Colony-Stimulating Factor Granulocyte-Macrophage Primary

Regulation of the Class II MHC Pathway in Primary Human Monocytes by Granulocyte-Macrophage Colony-Stimulating Factor This information is current as of September 27, 2021. Tara M. C. Hornell, Guy W. Beresford, Alyssa Bushey, Jeremy M. Boss and Elizabeth D. Mellins J Immunol 2003; 171:2374-2383; ; doi: 10.4049/jimmunol.171.5.2374 http://www.jimmunol.org/content/171/5/2374 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Regulation of the Class II MHC Pathway in Primary Human Monocytes by Granulocyte-Macrophage Colony-Stimulating Factor1

Tara M. C. Hornell,2* Guy W. Beresford,† Alyssa Bushey,† Jeremy M. Boss,† and Elizabeth D. Mellins*

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 stim- ulatory 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- Downloaded from 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␣. 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 http://www.jimmunol.org/ involved in the class II MHC pathway via induction of CIITA. The Journal of Immunology, 2003, 171: 2374–2383.

onocytes/macrophages play an important role in host Critical for the development of mature APC capability is the defense, acting as APCs to activate T cells and pro- expression of surface class II MHC molecules stably loaded with M ducing a variety of inflammatory mediators to influ- peptide. Expression of several molecules in addition to class II ence immune responses. Monocytes/Macrophages are important MHC molecules is important for this to occur. These include in- not only in lymphoid tissues, but are also critical for activation of variant chain (Ii),3 cathepsins, and, in particular, the nonclassical effector and memory T cells at the site of infection or inflamma- class II molecule HLA-DM. DM acts intracellularly on class II tion. Such activation can be beneficial in the case of a response to molecules loaded with class II-associated Ii-derived peptides by guest on September 27, 2021 pathogens or tumors, but deleterious in autoimmune responses. (CLIP) to catalyze peptide exchange and stabilize empty class II Thus, an understanding of the regulation of monocyte/macrophage molecules. Cell lines deficient in DM exhibit increased CLIP lev- Ag-presenting capability is critical to devising ways to manipulate els and, in some MHC alleles, decreased surface class II expres- immune responses. During inflammation or a local infection, che- sion (4). Another nonclassical class II molecule, HLA-DO, asso- mokines and/or inflammatory mediators are generated locally ciates tightly with DM and may modulate DM function (5Ð7). which stimulate monocytes to migrate into the site (1). In response Although DO expression has been reported in B cells and thymic to these environmental signals, monocytes, along with tissue mac- epithelial cells, the available data suggest that DO is not expressed rophages already present, develop mature Ag presentation func- by monocytes/macrophages (8Ð12). An understanding of the reg- tion, enabling efficient presentation to T cells. Before activation, ulation of class II MHC as well as molecules involved in class II however, these cells are not efficient stimulators of T cells. This is peptide loading is critical for efforts to manipulate Ag presentation. in part due to low cell surface expression of MHC molecules and Many of these molecules, class II MHC, DM, Ii, and DO␣ (but costimulatory molecules such as CD40, CD80, and CD86 (2, 3). not DO␤), are regulated coordinately, in part through dependence Although the action of some mediators, e.g., IFN-␥, of monocyte/ on expression of class II transactivator (CIITA) (13). CIITA ex- macrophage APC function has been well characterized, other me- pression is regulated transcriptionally and is under the control of diators important for their activation have been poorly studied. 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-␥ CIITA transcription has been found to be induced by activation of promoter *Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305; and †Department of Microbiology and Immunology, Emory University School IV and in some cases an element upstream of promoter III in var- of Medicine, Atlanta, GA 30322 ious human cell lines (14, 15). However, the CIITA promoter(s) Received for publication December 20, 2002. Accepted for publication June 26, 2003. responsible for CIITA expression in primary human monocytes in The costs of publication of this article were defrayed in part by the payment of page response to other inflammatory cytokines is unknown. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 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. 3 Abbreviations used in this paper: Ii, invariant chain; ChIP, chromatin immunopre- 2 Address correspondence and reprint requests to Dr. Tara M. C. Hornell, Department cipitation; CIITA, class II transactivator; CLIP, class II-associated Ii-derived peptide; of Pediatrics, Stanford University, 269 Campus Drive, CCSR, Room 2120, Stanford, GAS, IFN-␥-activated sequence; IRF-1, IFN regulatory factor-1; PI, promoter I; PIII, CA 94305-5164. E-mail address: [email protected] promoter III; PIV, promoter IV; SEB, staphylococcal enterotoxin B.

GM-CSF was first identified for its role in the proliferation of CA), and antiserum specific for DR (CHAMP) was a kind gift from L. hematopoietic progenitor cells and their differentiation into gran- Stern (University of Massachusetts Medical School, Worcester, MA). ulocytes and monocytes (16). GM-CSF is now viewed as a regu- Flow cytometry lator of granulocyte and monocyte lineage cells at all stages of For analysis of surface class II MHC and CLIP/class II expression, cells maturation, with effects on phagocytosis, oxidative metabolism, were first incubated with blocking buffer (PBS ϩ 5% human AB serum cytokine secretion, cytotoxicity, and Ag presentation capability (Gemini Bioproducts, Woodland, CA) ϩ 5% goat serum (Caltag)) for 15 (17). This has led to the clinical use of GM-CSF to treat fungal min on ice, washed with PBS ϩ .2% BSA (Sigma-Aldrich), and incubated infections and as a vaccine adjuvant, in addition to its use to mo- 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 bilize hematopoietic progenitors (18, 19). However, the precise Ј FITC-labeled goat F(ab )2 anti-mouse IgG (Caltag Laboratories), washed mechanisms by which GM-CSF mediates these immune potenti- again, and analyzed using a FACScan flow cytometer (BD Biosciences, ating effects are unknown. In this study, we hypothesized that GM- Mountain View, CA). Staining for CD1a, CD14, CD16, CD40, CD80, and CSF activates the class II MHC pathway, directly enhancing APC CD86 was performed as above, but without addition of the secondary Ab. function. This was tested by evaluating the effects of GM-CSF on Mixed leukocyte reaction purified human monocytes. Purified monocytes, incubated with either 800 U/ml GM-CSF or medium alone for 24Ð48 h, were irradiated (3000 rad). Various numbers of these Materials and Methods cells were mixed with 105 purified allogeneic T cells/well in a 96-well Cells plate. T cells were purified using RosetteSep T cell enrichment cocktail from StemCell Technologies (Vancouver, British Columbia, Canada), ac- Buffy coats were obtained from the Stanford Blood Center, and PBMCs

cording to the manufacturer’s instructions. Cells were incubated at 37¡C, Downloaded from were isolated by separation with Ficoll-Paque Plus (Amersham Bio- 5% CO2 for 4Ð6 days in RPMI (Life Technologies) supplemented with sciences, Piscataway, NJ). Primary human monocytes were isolated from 10% heat-inactivated human AB serum (Gemini Bioproducts), 2 mM glu- PBMC by negative selection using Miltenyi Biotec (Auburn, CA) MACS tamine (Life Technologies), 10 mM HEPES (Life Technologies), 1 mM monocyte isolation kit, as directed by the manufacturer. The purity of the sodium pyruvate (Life Technologies), and 0.1 mM nonessential amino ac- monocyte isolation was confirmed by flow cytometric analysis using anti- ids (Life Technologies). [3H]Thymidine (from PerkinElmer Life Sciences, Ͼ CD14 FITC, with 90% positive cells. Purified monocytes were incubated Boston, MA) incorporation was measured following 15- to 17-h pulses in IMDM (Life Technologies, Rockville, MD) supplemented with 10% using a Tomtec Harvestor (Hamden, CT) and a Wallac Microbeta Jet 1450 FBS (HyClone Laboratories, Logan, UT) and 2 mM L-glutamine (Life ␤ http://www.jimmunol.org/ o -reader (PerkinElmer). Results are shown as mean cpm of triplicates. Technologies) at 37 C, 5% CO2. Immature monocyte-derived dendritic cells were generated from puri- IL-10 ELISA fied monocytes, as previously described (20). Briefly, monocytes were cultured for 7 days in IMDM (Life Technologies) supplemented with 10% IL-10 levels in supernatant from monocytes grown in medium alone or FBS (HyClone Laboratories), 2 mM L-glutamine (Life Technologies), 800 GM-CSF were determined using the Quantikine human IL-10 immunoas- U/ml GM-CSF (R&D Systems, Minneapolis, MN), and 1000 U/ml IL-4 say from R&D Systems, according to the manufacturers’ instructions. (R&D Systems), with the medium replaced after 3 and 5 days of incubation Western blotting 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 tran- Cells were harvested and lysed in 50 mM Tris-HCL, (pH 7.5), 150 mM

script in real-time RT-PCR assays. NaCl, 1% Nonidet P-40, 1 mM MnCl2, 1 mM CaCl2, plus protease inhib- The Raji cell line was cultured in RPMI medium (Life Technologies) itors. Unextracted material was pelleted, and the amount of protein in the by guest on September 27, 2021 supplemented with 10% FBS (HyClone Laboratories) and 2 mM L-glu- supernatant was quantitated by Bradford assay and normalized between tamine (Life Technologies). samples. Equal protein equivalents of lysate were mixed with 20 ␮l Con A-Sepharose (Sigma-Aldrich). After rotating overnight at 4oC, Con A- Staphylococcal enterotoxin B (SEB) stimulation Sepharose pellets were washed four times in 0.75 ml lysis buffer. Glyco- proteins were boiled in Laemmli SDS-PAGE sample buffer with 2-ME. Blood from random donors was collected in sodium heparin and trans- The eluted precipitates were run on 12% SDS-PAGE gels and transferred ferred to polypropylene tubes. SEB (Sigma-Aldrich, St. Louis, MO) was to polyvinylidene difluoride membranes (immobilon P; Millipore, Bedford, added to a final concentration of 1 or 10 ␮g/ml, and the blood was incu- ␤ o MA). Alternatively, for -actin staining, equal amounts of lysate prepared bated for6hat37C with frequent mixing. RNA was harvested using and quantitated as described above were mixed with Laemmli SDS-PAGE TRIzol LS (Life Technologies), according to the manufacturer’s sample buffer with 2-ME, boiled, run on 12% SDS-PAGE gels, and trans- instructions. ferred to polyvinylidene difluoride membranes. Membranes were blocked o Cytokines 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%

Human rGM-CSF from R&D Systems or from Immunex (Seattle, WA) Tween 20, and 0.05% NaN3, and incubated with the appropriate Abs di- (Leukine) was used in all experiments at a dose of 800 U/ml. Human rIL-4 luted in blocking buffer. After washing in TBS ϩ 0.05% Tween 20, HRP- was obtained from R&D Systems. Human rIFN-␥ from Roche (Indianap- conjugated secondary Abs (donkey anti-rabbit Ig, Amersham; or goat anti- olis, IN) was used at a dose of 100 U/ml. mouse Ig, Caltag) were added in TBS-Tween containing 5% nonfat dry milk. Following additional washes, ECL substrate was added (Renais- Antibodies sance; DuPont NEN, Boston, MA), and the blots were exposed to film (Hyperfilm ECL; Amersham). Densitometric analysis was performed using L243 is a murine IgG2a mAb that recognizes a monomorphic determinant a Bio-Rad GS-710 Calibrated Imaging Densitometer (Hercules, CA). in the first domain of the DR␣ chain (21). The mAb CerCLIP, specific for CLIP-loaded class II molecules, was a kind gift from P. Cresswell (Yale RNA isolation and reverse transcription University, New Haven, CT). FITC-conjugated Abs to CD1a, CD14, CD16, CD80, and CD86 and the appropriate isotype controls were pur- RNA was isolated using TRIzol (Life Technologies) or Qiagen RNeasy chased from Caltag Laboratories (Burlingame, CA). Anti-CD40 PE and the kits (Valencia, CA), according to the manufacturer’s instructions. RNA ␮ appropriate isotype control were purchased from BD PharMingen (San was quantitated by absorbance at 260 nm, and 0.5 g of RNA was reverse Diego, CA). transcribed using Moloney murine leukemia virus reverse transcriptase Mouse mAbs with the following specificities were used for Western (Promega, Madison, WI). blotting: DA6.147 (DR␣), 5C1 (DM␣) was a kind gift from J. Trowsdale RT-PCR and real-time RT-PCR (University of Cambridge, Cambridge, U.K.), 47GS4 (DM␤) was a generous gift from S. Pierce (Northwestern University, Chicago, IL), anti-␤- PCR using IFN-␥-specific primers (5Ј-GCATCCAAAAGAGTGTGGAG actin Ab was obtained from Sigma-Aldrich, and Pin1.1 (Ii chain) was and 5Ј-GACAGTTCAGCCATCACTTGG) was performed under the fol- kindly provided by P. Cresswell. Rabbit antiserum specific for DO␣ lowing conditions: 3 min at 95oC and 40 cycles of 95oC for 60 s, 57oC for (SU66) was generated against the following peptide, CMGTYVSSVPR. 60 s, and 72oC for 90 s. PCR products were run on 2% agarose gels. Rabbit antiserum specific for DO␤ (K571) was a generous gift from L. For real-time PCR, primers were designed using Primer Express soft- Karlsson (R. W. Johnson Pharmaceutical Research Institute, San Diego, ware (Applied Biosystems, Foster City, CA). The primer sets used were: 2376 GM-CSF INDUCES CLASS II MHC PATHWAY IN MONOCYTES

DMA, 5Ј-TGATCCAGCAAATAGGGCCA and 5Ј-CTCTGGACACCG GGATTTTC; DMB, 5Ј-AAAGACACCCTGATGCAGCG and 5Ј-TGTG GCACAATTCTGAAGCC; DOA, 5Ј-TGGCCCAGACCAGCTTCTAT and 5Ј-GGAACTTGCGGAACAAATGG; DOB, 5Ј-GATTCAGGCAAA GGCTGACTG and 5Ј-TGCACCTTTTCTGTCCCGTT; DRA, 5Ј-GTCT GGCGGCTTGAAGAATT and 5Ј-ACCTTGAGCCTCAAAGCTGG; DRB, 5Ј-CGTGACAAGCCCTCTCACAG and 5Ј-TGTGCAGATTCAGACCG TGC; CIITA, 5Ј-GCTCTGAGTGGCGAAATCAAG and 5Ј-CAATGCT AGGTACTGCGGGAG; GAPDH, 5Ј-TGGGCTACACTGAGCACCAG and 5Ј-GGGTGTCGCTGTTGAAGTCA; type I CIITA, 5Ј-GGAGACCT GGATTTGGCCCT and 5Ј-GAAGCTCCAGGTAGCCACCTTCTA; type III CIITA, 5Ј-GGGGAAGCTGAGGGCACG and 5Ј-GAAGCTCCAGG TAGCCACCTTCTA; and type IV CIITA, 5Ј-GCGGCCCCAGAGCTGG and 5Ј-GAAGCTCCAGGTAGCCACCTTCTA. The 2ϫ SYBR Green PCR Master Mix (Applied Biosystems) was used in PCR with 200 nM of forward and reverse primers, 10Ð40 ng of the reverse-transcription product, and RNase/DNase-free water to 50 ␮l. 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 50oC, 10 min at 95oC, and 40 cycles of 95oC for 15 s and 60oC for 1 min. To determine relative quantity, control cDNA generated from Raji cell, monocyte-derived dendritic cell, or IFN-␥-stimulated monocyte total RNA was used to generate a standard curve. Relative FIGURE 1. GM-CSF increases surface class II MHC and costimulatory Downloaded from quantities of the gene of interest were determined for unknown samples by molecule expression in primary human monocytes. A, Expression of MHC comparison with this standard curve, and normalized to GAPDH quanti- class II, as detected by ﬂow cytometry using the HLA-DR-speciﬁc mAb ties. Fold changes in expression were determined by dividing the normal- L243, is shown after 24 h of incubation with medium alone (open) or 800 ized quantity of the gene of interest from stimulated (with GM-CSF or U/ml GM-CSF (shaded). Staining with the secondary Ab alone is shown. IFN-␥) monocytes by the normalized quantity of the gene of interest from B, Expression of CD86, CD40, and CD80 by human monocytes is shown monocytes incubated with medium alone. All real-time RT-PCR experi- after 24Ð48 h of incubation with medium alone (open) or 800 U/ml GM- ments were performed at least three times, and the average of these ex- CSF (shaded). Staining with isotype control Abs is shown. http://www.jimmunol.org/ periments is shown relative to levels of monocytes incubated with medium alone.

Chromatin immunoprecipitation (ChIP) assays due to activation of the cells or are part of a differentiation path- ChIPs were performed, as previously described (22). Briefly, formaldehyde way, expression of CD1a and CD16 was examined following GM- cross-linked chromatin was prepared from between 1.7 ϫ 107 and 4.1 ϫ CSF treatment. Incubation of human monocytes with GM-CSF and 7 10 cells for each experimental condition. The CIITA (anti-MBP-CIITA)- IL-4 for 7 days results in the generation of immature CD1aϩ den- specific antiserum used to precipitate CIITA complexed with chromatin ␮ dritic cells, while incubation of monocytes with medium alone or has been previously described (22). For each precipitation, 60 l of protein ϩ A-Sepharose beads was used. After the wash steps, cross-links were re- GM-CSF for 7 days leads to the generation of CD16 macro- versed, and the DNA was purified and analyzed by real-time PCR. phages (20). Thus, an increase in CD1aϩ or CD16ϩ cells follow- by guest on September 27, 2021 Quantitative PCR was performed using an iCycler unit with an optical ing GM-CSF treatment may reflect differentiation of monocytes assembly (BioRad Laboratories, Hercules, CA). For quantification of PCR product, SYBR Green incorporation was determined. The average values into dendritic cells or macrophages, respectively. However, as for each sample were normalized to the amount of input chromatin. The shown in Fig. 2, GM-CSF does not increase monocyte CD1a or primers used were: HLA-DRA forward, 5Ј-GATCTCTTGTGTCCTGG CD16 expression in the time frame in which changes in class II ACCCTTTGCAAGAACCCT-3Ј, and HLA-DRA reverse, 5Ј-CCCAAT MHC and costimulatory molecule expression are detected. In fact, TACTCTTTGGCCAATCAGAAAAATATTTTG-3Ј. 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 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 ϭ 17), surface class II expression is increased 2-fold ( p Ͻ 5 ϫ 10Ϫ8) by GM-CSF, and the response peaks after 24Ð48 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. FIGURE 2. GM-CSF does not induce expression of the differentiation ϭ On average, GM-CSF increases CD40 expression 3.6-fold (n 5, markers CD1a or CD16. Expression of CD1a (A) and CD16 (B) is shown p Ͻ 0.005) and CD86 2.5-fold (n ϭ 7, p Ͻ 0.05). after 48 h of incubation with medium alone (left) or 800 U/ml GM-CSF To examine whether GM-CSF-mediated effects on surface class (right). The open histograms are staining with the isotype control Ab, IgG1- II MHC and costimulatory molecule expression represent changes PE; shaded histograms are staining with anti-CD1a PE or anti-CD16 PE. The Journal of Immunology 2377

GM-CSF-treated monocytes express less CD16 compared with un- GM-CSF enhances monocyte APC function treated monocytes after 48 h (Fig. 2B). There is a small increase in To test whether GM-CSF-mediated increases in surface class II the percentage of CD1a-positive cells seen after GM-CSF treat- MHC, CD40, and CD86 expression and decreased relative CLIP- ment (Fig. 2A), but this varies with the donor, and no more than loaded class II molecules are associated with an altered ability of 10% of the cells are CD1a positive in the time period examined. In monocytes to stimulate an allogeneic T cell response, GM-CSF- addition, the morphology of GM-CSF-treated monocytes (up to treated or untreated monocytes were irradiated and incubated with 48 h) does not resemble either that of immature dendritic cells or purified allogeneic T cells and T cell proliferation measured after macrophages (data not shown). These data suggest that GM-CSF- 4Ð6 days. We found that allogeneic T cells consistently proliferate treated monocytes do not differentiate during the time frame in more in response to GM-CSF-treated monocytes compared with which GM-CSF increases class II MHC and costimulatory mole- monocytes incubated with medium alone. At stimulator to re- cule expression, and instead these changes likely represent an sponder ratios of 2000 or 8000 monocytes per 105 allogeneic T acute activation event. cells, GM-CSF-treated monocytes stimulate 2- to 3-fold more T GM-CSF treatment of human monocytes decreases relative cell proliferation than untreated monocytes (Fig. 4). At higher CLIP/class II levels stimulator to responder ratios, the difference between the T cell proliferation stimulated by GM-CSF-treated vs untreated mono- To determine whether GM-CSF affects the peptide loading of class cytes decreases (data not shown). The observed difference in T cell II MHC molecules, we examined the relative levels of CLIP- proliferation in response to GM-CSF-treated monocytes compared loaded surface class II MHC molecules in GM-CSF-treated and with untreated monocytes is likely due, at least in part, to the untreated monocytes. Release of CLIP from class II molecules is increase in surface class II MHC expression seen in GM-CSF- Downloaded from required for association with potentially antigenic peptides, and for treated vs untreated monocytes. Thus, we sought to examine the many MHC alleles, is also required for the generation of stable mechanisms responsible for GM-CSF-mediated increase in surface peptide-MHC complexes capable of survival at the cell surface. class II expression. Thus, a comparison of the relative level of CLIP-loaded class II molecules provides an indication of the potential peptide diversity GM-CSF increases total protein levels of DR, DM, Ii, and DO␣, presented on the surface of the monocytes. After 24Ð48hofin- but not DO␤ by human monocytes http://www.jimmunol.org/ cubation with GM-CSF, monocytes from five of nine experiments To test whether the increased surface class II MHC expression and express less surface CLIP-class II/total class II compared with decreased relative CLIP-class II/total class II levels are due to an monocytes incubated with medium alone, as measured by flow increase in total protein levels of MHC class II molecules and the cytometry, using mAb specific for CLIP and DR (Fig. 3). This accessory molecule HLA-DM, Western blot analysis was per- Ͻ modest, but significant, difference ( p 0.005) suggests that more formed. As shown in Fig. 5, an increase in cellular DR levels is efficient CLIP release and peptide loading occur in the presence of seen after GM-CSF treatment. Correlating with the time course of GM-CSF. On average, GM-CSF-treated monocytes from these do- increased DR surface expression, total DR levels peak between 24 nors express .57-fold of the level of relative CLIP/class II surface

and 48 h of incubation with GM-CSF. Densitometric analysis re- by guest on September 27, 2021 levels as untreated monocytes. Monocytes from the remaining four vealed that GM-CSF increases DR␣ and DR␤ levels on average experiments did not appreciably change or expressed higher rela- 8.5- and 4.6-fold, respectively. tive CLIP class II levels after treatment with GM-CSF, suggesting A similar analysis was done to examine the effect of GM-CSF that there is variability between donors. This is likely due to allelic on monocyte expression of the accessory molecules Ii, DM, and MHC differences that affect binding to CLIP and dependence on DO. Ii is crucial for proper trafﬁcking of class II molecules to HLA-DM (23, 24). The ﬁnding that GM-CSF decreases the rela- peptide-loading compartments (25). DM plays a critical role in tive level of CLIP-loaded class II molecules on the surface of stable peptide binding by class II molecules, while DO is thought primary human monocytes suggests that HLA-DM expression to play an inhibitory role, but has not been found to be expressed and/or function may be enhanced, leading to the generation of more stable peptide/MHC complexes on the cell surface.

FIGURE 4. GM-CSF enhances monocyte APC function. Monocytes FIGURE 3. GM-CSF decreases relative surface CLIP/class II levels. were incubated with 800 U/ml GM-CSF (shaded) or medium (open) for CLIP-loaded class II and total HLA-DR levels were measured by flow 48 h, irradiated (3000 rad), and used as stimulators for 1 ϫ 105 allogeneic cytometry using the mAbs CerCLIP and L243, respectively. CerCLIP/ purified T cells at the indicated concentrations. T cell proliferation was L243 mean fluorescence intensity ratios (ϫ100) of monocytes incubated measured after 5 days by pulsing with [3H]thymidine and measuring in- with medium alone, or 800 U/ml GM-CSF, for 24Ð48 h were determined. corporation, as described in Materials and Methods. The experiment The difference ,ء .The fold difference in CerCLIP/L243 levels between monocytes incubated shown is representative of three independent experiments with 800 U/ml GM-CSF compared with incubation with medium alone is in the T cell response to medium vs GM-CSF-treated monocytes is statis- shown. tically significant, p Ͻ 0.05. 2378 GM-CSF INDUCES CLASS II MHC PATHWAY IN MONOCYTES

quantitation, staining for ␤-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-␥ or decreasing IL-10 production in human monocytes As GM-CSF-mediated effects on class II expression are not im- mediate, 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-␥. Although monocytes are not tradi- tionally considered to be capable of producing IFN-␥, recent reports indicate that following treatment with IL-12 and/or IL-18, monocytes do produce measurable quantities of IFN-␥ (27). As IFN-␥ is a potent inducer of class II MHC and DM expression, it Downloaded from FIGURE 5. GM-CSF increases total protein expression of HLA-DR, was important to test whether GM-CSF mediates its effect indi- DM, Ii, and DO␣ by monocytes, but has no effect on DO␤. Total cellular rectly through induction of IFN-␥. RT-PCR was performed using DR␣,DR␤,DM␣,DM␤, Ii, DO␣, and DO␤ in monocytes incubated with primers that speciﬁcally amplify IFN-␥ RNA. However, as shown medium or 800 U/ml GM-CSF for 48 h were determined by Western blot in Fig. 6A, no detectable IFN-␥ RNA is found following GM-CSF analysis using the following Abs: DA6.147, CHAMP, 5C1, 47G.S4, treatment of monocytes. Thus, GM-CSF up-regulation of class II is Pin1.1, SU66, and K571, respectively. Expression of DO␤ by Raji cells is not mediated via production of IFN-␥. http://www.jimmunol.org/ shown as a positive control for the K571 Ab. Total cellular ␤-actin levels are also shown to control for equal protein quantitation. Results shown are We also tested whether GM-CSF acts to increase surface class representative of at least three independent experiments. 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 mono- in monocytes (4, 12). The decrease in relative CLIP/class II levels cytes (28). If GM-CSF decreases monocyte production of IL-10, seen following GM-CSF treatment may reﬂect a change in DM, Ii, this could indirectly lead to increased cell surface levels of class II and/or DO expression, and this was tested directly. Expression of MHC. To test this possibility, the level of IL-10 secreted by GM- both the ␣- and ␤-chains of DM and DO was examined, as, at least CSF-treated monocytes relative to monocytes incubated with me- by guest on September 27, 2021 in the case of DO, the two chains are known to be regulated dis- dium alone was determined by ELISA. As shown in Fig. 6B, GM- tinctly (26). As shown in Fig. 5, GM-CSF increases expression of CSF-treated monocytes do not express less IL-10 than untreated HLA-DM␣ 3-fold, DM␤ 10.5-fold, DO␣ 33-fold, and Ii 5.5-fold. monocytes, arguing that GM-CSF does not mediate its effects on However, no DO␤ is detectable in monocytes incubated with ei- surface class II MHC expression via reduction of IL-10 ther medium alone or GM-CSF. As a control for equal protein production.

FIGURE 6. GM-CSF does not mediate its effects on class II expression by inducing IFN-␥ 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-␥ for 24 h, and RNA was harvested. RT-PCR was performed using primers specific for IFN-␥, 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-␥-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 24Ð48 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 ϭ 0.12 (24 h) and p ϭ 0.2 (48 h). The Journal of Immunology 2379

GM-CSF increases DR, DM, and DO␣ mRNA levels in human monocytes To test whether the increase in protein levels of DR, DM, and DO␣ 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␣ expression through a transcriptional or posttranscriptional FIGURE 8. GM-CSF increases total CIITA mRNA levels in human mechanism. monocytes. Relative expression of total CIITA mRNA was determined by real-time RT-PCR and normalized to expression of GAPDH. The fold GM-CSF increases total CIITA mRNA levels in human change in total CIITA expression in monocytes treated with GM-CSF for monocytes 24 h compared with monocytes incubated with medium alone is shown and Ͻ Similarities have been found in the transcriptional regulation of is statistically significant, p 0.05. class II MHC genes as well as the genes encoding the accessory molecules DM and DO␣ (29). Coordinate regulation of the above Downloaded from genes has been shown to involve several transcription factors, in- a shared second exon. Real-time RT-PCR analysis, using primers cluding the transcriptional activator CIITA. CIITA expression is specific for each product, was performed to determine the CIITA correlated with class II expression, and its absence in humans leads promoter(s) induced by GM-CSF. As shown in Fig. 9, GM-CSF to severe immunodeficiency (13). We sought to address whether induces types I and III CIITA RNA, but has no effect on type IV GM-CSF induces class II MHC and accessory molecule expres- CIITA in primary human monocytes. This is in contrast to IFN-␥, sion via induction of CIITA. Real-time RT-PCR analysis was per-

which induces expression of types III and IV CIITA in primary http://www.jimmunol.org/ formed using RNA puriﬁed from untreated and GM-CSF-treated human monocytes (Fig. 9). monocytes. As shown in Fig. 8, GM-CSF increases CIITA RNA There are additional differences between GM-CSF- and IFN-␥- levels on average 5.5-fold in human monocytes. This is the ﬁrst induced increases in CIITA expression. Kinetic experiments dem- demonstration of a GM-CSF-mediated effect on CIITA expression. onstrate that the increase in CIITA transcript following GM-CSF ␥ GM-CSF increases types I and III CIITA mRNA in primary treatment occurs at the 18 h time point, while IFN- -mediated human monocytes, while IFN-␥ increases types III and IV CIITA increases in CIITA transcript levels are detected at the 1.5 h time ␥ transcripts point (Fig. 9, B–D). This suggests that GM-CSF and IFN- increase CIITA transcription through distinct mechanisms. Of note,

CIITA is a complexly regulated gene, with three promoters that are monocytes incubated with medium alone increase expression of by guest on September 27, 2021 known to display distinct cell type- and cytokine-speciﬁc re- type III CIITA slowly with time, expressing 144-fold more tran- sponses (13). To provide clues as to the mechanisms involved in script by 48 h compared with 1.5 h of incubation (Fig. 9C). In GM-CSF-mediated regulation of CIITA expression, we deter- contrast, time course experiments demonstrate that GM-CSF and mined the promoter(s) induced by GM-CSF. Transcription initi- IFN-␥ transiently increase CIITA levels. By 48 h, CIITA levels in ated by each of the CIITA promoters leads to the synthesis of GM-CSF- or IFN-␥-activated monocytes decrease substantially distinct CIITA mRNAs containing alternative ﬁrst exons spliced to 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␣ 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 ϭ 5, p Ͻ 0.05) (Fig. 10). This provides direct evidence that the increase in CIITA mes- sage 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. Together these data show that GM-CSF activates monocyte FIGURE 7. DR, DM, and DO␣ expression are regulated transcription- APC function by increasing expression of costimulatory molecules ally by GM-CSF, while DO␤ mRNA levels are unchanged following GM- as well as several molecules important for class II MHC Ag pre- CSF treatment. RNA was harvested from monocytes incubated with 800 sentation. This latter effect is mediated by GM-CSF induction of U/ml GM-CSF or medium alone for 24 h and reverse transcribed. Real- CIITA expression. 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 Discussion in expression of these transcripts in GM-CSF-stimulated monocytes com- This study elucidates the effects of GM-CSF on the expression of pared with unstimulated monocytes is shown. These changes, with the molecules important for class II MHC Ag presentation in human exception of DOB, are statistically signiﬁcant, p Ͻ 0.05. monocytes. GM-CSF induces expression of surface and total class 2380 GM-CSF INDUCES CLASS II MHC PATHWAY IN MONOCYTES Downloaded from http://www.jimmunol.org/

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-␥-stimulated monocyte RNA was used. Fold change in expression of types I, III, and IV CIITA by GM-CSF- and IFN-␥-treated monocytes compared with monocytes incubated with medium alone by guest on September 27, 2021 for 24 h is shown. GM-CSF-mediated increases in types I and III CIITA are statistically signiﬁcant, p Ͻ 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-␥ for the indicated times. Relative normalized levels of each CIITA transcript are shown.

II MHC, the class II-like molecule DM that is crucial for the for- via induction of CIITA by GM-CSF. We demonstrate that incu- mation of stable peptide/MHC complexes, Ii, and the ␣-chain of bation with GM-CSF increases the amount of CIITA associated DO. In addition, increased surface expression of the costimulatory with the DRA promoter. As the expression of DR␤,DM␣,DM␤, molecules CD40 and CD86 is seen following GM-CSF treatment. and DO␣ are similarly regulated by CIITA, this result further sug- Together these changes most likely contribute to enhanced recog- gests that the increased expression of these molecules induced by nition of GM-CSF-treated monocytes by T cells. Furthermore, we GM-CSF may be due to an increased amount of CIITA associated show that these effects on the class II pathway result from an with the promoters of these genes. ␣ increase in total protein and RNA levels of class II, DM, and DO These changes are seen in monocytes after 24Ð48 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 CD1aϩ, 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 24Ð48 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 mac- FIGURE 10. Association of CIITA with the DRA promoter increases rophages. This observed decrease in CD16 expression likely following GM-CSF treatment of human monocytes. Real-time ChIP anal- represents the presence of CD14ϩCD16ϩ monocytes, which com- ysis using Abs to CIITA. Fold increase compared with untreated cells is shown; data are mean Ϯ SEM of five separate experiments. The increase prise, on average, 10% of total monocytes, and whose expression of in CIITA associated with the DRA promoter observed following GM-CSF CD16 decreases following activation (Hornell, unpublished data). Un- treatment is statistically significant, p Ͻ 0.05. derstanding the pathways initiated through these in vitro processes The Journal of Immunology 2381 may have important implications for mechanisms involved in in vivo important for activation of PIII have been identified in B and T monocyte activation. In addition, GM-CSF-mediated activation of the cells (15, 35, 36, 39). In addition, a region upstream of PIII re- class II MHC pathway may also occur in other APCs, such as tissue sponsible for CIITA expression in response to IFN-␥ has been macrophages and dendritic cells. identified and involves binding of STAT-1, but not IRF-1 (40). We demonstrate in this study that GM-CSF is a potent up-reg- GM-CSF binding to the GM-CSF receptor results in the activation ulator of several molecules important for class II MHC Ag pre- of Janus kinase 2, which in turn activates STAT5 (41, 42). Acti- sentation: DR, DM, Ii, and DO␣. All of these molecules are co- vated STAT5 may bind IFN-␥-activated sequence (GAS) elements ordinately regulated by CIITA (29). Interestingly, while the found in CIITA PIII. In addition, there are reports in some cell expression of DO␣ is increased, no DO␤ is detected by Western types that GM-CSF can activate STAT1, which also could bind blot. In addition, sensitive real-time RT-PCR data indicate that GAS elements (43, 44). Our preliminary results suggest that in GM-CSF does not increase DO␤ transcript levels despite increas- addition to STAT5, STAT1 may be phosphorylated following ing DO␣ levels. It has been previously shown that the two chains GM-CSF treatment (T. M. C. Hornell, unpublished data). We also of DO are differentially regulated, and that in B cells DO␤ expres- have evidence that IRF-1 is not activated by GM-CSF. Together sion is influenced by, but not dependent upon, CIITA (26, 30). these results are consistent with GM-CSF acting through the pre- This raises the question of whether DO␣ expressed in the absence, viously identified region upstream of CIITA PIII, although this ␤ or in excess, of DO can function independently from, or with remains to be tested directly. ␤ another partner than, DO . The mechanisms responsible for activation of CIITA PI are cur- Our findings have important implications for understanding the rently under exploration. Initial reports indicate that the transcrip- regulation of CIITA expression. CIITA, which functions as a mas- tion factors PU.1 and NF-␬B are bound to CIITA PI in dendritic Downloaded from ter regulator of MHC class II transcription by coordinating the cells derived from monocytes incubated with GM-CSF and IL-4 (J. assembly of the transcriptional apparatus at MHC class II promot- Wu and K. Wright, Moffitt Cancer Center, Tampa, FL, personal ers, is itself regulated at the transcriptional and posttranscriptional communication). Intriguingly, PU.1 has been shown to be impor- levels (13). Importantly, we find that GM-CSF increases CIITA tant for GM-CSF-mediated effects in alveolar macrophages: PU.1 mRNA levels. Each of the three active CIITA promoters encodes levels are higher in alveolar macrophages isolated from GM- a distinct first exon to the CIITA protein. The use of promoter I CSFϩ/ϩ mice compared with GM-CSFϪ/Ϫ mice; PU.1 levels are http://www.jimmunol.org/ (PI) provides CIITA with the greatest transcriptional potential Ϫ Ϫ restored in alveolar macrophages from GM-CSF / mice by trans- (31). Understanding the regulation of CIITA in different cell types genic expression of GM-CSF in the lung; and retroviral expression and in response to different mediators will help uncover the sig- Ϫ Ϫ of PU.1 in GM-CSF / mice restores functions of alveolar mac- naling pathways involved and also the distinct functions that dif- rophages that are lacking in the absence of GM-CSF (45). Thus, ferent CIITA types may play. Previous studies using mouse and PU.1 is an attractive candidate transcription factor that may link human cell lines and primary cells have shown that PI is active in GM-CSF receptor activation and CIITA PI activation. dendritic cells as well as in murine macrophages and microglia in Although GM-CSF effects on expression of molecules involved response to IFN-␥; promoter III (PIII) is active in dendritic cells,

in the class II MHC pathway are mediated transcriptionally, we by guest on September 27, 2021 T and B lymphocytes, as well as IFN-␥-activated cells; and pro- have not determined at what level regulation of CD40 and CD86 moter IV (PIV) is IFN-␥ responsive in murine and human macro- surface expression by GM-CSF occurs. STAT5, or possibly phage and microglial cell lines as well as in non-bone marrow- STAT1, phosphorylated in response to GM-CSF may be respon- derived cells such as fibroblast, endothelial, and epithelial cells and cell lines, and astrocytes, and is also responsible for CIITA ex- sible for their induction, as both the CD86 and CD40 promoters pression in murine cortical thymic epithelial cells (14, 15, 32Ð36). contain GAS elements (46, 47). In this study, we demonstrate that freshly isolated monocytes GM-CSF also affects other functions of monocytes/macro- express low levels of CIITA and that GM-CSF specifically in- phages, including priming them for increased cytokine production ␣ creases types I and III CIITA. This is the first report examining the in response to LPS or TNF- (48). In addition, a recent study regulation of CIITA expression in primary human monocytes, and examined the genes in mouse microglia regulated by GM-CSF and the first evidence that PI is inducible in human cells. Previous found that many different genes are affected, including some im- studies in primary human monocytes are limited to a single ob- portant for APC function (49). Although the authors’ conclusion servation that type III CIITA is expressed in freshly isolated cells, that GM-CSF induces global changes affecting Ag presentation is albeit at low levels (37). Using primary cells, a recent study found similar to our findings, there are several notable differences in the that murine bone marrow-derived macrophages (derived in the details of the molecules affected by GM-CSF. These differences presence of M-CSF) and thioglycolate-elicited peritoneal macro- most likely reflect the different systems involved: while in this phages express low levels of type I CIITA, with types I and IV study we examine the acute effects of GM-CSF treatment on hu- CIITA induced in IFN-␥-activated cells (38). In addition, macro- man monocytes; Re et al. (49) examine the effects of prolonged phages from CIITA PIV knockout mice express CIITA driven GM-CSF treatment on murine microglia. from PI following IFN-␥ treatment (34). These differences in the GM-CSF is produced by a number of cell types in response to activation of particular CIITA promoters by IFN-␥ (PIII and PIV a variety of stimuli. In response to the proinflammatory cytokines in human cell lines; PI and PIV in murine systems) have been TNF and IL-1, fibroblasts, endothelial cells, and epithelial cells suggested to be due to species differences in promoter regulation, produce GM-CSF (17). Monocytes and macrophages produce but this remains to be determined. Indeed, in this study, we show GM-CSF in response to LPS, and GM-CSF is produced by some that PIII and PIV are induced in human monocytes in response to activated T cells and NK cells (17, 50). Based on our results, we IFN-␥, but not PI, in agreement with previous data in human cell propose that GM-CSF generated locally following infection or in- lines. flammation activates the APC function of recruited monocytes and The mechanisms responsible for GM-CSF-mediated increases resident macrophages, leading to the activation of T cells and an in CIITA expression remain to be revealed. However, candidate immune response. If the activated T cells are specific for autoan- intermediates can be predicted based on what is known of GM- tigens, this could lead to enhanced autoimmune disease symptoms. CSF signaling and CIITA promoter regulation. The cis regions Interestingly, increased levels of GM-CSF are found in the joints 2382 GM-CSF INDUCES CLASS II MHC PATHWAY IN MONOCYTES of patients with rheumatoid arthritis, although whether this is caus- 21. Lampson, L. A., and R. Levy. 1980. Two populations of Ia-like molecules on a ative or merely indicative of an inflammatory response is unclear (51). human B cell line. J. Immunol. 125:293. 22. Beresford, G. W., and J. M. Boss. 2001. CIITA coordinates multiple histone GM-CSF treatment in humans has not been implicated in the induc- acetylation modifications at the HLA-DRA promoter. Nat. Immun. 2:652. tion of autoimmune disease, but instead in the activation of pre-ex- 23. Sette, A., S. Southwood, J. Miller, and E. Appella. 1995. Binding of major his- isting disease (52Ð54). In addition, studies in mouse models per- tocompatibility complex class II to the invariant chain-derived peptide, CLIP, is regulated by allelic polymorphism in class II. J. Exp. Med. 181:677. formed in genetically susceptible stains have implicated a role for 24. Stebbins, C. C., G. E. Loss, Jr., C. G. Elias, A. Chervonsky, and A. J. Sant. 1995. GM-CSF in autoimmune disease severity (55, 56). Together these The requirement for DM in class II-restricted antigen presentation and SDS- studies argue that GM-CSF alone is insufficient to induce autoimmu- stable dimer formation is allele and species dependent. J. Exp. Med. 181:223. 25. Elliott, E. A., J. R. Drake, S. Amigorena, J. Elsemore, P. Webster, I. Mellman, nity, but that in the presence of pre-existing autoimmune disease or and R. A. Flavell. 1994. The invariant chain is required for intracellular transport predisposing factors, GM-CSF can aggravate disease symptoms. Such and function of major histocompatibility complex class II molecules. J. Exp. Med. speculations have led to proposals to block autoimmune disease 179:681. symptoms by treatment with Abs to GM-CSF. In animal models, 26. Taxman, D. J., D. E. Cressman, and J. P. Ting. 2000. Identification of class II transcriptional activator-induced genes by representational difference analysis: treatment with a GM-CSF Ab has been shown to be effective in pre- discoordinate regulation of the DN␣/DO␤ heterodimer. J. Immunol. 165:1410. venting progression of pre-established disease (57). 27. Munder, M., M. Mallo, K. Eichmann, and M. Modolell. 1998. Murine macro- ␥ In summary, we have identified a novel pathway by which GM- phages secrete interferon upon combined stimulation with interleukin (IL)-12 and IL-18: a novel pathway of autocrine macrophage activation. J. Exp. Med. CSF activates APC function. GM-CSF specifically induces types I 187:2103. and III CIITA, leading to increased expression of class II MHC, 28. Koppelman, B., J. J. Neefjes, J. E. de Vries, and R. de Waal Malefyt. 1997. ␣␤ DM, DO␣, and Ii. These changes along with increased expression Interleukin-10 down-regulates MHC class II peptide complexes at the plasma membrane of monocytes by affecting arrival and recycling. Immunity 7:861. of the costimulatory molecules CD86 and CD40 result in more 29. Ting, J. P., and J. Trowsdale. 2002. Genetic control of MHC class II expression. Downloaded from potent APCs. Cell 109:S21. 30. Nagarajan, U. M., J. Lochamy, X. Chen, G. W. Beresford, R. Nilsen, P. E. Jensen, and J. M. Boss. 2002. Class II transactivator is required for maximal expression References of HLA-DOB in B cells. J. Immunol. 168:1780. 1. Muller, W. A. 2001. New mechanisms and pathways for monocyte recruitment. 31. Nickerson, K., T. J. Sisk, N. Inohara, C. S. Yee, J. Kennell, M. C. Cho, J. Exp. Med. 194:F47. P. J. Yannie II, G. Nunez, and C. H. Chang. 2001. Dendritic cell-specific MHC 2. Fleischer, J., E. Soeth, N. Reiling, E. Grage-Griebenow, H. D. Flad, and M. Ernst. class II transactivator contains a caspase recruitment domain that confers potent 1996. Differential expression and function of CD80 (B7-1) and CD86 (B7-2) on transactivation activity. J. Biol. Chem. 276:19089. http://www.jimmunol.org/ human peripheral blood monocytes. Immunology 89:592. 32. O’Keefe, G. M., V. T. Nguyen, L. L. Ping Tang, and E. N. Benveniste. 2001. 3. Laupeze, B., O. Fardel, M. Onno, N. Bertho, B. Drenou, R. Fauchet, and IFN-␥ regulation of class II transactivator promoter IV in macrophages and mi- L. Amiot. 1999. Differential expression of major histocompatibility complex croglia: involvement of the suppressors of cytokine signaling-1 protein. J. Im- class Ia, Ib, and II molecules on monocytes-derived dendritic and macrophagic munol. 166:2260. cells. Hum. Immunol. 60:591. 33. Soos, J. M., J. Morrow, T. A. Ashley, B. E. Szente, E. K. Bikoff, and S. S. Zamvil. 4. Busch, R., and E. D. Mellins. 1996. Developing and shedding inhibitions: how 1998. Astrocytes express elements of the class II endocytic pathway and process MHC class II molecules reach maturity. Curr. Opin. Immunol. 8:51. central nervous system autoantigen for presentation to encephalitogenic T cells. 5. Liljedahl, M., T. Kuwana, W. P. Fung-Leung, M. R. Jackson, P. A. Peterson, and J. Immunol. 161:5959. L. Karlsson. 1996. HLA-DO is a lysosomal resident which requires association 34. Waldburger, J. M., T. Suter, A. Fontana, H. Acha-Orbea, and W. Reith. 2001. with HLA-DM for efficient intracellular transport. EMBO J. 15:4817. Selective abrogation of major histocompatibility complex class II expression on 6. Denzin, L. K., D. B. Sant’Angelo, C. Hammond, M. J. Surman, and P. Cresswell. extrahematopoietic cells in mice lacking promoter IV of the class II transactivator by guest on September 27, 2021 1997. Negative regulation by HLA-DO of MHC class II-restricted antigen pro- gene. J. Exp. Med. 194:393. cessing. Science 278:106. 35. Holling, T. M., N. van der Stoep, E. Quinten, and P. J. van den Elsen. 2002. 7. Van Ham, S. M., E. P. Tjin, B. F. Lillemeier, U. Gruneberg, Activated human T cells accomplish MHC class II expression through T cell- K. E. van Meijgaarden, L. Pastoors, D. Verwoerd, A. Tulp, B. Canas, D. Rahman, specific occupation of class II transactivator promoter III. J. Immunol. 168:763. et al. 1997. HLA-DO is a negative modulator of HLA-DM-mediated MHC class II peptide loading. Curr. Biol. 7:950. 36. Wong, A. W., N. Ghosh, K. P. McKinnon, W. Reed, J. F. Piskurich, K. L. Wright, 8. Karlsson, L., C. D. Surh, J. Sprent, and P. A. Peterson. 1991. A novel class II and J. P. Ting. 2002. Regulation and specificity of MHC2TA promoter usage in MHC molecule with unusual tissue distribution. Nature 351:485. human primary T lymphocytes and cell line. J. Immunol. 169:3112. 9. Tonnelle, C., R. DeMars, and E. O. Long. 1985. DO␤: a new ␤ chain gene in 37. Landmann, S., A. Muhlethaler-Mottet, L. Bernasconi, T. Suter, J. M. Waldburger, HLA-D with a distinct regulation of expression. EMBO J. 4:2839. K. Masternak, J. F. Arrighi, C. Hauser, A. Fontana, and W. Reith. 2001. Matu- 10. Surh, C. D., E. K. Gao, H. Kosaka, D. Lo, C. Ahn, D. B. Murphy, L. Karlsson, ration of dendritic cells is accompanied by rapid transcriptional silencing of class P. Peterson, and J. Sprent. 1992. Two subsets of epithelial cells in the thymic II transactivator (CIITA) expression. J. Exp. Med. 194:379. medulla. J. Exp. Med. 176:495. 38. Pai, R. K., D. Askew, W. H. Boom, and C. V. Harding. 2002. Regulation of class 11. Douek, D. C., and D. M. Altmann. 1997. HLA-DO is an intracellular class II II MHC expression in APCs: roles of types I, III, and IV class II transactivator. molecule with distinctive thymic expression. Int. Immunol. 9:355. J. Immunol. 169:1326. 12. Chen, X., O. Laur, T. Kambayashi, S. Li, R. A. Bray, D. A. Weber, L. Karlsson, 39. Ghosh, N., J. F. Piskurich, G. Wright, K. Hassani, J. P. Ting, and K. L. Wright. and P. E. Jensen. 2002. Regulated expression of human histocompatibility leu- 1999. A novel element and a TEF-2-like element activate the major histocom- kocyte antigen (HLA)-DO during antigen-dependent and antigen-independent patibility complex class II transactivator in B-lymphocytes. J. Biol. Chem. phases of B cell development. J. Exp. Med. 195:1053. 274:32342. 13. Reith, W., and B. Mach. 2001. The bare lymphocyte syndrome and the regulation 40. Piskurich, J. F., M. W. Linhoff, Y. Wang, and J. P. Ting. 1999. Two distinct ␥ of MHC expression. Annu. Rev. Immunol. 19:331. interferon-inducible promoters of the major histocompatibility complex class II 14. Muhlethaler-Mottet, A., L. A. Otten, V. Steimle, and B. Mach. 1997. Expression transactivator gene are differentially regulated by STAT1, interferon regulatory of MHC class II molecules in different cellular and functional compartments is factor 1, and transforming growth factor ␤. Mol. Cell. Biol. 19:431. controlled by differential usage of multiple promoters of the transactivator CIITA. 41. Quelle, F. W., N. Sato, B. A. Witthuhn, R. C. Inhorn, M. Eder, A. Miyajima, EMBO J. 16:2851. J. D. Griffin, and J. N. Ihle. 1994. JAK2 associates with the ␤c chain of the 15. Piskurich, J. F., Y. Wang, M. W. Linhoff, L. C. White, and J. P. Ting. 1998. receptor for granulocyte-macrophage colony-stimulating factor, and its activation Ј Identification of distinct regions of 5 flanking DNA that mediate constitutive, requires the membrane-proximal region. Mol. Cell. Biol. 14:4335. IFN-␥, STAT1, and TGF-␤-regulated expression of the class II transactivator 42. Mui, A. L., H. Wakao, A. M. O’Farrell, N. Harada, and A. Miyajima. 1995. gene. J. Immunol. 160:233. Interleukin-3, granulocyte-macrophage colony stimulating factor and interleu- 16. Nicola, N. A. 2000. GM-CSF. In Cytokine Reference. J. J. Oppenheim and M. kin-5 transduce signals through two STAT5 homologs. EMBO J. 14:1166. Feldmann, eds. Academic Press, London, p. 899. 17. Hamilton, J. A. 2002. GM-CSF in inflammation and autoimmunity. Trends Im- 43. Brizzi, M. F., M. G. Aronica, A. Rosso, G. P. Bagnara, Y. Yarden, and munol. 23:403. L. Pegoraro. 1996. Granulocyte-macrophage colony-stimulating factor stimulates 18. Mach, N., and G. Dranoff. 2000. Cytokine-secreting tumor cell vaccines. Curr. JAK2 signaling pathway and rapidly activates p93fes, STAT1 p91, and STAT3 Opin. Immunol. 12:571. p92 in polymorphonuclear leukocytes. J. Biol. Chem. 271:3562. 19. Jones, T. C. 1999. Use of granulocyte-macrophage colony stimulating factor 44. Welte, T., F. Koch, G. Schuler, J. Lechner, W. Doppler, and C. Heufler. 1997. (GM-CSF) in prevention and treatment of fungal infections. Eur. J. Cancer 35 Granulocyte-macrophage colony-stimulating factor induces a unique set of STAT (Suppl. 3):S8. factors in murine dendritic cells. Eur. J. Immunol. 27:2737. 20. Bertho, N., B. Drenou, B. Laupeze, C. L. Berre, L. Amiot, J. M. Grosset, 45. Shibata, Y., P. Y. Berclaz, Z. C. Chroneos, M. Yoshida, J. A. Whitsett, and O. Fardel, D. Charron, N. Mooney, and R. Fauchet. 2000. HLA-DR-mediated B. C. Trapnell. 2001. GM-CSF regulates alveolar macrophage differentiation and apoptosis susceptibility discriminates differentiation stages of dendritic/mono- innate immunity in the lung through PU.1. Immunity 15:557. cytic APC. J. Immunol. 164:2379. 46. Li, J., A. I. Colovai, R. Cortesini, and N. Suciu-Foca. 2000. Cloning and func- The Journal of Immunology 2383

tional characterization of the 5Ј-regulatory region of the human CD86 gene. Hum. 52. De Vries, E. G., P. H. Willemse, B. Biesma, A. C. Stern, P. C. Limburg, and Immunol. 61:486. E. Vellenga. 1991. Flare-up of rheumatoid arthritis during GM-CSF treatment 47. Nguyen, V. T., and E. N. Benveniste. 2000. Involvement of STAT-1 and ets after chemotherapy. Lancet 338:517. family members in interferon-␥ induction of CD40 transcription in microglia/ 53. Hansen, P. B., H. E. Johnsen, and E. Hippe. 1993. Autoimmune hypothyroidism macrophages. J. Biol. Chem. 275:23674. and granulocyte-macrophage colony-stimulating factor. Eur. J. Haematol. 48. Brissette, W. H., D. A. Baker, E. J. Stam, J. P. Umland, and R. J. Griffiths. 1995. 50:183. GM-CSF rapidly primes mice for enhanced cytokine production in response to 54. Hoekman, K., B. M. von Blomberg-van der Flier, J. Wagstaff, H. A. Drexhage, LPS and TNF. Cytokine 7:291. and H. M. Pinedo. 1991. Reversible thyroid dysfunction during treatment with GM-CSF. Lancet 338:541. 49. Re, F., S. L. Belyanskaya, R. J. Riese, B. Cipriani, F. R. Fischer, F. Granucci, 55. Biondo, M., Z. Nasa, A. Marshall, B. H. Toh, and F. Alderuccio. 2001. Local P. Ricciardi-Castagnoli, C. Brosnan, L. J. Stern, J. L. Strominger, and transgenic expression of granulocyte macrophage-colony stimulating factor ini- L. Santambrogio. 2002. Granulocyte-macrophage colony-stimulating factor in- tiates autoimmunity. J. Immunol. 166:2090. duces an expression program in neonatal microglia that primes them for antigen 56. Campbell, I. K., A. Bendele, D. A. Smith, and J. A. Hamilton. 1997. Granulocyte- presentation. J. Immunol. 169:2264. macrophage colony stimulating factor exacerbates collagen induced arthritis in 50. Cooper, M. A., T. A. Fehniger, and M. A. Caligiuri. 2001. The biology of human mice. Ann. Rheum. Dis. 56:364. natural killer-cell subsets. Trends Immunol. 22:633. 57. Cook, A. D., E. L. Braine, I. K. Campbell, M. J. Rich, and J. A. Hamilton. 2001. 51. Xu, W. D., G. S. Firestein, R. Taetle, K. Kaushansky, and N. J. Zvaifler. 1989. Blockade of collagen-induced arthritis post-onset by antibody to granulocyte- Cytokines in chronic inflammatory arthritis. II. Granulocyte-macrophage colony- macrophage colony-stimulating factor (GM-CSF): requirement for GM-CSF in stimulating factor in rheumatoid synovial effusions. J. Clin. Invest. 83:876. the effector phase of disease. Arthritis Res. 3:293. Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021