Decreased immediate inflammatory gene induction in activating transcription factor-2 mutant mice

Andreas M. Reimold, James Kim1, Robert Finberg2 and Laurie H. Glimcher1

Rheumatic Diseases Division, University of Texas Southwestern Medical Center, Dallas, TX 75390-8884, USA
1 Department of Immunology and Infectious Diseases, Harvard School of Public Health, and Department of Medicine, Harvard Medical School, 651 Huntington Avenue, Boston, MA 02115, USA
2 Department of Medicine, University of Massachusetts, Worcester, MA 01655, USA

Correspondence to: Correspondence to: L. Glimcher


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Transcription factor activating transcription factor (ATF)-2 is activated by inflammatory signals transduced by the JNK and p38 MAP kinase pathways. To better define the role of ATF-2 in inflammation, adult mice expressing small amounts of a mutant ATF-2 protein were challenged with lipopolysaccharide (LPS), anti-CD3 antibody or virus. Within 3 h of challenge by LPS, ATF-2 mutant mice had decreased induction of the adhesion molecules E-selectin, P-selectin and VCAM-1 as well as the cytokines tumor necrosis factor-{alpha}, IL-1ß and IL-6 compared with control mice. Stimulation of T lymphocytes by anti-CD3 antibody also showed less induction of IL-1 and IL-6 in ATF-2 mutant tissues. ATF-2 mutant thymocytes treated with anti-CD3 antibody in vitro demonstrated reduced induction of c-Jun, JunB, JunD and Fra-2. However, similar to what was observed after p38 kinase inhibition in normal mice, relative ATF-2 deficiency did not prevent the development of a mononuclear cell infiltrate in the week following an inflammatory stimulus. ATF-2 mutant mice proved more susceptible to death than control mice from LPS plus D-galactosamine injection or Coxsackievirus B3 infection and had a higher incidence of mononuclear pulmonary infiltrates after exposure to Herpes simplex virus-1. ATF-2 is essential for maximal immediate induction of adhesion molecules and cytokine genes, but at later time points may even protect against overactive immune responses.

Keywords: cytokines, gene regulation, inflammation, in vivo animal models, transcription factors


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The CREB/activating transcription factor (ATF) family of transcription factors consists of over 15 leucine zipper proteins that are active as homoor heterodimers (1). Although their consensus binding site is the widely found cyclic AMP-responsive element [(CRE) sequence TGACGTCA] only some family members (e.g. CREB, ATF-1) are cAMP-induced, while others (e.g. ATF-2) are not (2,3). Induction and activation of CREB/ATF family members can occur by different mechanisms, including transcriptional and translational increases, increased protein dimerization or increased protein phosphorylation (46). For the case of ATF-2, it has been determined that responses are limited by ubiquitination and proteasome digestion of the transcription factor (7,8). Some essential functions of CREB/ATF family members have been revealed by gene disruptions in mice. For CREB, complete deficiency is lethal at birth, while partial deficiency leads to impaired memory formation (9,10). For ATF-2, complete deficiency caused death immediately after birth due to a surfactant deficiency, while low-level expression of a mutant isoform additionally caused a chondrodysplasia, abnormal cerebellar Purkinje cell migration and impaired E-selectin induction (11,12). While these widely varying defects reflect the many promoters containing CRE sites, they also indicate that CREB/ATF proteins have many unique functions not compensated for by other family members.

Progress has also been made in defining the roles of CREB and ATF-2 in immune system activation. CRE or closely related AP-1 sites are found in the promoter regions of multiple cytokine, chemokines and adhesion molecules, including tumor necrosis factor (TNF)-{alpha}, IFN-{gamma}, IL-1, IL-6, MCP-1{alpha}, E-selectin and P-selectin (1318). In addition, cell proliferation is also at least partially regulated by these transcription factors through their action on the cyclin A and cyclin D genes (19,20).

During T cell activation, there is increased protein binding to CRE sites that peaks 48 h after anti-CD3 antibody treatment and remains elevated above baseline at 120 h (21). It was found that CREB homodimers are present in resting lymphocytes, while CREB/ATF-2 heterodimers predominate in activated cells. Among the potential CREB/ATF targets in activated lymphocytes studied to date are the promoter of PCNA, an obligate cofactor of DNA polymerase {delta} and the MEKK1 response element in the FasL gene, where ATF-2 and c-Jun bind and are phosphorylated (22,23).

The route of cell activation can also be important in the activation of particular intracellular signal transduction pathways. Stimulation of T cells by anti-CD3 antibody led to an increase in cAMP along with changes in the composition and phosphorylation state of CRE binding proteins, while the increase in CRE binding seen after mitogen stimulation of T cells was due mainly to increases in the phosphorylated dimerization partners of CREB rather than in the level or phosphorylation status of CREB itself (24). In addition, the G1/S progression seen in T cells induced by IL-2 is not associated with increased cAMP levels (22). However, G1/S progression does involve a gradual increase in ATF/CREB family member expression and activation of the cyclin A promoter by ATF-2 and Jun family members (25). Co-activation of T cells can also alter the activity of CREB/ATF factors. CREB phosphorylation at Ser133 was found to be essential for IL-2 gene expression but not for increased transcriptional activity by CREB, which required CD28 co-stimulation (26).

The reported involvement of ATF-2 in immune system activation can now be studied in vivo using ATF-2 mutant mice. The findings indicate that ATF-2 is critical for the full activation of a number of transcription factor, adhesion molecule, cytokine and chemokine genes early in an immune response, but the absence of ATF-2 allows an over-exuberant response after 1–2 days.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
ATF-2 mutant animals have been described (12) and were backcrossed at least eight generations onto a BALB/c background.

Injection and infection of animals
Six ATF-2 mutant and 11 wild-type or heterozygous age-matched control animals were injected i.p. with 3.4 ng/g body wt lipopolysaccharide (LPS; Sigma, St Louis, MO) or i.v. with anti-CD3 antibody (2C11; PharMingen, San Diego, CA). Lymphoid tissues were harvested 3–24 h later. For viral infection, animals were injected i.p. with Coxsackievirus B3 (100 p.f.u. injected into a total of 10 wild-type and 10 ATF-2 mutant animals) or with Herpes simplex virus-1 (5x107 p.f.u. injected into a total of 14 wild-type and 13 ATF-2 mutant animals) and observed for survival over the following week. Moribund animals were sacrificed for analysis and the experiments were terminated at 7 days. Tissues were fixed in Omnifix for histologic analysis by H & E stain. Statistical analysis was performed by using the {chi}2 test.

In vitro analysis of lymphoid tissue
Single-cell suspensions of thymocytes, splenocytes or lymph node cells from mice were plated in RPMI with 10% FCS. For stimulation of T cells, anti-CD3 antibody (1–10 µg/ml; clone 2C11; PharMingen) and anti-CD28 antibody (1 µg/ml; PharMingen) were added and supernatant was collected for cytokine determination after 24 and 48 h. Proliferation was measured by adding 50 µl [3H]thymidine to 100 µl of cell culture and harvesting 12 h later for detection of incorporated 3H by scintillation counting.

Northern blotting
Tissue from experimental animals was dissociated and RNA was isolated by centrifugation through cesium chloride, as described (12). RNA was isolated from lymphoid tissues by pelleting the cells and treating with Trizol (Gibco/BRL, Gaithersburg, MD). Northern blots were probed with random-primed 32P-labeled cDNAs. cDNAs for AP-1 family members were the gift of Jeffrey Leiden (Harvard School of Public Health, Boston, MA).

Western blotting
Western blots were performed on thymocytes cultured in vitro as described (27) or after immunoprecipitation of ATF-2 using a polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Detection of ATF-2 was performed with antibody C-17 to ATF-2 (Santa Cruz).

ELISA assays
Supernatants from cultured lymphoid cells or serum from mice was used in ELISA assays for IL-2, IFN- {gamma}, IL-4 and IL-6 as described (28).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
ATF-2 induction upon T cell stimulation
ATF-2 mutant animals (12) have a disruption in the ATF-2 gene leading to an absence of full-length ATF-2 and the appearance of low amounts of a novel splice form of ATF-2 (11). Since a more C-terminal disruption of ATF-2 leads to perinatal lethality in all cases (11), the ATF-2 splice form present in the mutant animals permits the survival to adulthood of a subset of ATF-2 mutant animals. For the studies presented here, adult ATF-2 mutant mice were used that had a mildly reduced body mass compared to wild-type animals, were able to reproduce and had a normal lifespan. These animals allow an analysis of ATF-2 deficiency in the adult immune system not possible when all immunoreactive ATF-2 is absent during development. ATF-2 mutant mice have no defects in cell numbers in their immune systems, as shown by normal counts of circulating granulocytes and normal lymphocyte cell numbers in lymph nodes, spleen and thymus when compared to wild-type animals (data not shown). Western blotting of proteins from adult mouse thymus, spleen and brain shows the highest levels of ATF-2 in wild-type brain, low levels of mutant ATF-2 in mutant brain, as well as low levels of mutant ATF-2 in spleen and thymus from mutant animals (Fig. 1Go and data not shown). Therefore, ATF-2 mutant animals that survive to adulthood express low levels of a mutant ATF-2 protein and provide a useful model for studies of immune responses.



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Fig. 1. Detection of ATF-2 on Western blotting. Immunoprecipitation was performed on mouse brain and spleen whole cell extracts, followed by Western blotting and probing with ATF-2 antibody. Wild-type cells had ATF-2 species of 72 (spleen and brain) and 64 (brain only) kDa, while ATF-2 mutant cells showed low amounts of a 68 kDa mutant ATF-2 protein. The dark band at ~55 kDa represents the heavy chain from the immunoprecipitation.

 
Abnormal AP-1 transcription factor induction in ATF-2 mutant mice
ATF-2 is known to interact with c-Jun and therefore has the potential to affect the activation of AP-1 target genes, including c-Jun itself. The AP-1 genes are rapidly up-regulated by signals that activate lymphocytes (29), and in turn regulate the promoters of numerous pro-inflammatory genes such as cytokines, chemokines and adhesion molecules. Due to its interaction with AP-1 proteins and its potential to regulate AP-1 genes, we asked if disruption of ATF-2 would affect AP-1 induction in thymocytes. Using thymocytes from BALB/c or ATF-2-mutant BALB/c mice, cells were stimulated in vitro for 0, 30, 60, 90 or 120 min (Fig. 2Go) and harvested for RNA extraction. Northern blots were then hybridized with probes for multiple AP-1 family members. It is clear that the kinetics of AP-1 gene induction are similar for wild-type and ATF-2 mutant samples, but that the strength of induction is not uniform. For the Jun family members, the most dramatic differences were seen for c-Jun. By densitometry, unstimulated wild-type cells contained 15-fold more c-Jun mRNA than did ATF-2 mutant cells. Stimulation caused a significant induction of c-Jun in both samples, but the induction was even more dramatic in the wild-type sample, which contained 40-fold higher levels by the 30 min time point. The induction of other Jun family members was also diminished in ATF-2 mutant samples, with 3.7-fold lower induction of JunB and 2.7-fold lower induction of JunD compared to wild-type. On the other hand, the induction of Fos family members c-Fos and FosB was identical in the wild-type and ATF-2 mutant samples, while Fra-2 up-regulation was 3.5-fold lower in ATF-2 mutant samples. These findings show that ATF-2 deficiency leads to subnormal induction of c-Jun, some blunting of JunB, JunD and Fra-2 up-regulation, and no defect in c-Fos and FosB induction.



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Fig. 2. Decreased AP-1 gene induction in ATF-2 mutant thymocytes. Thymocytes were harvested in a time course after in vitro anti-CD3 antibody stimulation. A Northern blot was prepared and probed sequentially with cDNAs for multiple Jun and Fos family members as well as the control gene {gamma}-actin.

 
Decreased cytokine gene induction in ATF-2 mutant tissues
We have shown previously that a deficiency of ATF-2 leads to poor induction of E-selectin 3 h after LPS injection (12). In light of the role of ATF-2 in T cell activation, we asked if a deficiency in ATF-2 and the associated alteration in AP-1 family member up-regulation affected other adhesion molecule and cytokine genes. Mice were injected with LPS and tissues were harvested for RNA after 3 h. Northern blots were probed sequentially and showed the expected minimal induction of E-selectin in ATF-2 mutant samples (Fig. 3AGo). In addition, two other adhesion molecules, P-selectin and VCAM, were also induced significantly less strongly in the ATF-2 mutant samples. Sluggish up-regulation of these adhesion molecules is expected to cause a delay in accumulation of an inflammatory infiltrate.



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Fig. 3. Decreased induction of adhesion molecule and cytokine genes in ATF-2-mutant mice. (A) Wild-type (WT) or mutant (MUT) ATF-2 mice were injected with saline or with 50 µg LPS, followed by harvest of lungs and kidneys 3 h later for RNA blotting. Blots were sequentially probed for E-selectin, P-selectin, VCAM-1 and {gamma}-actin. (B) Mice were injected with saline or LPS as in (A), and Northern blots of lung, kidney and spleen mRNA were probed for expression of TNF-{alpha}, IL-1ß, IL-6, chemokine KC and the control {gamma}-actin. (C) Mice were injected i.v. with anti-CD3 antibody and spleen was harvested for mRNA. The Northern blots was probed for TNF-{alpha}, IL-2, IL-4, IL-6, IFN-{gamma}, MIP-1{alpha} and the control {gamma}-actin.

 
LPS initiates a cytokine cascade beginning with TNF-{alpha} followed by up-regulation of IL-1, IL-6 and the chemokine KC (30). We examined the induction of these molecules 3 h after LPS injection. In all cases, the up-regulation of LPS-induced cytokines and chemokines was higher in wild-type compared to ATF-2 mutant samples (Fig. 3BGo). Interestingly, this effect was more pronounced for the induction of IL-6 and KC, which represent more distal events in the cascade, while the induction of TNF-{alpha} and IL-1ß was not as severely diminished in ATF-2 mutant samples.

Next, mice were injected i.v. with anti-CD3 antibody to activate T lymphocytes in vivo and splenic mRNA was studied at 3 h for cytokine gene induction. Anti-CD3 antibody treatment led to less induction of IL-2, IL-4 and IL-6 mRNA in ATF-2 mutant tissues (Fig. 3CGo). It is expected that at this time point, NK1 T cells are the major source of IL-4 production (reviewed in 31). The induction of TNF-{alpha} and IFN-{gamma} in ATF-2 mutant samples was normal or only slightly reduced. In addition, ATF-2 mutant mice showed normal induction of MIP-1{alpha}, a chemokine known to be essential for development of myocardial inflammation after Coxsackievirus infection (32). These results demonstrate that ATF-2 deficiency decreases the induction of selected cytokines 3 h after T cell activation or after broader immune activation by LPS.

ATF-2 mutant T lymphocytes produce normal cytokine levels after 24 h
Given the blunted transcription of multiple cytokine genes in ATF-2 mutant animals, one might predict low levels of cytokine elaboration as well. However, this was not observed in vitro. T lymphocytes from normal or ATF-2 mutant animals were stimulated in vitro with anti-CD3 antibody or with phorbol myristate acetate (PMA) plus ionomycin. The ATF-2 mutant T lymphocytes produced normal levels of IL-2 at 24 h and of IFN-{gamma}, IL-4, and IL-6 (Fig. 4AGo and not shown) at 48 h. In addition, cell proliferation was normal in cultures of splenocytes stimulated for 48 h with anti-CD3 antibody or with anti-CD3 plus anti-CD28 antibodies, and in thymocyte cultures stimulated with PMA plus ionomycin (Fig. 4BGo). This indicates that although the rate of cytokine gene transcription was low initially, it was adequate to produce normal levels of gene product after 1–2 days or showed a delayed recovery to normal levels.



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Fig. 4. Stimulation of ATF-2 mutant lymphocytes in vitro. (A) ELISA assays were used to measure cytokine production by CD4+ T lymphocytes from wild-type (WT) or ATF-2 mutant (MUT) animals. Cultures were stimulated with anti-CD3 antibody at 3 µg/ml or with PMA (10 ng/ml) and ionomycin (0.5 µg/ml). Supernatants were collected after 24 h of culture for IL-2 assays and after 48 h for all other cytokines. Results are shown for three to six determinations from at least three experiments and are presented as means ± SD. (B) Proliferation of wild-type or mutant lymphocytes was assayed by [3H]thymidine incorporation after 48 h of culture. Splenocytes were stimulated with control hamster Ig or with anti-CD3 antibody (0.1 µg/ml) without or with anti-CD28 antibody (1.0 µg/ml). Thymocytes were stimulated with PMA (10 ng/ml) and ionomycin (0.5 µg/ml).

 
ATF-2 mutant animals succumb to LPS or virus
Next, the effects of LPS injection were observed in ATF-2 mutant mice beyond the 3 h endpoint used above. To avoid using large doses of LPS, mice were injected with low doses of LPS along with D-galactosamine to sensitize the animals to the LPS effects (33). The LPS dose was titrated to produce death by 24 h in one-third of the control wild-type or heterozygous animals. Death occurred in some animals by 8 h after injection, but was most pronounced in ATF-2 mutant mice between 17 and 20 h (Fig. 5AGo). The death rate for ATF-2 mutant animals was higher than for control animals (P = 0.02). Animals that survived past 20 h recovered fully in all cases. From these findings, we conclude that ATF-2 mutant animals are more sensitive than wild-type animals to LPS-induced toxicity.



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Fig. 5. ATF-2 mutant animals succumb to LPS or virus. (A) Control animals (ATF-2 wild-type and heterozygous mice) or ATF-2 mutant animals were injected with LPS plus D-galactosamine and observed for survival. ATF-2 mutant animals died at a higher rate than the controls. (B) Survival of Coxsackievirus B3-infected wild-type or ATF-2 mutant mice, showing an increased death rate in ATF-2 mutant mice. Results are presented from two experiments.

 
To assess the in vivo response of ATF-2 mutant animals to further inflammatory agents, groups of mice were infected with Coxsackievirus B3 or with Herpes simplex virus-1. Using death as an endpoint, injection of 100 p.f.u. of Coxsackievirus B3 was fatal in 70% of ATF-2 mutant but in 0% of control mice (P = 0.001; Fig. 5BGo). Since Coxsackievirus produces a myocarditis, hearts from animals infected 7 days previously were examined histologically. Although animals dying earlier with potentially more significant myocarditis were not included in this analysis, surviving ATF-2 mutant animals still showed denser lymphocytic inflammatory infiltrates than did wild-type mice (Fig. 6A and BGo).



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Fig. 6. (A and B) Histologic sections of hearts from Coxsackievirus B3-infected mice. Specimens from ATF-2 mutant animals (B) showed areas of lymphocytic infiltrates, especially in perivascular areas, that were denser than those found in wild-type animals (A). (C and D) Histologic sections of lung tissue from Herpes simplex virus-1-infected mice. The ATF-2 mutant animals (D) demonstrated peribronchial and interstitial mononuclear infiltrates more frequently than wild-type mice (C).

 
Other groups of mice were injected with Herpes simplex virus and analyzed for development of pneumonitis. Again, ATF-2 mutant animals proved more susceptible to the infectious agent. At the end of 7 days, two of 14 wild-type animals had developed peribronchial and interstitial lymphocytic infiltrates, while six of 13 ATF-2 mutant mice showed this phenotype (P = 0.069; Fig. 6C and DGo), indicating a trend towards a more severe inflammatory infiltrate in the ATF-2 mutant animals.

These data provide evidence that the initial decreases in cytokine gene and adhesion molecule induction in ATF-2 mutant mice are not protective against the development of toxic shock or cytotoxicity from viral infections. Instead, the initial sluggish activation of the immune system is overcome and mononuclear inflammatory infiltrates form readily in the face of ATF-2 deficiency.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Transcription factor ATF-2 is known to be a target of the p38 MAP kinase as well as certain of the JNK kinases (6). T lymphocyte stimulation by anti-CD3 antibody with co-stimulation by anti-CD28 activates both p38 and JNK kinases, and therefore activates ATF-2. In this way, ATF-2 is positioned to carry out many of the changes seen after T cell activation, potentially influencing proliferation, induction of cytokines and chemokines, and induction of adhesion molecules. Promoter studies have identified several ATF-2 target genes in T cells, including the transcription factor c-Jun and the cytokines TNF-{alpha} and IFN-{gamma}.

However, the activation of the p38 kinase in T cells has more widespread effects than the induction of known ATF-2 target genes, suggesting that ATF-2 may act at still other promoters as well. In murine T cells stimulated by anti-CD3 plus anti-CD28, inhibition of p38 decreased cellular proliferation and induction of IL-2, IL-4 and IFN-{gamma} (34). A similar experiment performed in human cells revealed decreased induction of IL-10, with lesser effects on IL-4, IL-5, IL-13 and TNF-{alpha}, with no effect on proliferation or IL-2 secretion (35). Two other groups found that the main effect of p38 inhibition in T cells was a decrease in IL-4 or IL-5 secretion respectively (35,36). In murine macrophages and dendritic cells, inhibition of p38 diminished the release of IL-12 and TNF-{alpha} (37). ATF-2 has also been implicated in the regulation of the IL-6 promoter in studies performed in osteoblast cells (38). From these types of reports, it emerges that p38 activation is vital for the induction of Th2 (and possibly Th1) inflammatory responses, thus implicating ATF-2 in the regulation of a large group of cytokines as well.

In the model of LPS-induced inflammation, the specific role of p38 has recently been further elucidated (39). After p38 inhibition, there was loss of initial neutrophil recruitment to the airspaces of the lung, with intact accumulation of mononuclear cells at later time points. In vitro, p38 inhibition greatly reduced the release of TNF-{alpha} and MIP-2 from neutrophils, while alveolar macrophages were >1000-fold less sensitive to the inhibitor. These findings may help to explain the effects of inflammatory agents in ATF-2 mutant animals, where blunted initial induction of cytokine and adhesion molecule genes did not prevent the eventual development of an exuberant mononuclear infiltrate.

Since p38 activation peaks at 20 min post-stimulus and approaches baseline by 2 h, the best time to observe downstream consequences on transcription factor targets would also be in the first hour. This was seen for the induction of c-Jun, JunB and Fra-2, which showed decreased induction in the absence of ATF-2. These findings for the first time also identify JunB and Fra-2 as potential ATF-2 target genes.

Also within the first 3 h of a stimulus, our data show the importance of ATF-2 in the induction of multiple cytokine genes. The entire cascade of LPS-inducible cytokines was poorly up-regulated in the absence of ATF-2, beginning with TNF-{alpha}, and continuing with IL-1ß, IL-6 and the chemokine KC (which is similar to human IL-8). The LPS effects may predominantly act on cytokine elaboration from macrophages, but defects were also observed after injection of anti-CD3 antibody into ATF-2 mutant mice. Here, the most prominent abnormalities were in the induction of IL-2, IL-4 and IL-6, and reflect abnormal cytokine elaboration by NK1 T cells as well as conventional T cells. However, these effects were not seen after anti-CD3 plus anti-CD28 stimulation for 48 h in vitro, demonstrating that after the first few hours, there is compensation for the lack of ATF-2 to induce cytokines. These results were also found in vivo, where ATF-2 mutant animals were actually more susceptible to LPS, Coxsackievirus or Herpes simplex virus infection instead of showing resistance to inflammation. Histologic sections from virus-infected animals confirmed that inflammatory infiltrates were often more prominent in ATF-2 mutant than in control mice. Such findings would also be predicted from the inhibition of the p38 pathway, where accumulation of mononuclear cell infiltrates was unaffected despite earlier defects in cytokine elaboration and neutrophil chemotaxis (39). The normal induction in ATF-2 mutant tissues of MIP-1{alpha}, a chemokine essential for the development of myocardial inflammation from Coxsackievirus B3 infection, also indicates that ATF-2 deficiency does not interfere with the recruitment of a mononuclear inflammatory infiltrate. Therefore, ATF-2 is involved in immediate gene up-regulation after an inflammatory stimulus, but at later time points may even protect against overactivity of pro-inflammatory responses.


    Acknowledgments
 
We thank Steve Jean for performing mouse injections, Jeff Leiden for AP-1 plasmids and Joseph Paulauskis for the MIP-1{alpha} cDNA. This work was supported by grants from the American College of Rheumatology Research and Education Foundation and the NIH (A. M. R.) and by NIH grant AI 3241200 and the Harold and Leila Mathers Foundation (L. H. G.).


    Abbreviations
 
ATF activating transcription factor
CRE cyclic AMP-responsive element
LPS lipopolysaccharide
PMA phorbol myristate acetate
TNF tumor necrosis factor

    Notes
 
Transmitting editor: A. Singer

Received 12 September 2000, accepted 31 October 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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