ag presentation.

Many of these molecules, class II MHC, DM, Ii, and DO{alpha} (but not DO{beta}), are regulated coordinately, in part through dependence on expression of class II transactivator (CIITA) (13). CIITA expression is regulated transcriptionally and is under the control of four distinct promoters in human cells (13). These promoters are differentially used in different cell types and in response to inflammatory stimuli. For example, in response to IFN-{gamma} CIITA transcription has been found to be induced by activation of promoter IV and in some cases an element upstream of promoter III in various human cell lines (14, 15). However, the CIITA promoter(s) responsible for CIITA expression in primary human monocytes in response to other inflammatory cytokines is unknown.

GM-CSF was first identified for its role in the proliferation of hematopoietic progenitor cells and their differentiation into granulocytes and monocytes (16). GM-CSF is now viewed as a regulator of granulocyte and monocyte lineage cells at all stages of maturation, with effects on phagocytosis, oxidative metabolism, cytokine secretion, cytotoxicity, and Ag presentation capability (17). This has led to the clinical use of GM-CSF to treat fungal infections and as a vaccine adjuvant, in addition to its use to mobilize hematopoietic progenitors (18, 19). However, the precise mechanisms by which GM-CSF mediates these immune potentiating effects are unknown. In this study, we hypothesized that GM-CSF activates the class II MHC pathway, directly enhancing APC function. This was tested by evaluating the effects of GM-CSF on purified human monocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

Buffy coats were obtained from the Stanford Blood Center, and PBMCs were isolated by separation with Ficoll-Paque Plus (Amersham Biosciences, Piscataway, NJ). Primary human monocytes were isolated from PBMC by negative selection using Miltenyi Biotec (Auburn, CA) MACS monocyte isolation kit, as directed by the manufacturer. The purity of the monocyte isolation was confirmed by flow cytometric analysis using anti-CD14 FITC, with >90% positive cells. Purified monocytes were incubated in IMDM (Life Technologies, Rockville, MD) supplemented with 10% FBS (HyClone Laboratories, Logan, UT) and 2 mM L-glutamine (Life Technologies) at 37°C, 5% CO2.

Immature monocyte-derived dendritic cells were generated from purified monocytes, as previously described (20). Briefly, monocytes were cultured for 7 days in IMDM (Life Technologies) supplemented with 10% FBS (HyClone Laboratories), 2 mM L-glutamine (Life Technologies), 800 U/ml GM-CSF (R&D Systems, Minneapolis, MN), and 1000 U/ml IL-4 (R&D Systems), with the medium replaced after 3 and 5 days of incubation with freshly added 800 U/ml GM-CSF and 500 U/ml IL-4. RNA isolated from these cells was used as a positive control for the type I CIITA transcript in real-time RT-PCR assays.

The Raji cell line was cultured in RPMI medium (Life Technologies) supplemented with 10% FBS (HyClone Laboratories) and 2 mM L-glutamine (Life Technologies).

Staphylococcal enterotoxin B (SEB) stimulation

Blood from random donors was collected in sodium heparin and transferred to polypropylene tubes. SEB (Sigma-Aldrich, St. Louis, MO) was added to a final concentration of 1 or 10 µg/ml, and the blood was incubated for 6 h at 37°C with frequent mixing. RNA was harvested using TRIzol LS (Life Technologies), according to the manufacturer’s instructions.

Cytokines

Human rGM-CSF from R&D Systems or from Immunex (Seattle, WA) (Leukine) was used in all experiments at a dose of 800 U/ml. Human rIL-4 was obtained from R&D Systems. Human rIFN-{gamma} from Roche (Indianapolis, IN) was used at a dose of 100 U/ml.

Antibodies

L243 is a murine IgG2a mAb that recognizes a monomorphic determinant in the first domain of the DR{alpha} chain (21). The mAb CerCLIP, specific for CLIP-loaded class II molecules, was a kind gift from P. Cresswell (Yale University, New Haven, CT). FITC-conjugated Abs to CD1a, CD14, CD16, CD80, and CD86 and the appropriate isotype controls were purchased from Caltag Laboratories (Burlingame, CA). Anti-CD40 PE and the appropriate isotype control were purchased from BD PharMingen (San Diego, CA).

Mouse mAbs with the following specificities were used for Western blotting: DA6.147 (DR{alpha}), 5C1 (DM{alpha}) was a kind gift from J. Trowsdale (University of Cambridge, Cambridge, U.K.), 47GS4 (DM{beta}) was a generous gift from S. Pierce (Northwestern University, Chicago, IL), anti-{beta}-actin Ab was obtained from Sigma-Aldrich, and Pin1.1 (Ii chain) was kindly provided by P. Cresswell. Rabbit antiserum specific for DO{alpha} (SU66) was generated against the following peptide, CMGTYVSSVPR. Rabbit antiserum specific for DO{beta} (K571) was a generous gift from L. Karlsson (R. W. Johnson Pharmaceutical Research Institute, San Diego, CA), and antiserum specific for DR (CHAMP) was a kind gift from L. Stern (University of Massachusetts Medical School, Worcester, MA).

Flow cytometry

For analysis of surface class II MHC and CLIP/class II expression, cells were first incubated with blocking buffer (PBS + 5% human AB serum (Gemini Bioproducts, Woodland, CA) + 5% goat serum (Caltag)) for 15 min on ice, washed with PBS + .2% BSA (Sigma-Aldrich), and incubated for 30 min with mAbs to HLA-DR (L243) and CLIP-loaded class II molecules (CerCLIP) on ice diluted in blocking buffer, washed, incubated with FITC-labeled goat F(ab')2 anti-mouse IgG (Caltag Laboratories), washed again, and analyzed using a FACScan flow cytometer (BD Biosciences, Mountain View, CA). Staining for CD1a, CD14, CD16, CD40, CD80, and CD86 was performed as above, but without addition of the secondary Ab.

Mixed leukocyte reaction

Purified monocytes, incubated with either 800 U/ml GM-CSF or medium alone for 24–48 h, were irradiated (3000 rad). Various numbers of these cells were mixed with 105 purified allogeneic T cells/well in a 96-well plate. T cells were purified using RosetteSep T cell enrichment cocktail from StemCell Technologies (Vancouver, British Columbia, Canada), according to the manufacturer’s instructions. Cells were incubated at 37°C, 5% CO2 for 4–6 days in RPMI (Life Technologies) supplemented with 10% heat-inactivated human AB serum (Gemini Bioproducts), 2 mM glutamine (Life Technologies), 10 mM HEPES (Life Technologies), 1 mM sodium pyruvate (Life Technologies), and 0.1 mM nonessential amino acids (Life Technologies). [3H]Thymidine (from PerkinElmer Life Sciences, Boston, MA) incorporation was measured following 15- to 17-h pulses using a Tomtec Harvestor (Hamden, CT) and a Wallac Microbeta Jet 1450 {beta}-reader (PerkinElmer). Results are shown as mean cpm of triplicates.

IL-10 ELISA

IL-10 levels in supernatant from monocytes grown in medium alone or GM-CSF were determined using the Quantikine human IL-10 immunoassay from R&D Systems, according to the manufacturers’ instructions.

Western blotting

Cells were harvested and lysed in 50 mM Tris-HCL, (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 1 mM MnCl2, 1 mM CaCl2, plus protease inhibitors. Unextracted material was pelleted, and the amount of protein in the supernatant was quantitated by Bradford assay and normalized between samples. Equal protein equivalents of lysate were mixed with 20 µl Con A-Sepharose (Sigma-Aldrich). After rotating overnight at 4°C, Con A-Sepharose pellets were washed four times in 0.75 ml lysis buffer. Glycoproteins were boiled in Laemmli SDS-PAGE sample buffer with 2-ME. The eluted precipitates were run on 12% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (immobilon P; Millipore, Bedford, MA). Alternatively, for {beta}-actin staining, equal amounts of lysate prepared and quantitated as described above were mixed with Laemmli SDS-PAGE sample buffer with 2-ME, boiled, run on 12% SDS-PAGE gels, and transferred to polyvinylidene difluoride membranes. Membranes were blocked overnight at 4°C in 100 mM Tris-HCl (pH 7.7), 200 mM NaCl, 1% casein (Hammerstein grade; ICN Pharmaceuticals, Costa Mesa, CA), 0.05% Tween 20, and 0.05% NaN3, and incubated with the appropriate Abs diluted in blocking buffer. After washing in TBS + 0.05% Tween 20, HRP-conjugated secondary Abs (donkey anti-rabbit Ig, Amersham; or goat anti-mouse Ig, Caltag) were added in TBS-Tween containing 5% nonfat dry milk. Following additional washes, ECL substrate was added (Renaissance; DuPont NEN, Boston, MA), and the blots were exposed to film (Hyperfilm ECL; Amersham). Densitometric analysis was performed using a Bio-Rad GS-710 Calibrated Imaging Densitometer (Hercules, CA).

RNA isolation and reverse transcription

RNA was isolated using TRIzol (Life Technologies) or Qiagen RNeasy kits (Valencia, CA), according to the manufacturer’s instructions. RNA was quantitated by absorbance at 260 nm, and 0.5 µg of RNA was reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI).

RT-PCR and real-time RT-PCR

PCR using IFN-{gamma}-specific primers (5'-GCATCCAAAAGAGTGTGGAG and 5'-GACAGTTCAGCCATCACTTGG) was performed under the following conditions: 3 min at 95°C and 40 cycles of 95°C for 60 s, 57°C for 60 s, and 72°C for 90 s. PCR products were run on 2% agarose gels.

For real-time PCR, primers were designed using Primer Express software (Applied Biosystems, Foster City, CA). The primer sets used were: DMA, 5'-TGATCCAGCAAATAGGGCCA and 5'-CTCTGGACACCGGGATTTTC; DMB, 5'-AAAGACACCCTGATGCAGCG and 5'-TGTGGCACAATTCTGAAGCC; DOA, 5'-TGGCCCAGACCAGCTTCTAT and 5'-GGAACTTGCGGAACAAATGG; DOB, 5'-GATTCAGGCAAAGGCTGACTG and 5'-TGCACCTTTTCTGTCCCGTT; DRA, 5'-GTCTGGCGGCTTGAAGAATT and 5'-ACCTTGAGCCTCAAAGCTGG; DRB, 5'-CGTGACAAGCCCTCTCACAG and 5'-TGTGCAGATTCAGACCGTGC; CIITA, 5'-GCTCTGAGTGGCGAAATCAAG and 5'-CAATGCTAGGTACTGCGGGAG; GAPDH, 5'-TGGGCTACACTGAGCACCAG and 5'-GGGTGTCGCTGTTGAAGTCA; type I CIITA, 5'-GGAGACCTGGATTTGGCCCT and 5'-GAAGCTCCAGGTAGCCACCTTCTA; type III CIITA, 5'-GGGGAAGCTGAGGGCACG and 5'-GAAGCTCCAGGTAGCCACCTTCTA; and type IV CIITA, 5'-GCGGCCCCAGAGCTGG and 5'-GAAGCTCCAGGTAGCCACCTTCTA. The 2x SYBR Green PCR Master Mix (Applied Biosystems) was used in PCR with 200 nM of forward and reverse primers, 10–40 ng of the reverse-transcription product, and RNase/DNase-free water to 50 µl. The PCR mixtures were transferred to MicroAmp optical 96-well reaction plates and run on the Applied Biosystems GeneAmp 5700 Sequence Detection System under the following conditions: 2 min at 50°C, 10 min at 95°C, and 40 cycles of 95°C for 15 s and 60°C for 1 min. To determine relative quantity, control cDNA generated from Raji cell, monocyte-derived dendritic cell, or IFN-{gamma}-stimulated monocyte total RNA was used to generate a standard curve. Relative quantities of the gene of interest were determined for unknown samples by comparison with this standard curve, and normalized to GAPDH quantities. Fold changes in expression were determined by dividing the normalized quantity of the gene of interest from stimulated (with GM-CSF or IFN-{gamma}) monocytes by the normalized quantity of the gene of interest from monocytes incubated with medium alone. All real-time RT-PCR experiments were performed at least three times, and the average of these experiments is shown relative to levels of monocytes incubated with medium alone.

Chromatin immunoprecipitation (ChIP) assays

ChIPs were performed, as previously described (22). Briefly, formaldehyde cross-linked chromatin was prepared from between 1.7 x 107 and 4.1 x 107 cells for each experimental condition. The CIITA (anti-MBP-CIITA)-specific antiserum used to precipitate CIITA complexed with chromatin has been previously described (22). For each precipitation, 60 µl of protein A-Sepharose beads was used. After the wash steps, cross-links were reversed, and the DNA was purified and analyzed by real-time PCR.

Quantitative PCR was performed using an iCycler unit with an optical assembly (BioRad Laboratories, Hercules, CA). For quantification of PCR product, SYBR Green incorporation was determined. The average values for each sample were normalized to the amount of input chromatin. The primers used were: HLA-DRA forward, 5'-GATCTCTTGTGTCCTGGACCCTTTGCAAGAACCCT-3', and HLA-DRA reverse, 5'-CCCAATTACTCTTTGGCCAATCAGAAAAATATTTTG-3'.

Statistics

The statistical significance of differences among results between medium and GM-CSF-treated monocytes was evaluated by the Student’s t test (analysis toolpack; Excel). Values of p were determined using the one-tailed t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GM-CSF increases surface class II MHC and costimulatory molecule expression in primary human monocytes without evidence of differentiation

The effect of GM-CSF on surface expression of MHC class II (HLA-DR) and costimulatory molecules by primary human monocytes was assessed by flow cytometry. As shown in Fig. 1A, incubation with GM-CSF leads to an increase in surface DR expression. On average (n = 17), surface class II expression is increased 2-fold (p < 5 x 10-8) by GM-CSF, and the response peaks after 24–48 h of incubation. As shown in Fig. 1B, GM-CSF increases surface expression of the costimulatory molecules CD40 and CD86 by human monocytes, while in most donors no CD80 expression is detected either in the presence or absence of GM-CSF. On average, GM-CSF increases CD40 expression 3.6-fold (n = 5, p < 0.005) and CD86 2.5-fold (n = 7, p < 0.05).



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  		FIGURE 1. GM-CSF increases surface class II MHC and costimulatory molecule expression in primary human monocytes. A, Expression of MHC class II, as detected by flow cytometry using the HLA-DR-specific mAb L243, is shown after 24 h of incubation with medium alone (open) or 800 U/ml GM-CSF (shaded). Staining with the secondary Ab alone is shown. B, Expression of CD86, CD40, and CD80 by human monocytes is shown after 24–48 h of incubation with medium alone (open) or 800 U/ml GM-CSF (shaded). Staining with isotype control Abs is shown.

 
To examine whether GM-CSF-mediated effects on surface class II MHC and costimulatory molecule expression represent changes due to activation of the cells or are part of a differentiation pathway, expression of CD1a and CD16 was examined following GM-CSF treatment. Incubation of human monocytes with GM-CSF and IL-4 for 7 days results in the generation of immature CD1a+ dendritic cells, while incubation of monocytes with medium alone or GM-CSF for 7 days leads to the generation of CD16+ macrophages (20). Thus, an increase in CD1a+ or CD16+ cells following GM-CSF treatment may reflect differentiation of monocytes into dendritic cells or macrophages, respectively. However, as shown in Fig. 2, GM-CSF does not increase monocyte CD1a or CD16 expression in the time frame in which changes in class II MHC and costimulatory molecule expression are detected. In fact, GM-CSF-treated monocytes express less CD16 compared with untreated monocytes after 48 h (Fig. 2B). There is a small increase in the percentage of CD1a-positive cells seen after GM-CSF treatment (Fig. 2A), but this varies with the donor, and no more than 10% of the cells are CD1a positive in the time period examined. In addition, the morphology of GM-CSF-treated monocytes (up to 48 h) does not resemble either that of immature dendritic cells or macrophages (data not shown). These data suggest that GM-CSF-treated monocytes do not differentiate during the time frame in which GM-CSF increases class II MHC and costimulatory molecule expression, and instead these changes likely represent an acute activation event.



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  		FIGURE 2. GM-CSF does not induce expression of the differentiation markers CD1a or CD16. Expression of CD1a (A) and CD16 (B) is shown after 48 h of incubation with medium alone (left) or 800 U/ml GM-CSF (right). The open histograms are staining with the isotype control Ab, IgG1-PE; shaded histograms are staining with anti-CD1a PE or anti-CD16 PE.

 
GM-CSF treatment of human monocytes decreases relative CLIP/class II levels

To determine whether GM-CSF affects the peptide loading of class II MHC molecules, we examined the relative levels of CLIP-loaded surface class II MHC molecules in GM-CSF-treated and untreated monocytes. Release of CLIP from class II molecules is required for association with potentially antigenic peptides, and for many MHC alleles, is also required for the generation of stable peptide-MHC complexes capable of survival at the cell surface. Thus, a comparison of the relative level of CLIP-loaded class II molecules provides an indication of the potential peptide diversity presented on the surface of the monocytes. After 24–48 h of incubation with GM-CSF, monocytes from five of nine experiments express less surface CLIP-class II/total class II compared with monocytes incubated with medium alone, as measured by flow cytometry, using mAb specific for CLIP and DR (Fig. 3). This modest, but significant, difference (p < 0.005) suggests that more efficient CLIP release and peptide loading occur in the presence of GM-CSF. On average, GM-CSF-treated monocytes from these donors express .57-fold of the level of relative CLIP/class II surface levels as untreated monocytes. Monocytes from the remaining four experiments did not appreciably change or expressed higher relative CLIP class II levels after treatment with GM-CSF, suggesting that there is variability between donors. This is likely due to allelic MHC differences that affect binding to CLIP and dependence on HLA-DM (23, 24). The finding that GM-CSF decreases the relative level of CLIP-loaded class II molecules on the surface of primary human monocytes suggests that HLA-DM expression and/or function may be enhanced, leading to the generation of more stable peptide/MHC complexes on the cell surface.



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  		FIGURE 3. GM-CSF decreases relative surface CLIP/class II levels. CLIP-loaded class II and total HLA-DR levels were measured by flow cytometry using the mAbs CerCLIP and L243, respectively. CerCLIP/L243 mean fluorescence intensity ratios (x100) of monocytes incubated with medium alone, or 800 U/ml GM-CSF, for 24–48 h were determined. The fold difference in CerCLIP/L243 levels between monocytes incubated with 800 U/ml GM-CSF compared with incubation with medium alone is shown.

 
GM-CSF enhances monocyte APC function

To test whether GM-CSF-mediated increases in surface class II MHC, CD40, and CD86 expression and decreased relative CLIP-loaded class II molecules are associated with an altered ability of monocytes to stimulate an allogeneic T cell response, GM-CSF-treated or untreated monocytes were irradiated and incubated with purified allogeneic T cells and T cell proliferation measured after 4–6 days. We found that allogeneic T cells consistently proliferate more in response to GM-CSF-treated monocytes compared with monocytes incubated with medium alone. At stimulator to responder ratios of 2000 or 8000 monocytes per 105 allogeneic T cells, GM-CSF-treated monocytes stimulate 2- to 3-fold more T cell proliferation than untreated monocytes (Fig. 4). At higher stimulator to responder ratios, the difference between the T cell proliferation stimulated by GM-CSF-treated vs untreated monocytes decreases (data not shown). The observed difference in T cell proliferation in response to GM-CSF-treated monocytes compared with untreated monocytes is likely due, at least in part, to the increase in surface class II MHC expression seen in GM-CSF-treated vs untreated monocytes. Thus, we sought to examine the mechanisms responsible for GM-CSF-mediated increase in surface class II expression.



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  		FIGURE 4. GM-CSF enhances monocyte APC function. Monocytes were incubated with 800 U/ml GM-CSF (shaded) or medium (open) for 48 h, irradiated (3000 rad), and used as stimulators for 1 x 105 allogeneic purified T cells at the indicated concentrations. T cell proliferation was measured after 5 days by pulsing with [3H]thymidine and measuring incorporation, as described in Materials and Methods. The experiment shown is representative of three independent experiments. *, The difference in the T cell response to medium vs GM-CSF-treated monocytes is statistically significant, p < 0.05.

 
GM-CSF increases total protein levels of DR, DM, Ii, and DO{alpha}, but not DO{beta} by human monocytes

To test whether the increased surface class II MHC expression and decreased relative CLIP-class II/total class II levels are due to an increase in total protein levels of MHC class II molecules and the accessory molecule HLA-DM, Western blot analysis was performed. As shown in Fig. 5, an increase in cellular DR levels is seen after GM-CSF treatment. Correlating with the time course of increased DR surface expression, total DR levels peak between 24 and 48 h of incubation with GM-CSF. Densitometric analysis revealed that GM-CSF increases DR{alpha} and DR{beta} levels on average 8.5- and 4.6-fold, respectively.



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  		FIGURE 5. GM-CSF increases total protein expression of HLA-DR, DM, Ii, and DO{alpha} by monocytes, but has no effect on DO{beta}. Total cellular DR{alpha}, DR{beta}, DM{alpha}, DM{beta}, Ii, DO{alpha}, and DO{beta} in monocytes incubated with medium or 800 U/ml GM-CSF for 48 h were determined by Western blot analysis using the following Abs: DA6.147, CHAMP, 5C1, 47G.S4, Pin1.1, SU66, and K571, respectively. Expression of DO{beta} by Raji cells is shown as a positive control for the K571 Ab. Total cellular {beta}-actin levels are also shown to control for equal protein quantitation. Results shown are representative of at least three independent experiments.

 
A similar analysis was done to examine the effect of GM-CSF on monocyte expression of the accessory molecules Ii, DM, and DO. Ii is crucial for proper trafficking of class II molecules to peptide-loading compartments (25). DM plays a critical role in stable peptide binding by class II molecules, while DO is thought to play an inhibitory role, but has not been found to be expressed in monocytes (4, 12). The decrease in relative CLIP/class II levels seen following GM-CSF treatment may reflect a change in DM, Ii, and/or DO expression, and this was tested directly. Expression of both the {alpha}- and {beta}-chains of DM and DO was examined, as, at least in the case of DO, the two chains are known to be regulated distinctly (26). As shown in Fig. 5, GM-CSF increases expression of HLA-DM{alpha} 3-fold, DM{beta} 10.5-fold, DO{alpha} 33-fold, and Ii 5.5-fold. However, no DO{beta} is detectable in monocytes incubated with either medium alone or GM-CSF. As a control for equal protein quantitation, staining for {beta}-actin is also shown. These results indicate that increased surface expression of MHC class II molecules and decreased levels of surface CLIP-loaded class II molecules relative to total class II molecules following GM-CSF treatment are likely due to increased protein levels of DR and DM.

GM-CSF does not mediate its effects on class II MHC expression by inducing IFN-{gamma} or decreasing IL-10 production in human monocytes

As GM-CSF-mediated effects on class II expression are not immediate, but occur in a time frame that would allow synthesis of additional mediators, we tested whether GM-CSF increases expression of class II MHC and accessory molecules indirectly by inducing expression of IFN-{gamma}. Although monocytes are not traditionally considered to be capable of producing IFN-{gamma}, recent reports indicate that following treatment with IL-12 and/or IL-18, monocytes do produce measurable quantities of IFN-{gamma} (27). As IFN-{gamma} is a potent inducer of class II MHC and DM expression, it was important to test whether GM-CSF mediates its effect indirectly through induction of IFN-{gamma}. RT-PCR was performed using primers that specifically amplify IFN-{gamma} RNA. However, as shown in Fig. 6A, no detectable IFN-{gamma} RNA is found following GM-CSF treatment of monocytes. Thus, GM-CSF up-regulation of class II is not mediated via production of IFN-{gamma}.



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  		FIGURE 6. GM-CSF does not mediate its effects on class II expression by inducing IFN-{gamma} production or decreasing IL-10 production in human monocytes. A, Purified human monocytes were incubated with medium alone, 800 U/ml GM-CSF, or 100 U/ml IFN-{gamma} for 24 h, and RNA was harvested. RT-PCR was performed using primers specific for IFN-{gamma}, and the products were run on a 2% agarose gel. As a positive control, RNA from unstimulated and SEB-stimulated whole blood was used. The size of the IFN-{gamma}-specific band (169 bp) is indicated by the arrow on the right. B, Purified human monocytes were incubated with medium alone or 800 U/ml GM-CSF for 24–48 h, and culture supernatant was harvested. IL-10 levels were determined using R&D Systems Quantikine IL-10 Immunoassay kit. Data are shown as fold change in IL-10 following GM-CSF treatment for the indicated time. Experiments were performed at least three times. The difference in IL-10 production between medium and GM-CSF-treated monocytes is not significant, p = 0.12 (24 h) and p = 0.2 (48 h).

 
We also tested whether GM-CSF acts to increase surface class II levels indirectly by decreasing monocyte production of IL-10. Reduction of surface class II expression is an autocrine effect of IL-10 on monocytes, which is observed in cultured human monocytes (28). If GM-CSF decreases monocyte production of IL-10, this could indirectly lead to increased cell surface levels of class II MHC. To test this possibility, the level of IL-10 secreted by GM-CSF-treated monocytes relative to monocytes incubated with medium alone was determined by ELISA. As shown in Fig. 6B, GM-CSF-treated monocytes do not express less IL-10 than untreated monocytes, arguing that GM-CSF does not mediate its effects on surface class II MHC expression via reduction of IL-10 production.

GM-CSF increases DR, DM, and DO{alpha} mRNA levels in human monocytes

To test whether the increase in protein levels of DR, DM, and DO{alpha} seen following GM-CSF treatment is associated with an increase in RNA levels, real-time RT-PCR was performed using primers specific for DRA, DRB, DMA, DMB, DOA, and DOB transcripts. As shown in Fig. 7, after 24 h of incubation GM-CSF increases expression of DRA, DRB, DMA, DMB, and DOA RNA 2.5- to 3-fold. DOB RNA levels are unchanged, or decrease slightly. Thus, GM-CSF likely mediates enhanced class II MHC, DM, and DO{alpha} expression through a transcriptional or posttranscriptional mechanism.



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  		FIGURE 7. DR, DM, and DO{alpha} expression are regulated transcriptionally by GM-CSF, while DO{beta} mRNA levels are unchanged following GM-CSF treatment. RNA was harvested from monocytes incubated with 800 U/ml GM-CSF or medium alone for 24 h and reverse transcribed. Real-time PCR was performed using primers specific for DRA, DRB, DMA, DMB, DOA, and DOB, and the relative quantity of each transcript was determined after normalization to the quantity of GAPDH. The fold change in expression of these transcripts in GM-CSF-stimulated monocytes compared with unstimulated monocytes is shown. These changes, with the exception of DOB, are statistically significant, p < 0.05.

 
GM-CSF increases total CIITA mRNA levels in human monocytes

Similarities have been found in the transcriptional regulation of class II MHC genes as well as the genes encoding the accessory molecules DM and DO{alpha} (29). Coordinate regulation of the above genes has been shown to involve several transcription factors, including the transcriptional activator CIITA. CIITA expression is correlated with class II expression, and its absence in humans leads to severe immunodeficiency (13). We sought to address whether GM-CSF induces class II MHC and accessory molecule expression via induction of CIITA. Real-time RT-PCR analysis was performed using RNA purified from untreated and GM-CSF-treated monocytes. As shown in Fig. 8, GM-CSF increases CIITA RNA levels on average 5.5-fold in human monocytes. This is the first demonstration of a GM-CSF-mediated effect on CIITA expression.



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  		FIGURE 8. GM-CSF increases total CIITA mRNA levels in human monocytes. Relative expression of total CIITA mRNA was determined by real-time RT-PCR and normalized to expression of GAPDH. The fold change in total CIITA expression in monocytes treated with GM-CSF for 24 h compared with monocytes incubated with medium alone is shown and is statistically significant, p < 0.05.

 
GM-CSF increases types I and III CIITA mRNA in primary human monocytes, while IFN-{gamma} increases types III and IV CIITA transcripts

CIITA is a complexly regulated gene, with three promoters that are known to display distinct cell type- and cytokine-specific responses (13). To provide clues as to the mechanisms involved in GM-CSF-mediated regulation of CIITA expression, we determined the promoter(s) induced by GM-CSF. Transcription initiated by each of the CIITA promoters leads to the synthesis of distinct CIITA mRNAs containing alternative first exons spliced to a shared second exon. Real-time RT-PCR analysis, using primers specific for each product, was performed to determine the CIITA promoter(s) induced by GM-CSF. As shown in Fig. 9, GM-CSF induces types I and III CIITA RNA, but has no effect on type IV CIITA in primary human monocytes. This is in contrast to IFN-{gamma}, which induces expression of types III and IV CIITA in primary human monocytes (Fig. 9).



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  		FIGURE 9. GM-CSF induces types I and III CIITA but not type II transcripts, in monocytes. A, Relative expression of types I, III, and IV CIITA was determined by real-time RT-PCR and normalized to expression of GAPDH. Relative expression of each transcript was determined by generating standard curves. For type I CIITA, cDNA generated from immature monocyte-derived dendritic cell RNA was used to generate the standard curve, while for type III CIITA, cDNA generated from Raji RNA was used, and for type IV CIITA, cDNA generated from IFN-{gamma}-stimulated monocyte RNA was used. Fold change in expression of types I, III, and IV CIITA by GM-CSF- and IFN-{gamma}-treated monocytes compared with monocytes incubated with medium alone for 24 h is shown. GM-CSF-mediated increases in types I and III CIITA are statistically significant, p < 0.05. B–D, Time courses of type I (B), type III (C), and type IV (D) CIITA expression in monocytes incubated with medium, GM-CSF, or IFN-{gamma} for the indicated times. Relative normalized levels of each CIITA transcript are shown.

 
There are additional differences between GM-CSF- and IFN-{gamma}-induced increases in CIITA expression. Kinetic experiments demonstrate that the increase in CIITA transcript following GM-CSF treatment occurs at the 18 h time point, while IFN-{gamma}-mediated increases in CIITA transcript levels are detected at the 1.5 h time point (Fig. 9, B–D). This suggests that GM-CSF and IFN-{gamma} increase CIITA transcription through distinct mechanisms. Of note, monocytes incubated with medium alone increase expression of type III CIITA slowly with time, expressing 144-fold more transcript by 48 h compared with 1.5 h of incubation (Fig. 9C). In contrast, time course experiments demonstrate that GM-CSF and IFN-{gamma} transiently increase CIITA levels. By 48 h, CIITA levels in GM-CSF- or IFN-{gamma}-activated monocytes decrease substantially from peak levels (Fig. 9, B–D).

GM-CSF increases the amount of CIITA associated with the DRA promoter

The increase in CIITA RNA seen in response to GM-CSF treatment may be responsible for the transcriptional increase in DR, DM, and DO{alpha} expression observed. To test this, we performed ChIP assays to directly quantitate the amount of CIITA associated with the DRA promoter in the presence or absence of GM-CSF. Incubation with GM-CSF increases the amount of CIITA associated with the DRA promoter by 3.3-fold (n = 5, p < 0.05) (Fig. 10). This provides direct evidence that the increase in CIITA message seen following GM-CSF treatment leads to an increase in the amount of CIITA associated with the DRA promoter, leading to increased transcription of DRA.



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  		FIGURE 10. Association of CIITA with the DRA promoter increases following GM-CSF treatment of human monocytes. Real-time ChIP analysis using Abs to CIITA. Fold increase compared with untreated cells is shown; data are mean ± SEM of five separate experiments. The increase in CIITA associated with the DRA promoter observed following GM-CSF treatment is statistically significant, p < 0.05.

 
Together these data show that GM-CSF activates monocyte APC function by increasing expression of costimulatory molecules as well as several molecules important for class II MHC Ag presentation. This latter effect is mediated by GM-CSF induction of CIITA expression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study elucidates the effects of GM-CSF on the expression of molecules important for class II MHC Ag presentation in human monocytes. GM-CSF induces expression of surface and total class II MHC, the class II-like molecule DM that is crucial for the formation of stable peptide/MHC complexes, Ii, and the {alpha}-chain of DO. In addition, increased surface expression of the costimulatory molecules CD40 and CD86 is seen following GM-CSF treatment. Together these changes most likely contribute to enhanced recognition of GM-CSF-treated monocytes by T cells. Furthermore, we show that these effects on the class II pathway result from an increase in total protein and RNA levels of class II, DM, and DO{alpha} via induction of CIITA by GM-CSF. We demonstrate that incubation with GM-CSF increases the amount of CIITA associated with the DRA promoter. As the expression of DR{beta}, DM{alpha}, DM{beta}, and DO{alpha} are similarly regulated by CIITA, this result further suggests that the increased expression of these molecules induced by GM-CSF may be due to an increased amount of CIITA associated with the promoters of these genes.

These changes are seen in monocytes after 24–48 h of culture with GM-CSF, and may represent the initial stages of differentiation or, alternatively, activation. Culturing of monocytes for 7 days in the presence of GM-CSF yields CD16bright macrophages, while incubation with GM-CSF and IL-4 for 7 days results in CD1a+, CD14dim dendritic cells (20). We observe a decrease in surface CD14 expression on monocytes following GM-CSF treatment (Hornell, unpublished data); however, overall our data suggest that GM-CSF activates monocytes during this brief culture period. GM-CSF-mediated effects peak by 24–48 h of culture and are transient. In addition, CD1a levels remain low in GM-CSF-treated monocytes even after 48 h, and CD16 levels actually decrease, indicating that the cells are not becoming CD16bright macrophages. This observed decrease in CD16 expression likely represents the presence of CD14+CD16+ monocytes, which comprise, on average, 10% of total monocytes, and whose expression of CD16 decreases following activation (Hornell, unpublished data). Understanding the pathways initiated through these in vitro processes may have important implications for mechanisms involved in in vivo monocyte activation. In addition, GM-CSF-mediated activation of the class II MHC pathway may also occur in other APCs, such as tissue macrophages and dendritic cells.

We demonstrate in this study that GM-CSF is a potent up-regulator of several molecules important for class II MHC Ag presentation: DR, DM, Ii, and DO{alpha}. All of these molecules are coordinately regulated by CIITA (29). Interestingly, while the expression of DO{alpha} is increased, no DO{beta} is detected by Western blot. In addition, sensitive real-time RT-PCR data indicate that GM-CSF does not increase DO{beta} transcript levels despite increasing DO{alpha} levels. It has been previously shown that the two chains of DO are differentially regulated, and that in B cells DO{beta} expression is influenced by, but not dependent upon, CIITA (26, 30). This raises the question of whether DO{alpha} expressed in the absence, or in excess, of DO{beta} can function independently from, or with another partner than, DO{beta}.

Our findings have important implications for understanding the regulation of CIITA expression. CIITA, which functions as a master regulator of MHC class II transcription by coordinating the assembly of the transcriptional apparatus at MHC class II promoters, is itself regulated at the transcriptional and posttranscriptional levels (13). Importantly, we find that GM-CSF increases CIITA mRNA levels. Each of the three active CIITA promoters encodes a distinct first exon to the CIITA protein. The use of promoter I (PI) provides CIITA with the greatest transcriptional potential (31). Understanding the regulation of CIITA in different cell types and in response to different mediators will help uncover the signaling pathways involved and also the distinct functions that different CIITA types may play. Previous studies using mouse and human cell lines and primary cells have shown that PI is active in dendritic cells as well as in murine macrophages and microglia in response to IFN-{gamma}; promoter III (PIII) is active in dendritic cells, T and B lymphocytes, as well as IFN-{gamma}-activated cells; and promoter IV (PIV) is IFN-{gamma} responsive in murine and human macrophage and microglial cell lines as well as in non-bone marrow-derived cells such as fibroblast, endothelial, and epithelial cells and cell lines, and astrocytes, and is also responsible for CIITA expression in murine cortical thymic epithelial cells (14, 15, 32, 33, 34, 35, 36).

In this study, we demonstrate that freshly isolated monocytes express low levels of CIITA and that GM-CSF specifically increases types I and III CIITA. This is the first report examining the regulation of CIITA expression in primary human monocytes, and the first evidence that PI is inducible in human cells. Previous studies in primary human monocytes are limited to a single observation that type III CIITA is expressed in freshly isolated cells, albeit at low levels (37). Using primary cells, a recent study found that murine bone marrow-derived macrophages (derived in the presence of M-CSF) and thioglycolate-elicited peritoneal macrophages express low levels of type I CIITA, with types I and IV CIITA induced in IFN-{gamma}-activated cells (38). In addition, macrophages from CIITA PIV knockout mice express CIITA driven from PI following IFN-{gamma} treatment (34). These differences in the activation of particular CIITA promoters by IFN-{gamma} (PIII and PIV in human cell lines; PI and PIV in murine systems) have been suggested to be due to species differences in promoter regulation, but this remains to be determined. Indeed, in this study, we show that PIII and PIV are induced in human monocytes in response to IFN-{gamma}, but not PI, in agreement with previous data in human cell lines.

The mechanisms responsible for GM-CSF-mediated increases in CIITA expression remain to be revealed. However, candidate intermediates can be predicted based on what is known of GM-CSF signaling and CIITA promoter regulation. The cis regions important for activation of PIII have been identified in B and T cells (15, 35, 36, 39). In addition, a region upstream of PIII responsible for CIITA expression in response to IFN-{gamma} has been identified and involves binding of STAT-1, but not IRF-1 (40). GM-CSF binding to the GM-CSF receptor results in the activation of Janus kinase 2, which in turn activates STAT5 (41, 42). Activated STAT5 may bind IFN-{gamma}-activated sequence (GAS) elements found in CIITA PIII. In addition, there are reports in some cell types that GM-CSF can activate STAT1, which also could bind GAS elements (43, 44). Our preliminary results suggest that in addition to STAT5, STAT1 may be phosphorylated following GM-CSF treatment (T. M. C. Hornell, unpublished data). We also have evidence that IRF-1 is not activated by GM-CSF. Together these results are consistent with GM-CSF acting through the previously identified region upstream of CIITA PIII, although this remains to be tested directly.

The mechanisms responsible for activation of CIITA PI are currently under exploration. Initial reports indicate that the transcription factors PU.1 and NF-{kappa}B are bound to CIITA PI in dendritic cells derived from monocytes incubated with GM-CSF and IL-4 (J. Wu and K. Wright, Moffitt Cancer Center, Tampa, FL, personal communication). Intriguingly, PU.1 has been shown to be important for GM-CSF-mediated effects in alveolar macrophages: PU.1 levels are higher in alveolar macrophages isolated from GM-CSF+/+ mice compared with GM-CSF-/- mice; PU.1 levels are restored in alveolar macrophages from GM-CSF-/- mice by transgenic expression of GM-CSF in the lung; and retroviral expression of PU.1 in GM-CSF-/- mice restores functions of alveolar macrophages that are lacking in the absence of GM-CSF (45). Thus, PU.1 is an attractive candidate transcription factor that may link GM-CSF receptor activation and CIITA PI activation.

Although GM-CSF effects on expression of molecules involved in the class II MHC pathway are mediated transcriptionally, we have not determined at what level regulation of CD40 and CD86 surface expression by GM-CSF occurs. STAT5, or possibly STAT1, phosphorylated in response to GM-CSF may be responsible for their induction, as both the CD86 and CD40 promoters contain GAS elements (46, 47).

GM-CSF also affects other functions of monocytes/macrophages, including priming them for increased cytokine production in response to LPS or TNF-{alpha} (48). In addition, a recent study examined the genes in mouse microglia regulated by GM-CSF and found that many different genes are affected, including some important for APC function (49). Although the authors’ conclusion that GM-CSF induces global changes affecting Ag presentation is similar to our findings, there are several notable differences in the details of the molecules affected by GM-CSF. These differences most likely reflect the different systems involved: while in this study we examine the acute effects of GM-CSF treatment on human monocytes; Re et al. (49) examine the effects of prolonged GM-CSF treatment on murine microglia.

GM-CSF is produced by a number of cell types in response to a variety of stimuli. In response to the proinflammatory cytokines TNF and IL-1, fibroblasts, endothelial cells, and epithelial cells produce GM-CSF (17). Monocytes and macrophages produce GM-CSF in response to LPS, and GM-CSF is produced by some activated T cells and NK cells (17, 50). Based on our results, we propose that GM-CSF generated locally following infection or inflammation activates the APC function of recruited monocytes and resident macrophages, leading to the activation of T cells and an immune response. If the activated T cells are specific for autoantigens, this could lead to enhanced autoimmune disease symptoms. Interestingly, increased levels of GM-CSF are found in the joints of patients with rheumatoid arthritis, although whether this is causative or merely indicative of an inflammatory response is unclear (51). GM-CSF treatment in humans has not been implicated in the induction of autoimmune disease, but instead in the activation of pre-existing disease (52, 53, 54). In addition, studies in mouse models performed in genetically susceptible stains have implicated a role for GM-CSF in autoimmune disease severity (55, 56). Together these studies argue that GM-CSF alone is insufficient to induce autoimmunity, but that in the presence of pre-existing autoimmune disease or predisposing factors, GM-CSF can aggravate disease symptoms. Such speculations have led to proposals to block autoimmune disease symptoms by treatment with Abs to GM-CSF. In animal models, treatment with a GM-CSF Ab has been shown to be effective in preventing progression of pre-established disease (57).

In summary, we have identified a novel pathway by which GM-CSF activates APC function. GM-CSF specifically induces types I and III CIITA, leading to increased expression of class II MHC, DM, DO{alpha}, and Ii. These changes along with increased expression of the costimulatory molecules CD86 and CD40 result in more potent APCs.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health and The Arthritis Foundation. T.M.C.H. is funded by the Cancer Research Institute. Back

2 Address correspondence and reprint requests to Dr. Tara M. C. Hornell, Department of Pediatrics, Stanford University, 269 Campus Drive, CCSR, Room 2120, Stanford, CA 94305-5164. E-mail address: hornell{at}stanford.edu Back

3 Abbreviations used in this paper: Ii, invariant chain; ChIP, chromatin immunoprecipitation; CIITA, class II transactivator; CLIP, class II-associated Ii-derived peptide; GAS, IFN-{gamma}-activated sequence; IRF-1, IFN regulatory factor-1; PI, promoter I; PIII, promoter III; PIV, promoter IV; SEB, staphylococcal enterotoxin B. Back

Received for publication December 20, 2002. Accepted for publication June 26, 2003.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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