Interleukin-4 regulates proteoglycan-induced arthritis by specifically suppressing the innate immune response.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 56, No. 3, March 2007, pp 861–870 DOI 10.1002/art.22422 © 2007, American College of Rheumatology Interleukin-4 Regulates Proteoglycan-Induced Arthritis by Specifically Suppressing the Innate Immune Response Yanxia Cao,1 Frank Brombacher,2 Miklos Tunyogi-Csapo,1 Tibor T. Glant,1 and Alison Finnegan1 flammatory cytokines IL-1␤ and IL-6 and with elevated cytokine (IL-1␤ and IL-6) and chemokine (macrophage inflammatory protein 1␣ [MIP-1␣] and MIP-2) transcripts from joints. However, arthritis susceptibility did not correlate with IL-2 or interferon-␥ (IFN␥) concentrations or with PG-specific antibody IgG2a isotype, since levels of IL-2, IFN␥, or PG-specific antibody IgG2a isotype in control (IL-4R␣flox/ⴚ) and LysMcreIL4R␣flox/ⴚ mice were reduced in comparison with those in IL-4R␣ⴚ/ⴚ mice. Conclusion. These findings indicate that IL-4 functions as a major antiinflammatory cytokine in PGIA by governing the activity of macrophages/ neutrophils and less so by controlling T cell activity and autoantibody isotype expression. Objective. Interleukin-4 (IL-4) is an antiinflammatory cytokine that inhibits the onset and severity of proteoglycan-induced arthritis (PGIA). To distinguish the role of IL-4 in the innate immune response versus the adaptive immune response, we generated mice with a specific deletion of the IL-4 receptor ␣-chain (IL-4R␣) in macrophages and neutrophils. Methods. To obtain mice in which IL-4R␣ is deleted in macrophages and neutrophils, we intercrossed mice carrying a loxP-flanked (floxed) IL-4R␣ allele and Cre recombinase expressed under control of the regulatory region for the lysozyme M gene (LysMcre mice) with conditional IL-4R␣flox/flox mice and then mated them to complete IL-4R␣ⴚ/ⴚ mice to obtain hemizygous LysMcreIL-4R␣flox/ⴚ mice. LysMcre-negative IL-4R␣flox/ⴚ mice (IL-4R␣flox/ⴚ mice) were used as control mice. PGIA was induced by immunization with human PG in adjuvant. The onset, incidence, and severity of arthritis were monitored over time. Levels of proinflammatory cytokines were measured in the sera of PG-immunized mice, and cytokine and chemokine transcripts were measured in joints. Results. The severity of PGIA was exacerbated in IL-4R␣ⴚ/ⴚ and LysMcreIL-4R␣flox/ⴚ mice in comparison with control (IL-4R␣flox/ⴚ) mice. The increase in arthritis susceptibility in IL-4R␣ⴚ/ⴚ and LysMcreIL-4R␣flox/ⴚ mice correlated with elevated serum levels of the proin- Rheumatoid arthritis (RA) is a chronic inflammatory joint disease characterized by synovial proliferation and infiltration of leukocytes, which leads over time to the destruction of cartilage and bone. It is well accepted that tumor necrosis factor ␣ (TNF ␣ ), interleukin-1 (IL-1), and IL-6 are key cytokines that drive inflammation and cartilage destruction (1). Reciprocally, there are antiinflammatory cytokines such as IL-4 that regulate inflammation. Many of the IL-4 regulatory effects are directed at modulating macrophage activity. IL-4 suppresses proinflammatory cytokines (TNF␣, IL-1␤, and IL-12) (2–4) and chemokines (interferon-␥–inducible protein 10 and macrophage migration inhibitory factor) (2,5) as well as the cell surface molecules intercellular adhesion molecule 1, Fc␥ receptor, and IL-12 receptor (IL-12R) (3,4). In addition, IL-4 directly induces the differentiation of T cells playing a central role in the development of committed Th2 effector cells (5). IL-4 facilitates Th2 differentiation by activation of the transcription factor GATA-3, which attenuates Th1 responses by down-regulating STAT-4 (6). Immunoglobulin isotypes are also regulated by IL-4. Drs. Glant and Finnegan’s work was supported by the NIH (grant AR-045652 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases). 1 Yanxia Cao, MS, Miklos Tunyogi-Csapo, MD, Tibor T. Glant, MD, PhD, Alison Finnegan, PhD: Rush University Medical Center, Chicago, Illinois; 2Frank Brombacher, MD: University of Cape Town, Cape Town, South Africa. Address correspondence and reprint requests to Alison Finnegan, PhD, Department of Medicine, Section of Rheumatology, Rush University Medical Center, 1735 West Harrison Street, Cohn Research Building, Room 726, Chicago, IL 60612. E-mail: Alison_Finnegan@rush.edu. Submitted for publication August 30, 2006; accepted in revised form November 20, 2006. 861 862 Specifically, interferon-␥ (IFN␥)–mediated IgG2a responses are suppressed by IL-4 (7,8). The antiinflammatory activity of IL-4 was confirmed in vivo in several models of arthritis. We and others have demonstrated that systemic administration of IL-4 effectively suppresses disease in proteoglycaninduced arthritis (PGIA) and collagen-induced arthritis (CIA) (9,10). Treatment with IL-4 or local expression of IL-4 in CIA prevents cartilage and bone destruction (11,12). In PGIA, we further showed that arthritis is exacerbated in IL-4⫺/⫺ mice, and IL-4 mediates this effect through activation of STAT-6 (7). Aggravated arthritis in IL-4⫺/⫺ mice is associated with an increase in chemokine (macrophage inflammatory protein 1␤ [MIP-1␤], MIP-1␣, MIP-2, and monocyte chemoattractant protein 1 [MCP-1]) and cytokine (IL-12p35, IL-1␤, IL-6, and TNF␣) transcripts in joints before development of any signs of inflammation or cellular infiltration (7). Thus, IL-4 may regulate the activation of resident synovial macrophages reducing infiltration of other leukocytes into the joint. In addition, elevated production of IFN␥ by T cells and a dramatic increase in the PG-specific antibody of the IgG2a isotype were observed in IL-4⫺/⫺ mice (7,8). Consequently, there are several ways in which IL-4 may regulate the onset and severity of PGIA. IL-4 may directly suppress macrophage chemokine and cytokine production, inhibit Th1 differentiation and the production of IFN␥, or control the IgG isotype of the autoantibodies produced. To distinguish between the regulatory role of IL-4 in cells of the innate immune system (macrophages and neutrophils) and in those of the adaptive immune system (T cells and B cells) in PGIA, we engineered mice with selective deletion of the IL-4R ␣-chain (IL4R␣) in macrophages and neutrophils. To obtain these mice, we intercrossed mice carrying a loxP-flanked (floxed) IL-4R␣ allele and Cre recombinase expressed under control of the regulatory region for the lysozyme M gene (LysMcre mice) with conditional IL-4R␣flox/flox mice and then mated them to complete IL-4R␣⫺/⫺ mice to obtain hemizygous LysMcreIL-4R␣flox/⫺ mice. It has been previously demonstrated that LysMcreIL-4R␣flox/⫺ mice have an impairment in alternative macrophage activation that is necessary for down-regulating cellmediated immune responses to infectious agents (13). In the present study, we demonstrated that the severity of PGIA was significantly enhanced in IL4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice in comparison with control IL-4R␣flox/⫺ mice. Exacerbated arthritis in IL-4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice was associated with an increase in proinflammatory cytokines and che- CAO ET AL mokines and not with increased Th1 IFN␥ production or an alternation in PG-specific antibody isotype. MATERIALS AND METHODS Mice. BALB/c and C3H mice are susceptible to PGIA (14,15). Female wild-type (WT) BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were backcrossed to BALB/c for at least 9 generations. Breeding pairs of IL-4⫺/⫺ mice were generously provided by Professor H. Eibel (Klinische Forschergruppe fur Rheumatologie, Freiburg, Germany). Cell-specific IL-4R␣–deficient mice were obtained by intercrossing LysMcre mice with conditional IL4R␣flox/flox mice and then further mated to complete IL4R␣⫺/⫺ mice to generate hemizygous LysMcreIL-4R␣flox/⫺ mice (13). LysMcre-negative IL-4R␣flox/⫺ mice (IL-4R␣flox/⫺ mice) were used as control mice. All animal procedures were conducted under a protocol approved by the Institutional Animal Care and Use Committee of Rush University Medical Center. Clinical evaluation of arthritis. Female mice ages 12–16 weeks were immunized intraperitoneally (IP) with 150 g human PG emulsified in Freund’s complete adjuvant (CFA), as described (7,10). At weeks 3 and 6, mice received booster immunizations with 100 g PG in Freund’s incomplete adjuvant. Mice were monitored biweekly for signs of arthritis subsequent to the final immunization, and the same investigator inspected and scored the paws in a blinded manner. Paws were scored for clinical signs of arthritis on a scale of 1–4, as follows: 0 ⫽ normal; 1 ⫽ mild swelling and erythema; 2 ⫽ moderate erythema and swelling of the paw; 3 ⫽ more intense erythema, swelling, and redness affecting a greater proportion of the paw; 4 ⫽ severe erythema, swelling, and redness affecting the entire paw. A cumulative score ranging from 0 to 16, based on individual paw scores of 0 to 4 was assigned for each animal. Assay of PG-specific immune responses. IL-2 was measured from T cells purified from peritoneal exudates of PG-immunized mice by passage through nylon wool. T cells (2.5 ⫻ 105/well) were cultured with irradiated (2,500 rads) naive WT spleen cells (2.5 ⫻ 105/well) in quadruplicate in 96-well Falcon plates (Fisher Scientific, Fair Lawn, NJ) in serum-free HL-1 media containing 100 g/ml penicillin, 100 g/ml streptomycin, and 2 mM L-glutamine, in the presence and absence of human PG (10 g/ml). Supernatants were collected on day 1. To determine the production of IFN␥ and IL-15, spleen cells (2 ⫻ 106/well) were cultured for 4 days in 24-well plates in complete media in the presence of human PG (10 g/ml). To determine the amount of IL-1␤, IL-6, and TNF␣ in the serum, mice were bled at the time that they were killed, and serum samples were stored at ⫺20°C. The concentrations of serum IL-1␤, IL-6, and TNF␣ and of supernatant IL-2 and IFN␥ (BD Biosciences, San Jose, CA) and IL-15 (R&D Systems, Minneapolis, MN) were measured by enzymelinked immunosorbent assay (ELISA). PG-specific serum antibody concentrations were measured by ELISA. Mice were bled, and serum samples were stored at ⫺20°C. Ninety-six enzyme immunoassay tissue culture “half area” plates (Costar, Corning, NY) were coated overnight at 4°C with 0.5 g human PG or with 0.75 g native IL-4 RECEPTOR EXPRESSION ON MACROPHAGES/NEUTROPHILS CONTROLS PGIA SEVERITY mouse PG in carbonate buffer. Serially diluted samples were incubated with the immobilized PGs, and plate-bound human PG– or native mouse PG–specific antibodies were detected using peroxidase-conjugated rabbit IgG against mouse IgG1 and IgG2a (Zymed, South San Francisco, CA), respectively. Colorimetric change in each sample was measured with a spectrophotometer at 490 nm and compared with a standard curve of known concentrations of unlabeled murine IgG1 and IgG2a. Assay for IL-4 inhibition of macrophage and neutrophil cytokine production. Macrophages from WT (IL4R␣flox/⫺), LysMcreIL-4R␣flox/⫺, and IL-4R␣⫺/⫺ mice were elicited by injection of 3% thioglycolate (1 ml) IP, and peritoneal exudate cells were harvested 5 days later. Peritoneal exudate cells (2 ⫻ 106) were cultured for 2 hours at 37°C, then extensively washed with ice-cold RPMI 1640 to remove nonadherent cells. Adherent cells were incubated overnight and washed again to remove additional nonadherent cells. Neutrophils were elicited by injection of PG in CFA, and peritoneal exudate cells were harvested at 24 hours. Neutrophils comprised ⬃70% of the peritoneal exudate cells at this point. Adherent macrophages and neutrophils were pretreated with IL-4 (100 units/ml) for 2 hours and then stimulated with lipopolysaccharide (LPS; 10 ng/ml) for 24 hours. Supernatants were harvested and assayed for IL-12 and TNF␣ by ELISA. Quantitative reverse transcriptase–polymerase chain reaction (RT-PCR). Splenic T cells and B cells were negatively selected using a mixture of biotinylated monoclonal antibodies (mAb) directed against cell surface antigens on unwanted cells (Miltenyi Biotec, Auburn, CA), followed by incubation with antibiotin mAb–conjugated microbeads, and were then passed through a magnetic-activated cell sorter (MACS) separation column in an autoMACS cell separator (Miltenyi Biotec). Cells were ⬎95% CD3⫹ and ⬎98% B220⫹, respectively, by flow cytometry (FACSCalibur instrument with CellQuest software; BD Biosciences). Thioglycolate-induced macrophages were isolated as described above. RNA was isolated from T cells, B cells, and macrophages or hind paw joints using the Tri-Reagent (Molecular Research Center, Cincinnati, OH). Reverse transcription was performed with random hexamers for priming and SuperScript II (Invitrogen, Carlsbad, CA). Optimum concentration of probe, primers, and MgCl2 was determined by preliminary experiments. The PCR reaction volume was 25 l, containing 0.3 l of each primer and 0.3 l of Taq (Invitrogen), and the annealing temperature was 55°C (35 seconds). Complementary DNA (cDNA) was analyzed for the expression of cytokine PCR products by the DNAintercalating SYBR Green I fluorescent dye using a GeneAmp 5700 Sequence Detection System (Applied Biosystems, Foster City, CA). The following primers were used: for murine IL-6, 5⬘-TTC-CAT-CCA-GTT-GCC-TTC-TT-3⬘ (forward) and 5⬘TTT-CCA-CGA-TTT-CCC-AGA-GA-3⬘ (reverse); for murine IL-1␤, 5⬘-TTG-ACG-GAC-CCA-AAA-GAT-G-3⬘ (forward) and 5⬘-AGA-AGG-TGC-TCA-TGT-CCT-CA-3⬘ (reverse); for murine TNF␣, 5⬘-ACG-GCA-TGG-ATC-TCAAAG-AC-3⬘ (forward) and 5⬘-GTG-GGT-GAG-GAG-CACGTA-GT-3⬘ (reverse); for murine IL-4R␣, 5⬘-CCT-CACACT-CCA-CAC-CAA-TG-3⬘ (forward) and 5⬘-AGC-CTGGGT-TCC-TTG-TAG-GT-3⬘ (reverse); for murine MIP-1␣, 5⬘-ATG-AAG-GTC-TCC-ACC-ACT-GC-3⬘ (forward) and 863 5⬘-GAT-GAA-TTG-GCG-TGG-AAT-CT-3⬘ (reverse); for murine MIP-2, 5⬘-AGT-GAA-CTG-CGC-TGT-CAA-TG-3⬘ (forward) and 5⬘-GCC-CTT-GAG-AGT-GGC-TAT-GA-3⬘ (reverse); and for MCP-1, 5⬘-CCC-AAT-GAG-TAG-GCTGGA-GA-3⬘ (forward) and 5⬘-TCT-GGA-CCC-ATT-CCTTCT-TG-3⬘ (reverse). To verify that equivalent amounts of RNA were added to each PCR reaction, PCR amplification of the murine ␤-actin was performed for each sample. All samples were run in triplicate. The relative differences among the samples were determined using the ⌬⌬Ct method, where Ct ⫽ threshold cycle. The average ‚Ct value was calculated for each sample using the average Ct value for murine ␤-actin to account for loading differences in the RT-PCR and the average Ct value for the input DNA samples to normalize the IL-4R␣ or cytokine/ chemokine results. The ⌬⌬Ct value for LysMcreIL-4R␣flox/⫺ DNA samples was then calculated by subtracting the ⌬Ct for the WT (IL-4R␣flox/⫺) samples. The ⌬⌬Ct values for the joint DNA samples were calculated by subtracting the ⌬Ct for the control samples (joints from naive, nonimmune mice) from the ⌬Ct for the WT (IL-4R␣flox/⫺), IL-4R␣⫺/⫺, and LysMcreIL4R␣flox/⫺ (PG-immunized) samples. The ⌬⌬Ct values were converted to fold differences compared with the control by raising 2 to the ⫺⌬⌬Ct power (2⫺⌬⌬Ct). Statistical analysis. The Mann-Whitney U test was used to compare nonparametric data for statistical significance. P values less than 0.05 were considered significant. RESULTS Regulation of PGIA severity by IL-4. We have previously reported that PGIA develops more rapidly, with increased severity, in IL-4⫺/⫺ mice (7). We confirmed these findings and also examined the induction of arthritis in IL-4R␣⫺/⫺ mice. BALB/c (WT), IL-4⫺/⫺, and IL-4R␣⫺/⫺ mice were immunized with human PG, and development of arthritis was monitored over time for the onset and severity of disease. As shown in Figures 1A and C, the onset of arthritis was exacerbated in IL-4⫺/⫺ mice but did not reach statistical significance in IL-4R␣⫺/⫺ mice in comparison with age-matched WT mice. The severity of arthritis was significantly enhanced in IL-4⫺/⫺ and IL-4R␣⫺/⫺ mice (Figures 1B and D). These differences were significant between days 52 and 75 comparing WT and IL-4⫺/⫺ mice and between days 58 and 70 comparing WT and IL-4R␣⫺/⫺ mice (P ⬍ 0.05 for both). Characterization of LysMcreIL-4R␣flox/⫺ mice. To determine whether cells of the innate immune system are involved in IL-4 suppression of arthritis, mice were generated with selective depletion of IL-4R␣ in macrophages and neutrophils. To disrupt the IL-4R␣ gene, IL-4R␣flox/flox mice were intercrossed with transgenic BALB/c mice in which a Cre recombinase–specific cDNA was introduced into the mouse lysozyme M gene 864 CAO ET AL Figure 1. Exacerbation of proteoglycan-induced arthritis (PGIA) in interleukin-4–knockout (IL-4⫺/⫺) mice and mice with selective deletion of the IL-4 receptor ␣-chain (IL-4R␣⫺/⫺ mice) in comparison with wild-type (WT) mice. BALB/c (WT), IL-4⫺/⫺, and IL-4R␣⫺/⫺ mice were immunized with human PG 3 times at 3-week intervals and monitored for the incidence and severity of PGIA at given time points. A and C, Incidence denotes the percentage of mice that developed PGIA. ⴱ ⫽ P ⬍ 0.05 for WT mice versus IL-4⫺/⫺ mice. B and D, Severity (cumulative arthritis score) is the sum of paw inflammation scores in individual mice divided by the number of arthritic mice. Values are the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05 for WT mice (n ⫽ 8) versus IL-4⫺/⫺ mice (n ⫽ 9); ⴱ ⫽ P ⬍ 0.05 for WT mice (n ⫽ 14) versus IL-4R␣⫺/⫺ mice (n ⫽ 17). by a knockout approach (16). Lysozyme M has been shown to be exclusively expressed in cells of the macrophage and granulocyte lineages after hematopoietic differentiation (17). To increase the efficiency of cellspecific deletion, we used hemizygous mice bearing 1 floxed IL-4R␣ allele and 1 already disrupted IL-4R␣ allele (IL-4R␣flox/⫺). IL-4R␣flox/⫺ mice intercrossed with transgenic LysMcre mice on an IL-4R␣⫺/⫺ background yielded LysMcreIL-4R␣flox/⫺ mice. These mice have normal numbers and distributions of T and B cells in spleen and lymph nodes as described (13). To confirm that the disruption of the IL-4R␣ gene was selective in macrophages and not T or B cells, expression of IL-4R␣ messenger RNA (mRNA) from peritoneal macrophages and splenic T and B cells was evaluated in LysMcreIL-4R␣flox/⫺ mice. As shown in Figure 2A, mean ⫾ SD expression of IL-4R␣ mRNA in LysMcreIL-4R␣flox/⫺ macrophages was reduced to 0.176 ⫾ 0.14 (82.4% suppressed) relative to that in WT (IL-4R␣flox/⫺) macrophages (set at 1.0). Furthermore, there was no reduction in IL-4R␣ expression in LysMcreIL-4R␣flox/⫺ T or B cells. To assess whether the IL-4R␣ was functional in LysMcreIL-4R␣flox/⫺ macrophages and neutrophils, suppression of LPS-induced cytokine secretion by IL-4 was evaluated. Adherent macrophages or neutrophils from WT (IL-4R␣flox/⫺), IL-4 RECEPTOR EXPRESSION ON MACROPHAGES/NEUTROPHILS CONTROLS PGIA SEVERITY 865 Figure 2. Substantial reduction of IL-4R␣ mRNA in macrophages from LysMcreIL-4R␣flox/⫺ mice, and inability of IL-4 to inhibit lipopolysaccharide (LPS)–induced IL-12 from macrophages or LPS-induced tumor necrosis factor ␣ (TNF␣) from neutrophils in IL-4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice. A, RNA was isolated from purified T cells, B cells, and macrophages. The relative fold difference in IL-4R␣ mRNA between IL-4R␣flox/⫺ and LysMcreIL-4R␣flox/⫺ mice was determined by quantitative reverse transcriptase–polymerase chain reaction. The relative differences among the samples were determined using the ⌬⌬Ct method, where Ct ⫽ threshold cycle (see Materials and Methods). Values are the mean and SD of triplicate samples. ⴱ ⫽ P ⬍ 0.05 for macrophages from control (IL-4R␣flox/⫺) mice (set at 1.0) versus macrophages from LysMcreIL-4R␣flox/⫺ mice. B, Adherent macrophages or C, adherent neutrophils from WT (IL-4R␣flox/⫺), IL-4R␣⫺/⫺, and LysMcreIL-4R␣flox/⫺ mice were pretreated with IL-4 for 2 hours and then stimulated for 24 hours with LPS. Supernatants were assayed for IL-12 (B) and TNF␣ (C) by enzyme-linked immunosorbent assay. Values are the mean and SD of triplicate cultures. Data are representative of 2 separate experiments. ⴱ ⫽ P ⬍ 0.05 versus macrophages or neutrophils from IL-4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice. See text for description of LysMcreIL-4R␣flox/⫺ mice. See Figure 1 for other definitions. IL-4R␣⫺/⫺, and LysMcreIL-4R␣flox/⫺ mice were treated with IL-4 for 2 hours and then stimulated with LPS for 24 hours. IL-4 suppressed the production of IL-12 in WT macrophages, but not in IL-4R␣⫺/⫺ or LysMcreIL- 4R␣flox/⫺ macrophages (Figure 2B). Similarly, IL-4 reduced the secretion of TNF␣ in WT neutrophils, but not in IL-4R␣⫺/⫺ or LysMcreIL-4R␣flox/⫺ neutrophils (Figure 2C). These data demonstrate that IL-4R␣ was Figure 3. Enhancement of PGIA onset and severity in IL-4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice. WT (IL-4R␣flox/⫺) (n ⫽ 17), IL-4R␣⫺/⫺ (n ⫽ 19), and LysMcreIL-4R␣flox/⫺ (n ⫽ 25) mice were immunized with human PG as described in Figure 1. A, Incidence represents the percentage of mice that developed PGIA. ⴱ ⫽ P ⬍ 0.05 for WT mice versus IL-4R␣⫺/⫺ mice; # ⫽ P ⬍ 0.05 for WT mice versus LysMcreIL-4R␣flox/⫺ mice. B, Severity (cumulative arthritis score) is the sum of paw inflammation scores in individual mice divided by the total number of arthritic mice. Values are the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05 for WT mice versus IL-4R␣⫺/⫺ mice; # ⫽ P ⬍ 0.05 for WT mice versus LysMcreIL-4R␣flox/⫺ mice. See text for description of LysMcreIL-4R␣flox/⫺ mice. See Figure 1 for definitions. 866 CAO ET AL Figure 4. Increased serum levels of proinflammatory cytokines and increased cytokine/chemokine transcripts from joints of IL-4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice. A, Serum concentrations of IL-6, IL-1␤, and tumor necrosis factor ␣ (TNF␣) were measured by enzyme-linked immunosorbent assay (n ⫽ 6 mice per group). B and C, The relative fold increase in cytokine mRNA for IL-6, IL-1␤, and TNF␣ (B) and chemokine mRNA for macrophage inflammatory protein 1␣ (MIP-1␣), MIP-2, and monocyte chemoattractant protein 1 (MCP-1) (C) in PG-immunized WT (IL-4R␣flox/⫺), IL-4R␣⫺/⫺, and LysMcreIL-4R␣flox/⫺ mice in comparison with nonimmune mice was determined by quantitative reverse trascriptase–polymerase chain reaction. Data are representative of 2 separate experiments. The relative differences among the samples were determined using the ⌬⌬Ct method (n ⫽ 4 mice per group), where Ct ⫽ threshold cycle (see Materials and Methods). Values are the mean and SD of triplicate samples. ⴱ ⫽ P ⬍ 0.05 versus WT mice. See text for description of LysMcreIL-4R␣flox/⫺ mice. See Figure 1 for other definitions. functionally disrupted in macrophages and neutrophils of LysMcreIL-4R␣flox/⫺ mice. Exacerbation of PGIA in LysMcreIL-4R␣flox/⫺ mice. To determine if IL-4 regulates the severity of PGIA through its effect on innate immune cells, macrophage/neutrophil-specific IL-4R ␣ –deficient (LysMcreIL-4R␣flox/⫺) mice were immunized with human PG. Development of arthritis was compared in mice with a complete deficiency in IL-4R␣ (IL-4R␣⫺/⫺) and mice hemizygous for IL-4R␣ expression (IL4R␣flox/⫺), here designated WT control mice. Paw erythema and swelling developed in IL-4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice more rapidly and with greater severity than in control mice (Figures 3A and B). However, over time, control mice developed a degree of paw erythema and swelling similar to that in IL-4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice. These data suggest that IL-4R ␣ –mediated mechanisms in macrophages/ neutrophils are responsible for the rapid onset of arthritis in LysMcreIL-4R␣flox/⫺ mice. Correlation of an increase in proinflammatory cytokines and chemokines with enhanced PGIA in IL4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice. IL-4 is recognized as an antiinflammatory cytokine that modulates macrophage activity by suppressing proinflammatory cytokines and chemokines. To determine whether the intensity of cytokine and chemokine expression correlated with exacerbation of PGIA in IL-4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice, we examined serum cytokines and cytokine and chemokine transcripts from paws of WT (IL-4R␣flox/⫺), IL-4R␣⫺/⫺, and LysMcreIL-4R␣flox/⫺ mice immunized with human PG. Mice were killed between days 45 and 55 after the initial immunization with PG. Serum levels of the cytokines IL-1␤ and IL-6 were significantly increased in IL-4R ␣ ⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice in comparison with those in WT (IL-4R␣flox/⫺) mice (Figure 4A), whereas the TNF␣ level was low and no differences were detected between the different groups of mice. Our previous studies showed that an increase in cytokine and chemokine transcripts in joints of PG-immunized mice correlates with the early onset of paw redness and swelling. Similarly, we assessed whether enhanced arthritis in IL4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice correlated with an amplified expression of IL-6, IL-1␤, and TNF␣ transcripts in joints of these mice. IL-6, IL-1␤, and TNF␣ transcripts were increased in PG-immunized WT (IL-4R␣flox/⫺), IL-4R␣⫺/⫺, and LysMcreIL-4R␣flox/⫺ mice in comparison with nonimmune mice (Figure 4B). IL-1 ␤ transcripts were significantly increased in LysM cre IL-4R ␣ flox/⫺ mice compared with PGimmunized WT mice, whereas IL-6 transcripts in LysMcreIL-4R␣flox/⫺ mice were increased, but not significantly, compared with PG-immunized WT mice (Figure IL-4 RECEPTOR EXPRESSION ON MACROPHAGES/NEUTROPHILS CONTROLS PGIA SEVERITY 867 Figure 5. Similarity of T cell cytokines and PG-specific antibody isotypes in control and LysMcreIL-4R␣flox/⫺ mice. A, The concentration of IL-2 was measured by enzyme-linked immunosorbent assay (ELISA) in supernatants of T cells stimulated for 24 hours in the absence (media control) or presence of human PG. B, The concentration of interferon-␥ (IFN␥) was measured in supernatants of spleen cells stimulated for 4 days with human PG or left unstimulated (as in A) (n ⫽ 9 mice per group). C, The concentrations of PG-specific anti-mouse IgG1 (mIgG1) and IgG2a (mIgG2a) and D, PG-specific anti-human IgG1 (hIgG1) and IgG2a (hIgG2a) were measured by ELISA (n ⫽ 9 mice per group). Values are the mean and SEM. ⴱ ⫽ P ⬍ 0.05 versus concentrations of cytokines and PG-specific antibody isotypes in WT (IL-4R␣flox/⫺) and LysMcreIL-4R␣flox/⫺ mice. Data are representative of 2 separate experiments. See text for description of LysMcreIL-4R␣flox/⫺ mice. See Figure 1 for other definitions. 4B). Both IL-1␤ and IL-6 transcripts were significantly increased in IL-4R␣⫺/⫺ mice compared with WT mice. When we assessed chemokine expression in joints, MIP-1␣ (CCL3), MIP-2 (CXCL1), and MCP-1 (CCL2) were increased in IL-4R␣⫺/⫺ and LysMcreIL4R␣flox/⫺ mice in comparison with WT mice (Figure 4C). The differences were statistically significant for MIP-1␣ and MIP-2, but not for MCP-1. The reduced levels of cytokines and transcripts in LysMcreIL4R␣flox/⫺ mice in comparison with IL-4R␣⫺/⫺ mice may be due to the incomplete deletion of IL-4R␣ in macrophages, as depicted in Figure 2A. These results demonstrate that augmented PGIA in IL-4R ␣ ⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice is associated with elevated levels of proinflammatory cytokines and chemokines. No correlation of enhanced arthritis severity in LysMcreIL-4R␣flox/⫺ mice with T cell cytokine production or PG-specific antibody isotype. We previously reported that PG-specific IFN␥ and PG-specific IgG2a antibody isotype were amplified in IL-4⫺/⫺ mice. We speculated at the time that either an increase in Th1 cells secreting IFN␥ or an increase in PG-specific IgG2a pathogenic antibodies could contribute to the enhanced arthritis in IL-4⫺/⫺ mice (8). We therefore assessed whether T cell cytokines or antibody isotype correlated with the severity of PGIA in IL-4R␣⫺/⫺ and LysMcreIL- 868 CAO ET AL 4R␣flox/⫺ mice. We specifically tested the requirement for IFN␥ because development of PGIA is dependent on IFN␥ and STAT-4, and IL-4 has been shown to inhibit several components of the Th1 pathway, in particular STAT-4, IL-12R␤2, and T-box expressed in T cells (T-bet) (7,18). T cell IL-2 production was assessed in culture supernatants from T cells activated with human PG in the presence of irradiated naive splenocytes. IFN␥ was evaluated from supernatant of splenocytes stimulated in the presence and absence of human PG. Analysis of cytokine levels revealed that cells obtained from IL-4R␣⫺/⫺ mice produced significantly more PG-specific IFN␥ and IL-2 than either control (IL-4R␣flox/⫺) or LysMcreIL-4R␣flox/⫺ cells (Figures 5A and B). IL-15 levels in supernatants were undetectable (data not shown). PG-specific antibody isotypes, IgG1 and IgG2a, were measured in serum of PG-immunized mice at the time that they were killed. When we measured the PG-specific serum antibody titers, we found that both the anti-mouse PG IgG2a and the anti-human PG IgG2a responses were significantly increased in IL-4R␣⫺/⫺ mice, but not in either WT (IL-4R␣flox/⫺) or LysMcreIL-4R␣flox/⫺ mice (Figures 5C and D). These data demonstrate that similar to our reported findings in the IL-4⫺/⫺ mice, PG-specific T cell IFN␥ and IgG2a antibody are increased in IL-4R␣⫺/⫺ mice. However, despite the exacerbated arthritis in IL-4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice, IFN␥ levels and PG-specific IgG2a isotype were not increased in LysMcreIL4R␣flox/⫺ mice. These results suggest that augmented PGIA in LysMcreIL-4R␣flox/⫺ mice was not associated with a detectable increase in Th1 IFN␥ production or PG-specific IgG2a production. DISCUSSION The intent of this study was to determine whether IL-4 regulation of autoimmune arthritis was directed at cells of the innate immune system. We previously reported that administration of recombinant IL-4 completely prevents development of inflammation and histologic evidence of arthritis and suppresses acute clinical signs of arthritis (10). Reciprocally, we showed that arthritis onset and severity are exacerbated in IL-4⫺/⫺ mice (7). The pleiotropic effects of IL-4, involving macrophages, dendritic cells, and T cells, imply that IL-4 may suppress arthritis by a number of different mechanisms. Augmented proinflammatory cytokine and chemokine production in IL-4⫺/⫺ mice positively correlated with the early onset and severity of PGIA suggests that macrophages are an important target of IL-4 regulation (7). To assess this directly, we engineered mice with IL-4R␣ depleted selectively in macrophages and neutrophils. The enhanced susceptibility of LysMcre IL4R␣flox/⫺ mice to PGIA directly demonstrates an important role for IL-4 in controlling the activity of macrophages and/or neutrophils and suggests that these cells are the primary targets of IL-4 regulation in PGIA. It is important to note that IL-4 and IL-13 utilize the IL-4R ␣-chain for cell activation (13). The development of arthritis in IL-4⫺/⫺ and IL-4R␣⫺/⫺ mice was not significantly different, suggesting that IL-13 may not play a major role in PGIA. However, in the absence of testing the induction of arthritis in IL-13⫺/⫺ mice, we cannot completely rule out the possibility of such a role for IL-13. The increase in proinflammatory cytokines and chemokines in IL-4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice in vivo parallels the increase in rapid induction of arthritis in these mice. It is well accepted that proinflammatory cytokines and chemokines play a critical role in the pathogenesis of RA (19,20). Resident synovial macrophages and fibroblasts as well as infiltrating cells produce proinflammatory cytokines, mainly TNF␣, IL-1, and IL-6, that drive disease pathogenesis. These cytokines initiate a cascade of events that induce cartilagedegrading enzymes such as metalloproteases and that stimulate osteoclastogenesis and angiogenesis (21–27). Therapeutic blockade of TNF␣, IL-1, and IL-6 has proven efficacious in RA. Recruitment of cells to the synovial tissue involves several different chemokine families (20). The CC chemokines MIP-1␣ and MCP-1 are responsible for recruitment of mononuclear cells to the inflamed synovium, whereas the CXC chemokine MIP-2 acts on neutrophils. These chemokines are highly expressed in synovial tissue of RA patients. In animal models, targeted deletion of MCP-1, MIP-1␣, or MIP-2 inhibits arthritis (28,29). The ability of IL-4 to inhibit proinflammatory mediator expression has led investigators to test therapeutic gene transfer to deliver IL-4 directly to joints in experimental arthritis. In these studies, either directly delivering the IL-4 gene to the joint or transducing cells to express IL-4 suppresses CIA (11,12, 30,31). Our studies do not distinguish between the inhibitory effects of IL-4 on macrophages versus those on neutrophils. In RA and animal models of RA, neutrophils are the most abundant cell found in the synovial fluid (32,33). At one time, neutrophils were considered to be terminally differentiated at sites of inflammation and to survive there for only a short period of time. IL-4 RECEPTOR EXPRESSION ON MACROPHAGES/NEUTROPHILS CONTROLS PGIA SEVERITY However, more recently, neutrophils have been shown to persist for longer periods of time upon activation (34,35) and to secrete the proinflammatory cytokines IL-1 and TNF␣, which may contribute to joint inflammation (36,37). Neutrophils are essential for maintaining inflammation in PGIA, since in vivo depletion with anti–granulocyte-differentiation antigen mAb suppresses established disease (38). IL-4 has the capacity to modulate proinflammatory activity of neutrophils (39– 41), so it is possible that IL-4 regulates both macrophage and neutrophil activity in PGIA. However, our previous data demonstrate that cytokine and chemokine transcripts are increased in joints before any signs of cellular infiltration, which suggests that a resident cell population, probably synovial macrophages, is activated before infiltration of neutrophils into the joint. In the absence of IL-4 (in the case of IL-4⫺/⫺ mice), this resident population is activated more rapidly, correlating with earlier onset of PGIA. The ability of IL-4 to suppress Th1 cell differentiation and IFN␥ production has suggested that a switch from a Th1 response to a Th2 response might be responsible for the ability of IL-4 to control disease (7,10). IL-4 has been shown to inhibit STAT-4 induction, IL-12R␤2 expression, and the transcription factor T-bet (18). Moreover, the quantity of IFN␥ produced was associated with the magnitude of the PG-specific IgG2a isotype (8). Thus, inflammation mediated by IFN␥ and/or pathogenic IgG2a autoantibodies could be responsible for arthritis severity. However, we observed a similar increase in PGIA onset and severity in IL4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice, despite the fact that IL-4R␣⫺/⫺ mice produced significantly more IFN␥ and PG-specific IgG2a antibodies than LysMcre IL-4R␣flox/⫺ mice. The lack of association between IL-4R␣⫺/⫺ and LysMcreIL-4R␣flox/⫺ mice for IFN␥ production suggests that the ability of IL-4 to regulate IFN␥ is not sufficient to alter arthritis severity in PGIA. In summary, IL-4 is an important regulator of disease severity in arthritis principally by inhibiting cells of the innate immune system. 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