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Lexicon Genetics Incorporated, The Woodlands, TX 77381
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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, IL-6, IL-12, MCP-1, IFN-
, and IL-10 levels after systemic administration of LPS. Furthermore, absence of MKP-1 increases systemic levels of proinflammatory cytokines and exacerbates disease development in a mouse model of rheumatoid arthritis. When activated through TLR2, TLR3, TLR4, TLR5, and TLR9, bone marrow-derived MKP-1–/– macrophages exhibit increased cytokine production and elevated expression of the differentiation markers B7.2 (CD86) and CD40. MKP-1-deficient macrophages also show enhanced constitutive and TLR-induced activation of p38 MAPK. Based on these findings, we propose that MKP-1 is an essential component of the intracellular homeostasis that controls the threshold and magnitude of p38 MAPK activation in macrophages, and inflammatory conditions accentuate the significance of this regulatory function. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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, IL-1
, IL-6, IL-12, and IFN-, play a key role in sepsis and septic shock during bacterial infections, which account for 100,000 deaths annually in the United States alone (1, 2, 3). Induction of the inflammatory cascade by Gram-negative bacteria is initiated by binding of the bacterial endotoxin LPS to TLR4 on the cell surface, which leads to the formation of the MyD88/IL-1R-associated protein kinase/TNFR-associated factor 6 complex inside the cell. Subsequently, the signal transduction events trigger activation of Ser/Thr protein kinases that belong to the three MAPK subfamilies (4, 5, 6, 7). Several lines of evidence demonstrate that the activation status of these kinases determines the outcome of downstream events that regulate proinflammatory cytokine production. Transient activation of ERK1/2, JNK, and p38 MAPK was observed in both immortalized and primary murine macrophages after stimulation via TLR4 (8). Dual phosphorylation of p38 MAPK on tyrosine and threonine is a key event in the activation of multiple transcription factors (e.g., NF-
B, activating transcription factor-2, Elk-1, and C/EBP homologous protein that control TLR-induced expression of proinflammatory cytokine genes (5, 9). The JNK and p38 MAPK signaling pathways are also important contributors to the LPS-induced expression of TNF- by stabilizing the TNF- mRNA and relieving its translational silencing (9). In addition, a targeted deletion of MAPK-activated protein kinase-2, one of several kinases that are regulated through direct phosphorylation by p38 MAPK, results in a 90% reduction in the production of TNF-, and renders mice resistant to LPS/D-galactosamine-induced shock (6). Finally, one of the enzymes that phosphorylate MAPK kinase, MAPK kinase kinase 3, has been described as an essential signal transducer of the MyD88/IL-1R-associated kinase/TNFR-associated factor 6 complex in TLR4 signaling. Embryonic fibroblasts from MAPK kinase kinase 3-deficient mice exhibit deficient TLR4-induced IL-6 production and defective IL-1- and LPS-induced activation of NF-B, JNK, and p38 MAPK (10). Hence, multiple lines of evidence point to the importance of MAPK signaling in the regulation of the innate immune response. Moreover, TLR signals and the MAPK pathway also control cytokine release during the activation and effector phases of adoptive immune responses (11, 12). Effective termination of proinflammatory cytokine production during innate and adoptive immune responses is essential to prevent potentially detrimental systemic effects, including septic shock and autoimmunity. Disregulation of signaling mechanisms that limit the release of proinflammatory cytokines is implicated in the pathogenesis of a variety of inflammatory, allergic, and autoimmune diseases, including rheumatoid arthritis (RA),2 atherosclerosis, asthma, Crohn’s disease, and systemic lupus erythematosus (2, 12). Therefore, activation of the proinflammatory MAPK signaling cascade also triggers negative feedback mechanisms that can restrain and terminate MAPK signaling. One such mechanism involves phosphatases that are up-regulated by stress-induced stimuli and display promiscuous substrate specificities to ERK1/2, p38 MAPK, and JNK. The MAPK phosphatases MKP-1, MKP-2, and MKP-5, as well as M3/6, phosphatase of activated cells 1, protein phosphatase 2C, wild-type p53-induced phosphatase 1, and hemopoietic protein tyrosine phosphatase have all been implicated in the regulation of stress-activated protein kinase signaling pathways (13, 14).
The relative contribution of these enzymes to protection against harmful effects of the innate and adaptive cellular immune response, cytokine synthesis, and inflammatory reactions in vivo is not yet defined. Ex vivo studies, however, suggest a key role for at least two phosphatases, MKP-1 and MKP-5, in terminating proinflammatory gene expression and cytokine production in various immune cell types (7, 8, 15, 16, 17, 18). MKP-1, which is also known as dual specificity phosphatase 1, and MKP-5 are members of the dual (tyrosine/threonine) specificity protein phosphatase family. The expression of these MPKs is induced in response to various stimuli, including growth factors, LPS, p53, osmotic and heat shock, anisomycin, UV, 12-O-tetradecanoylphorbol 13-acetate, and Ca2+ ionophores (13, 14, 19, 20).
Knockout technology is a powerful tool for studying gene function and delineating the redundancy in the physiological role of proteins in vivo. In this study, we examine the contribution of MKP-1 to the negative regulation of innate immune and inflammatory responses in mice with a targeted disruption of the MKP-1 gene expression. Our studies demonstrate for the first time that MKP-1 has a pivotal and nonredundant role in controlling cytokine release and development of autoimmunity in a mouse model of RA. The present results also define MKP-1 as a key negative regulator of TLR-mediated proinflammatory cytokine production and innate immunity mediated by primary macrophages.
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Materials and Methods |
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To generate MKP-1-deficient mice, the 
knockout shuttle (KOS) system (21) was used to derive an MKP-1 targeting vector. The KOS phage library, arrayed into 96 superpools, was screened by PCR using exon 1-specific primers (MKP1–1 (5'-CCGGGCCAGCTGAGAACGTGA-3')) and (MKP1–2 (5'-CCGGCGTTGAAAGCGAAGA-3')). The PCR-positive phage superpool was plated and screened by filter hybridization using the 637-bp amplicon derived from primers MKP1–1 and MKP1–2 as a probe. One pKOS genomic clone, pKOS-82, was isolated from the library screen and confirmed by sequence and restriction analysis. Gene-specific arms (5'-GCAGCGAGCACTTGGGGACTTAGGGCCACAGGACA-3' and 5'-TGCCACTTGCTCAAGAGTGGGGTCACCAAAGAAAGC-3') were appended by PCR to a yeast selection cassette containing the orotidine-5'-phosphate decarboxylase (URA3) marker. The yeast selection cassette and pKOS-82 were cotransformed into yeast, and clones that had undergone homologous recombination to replace a 2812-bp region containing exons 1–4 with the yeast selection cassette were isolated. The yeast cassette was subsequently replaced with a LacZ/Neo selection cassette to complete the MKP-1 targeting vector. The NotI-linearized targeting vector was electroporated into 129/SvEvBrd (Lex-1) embryonic stem cells (ESCs). G418/FIAU-resistant ESC clones were isolated, and correctly targeted clones were identified and confirmed by Southern analysis using a 417-bp 5' external probe (18/19) generated by PCR using primers MKP1–18 (5'-AAGCCTGCACACCGACTGTC-3') and MKP1–19 (5'-TCCAGGTGCCAGAGAGACTTACA-3'), and a 418-bp 3' internal probe (20/21), amplified by PCR using primers MKP1–20 (5'-GCAGAGGGAAACGGGTAAGCTG-3') and MKP1–21 (5'-GGCCTGGGACTTGGGAGTAGGTG-3'). Southern analysis using probe 18/19 detected a 7.5-kb wild-type band and 2.8-kb mutant band in BamHI-digested genomic DNA, while probe 20/21 detected a 12.4-kb wild-type band and 8.4-kb mutant band in Apa L1-digested genomic DNA.
Two targeted ESC clones were microinjected into C57BL/6 (albino) blastocysts to generate chimeric animals, which were bred to C57BL/6 mice, and the resulting heterozygous offspring were interbred to produce homozygous MKP-1-deficient mice (22). Determination of the genotype of mice at the MKP-1 locus was performed by screening of DNA isolated from tail biopsy samples, using quantitative PCR for the Neo cassette. This strategy enabled discrimination of zero, one, or two gene disruptions, representing MKP-1+/+, MKP-1+/–, and MKP-1–/– mice, respectively.
Comprehensive phenotypic analysis of MKP-1-deficient mice
Details of the phenotypic analysis protocol, which is applied to all lines generated in our facilities, have been described previously (23). It includes clinical diagnostic and pathology tests designed to identify modulation of therapeutically relevant aspects of mammalian physiology. All experiments were conducted on 10- to 16-wk-old mice representing both sexes. The Animal Care and Use Committee at Lexicon Genetics reviewed and approved all experimental procedures involving mice. Guidelines established by the "Guide for the Care and Use of Laboratory Animals" were followed in all animal work.
In vivo LPS challenge and measurement of inflammatory mediators
For the low-dose endotoxin shock model (24), mice were injected i.p. with LPS from Salmonella minnesota (Sigma-Aldrich) at a dose of 1 mg/kg in 0.2 ml of saline. In a pilot experiment, we determined that cytokine production in this model peaks 2 h after LPS challenge. Therefore, 2 h after injection, peripheral blood was drawn from each mouse via retroorbital bleed, and serum levels of the inflammatory cytokines TNF-, IL-12p70, IL-6, IFN-, IL-10, and MCP-1 were quantitated using a mouse inflammation cytometric bead array (CBA) kit (BD Biosciences), according to the manufacturer’s instructions. Data were acquired with a FACSCalibur flow cytometer and analyzed with BD CBA Software (BD Biosciences). Plasma PGE2 levels were measured by solid-phase enzyme immunoassay, using a commercially available kit (Cayman Chemical). The percentage of surviving animals was also recorded every 24 h for 7 days after LPS challenge; Fig. 2C shows the data for the first 72 h.
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BM cells were harvested from 15 animals of each genotype and combined into three samples containing cells from five animals per sample. Murine macrophages were obtained from these cells by culturing the nonadherent mononuclear cell population for 5 days in the presence of 50 ng/ml M-CSF (R&D Systems). To confirm the purity of isolated macrophages, the cells were double stained with fluorochrome-conjugated mAbs to the macrophage lineage-specific markers F4/80 and CD11b, or isotype control rat IgG2b (BD Pharmingen), and analyzed using a FACSCalibur flow cytometer with CellQuest software (BD Biosciences).
The cells were plated at a density of 0.5–1 x 106 cells/ml at 37°C in 5% CO2 in 24-well plates (Costar), and stimulated with LPS from S. minnesota (Sigma-Aldrich), bacterial lipoprotein Pam3Cys-SK4 (InvivoGen), zymosan (InvivoGen), flagellin from Salmonella typhimurium (InvivoGen), CpG (InvivoGen; 5 µM), or poly(I:C) (InvivoGen) for 24 h. In some experiments, cultures were preincubated for 1 h with a highly selective inhibitor of p38 MAPK, SB-203580 (4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole; Calbiochem). The optimal concentrations of the stimuli needed to induce cytokine production by BM-derived macrophages were determined in a pilot experiment and are indicated in Fig. 4. Culture supernatants were harvested at 24 h, and inflammatory cytokine levels were quantitated, as described above. Activated adherent macrophages were collected by rinsing and incubating the cells in prewarmed endotoxin-free Versene 1:5000 (Invitrogen Life Technologies) at 37°C for 5 min. After two washes in PBS, the cells were incubated with 1 µg of Fc block (BD Pharmingen) for 15 min at 4°C, and then stained for 30 min at 4°C in the dark with a mixture of fluorochrome-conjugated CD11b mAb and either CD86 (anti-B7.2), or CD40, or isotype control rat IgG1 mAbs (BD Pharmingen). Samples were analyzed by flow cytometry, as described above.
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Macrophages derived from 5-day BM cultures were either left unstimulated or stimulated with LPS from S. minnesota (Sigma-Aldrich) or CpG (InvivoGen) for 5 min at 37°C, using concentrations indicated in Fig. 5. Reactions were stopped by addition of an equal volume of PhosFlow Fix Buffer I solution (BD Biosciences) to the cell suspensions. After 10 min at 37°C, cells were permeabilized by washing twice at 4°C in PhosFlow Perm/Wash Buffer I (BD Biosciences). A total of 1 x 106 cells was then stained for 1 h at room temperature (RT) with a mixture of FITC-conjugated CD11b mAb and Alexa Fluor 647-conjugated mAb to either phospho-p38 MAPK (T180/Y182) or phospho-ERK1/2 (T202/Y204) (BD Biosciences) (25). Live CD11b-positive cells were gated and analyzed by flow cytometry, as described above.
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To elicit a synovial autoimmune response, mice were injected intradermally at the base of the tail with 100 µg of chicken type II collagen (CII; Sigma-Aldrich) and 250 µg of Mycobacterium tuberculosis emulsified in 50 µl of IFA (Sigma-Aldrich), followed by a repeat booster injection of the same emulsion 3 wk after the primary immunization. Mice were monitored for signs of arthritis by clinical scoring and measuring the width of the forelimbs at the time points indicated in Fig. 3. For assessment of disease severity, the following clinical scoring scale was used: 0, no evidence of erythema and swelling; 1, erythema and mild swelling confined to the mid-foot or ankle joint; 2, erythema and mild swelling extending to the ankle and the mid-foot; 3, erythema and moderate swelling extending from the ankle to the metatarsal joints; 4, erythema and severe swelling encompassing the ankle, foot, and digits. Total disease severity scores were recorded as a sum of clinical scores for four limbs. Forelimb thickness was measured by using a Käfer thickness micrometer gauge. Thickness values for the two forelimbs were averaged, and the extent of swelling was calculated by subtracting the baseline values of the first measurement from the values of subsequent measurements. Serum concentration of the inflammatory cytokines TNF- and IL-6 was also measured in the study on days 0, 34, and 59 after immunization by using CBA, as described above.
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Serum levels of anti-collagen IgG1 Abs were measured in animals on days 0, 34, and 59 after immunization, as described previously (26). In short, 96-well Maxisorb ELISA plates (Nunc) were coated overnight at 4°C with 2 µg/ml CII in PBS. After three washes with PBS containing 0.05% Tween 20, the plates were blocked for 1 h at RT with PBS supplemented with 10% heat-inactivated FBS. Serial dilutions of serum samples were applied to the coated plates and incubated for 2 h at RT. Bound anti-collagen IgG1 was detected with HRP-conjugated polyclonal goat anti-mouse IgG1 Abs (Southern Biotechnology Associates), and developed by the addition of 3,3',5,5'-tetramethylbenzidine solution as substrate (Sigma-Aldrich). Absorbance was measured at 450 nm with a VMax plate reader using SoftMax Pro software (Molecular Devices).
Statistical analysis
The normality of results was evaluated by the Kolmogorov-Smirnov test. Analysis of statistical significance of group differences between wild-type and knockout mice was performed using the two-sample t test or the Wilcoxon rank sum test for normal and non-Gaussian distributions (Kolmogorov-Smirnov test; p < 0.05), respectively. A repeated measures ANOVA was used to compare disease incidence and the severity of CIA in knockout mice and wild-type controls. In all tests, a p value of <0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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We generated MKP-1-deficient mice by targeted disruption of all four coding exons of the murine homologue of the MKP-1 gene (GenBank accession no. NM_013642) by homologous recombination in ESCs (21), followed by germline transmission of the mutant gene into chimeric, heterozygous (MKP-1+/–), and homozygous (MKP-1–/–) mice (Fig. 1A). Southern hybridization analysis demonstrated the targeted mutation in ESCs (Fig. 1B). MKP-1 transcripts can be detected by RT-PCR in all tissues of wild-type (MKP-1+/+) mice, except for total bone tissue, including BM (Fig. 1C). Our wild-type tissue expression panel also contains liver tissue of LPS-treated mice and lung tissue of mice, which were rendered asthmatic by OVA challenge. The level of MKP-1 message does not show significant differences between the tissues obtained after the in vivo inflammatory challenges and tissues from mock-challenged mice.
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Increased innate immune responses in MKP-1-deficient mice
To study the role of MKP-1 in innate immunity, we first analyzed serum levels of proinflammatory cytokines in MKP-1–/– mice after low-dose LPS challenge. The innate immune response in this inflammatory stress model is mediated by the dimerization of the TLR4 complex on macrophages, leading to the release of macrophage-derived cytokines such as TNF-, IL-6, and IL-12 (24). These cytokines are also the primary mediators of the LPS-associated hepatotoxicity and shock following administration of LPS. MCP-1 is a proinflammatory chemokine that potentiates the recruitment of monocytes to the site of inflammation after LPS challenge (27).
In response to bacterial LPS challenge, MKP-1–/– mice exhibit dramatically increased mean serum levels of all the measured proinflammatory mediators when compared with wild-type littermates (Fig. 2A). The knockout animals register 2- to 5-fold higher serum levels of IL-12, IL-6, and TNF-, compared with wild-type controls after the inflammatory challenge. Additionally, the serum levels of MCP-1 are 50% higher in MKP-1–/– mice than in the MKP-1+/+ animals. The baseline serum levels of these cytokines are below the assay sensitivity threshold in unchallenged animals, regardless of the MKP-1 genotype (data not shown). In agreement with the ability of IL-12 to stimulate the production of IFN- by T and NK cells (12), the serum IFN- levels after LPS challenge are also markedly elevated in MKP-1–/– mice, compared with wild-type littermates. Similarly, the same stimulus leads to 6-fold higher serum IL-10 levels in MKP-1–/– mice than in the control cohort. IL-10, which is produced mainly by activated macrophages, exerts a negative feedback control of IFN- production during innate and cell-mediated immune reactions by inhibiting the secretion of macrophage-derived IL-12 (28).
Another inflammatory mediator produced by many cell types after exposure to LPS is PGE2, which is a product of the arachidonic acid metabolic pathway. Following stimulation with IL-1 and TNF-, up-regulation of cyclooxygenase-2 augments PGE2 production in macrophages and endothelial cells (29). In response to LPS challenge, the mean plasma level of PGE2 in wild-type animals increases by 5-fold (142 ± 38 pg/ml before and 679 ± 123 pg/ml after challenge), whereas in MKP-1–/– mutants the increase is 7-fold (147 ± 33 pg/ml before and 975 ± 202 pg/ml after challenge) (Fig. 2B). Although perhaps indicative of slightly increased sensitivity, this overproduction of PGE2 observed in MKP-1-deficient mice is not statistically significant at the time point examined.
The low-dose endotoxin challenge model that we used to mimic inflammatory stress induced by bacterial infection normally does not lead to death in wild-type cohorts. Notwithstanding this, all of the MKP-1–/– mice treated with a single low dose of LPS (1 mg/kg) died within the first 48 h after LPS challenge, whereas all of their MKP-1+/+ littermates survived (Fig. 2C). Thus, MKP-1–/– mice are hyperresponsive to low-dose LPS-induced toxicity, which is primarily mediated by the release of proinflammatory cytokines from activated macrophages.
MKP-1–/– mice exhibit a marked increase in the incidence and severity of autoimmune arthritis
CIA is a mouse experimental model of RA, a systemic inflammatory autoimmune disease characterized by inflammation in the synovium, which leads to destruction of cartilage and the underlying bone (30). Proinflammatory cytokines produced by infiltrating cells play a critical role in the pathogenesis of RA by increasing osteoclast activity in the joints. Severe CD4+ T and B cell-dependent arthritis, which resembles RA in humans, can be induced in genetically susceptible mouse strains bearing the MHC class II H-2q or H-2r haplotypes by immunization with CII (26). A modification of the standard immunization procedure was successfully used to induce CIA in mice derived from the C57BL/6 (H-2b) genetic background, including C57BL/6 x 129/Sv hybrids, previously considered as CIA resistant (26). Mice of this background develop first clinical signs of arthritis by 20–25 days after primary immunization, with a high incidence that approaches 70–80% (26). However, the severity of arthritis in mice bearing H-2b is generally lower than that observed in CIA-susceptible DBA/1 (H-2q) mice. This slightly increased threshold of responsiveness to CIA in H-2b C57BL/6 x 129/Sv hybrids can be useful for identification and characterization of genes that increase susceptibility to CIA.
We have studied the role of MKP-1 in autoimmunity by assessing the kinetics of arthritis development in MKP-1-deficient mice after immunization with CII according to the protocol previously applied by Campbell et al. (26) to H-2b mouse strains. Consistent with the above findings of increased production of proinflammatory cytokines in the absence of MKP-1, the progress of the disease is markedly accelerated in CII-immunized MKP-1–/– mice, compared with their wild-type littermates (Fig. 3). Joint swelling and clinical signs of inflammation in the ankle and wrist joints are evident in the MKP-1-deficient mice from day 21 after the initial immunization, whereas the first signs of arthritis in wild-type controls develop 1 wk later (Fig. 3, A and B). Moreover, the incidence of arthritis and severity of disease as measured by the arthritis severity score are significantly elevated in the MKP-1-deficient animals. MKP-1–/– mice reached a maximum mean cumulative arthritis score of 8 by day 44, while the same scores for wild-type controls remained 
4 throughout the study. This dramatic increase in the severity of arthritis in the MKP-1–/– mice persists until day 59 after the first immunization, whereas in wild-type mice the disease incidence and activity scores already start to decline 1 wk earlier. Further analysis revealed significantly increased serum levels of TNF- in the MKP-1–/– mice immunized with CII in CFA throughout the entire study period (Fig. 3C). Similarly, another proinflammatory cytokine, IL-6, appears to show a strong trend of overproduction in the knockout animals, compared with control mice, although the difference does not reach statistical significance.
In the same studies, we have also examined the effect of the MKP-1 deletion on the B cell-dependent anti-collagen immune response. In agreement with a previously published study (26), the levels of circulating anti-collagen IgG isotypes in C57BL/6 x 129/Sv wild-type cohorts do not correlate with arthritis scores and disease incidence (data not shown). This suggests that the relative magnitude of the anti-collagen B cell response does not directly influence the degree of joint inflammation in mice of this genetic background. The mean anti-collagen IgG1 levels, however, are slightly higher in MKP-1–/– mice, compared with wild-type controls, on both days 34 and 59 after immunization, but the differences do not reach statistical significance (Fig. 3D). These findings indicate that uncontrolled up-regulation of proinflammatory cytokines results in exacerbated inflammatory immune responses in MKP-1–/– mice immunized with CII, and implicate MKP-1 as a negative regulator of autoimmunity and susceptibility to CIA.
Absence of MKP-1 enhances TLR-dependent inflammatory responses in BM-derived macrophages
We further addressed the role of MKP-1 in inflammatory signaling by comparing the ex vivo cellular response of MKP-1-deficient and wild-type macrophages after activation of the cells with various TLR-dependent stimuli. The activation agents in this study included zymosan and bacterial lipoprotein, which both act via TLR2 (4, 31); poly(I:C), which triggers TLR3 (4, 32); LPS, which binds to TLR-4 (12, 33); and flagellin and CpG, which trigger TLR5 and TLR9, respectively (4, 32). Treatment of BM-derived macrophages (Fig. 4A) for 24 h with each of the above stimuli induces TNF- production (Fig. 4B) and expression of the cell activation molecules CD86 and CD40 (Fig. 4C). CD86 is a costimulatory ligand for T cell activation via its interaction with CD28, whereas CD40 and its ligand play a critical role in the induction of CD86 expression and in the stimulation of proinflammatory cytokine production by macrophages. The results presented in Fig. 4A demonstrate that the amount of TNF- produced by MKP-1–/– macrophages in response to the TLR-binding agents is markedly increased, compared with MKP-1+/+ macrophages, irrespective of the stimulators’ TLR specificity. This elevated TNF- release is accompanied by a similar increase in the production of IL-10 in the LPS and flagellin-activated MKP-1–/– cell cultures (Fig. 4B), while in the other culture conditions the IL-10 level is below the sensitivity of detection (data not shown). In addition, LPS- and flagellin-activated MKP-1–/– macrophage cultures consistently contain more cells that are positive for expression of the CD86 and CD40 molecules, compared with MKP-1+/+ cell cultures (Fig. 4C).
We also designed these experiments with the aim to identify the molecular mechanisms behind the increased inflammatory response of MKP-1–/– cells by examining whether inhibition of p38-MAPK can negate this response in MKP-1-deficient macrophages. The p38 kinase inhibitor, SB 203580, has been shown to selectively inhibit the production of TNF- and IL-1 by LPS-stimulated human monocytes in vitro with an IC50 of 200 nM (5, 34, 35). SB 203580 also interferes with the systemic release of proinflammatory cytokines in a number of in vivo models of endotoxic shock (36), and mouse and rat models of RA (36, 37). Therefore, we stimulated MKP-1-deficient macrophages with LPS after preincubation for 1 h with SB-203580. As shown in Fig. 4, both the LPS-induced cytokine production and expression of costimulatory molecules were inhibited by SB-203580 in BM-derived macrophages, and no residual cell activation is evident in MKP-1-deficient cells.
These ex vivo studies strongly implicate MKP-1 as a negative regulator of the cellular inflammatory response of macrophages via diverse TLR- and p38 MAPK-mediated signaling pathways. The results are consistent with the LPS and CII challenge data, which establish an essential role for the enzyme in limiting inflammatory reactions in vivo.
MKP-1-deficient macrophages exhibit enhanced activation of the p38 and ERK1/2 MAPK pathways
Previous in vitro studies revealed that MKP-1 inhibits activation of p38 MAPK and ERK1/2 in a variety of mouse and human cell types (8, 15, 16, 17, 18). We investigated the effect of genetic deletion of MKP-1 on p38 MAPK and ERK1/2 activation by measuring the phosphorylation status of these enzymes at the single cell level in LPS- and CpG-activated macrophages, using flow cytometry.
In a pilot experiment, we found that LPS- and CpG-induced phosphorylation of p38 MAPK is transient and peaks at 5–15 min in BM-derived macrophages (data not shown). Therefore, the 5-min postactivation time point was selected for intracellular staining of resting and activated cells, using fluorochrome-conjugated mAbs that recognize phosphorylated activation sites of p38 MAPK and ERK1/2 (25, 38). As shown in Fig. 5, MKP-1-deficient macrophages exhibit greatly increased levels of phospho-p38 MAPK after ligation of TLR4 and TLR9 with LPS and CpG, respectively, compared with wild-type cells. The increased activation status of this enzyme in the mutant cells is evident both in terms of fluorescence intensity (Fig. 5A) and percentage of cells (Fig. 5B), displaying strong anti-phospho-p38 MAPK reactivity in the two activated cell populations. Further kinetic analysis of LPS stimulation (1 µg/ml) showed a gradual decline of p38 MAPK phosphorylation after the peak, but the percentages of phospho-p38 MAPK-positive cells remained significantly higher in MKP-1–/– macrophages, compared with wild-type cells, at all time points analyzed: 63 vs 48% at 15 min, 58 vs 39% at 30 min, and 51 vs 28% at 60 min, respectively. Furthermore, we consistently observed higher basal levels of phopsho-p38 MAPK in MKP-1–/– macrophages in the absence of stimuli as well, which most likely reflects background activation of the MAPK pathways in cells exposed to the ex vivo cell culture conditions.
No ERK1/2 phosphorylation is detectable by flow cytometry in wild-type BM-derived macrophages when activated with the same concentrations of TLR ligands (Fig. 5A). However, we routinely observed a slight increase in phospho-ERK1/2 fluorescence in MKP-1–/– macrophages treated with 10 µg/ml LPS, suggesting that LPS stimulation of mouse macrophages can trigger limited ERK1/2 phosphorylation, and that MKP-1 may also control its magnitude. In contrast, the degree of LPS-induced phosphorylation of JNK in MKP-1–/– BM-derived macrophages was similar to that in MKP-1+/+ cells (data not shown).
In summary, our results indicate that the activation status of the p38 and ERK1/2 MAPK pathways is greatly influenced by the presence or absence of MKP-1 in BM-derived macrophages, whereas the activity of JNK is controlled by other phosphatases, such as MKP-5 (7, 13, 14).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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and IL-6 by accelerating JNK and p38 inactivation (8, 18). MKP-1 can also inactivate p38 MAPK and JNK in primary fibroblasts stimulated with serum, anisomycin, or osmotic stress (15). Furthermore, transient overexpression of MKP-1 in THP-1 cells and peritoneal macrophages can induce endotoxin tolerance and down-regulation of TNF- production via inhibition of p38 MAPK phosphorylation (16).
In the present study, we demonstrate that mice homozygous for disruption of the MKP-1 gene are hyperresponsive to inflammatory signals, as shown by markedly elevated proinflammatory cytokine response to in vivo LPS and CII challenge, by increased susceptibility to endotoxin-induced lethality, and by exacerbated disease course in a model of autoimmune arthritis. Thus, during innate immune responses to bacterial endotoxin, MKP-1 moderates the production of proinflammatory cytokines and negatively regulates systemic levels of effector and regulatory cytokines produced by activated macrophages, T and NK cells, including TNF-, IL-6, IL-12, MCP-1, IL-10, and IFN-. In addition, our functional data reveal that MKP-1 controls inflammatory cytokine production and expression of costimulatory ligands by primary BM-derived macrophages activated through various TLR. This control is associated with the activation status of the p38 and ERK1/2 MAPK pathways.
Recently, another phosphatase, MKP-5, has been implicated in the regulation of both innate and adaptive immune responses (7). MKP-5-deficient cells exhibit increased JNK activity and produce increased levels of proinflammatory cytokines in response to LPS challenge ex vivo, but to date no evidence identifies MKP-5 as a critical regulator of p38 MAPK-mediated inflammatory responses in vivo. Despite the previously demonstrated ability of MKP-5 to dephosphorylate both JNK and p38 MAPK (13, 14), MKP-5-deficient Th1 and Th2 cells show no significant up-regulation of p38 MAPK activity, which suggests that another MKP such as MKP-1 may play a more significant physiologic role in controlling the activation of the p38 MAPK inflammation pathway.
In vitro studies indicate that JNK, p38 MAPK, and ERK1/2 are substrates of a multitude of phosphatases (13, 14, 19, 39, 40). A certain degree of selectivity in the phosphatase-mediated regulation of MAPKs can be achieved through temporal variations in the expression kinetics and compartmentalization of phosphatases during cell activation. The sequential phosphatase model (14) proposes that these regulatory enzymes act sequentially, responding to different extracellular stimuli. Notably, expression of the MKP-5 message shows some degree of tissue specificity with relative abundance in liver and skeletal muscle (41, 42), but not in unchallenged immune tissues. However, it is strongly induced in mouse macrophages after LPS treatment (7). In contrast, we observed high constitutive expression of MKP-1 in all tissues tested, including monocytes and neutrophils, which is consistent with previously published data (43). Our wild-type tissue expression panel also contains liver tissue of LPS-treated mice and lung tissue of mice, which were rendered asthmatic by OVA challenge. Comparison of the abundance of MKP-1 message between the tissues obtained after the in vivo inflammatory challenges and tissues from mock-challenged mice did not reveal significant up-regulation. Furthermore, our data also show that even in a relatively quiescent state, MKP-1-deficient macrophages maintain a higher basal level of phosphorylated p38 MAPK. Based on the above findings, we propose that MKP-1 is an important component of the intracellular homeostasis that controls the threshold and magnitude of p38 MAPK activation in macrophages, and that inflammatory conditions accentuate the significance of this regulatory function. Of note, Gadd45, a small p38 MAPK-binding molecule, has recently been demonstrated to serve a similar function in T cells. p38 MAPK from resting Gadd45-deficient T cells is spontaneously phosphorylated, and mice lacking Gadd45 exhibit signs of T cell hyperproliferation and lupus-like autoimmune disease (44).
Comparative studies using wild-type and TLR4 knockout mice demonstrate that innate immune responses have a significant impact on the development of arthritis induced by anti-CII Ab and LPS (45, 46). In this arthritis model, TLR4-deficient mice register delayed disease progression and decreased production of the proinflammatory mediators TNF- and cyclooxygenase-2 in the synovial tissue, compared with wild-type animals. Immune responses provoked by bacterial DNA and CpG have been implicated in the onset of a Th1 cell-dependent and IL-1/IFN--mediated joint-specific inflammation in LEW and LEW.1AV1 rat strains (47). In addition, numerous studies have shown expression of TLR4 and TLR2 in RA synovium (46, 48). Finally, mice deficient for MyD88, a critical adaptor molecule involved in TLR signaling, fail to develop joint inflammation induced by streptococcal cell wall and display lower levels of proinflammatory cytokines and chemokines in synovial tissue (49). Our data demonstrate that MKP-1 negatively regulates susceptibility to the development of autoimmune arthritis and down-regulates systemic levels of proinflammatory cytokines in the mouse model of RA. We observed a dramatic increase in the incidence and severity of arthritic inflammation in MKP-1–/– mice immunized with CII. The MKP-1–/– mice were more susceptible to the development of autoimmunity in joints, as revealed by significantly accelerated synovial inflammation and enhanced production of proinflammatory cytokines sustained throughout the entire study period. These observations are consistent with the model of MKP-1-mediated negative regulation of p38 MAPK signaling, which is thought to control LPS-induced release of TNF- in mononuclear phagocytes, as well as the disease onset and the progression of IL-1- and TNF-induced bone resorption and joint destruction in patients with RA (50).
In conclusion, our study provides the first in vivo genetic evidence of a critical and nonredundant role of MKP-1 in the negative control of p38 MAPK-regulated proinflammatory cytokine release. Our results demonstrate that MKP-1 modulates innate and adoptive immunity by suppression of TLR-induced activation of p38 MAPK and production of proinflammatory cytokines by TLR-activated macrophages. Failure to down-regulate cytokine production in the absence of MKP-1 may contribute to the enhanced inflammatory immune response of MKP-1–/– animals observed in the mouse model of arthritis. These results implicate MKP-1 as a vital component of the protective immune mechanisms that control inflammatory reactions during the course of autoimmune disease development. Targeting MKP-1 activity may offer an attractive new opportunity for therapeutic intervention aimed at modulating the inflammatory immune response.
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1 Address correspondence and reprint requests to Dr. Tamas Oravecz, Lexicon Genetics Incorporated, 8800 Technology Forest Place, The Woodlands, TX 77381. E-mail address: toravecz{at}lexgen.com 
2 Abbreviations used in this paper: RA, rheumatoid arthritis; BM, bone marrow; CBA, cytometric bead array; CIA, collagen-induced arthritis; CII, chicken type II collagen; ESC, embryonic stem cell; MKP, MAPK phosphatase; RT, room temperature; KOS, knockout shuttle.
Received for publication September 7, 2005. Accepted for publication November 12, 2005.
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biosynthesis. Nat. Cell Biol. 1: 94-97. [Medline] KOS genomic libraries. BioTechniques 26: 1150-1156. 1158. 1160.. [Medline]B by Toll-like receptor 3. Nature 413: 732-738. [Medline]: role of the p38 and p42/44 mitogen-activated protein kinases. J. Immunol. 160: 920-928. transcription. J. Immunol. 162: 5367-5373. inhibits the T cell alternative p38 activation pathway. Nat. Immunol. 6: 396-402. [Medline]Related articles in The JI:
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