Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo, Norway
Received April 19, 2004; accepted July 4, 2004
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ABSTRACT |
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Key Words: quartz; mitogen activated protein kinases; protein tyrosine kinases; interleukin-8; macrophage inflammatory protein-2.
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INTRODUCTION |
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Presently, numerous environmental stimuli are known to activate intracellular signaling cascades in the bronchial epithelium, triggering the release of pro-inflammatory mediators. Among the key enzymes in these events are the mitogen-activated protein kinase (MAPK) family of serine/threonine kinases (Puddicombe and Davies, 2000). MAPKs are activated in response to a range of extracellular stimuli (growth factors, cytokines, hormones, oxidants, toxins, physical stress) and regulate a variety of cellular responses including apoptosis, immune activation, and inflammation (Puddicombe and Davies, 2000
). Currently, the best described MAPK members are the extracellular signal-regulated kinase-1 and -2 (ERK1/2), the c-Jun-N-terminal kinases (JNKs), and the p38 MAPKs. Activation of ERK1/2, JNK, and p38 appears to be crucial for optimal IL-8 gene expression, as they contribute through both activation of transcription factors and posttranscriptional mechanisms such as IL-8 mRNA stabilization (Hoffmann et al., 2002
). Crystalline silica may activate all the three MAPK cascades. This activation seems to control silica-induced activation of activator protein (AP)-1 (Ding et al., 1999
, 2001
; Shukla et al., 2001
), which is an important transcription factor in the regulation of basal and induced cytokine expression (Hoffmann et al., 2002
). Taken together, this suggests the involvement of MAPKs in silica-induced cytokine release.
Another group of signaling molecules potentially important in silica-induced inflammation are the protein tyrosine kinases (PTKs). PTKs are roughly divided into the receptor PTKs and the non-receptor PTKs, which both are involved in the early stages of intracellular signaling. Silica particles have been shown to induce NF-B activation through tyrosine phosphorylation of I
B-
and this phosphorylation was attenuated by PTK inhibitors (Kang et al., 2000
). It has also recently been shown that PTK inhibitors may block silica-induced generation of reactive oxygen species (ROS) in fibroblasts (Kim et al., 2002
). However, the participation of specific PTKs in silica-induced signaling cascades has yet to be established. Of special interest are the Src family kinases (SFKs), which belong to the non-receptor PTKs. SFKs are activated following engagement of several different classes of cell surface receptors, including immuno-receptors, cytokine receptors, G protein-coupled receptors (GPCRs), and receptor PTKs (Thomas and Brugge, 1997
). Moreover, SFKs have been shown to mediate MAPK activation in response to various extracellular stimuli, including ROS, UV irradiation, monosodium urate (MSU) crystals, and ligands of scavenger receptors (Hsu et al., 2001
; Kitagawa et al., 2002
; Liu et al., 2001
; Nishida et al., 2000
).
The aim of this study was to investigate intracellular signaling pathways regulating crystalline silica-induced IL-8 release from a human lung epithelial cell line (A549), and to compare this with the mechanisms regulating chemokine release from primary rat type 2 (T2) cells. We have focused on determining whether the different MAPK cascades are involved in silica-induced chemokine release, and by what mechanisms MAPK activation is regulated. On the basis of the results presented below, we suggest the presence of two separate signaling pathways, which are important in the regulation of crystalline silica-induced IL-8 release from A549 cells; one involving SFK-dependent activation of ERK1/2, and the other activation of p38, at least partly independent of SFKs. We further suggest that similar mechanisms may be involved in silica-induced macrophage inflammatory protein (MIP)-2 release from primary rat T2 cells.
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MATERIALS AND METHODS |
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Crystalline silica particles. The commercially available crystalline silica particle MIN-U-SIL 5 Ground Silica (U.S. Silica Company, Berkley Springs, WV) was kindly provided by Dr. Paul Borm, Medical Institute of Environmental Hygiene, Düsseldorf, Germany. MIN-U-SIL 5 is a high purity, natural crystalline silica. According to the manufacturer, this ground silica is at least 98% SiO2 and a size distribution with typically 96% passing 5 µm and a median diameter of 1.6 µm. The crystalline silica particles were prepared and exposed to the cell cultures as previously described by Hetland et al. (2000b).
Cell cultures. The human epithelial lung cell line A549 from American Tissue Type Culture Collection (ATCC, Rockville, MD) was cultured in F12K medium, supplemented with ampicillin (100 µg/ml), penicillin/streptomycin (100 µg/ml), fungizone (0.25 µg/ml) and 10% heat-inactivated FBS. The A549 cells were plated in 35 mm six-well culture dishes (2 x 104 cells/well) or 90 mm dishes (2 x 105 cells/dish) and grown to confluence at 37°C in a humidified atmosphere of 5% CO2 in air (prior to exposure). T2 cells were isolated from rat lungs by the method described by Lag et al. (1996). In short, lungs were removed from six anaesthetized male, inbred Wistar Kyoto (Wky/NHds) rats between six and eight weeks old. Cells were isolated by sequential use of enzymatic digestion, centrifugal elutriation, and differential attachment. The isolated T2 cells were cultured in William's medium E supplemented with insulin (5 µg/ml), hydrocortisone (0.087 µg/ml), transferrin (5 µg/ml), EGF (10 ng/ml), sodium selenite (6.2 ng/ml), ascorbic acid (5 µg/ml), gluthatione (5 µg/ml), ampicillin (100 µg/ml), penicillin/streptomycin (100 µg/ml), fungizone (0.25 µg/ml), Hepes (15 mM), and 5% heat-inactivated FBS. The T2 cells were plated at a density of 4 x 105/cm2 and grown for two days at 37°C in a humidified atmosphere of 5% CO2 in air, prior to exposure.
IL-8 and MIP-2 assays. Cells were grown in 35 mm, six-well culture dishes, exposed to crystalline silica and incubated at 37°C. When inhibitors of MAPKs or PTKs were used, the cells were pre-incubated with the inhibitor for 1 h prior to crystalline silica exposure. All inhibitors were kept in the culture throughout the particle exposure period. After particle exposure supernatants were removed and centrifuged in two steps, first at 250 x g to remove cells, then at 2500 x g to remove the remaining silica particles. The final supernatants were stored at 70°C. IL-8 and MIP-2 protein levels were determined by enzyme-linked immunosorbant assay (ELISA) according to the manufacturer's guideline. Absorbance was measured and quantified using a plate reader (TECAN Sunrise, Phenix Research Products, Hayward, CA) complete with software (Magellan V 1.10).
Cell viability assay. Cell suspensions were stained with PI (5 µg/ml). Uptake of PI by damaged cells was analyzed using fluorescence microscopy, and 300400 cells from each sample were counted.
Immunoblotting. Cells were grown to confluence in 90 mm culture dishes and serum starved for 24 h prior to crystalline silica exposure. Inhibitors were applied in the same manner as for the cytokine assay. Cells were harvested in ice-cold PBS containing PMSF (1 mM) and resuspended in sample-buffer (3.1 ml destilled H2O, 1.0 ml 0.5 M Tris-HCl pH 6.8, 0.8 ml glycerol and 2.5 ml 10% SDS) prior to protein determination by the BioRad DC Protein Assay (BioRad Life Science, Camarillo, CA). Proteins (12.5 µg/well for all analyses except total and phosphorylated p38 with 25 µg/well) from whole-cell lysates were separated by 10% SDS-PAGE and blotted onto nitrocellulose membranes. To ensure that the protein levels of each well were equal, Ponceau-staining was used for loading control. The membranes were then probed for phosphorylated ERK1/2, p38, JNK, and ERK5 as well as phosphorylated SFKs (Tyr416), prior to incubation with horseradish peroxidase-conjugated secondary antibodies. The blots were developed using the SuperSignal West Dura chemoluminescence system (Pierce, Perbio Science, Sweden) according to the manufacturer's instructions. Finally the membranes were stripped by incubation for 1 h at 60°C in stripping-buffer (62.5 mM Tris-HCl, 100 mM 2-mercaptoethanol and 2% SDS) and re-probed for total ERK2, p38, JNK, ERK5, and Src. Quantification of optical densities and estimation of molecular weights of the protein bands, were performed by the use of KODAK 1D Image Analysis Software.
Statistical analysis. Statistical significance of treatments where evaluated using one-way ANOVA with Dunnett's post-test for multiple comparisons (Figs. 1A, 1B, 2A, 2B, 3, 4, 5, and 6), or two-way ANOVA with Bonferroni post-test (Figs. 1C and 2C). Values are presented as means ± SEM.
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RESULTS |
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Involvement of MAPKs in Crystalline Silica-Induced IL-8 Release from A549 Cells
MAPK signaling is important in various cellular stress responses including cytokine expression (Puddicombe and Davies, 2000). To investigate the involvement of MAPKs in crystalline silica-induced IL-8 release, A549 cell cultures were pre-incubated with the p38 inhibitor SB202190 or the MAPK/ERK kinase (MEK) 1 inhibitor PD98059, for 1 h prior to particle exposure. The presence of SB202190 significantly attenuated the silica-induced IL-8 release from A549 cells at the tested concentrations (Fig. 2A). Inhibition of MEK1, an activator protein located upstream of ERK1/2 (Puddicombe and Davies, 2000
), also significantly inhibited the chemokine induction (Fig. 2B). To investigate the p38 and ERK1/2 pathways, SB202190 is recommended to be used at 10 µM and PD98059 at 50 µM (Cuenda and Alessi, 2000
). At these concentrations SB202190 and PD98059 inhibited silica-induced IL-8 release by approximately 65 and 60%, respectively, when corrected for basal IL-8 levels. In the presence of both SB202190 (10 µM) and PD98059 (50 µM) crystalline silica-induced chemokine release was reduced to near-control levels (Fig. 2C). Basal IL-8 levels in controls were not significantly affected by inhibitor treatments (p > 0.05). We did not observe any obvious changes in cell viability due to the presence of the inhibitors (not shown).
Involvement of PTKs in Crystalline Silica-Induced IL-8 Release from A549 Cells
Having established that chemical inhibitors of the p38 and ERK1/2 pathways attenuate crystalline silica induced IL-8 release, we investigated the involvement of signaling events potentially located upstream of MAPK activation. In the presence of a general PTK inhibitor, genistein (50200 µM), silica-induced IL-8 release from A549 cells was significantly attenuated (Fig. 3A). Since SFKs may be important in MAPK activation and cytokine release (Hsu et al., 2001; Kitagawa et al., 2002
; Liu et al., 2001
), we also examined whether SFKs were involved in silica-induced IL-8 release. Pre-incubation with the SFK inhibitor, PP2 (1100 µM), led to a concentration-dependent reduction of crystalline silica-induced IL-8 release from A549 cells (Fig. 3B). At 10 µM, PP2 reduced silica-induced IL-8 release by approximately 80%, when corrected for basal IL-8 levels. Basal IL-8 levels in controls where not significantly affected by inhibitor treatments (p > 0.05). We did not observe any obvious changes in cell viability due to the presence of the inhibitors (not shown).
Activation of MAPKs and PTKs by Crystalline Silica in A549 Cells
To further explore the involvement of MAPKs and SFKs in crystalline silica-induced IL-8 release, we examined the effect of the particles on phosphorylation of MAPKs and SFKs. Compared to the IL-8 ELISA assay, detection of protein phosphorylation by Western blotting was less sensitive to silica exposure. This might be partly because IL-8 accumulates in the growth medium throughout the exposure time, whereas protein phosphorylations do not, but also because of the less quantitative nature of the Western technique. Therefore, the silica concentration was increased to 60 µg/cm2 in these experiments. As assessed with specific antibodies, silica exposure induced phosphorylation of p38 and ERK1/2 from 15 min after the particles were added to the cell cultures (Figs. 4A and 4B). We did not, however, observe phosphorylation of neither JNK nor ERK5 (also known as big mitogen-activated protein kinase [BMK]-1) in response to the silica concentrations tested (not shown). Activation of the SFKs was examined by using a phospho-specific (Tyr416) antibody which cross-reacts with several SFK members (c-Src, Lyn, Fyn, Lck, Yes, and Hck). Crystalline silica induced a significant phosphorylation of a protein estimated to approximately 61 kDa, corresponding to c-Src (60 kDa), visible from the earliest time-point tested (Fig. 4C). The antibody also revealed induction of additional phospho-proteins, mainly a 53 kDa protein, but also a band at approximately 57 kDa (only visible in some blotsnot shown), possibly corresponding to Lyn (53 and 56 kDa isoforms). Indeed, recent work has shown that c-Src and Lyn, but not Lck and Fyn, were abundantly expressed in A549 cells (Huang et al., 2003). The phosphorylation levels of the 53 and 57 kDa bands were, however, considerably weaker than the 61 kDa band and were not quantified. As seen from the figure, the antibody against total c-Src also seemed to cross-react with other SFK-members, giving rise to protein bands at approximately 61, 57, and 53 kDa (Fig. 2C).
Optical band quantification showed that the kinetics of phospho-p38 followed a biphasic pattern with a short transient peak at 15 min followed by a longer sustained phase, with a second peak at 2 h (Fig. 4A). In comparison, phosphorylation of ERK1/2 increased more gradually, peaked at 30 min and then decreased until 2 h of silica exposure, where it stabilized at approximately twice the basal phospho-ERK1/2 level (Fig. 4B). The kinetics of silica-induced phospho-c-Src had more resemblance with ERK1/2 than with p38. Src phosphorylation peaked after 1 h of silica exposure before it gradually decreased until 4 h (Fig. 4C). Initial screenings suggested that ERK1/2 and SFKs were phosphorylated for up to 16 h, whereas p38 phosphorylation disappeared between 8 and 16 h (not shown).
Involvement of SFKs in Crystalline Silica-Induced MAPK Activation in A549 Cells
SFKs have been shown to activate MAPKs in response to various stimuli (Kitagawa et al., 2002; Liu et al., 2001
; Nishida et al., 2000
), and it was therefore of interest to determine whether SFK activation was located upstream of MAPKs in the signaling pathway leading to IL-8 release. Thus, we examined the effect of PP2 on crystalline silica-induced MAPK activation in A549 cells. Pre-incubation with PP2 (10 µM) did not affect silica-induced p38 phosphorylation significantly (Fig. 5A). ERK1/2-phosphorylation was, however, inhibited to near-control levels in the presence of PP2 (Fig. 5B). PP2 did not affect basal phosphorylation levels of ERK1/2 and p38 (not shown).
MIP-2 Release by Crystalline Silica-Exposed Primary Rat T2 Cells
We examined if crystalline silica exposure of primary epithelial rat T2 cells would elicit a similar response as observed with the A549 cells. The release of MIP-2, a chemokine which in rats has an analogous function to IL-8 in humans (Driscoll, 2000; Harada et al., 1996
), was thus measured. T2 cells exposed to silica particles produced a concentration-dependent increase in MIP-2 release, with a significant increase at a particle concentration of 20 µg/cm2 and peaking at 30 µg/cm2 (Fig. 6A). We also examined whether signaling pathways similar to the above described for A549 cells might be involved in the observed MIP-2 release from crystalline silica-exposed T2 cells. The inhibitors SB202190 (10 µM) and PD98059 (50 µM) almost completely prevented silica-induced MIP-2 release from T2 cells (Fig. 6B). Furthermore, PP2 (10 µM) strongly attenuated silica-induced MIP-2 (Fig. 6B). Basal MIP-2 levels in controls were, however, not significantly affected by inhibitor treatments (p > 0.05). Western blotting revealed that crystalline silica induced phosphorylation of the MAPKs p38 and ERK1/2, as well as SFKs (Fig. 7). However, use of the phospho-Src family (Tyr416) antibody only revealed phosphorylation of a 52 and a 57 kDa band, but no band corresponding to the 60 kDa c-Src as observed in the A549 cells.
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DISCUSSION |
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In accordance with previous findings (Desaki et al., 2000; Hetland et al., 2001
; Schins et al., 2000
; Stringer et al., 1996
), our results show that crystalline silica induced a significant increase in IL-8 release from A549 cells. Our results further show that the IL-8 induction was significantly attenuated by the p38 inhibitor SB202190 and by the MEK inhibitor PD98059. Although PD98059 has been considered a relatively specific inhibitor of the ERK1/2 pathway, recent studies have shown that it may also inhibit other pathways such as JNK and in particular the MEK5-ERK5 pathway (Kamakura et al., 1999
; Mody et al., 2001
; Salh et al., 2000
). However, since crystalline silica did only induce phosphorylation of p38 and ERK1/2 but not JNK or ERK5, the observed effect of PD98059 was presumably due to inhibition of the ERK1/2 pathway. Thus, our findings suggest that crystalline silica induces IL-8 release through activation of the MAPKs p38 and ERK1/2, but not JNK or ERK5.
The role of MAPK pathways in IL-8 regulation seems to be highly cell type and stimulus specific. Previous studies have shown that IL-8 may be regulated by p38, ERK1/2, or JNK, alone (Jung et al., 2002; Kawaguchi et al., 2002
; Li et al., 2003
) or in different combinations (Furuichi et al., 2002
; Kumar et al., 2003
; Li et al., 2002
; Wu et al., 2002
). However, it appears that a maximal production of IL-8 and other cytokines may require a combined activation of all the three major MAPK cascades (Li et al., 2002
; Zhu et al., 2000
). Therefore, the potential to induce IL-8 of a given substance may partly depend on the ability to induce multiple MAPK-pathways. The IL-8 promoter region, which has been extensively studied, contains binding sites for NF-
B, CAAT/enhancer-binding protein (C/EBP), and AP-1 (see Hoffmann et al., 2002
). Whereas NF-
B binding seems to be pivotal to the IL-8 promoter activity for most cell types studied, the AP-1 and C/EBP (also called NF-IL-6) sites are not essential for induction but required for maximal gene expression (Hoffmann et al., 2002
). It appears from the literature that MAPKs primarily regulate IL-8 through activation of AP-1 and C/EBP (Jung et al., 2002
; Kumar et al., 2003
; Wu et al., 2002
). However, MAPKs may also regulate IL-8 through activation of NF-
B and through posttranscriptional mechanisms such as mRNA stabilization (Jijon et al., 2002
; Li et al., 2002
).
The knowledge on silica-induced MAPK activation and the intracellular processes it affects is limited. Studies by Ding et al. (1999, 2001
) have shown that crystalline silica induced phosphorylation of ERK1/2 and p38, but not JNK, and that both the ERK1/2 and the p38 pathways are involved in AP-1 induction in a mouse epidermal cell line (JB6). In a non-transformed alveolar T2 epithelial cell line (C10) crystalline silica-induced phosphorylation ERK1/2 and JNK, but not p38, were reported to be associated with AP-1 activation (Shukla et al., 2001
). Whether this slightly contradicting MAPK activation pattern may be a result of different cell systems or silica qualities remains unclear. However, more importantly these findings show that crystalline silica have the potential to activate all three MAPK cascades, and that these may regulate silica-induced AP-1 activity.
In our study we observed that crystalline silica induced a biphasic phosphorylation of p38. Also, the silica-induced kinetics of phospho-ERK1/2 displayed resemblance of a biphasic activation with a rapid increasing peak followed by a second sustained phase. Information on the role of transient versus sustained MAPK activation is mostly limited to the role of ERK1/2 in cell growth and proliferation (see Marshall, 1995, for review). Initially, it was believed that a sustained ERK1/2 activation was required for translocation of ERK1/2 to the nucleus, a process that is necessary for MAPKs to activate transcription factors, thus suggesting that transient and sustained MAPK activation would have different effects on gene expression (Marshall, 1995
). However, recent studies have also shown that a transient activation is sufficient both to translocate ERK1/2 to the nucleus and to induce gene expression (Chen et al., 2003
; Horgan and Stork, 2003
). Thus, it is uncertain what roles the different phases of MAPK activation may play in the regulation of cytokine production. However, silica appears to induce AP-1 activity in a slow and sustained manner, similar to the IL-8 release observed in our study (Ding et al., 1999
; Shukla et al., 2001
). Also, others have shown that silica-induced IL-8 mRNA levels peak at 3 h, and that IL-8 protein release appears later, at 24 h (Desaki et al., 2000
). Therefore, it seems likely that the late, sustained phases of silica-induced MAPK activity are more closely linked to regulation of the IL-8 induction, than the early peaks.
Genistein attenuated IL-8 release after crystalline silica exposure, suggesting the involvement of PTKs. More interestingly, the SFK inhibitor PP2, strongly attenuated silica-induced IL-8 release. Indeed, PP2 was a more efficient inhibitor of IL-8 induction than both the MAPK inhibitors tested, suggesting a crucial role of SFKs in silica-induced chemokine release. Although previous studies have implicated the involvement of PTKs in silica-induced signaling pathways such as NF-B activation (Kang et al., 2000
) and intracellular ROS generation (Kim et al., 2002
), the involvement of specific PTKs have not previously been described. SFKs are known to regulate MAPK activation, for example through phosphorylation of Shc which stimulates the Ras-MAPK pathway (see Thomas and Brugge, 1997
, for review). Here, we observed that although the overall silica-induced kinetics of phospho-ERK1/2 and phospho-SFKs differs, the early phases seem to parallel. Since PP2 strongly attenuated ERK1/2 phosphorylation, it is likely that SFK activation precedes and regulates ERK1/2 activity in response to silica exposure. In contrast, PP2 exposure did not affect silica-induced p38 phosphorylation significantly, indicating that p38 was mainly activated independently of SFKs, through a signaling pathway separate from the one activating ERK1/2. Differences in phosphorylation kinetics, between p38 and ERK1/2, support the idea that the two MAPK cascades are regulated through different mechanisms in response to silica exposure. Although our results suggest that SFKs regulate ERK1/2 activity in response to silica exposure, inhibition of silica-induced IL-8 release by the SFK inhibitor PP2 appears to be much more complete than with the ERK1/2-pathway inhibitor PD98059. This apparent discrepancy may be explained by the ability of SFKs to regulate a variety of signaling pathways involved in IL-8 induction. Indeed, we did observe a partial but nonsignificant effect of PP2 on phospho-p38, which could contribute to the observed effect on IL-8. Furthermore, SFKs have been shown to regulate NF-
B activity (Huang et al., 2003
; Liu et al., 2001
). Thus, the potent effect of PP2 compared to PD98059 suggests that silica-induced SFK activation may regulate other signaling pathways in addition to the ERK1/2 cascade, which contribute to the IL-8 release (Fig. 8).
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The origin of silica-induced signal transduction remains uncertain. Several reports have suggested that the observed effects of crystalline silica are largely due to the generation of ROS (Ding et al., 2001; Kang et al., 2000
; Shukla et al., 2001
), while others have focused on interactions between silica particles and cell surface receptors such as scavenger receptors or Fc-receptors (Hamilton et al., 2000
; Hetland et al., 2000a
; Stringer et al., 1996
). Interestingly, both of these scenarios may in theory lead to activation of SFKs. Both GPCRs (Luttrell et al., 1997
) and scavenger receptors (Hsu et al., 2001
) may signal through SFK activation, but evidence also indicates that SFKs can be activated by ROS, independent of receptor activation. H2O2 has been shown to activate
-subunits of small G-proteins directly, leading to dissociation of the ß
-subunits (Gß
) and activation of ERK1/2 (Nishida et al., 2000
). This Gß
-responsive ERK1/2 activation by H2O2 was independent of ligands binding to Gi-coupled receptors, but required Src activity (Nishida et al., 2000
). A future challenge will be to clarify whether silica-induced SFK activation is due to ROS generation or receptor interactions. To characterize the initiating mechanism(s) of the signaling pathways described in this work will be important for the understanding of crystalline silica-induced inflammation, and this is thus a prioritized focus in our current studies.
In summary, our results suggest the presence of two separate signaling pathways which are important in the regulation of silica-induced IL-8 release from A549 cells. One pathway involves SFK-dependent activation of ERK1/2, and the other activation of p38, at least partly independent of SFKs. The results further suggest that similar mechanisms may be involved in MIP-2 release from primary rat T2 cells.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo, Norway. Fax: +47 22 04 26 86. E-mail: johan.ovrevik{at}fhi.no.
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