The Differential Time-course of Extracellular-regulated Kinase Activity Correlates with the Macrophage Response toward Proliferation or Activation*

Bone marrow-derived macrophages proliferate in response to specific growth factors, including macrophage colony-stimulating factor (M-CSF). When stimulated with activating factors, such as lipopolysaccharide (LPS), macrophages stop proliferating and produce proinflammatory cytokines. Although triggering opposed responses, both M-CSF and LPS induce the activation of extracellular-regulated kinases (ERKs) 1 and 2. However, the time-course of ERK activation is different; maximal activation by M-CSF and LPS occurred after 5 and 15 min of stimulation, respectively. Granulocyte/macrophage colony-stimulating factor, interleukin 3, and TPA, all of which induced macrophage proliferation, also induced ERK activity, which was maximal at 5 min poststimulation. The use of PD98059, which specifically blocks ERK 1 and 2 activation, demonstrated that ERK activity was necessary for macrophage proliferation in response to these factors. The treatment with phosphatidylcholine-specific phospholipase C (PC-PLC) inhibited macrophage proliferation, induced the expression of cytokines, and triggered a pattern of ERK activation equivalent to that induced by LPS. Moreover, PD98059 inhibited the expression of cytokines induced by LPS or PC-PLC, thus suggesting that ERK activity is also required for macrophage activation by these two agents. Activation of the JNK pathway did not discriminate between proliferative and activating stimuli. In conclusion, our results allow to correlate the differences in the time-course of ERK activity with the macrophagic response toward proliferation or activation.

Macrophages perform critical functions in the immune system. They act as regulators of homeostasis and as effector cells in infection, wounding, and tumor growth (1). Macrophages originate in the bone marrow and, through the blood stream, reach all the tissues in the organism. According to the specific needs, tissue macrophages either proliferate, further differentiate to more specialized macrophagic populations, or become activated. When there is no need of macrophages and macro-phage colony-stimulating factor (M-CSF) 1 is not locally produced, these cells undergo a process of apoptotic death (2).
M-CSF is the major and the only specific growth factor for this cell type (3). The receptor for M-CSF is the product of the proto-oncogene c-fms (4). The binding of M-CSF induces the autophosphorylation of the receptor and the subsequent recruitment of different signal transducing molecules (reviewed in Ref. 5). One of the signaling cascades activated by M-CSF is the Raf/MEK/extracellular-regulated kinase (ERK) pathway (6,7). Raf-1, a serine/threonine protein kinase, phosphorylates and activates the threonine/tyrosine protein kinase MEK-1 (8), which in turn phosphorylates and activates ERKs 1 and 2 (9). These are proline-directed serine/threonine protein kinases, also known as p44-and p42-mitogen-activated protein kinases (MAPKs), respectively (10). Active ERKs phosphorylate and regulate several cellular proteins, including additional protein kinases, cytoskeletal components, phospholipase A 2 , and nuclear transcription factors, such as Elk1/TCF and c-Jun, which regulate the expression of immediate early genes (10,11). Lipopolysaccharide (LPS) or endotoxin, a major component of the outer membrane of Gram-negative bacteria, activates macrophages and induces the secretion of arachidonic acid metabolites (e.g. prostaglandins, leukotrienes, and platelet-activating factor), nitrogen intermediates, and cytokines, such as tumor necrosis factor ␣ (TNF-␣) and interleukins 1 and 6 (12,13), which play important roles in the immune response. LPS triggers the activation of the Raf/MEK/ERK pathway in macrophages (14,15).
Our aim is to determine the mechanism(s) that induces macrophages to either proliferate or become activated. In this study, we have found that both proliferating and activating processes in macrophages require the activation of the ERK cascade. However, the time-course of this activation is markedly different. Because the time-course of ERK activity may determine the fate of certain cellular responses (16 -18), we were interested in finding out a correlation between the pattern of ERK activation and the cellular response (proliferation versus activation) induced in macrophages. In this regard, the proliferating agents M-CSF, granulocyte/macrophage colonystimulating factor (GM-CSF), IL-3, and TPA induced a peak of ERK activity at 5 min of stimulation. In contrast, the activating agents LPS and exogenous PC-PLC, both of which blocked macrophage proliferation, induced ERK activation more slowly, with maximal induction at 15 min poststimulation. In contrast, we could not correlate the pattern of JNK activity with any particular macrophagic fate. In conclusion, in this report we show that differences in the time-course of ERK activity may be crucial in determining the macrophage response toward proliferation or activation; the macrophage response is the initial peak of ERK activity common to all the proliferative processes and the more delayed peak induced by activating agents.

EXPERIMENTAL PROCEDURES
Materials-Recombinant M-CSF was provided as a gift by DNAX (Palo Alto, CA). In some experiments, we used L-cell conditioned me-dium as the source of this growth factor. PC-PLC from Bacillus cereus and TPA were purchased from Calbiochem (San Diego, CA). Recombinant GM-CSF and LPS were obtained from Sigma. Recombinant IL-3 was purchased from R&D systems Inc. (Minneapolis, MN). PD98059 was purchased from New England Biolabs Inc. (Beverly, MA). All reagents were used following the manufacturer's recommendations.
Cell Culture-Bone marrow-derived macrophages were obtained from 6 -10-week-old Balb/c mice (Charles River Laboratories Inc., Wilmington, MA) as described (19). Macrophages were cultured in Dulbecco's modified Eagle's medium (Sigma), supplemented with 20% fetal bovine serum (FBS) (Sigma) and 30% L-cell conditioned medium as a source of M-CSF. Once macrophages were 80% confluent, normally after 6 days of culture, they were deprived of L-cell conditioned medium FIG. 1. Activation of ERK 1 and 2 is required both for macrophage proliferation in response to M-CSF and for the correct induction of cytokines by LPS. A, activation of ERK 1 and 2 by M-CSF was blocked by the inhibitor PD98059. The cells were untreated or preincubated with the indicated concentrations of PD98059 for 1 h and then stimulated with M-CSF (1200 units/ml) for 5 min. ERK 1 and 2 activity was analyzed by an in-gel kinase assay. B, quiescent macrophages were incubated for 24 h with M-CSF (1200 units/ml) in the presence or absence of the indicated doses of PD98059. The incorporation of [ 3 H]thymidine from triplicates was determined as described under "Experimental Procedures" and interpreted as a measure of macrophage proliferation. C, quiescent cells were either not treated (starved) or incubated with M-CSF (1200 units/ml) for 24 h in the presence of either vehicle or PD98059 (50 M). Viable cells were counted after trypan blue staining, and the mean Ϯ S.D. of three independent experiments is represented. D, ERK activation by LPS was blocked by PD98059. The cells were preincubated with vehicle or PD98059 (50 M) for 1 h and then treated or not treated with LPS (100 ng/ml) for 15 min. ERK activity was measured by an in-gel kinase assay. E, PD98059 inhibits the induction of proinflammatory cytokines by LPS. The cells were not treated or preincubated with either PD98059 (50 M) or vehicle (0.1% Me 2 SO) for 1 h and then stimulated with LPS (100 ng/ml) for 1 or 3 h. The expression of TNF-␣, IL-1␤, and IL-6 was determined by Northern blotting (15 g of total RNA per lane). F, normalized values of cytokine expression were represented. All images are representative of three independent experiments. for 16 -18 h and treated with either growth or activating factors in the presence or absence of selective inhibitors/activators. Name of the treatments were not toxic for the cells, as determined by trypan blue exclusion or by flow cytometry analysis.
Proliferation Assay-Cell proliferation was measured as described previously (20,21) with minor modifications. Quiescent cells (10 5 ) were incubated for 24 h in 24-well plates (3424 MARK II; Costar Corp., Cambridge, MA) in 1 ml of medium with the indicated concentrations of M-CSF. The medium was aspirated and replaced with 0.5 ml of medium containing [ 3 H]thymidine (1 Ci/ml) (ICN Pharmaceuticals Inc., Costa Mesa, CA). After 4 -6 h of incubation at 37°C, the medium was removed, and the cells were fixed in ice-cold 70% methanol. After three washes in ice-cold 10% trichloroacetic acid, the cells were solubilized in 1% SDS and 0.3 M NaOH at room temperature. Radioactivity was counted by liquid scintillation using a 1400 Tri-Carb Packard scintillation counter. Each point was performed in triplicate, and the results were expressed as the mean Ϯ S.D.
RNA Extraction and Northern Blot Analysis-The cells were washed twice in cold phosphate-buffered saline, and extraction of total RNA was performed as described (22). Total RNA samples (15 g) were separated on 1.2% agarose gels containing formaldehyde and transferred to nylon membranes (Genescreen, NEN Life Science Products). For TNF-␣ mRNA detection, we used the EcoRI/HindIII fragment of pSP65/TNF-␣ (kindly supplied by Dr. M. Nabholz, ISREC, Epalinges, Switzerland). To study the expression of IL-1␤, we obtained a probe by digesting the construct pGEM1/IL-1␤ (kindly provided by Dr. R. Wilson, Glaxo Research and Development Ltd., Greenford, United Kingdom) with EcoRI/PstI. The expression of IL-6 mRNA was analyzed by using as a probe the EcoRI/BglII fragment of pBS/IL-6 (kindly supplied by Dr. S. Rohatgi, Center for Blood Research, Boston, MA). To detect the L32 transcript, we used the EcoRI/HindIII fragment of pGEM1/L32 as a probe (23). All probes were labeled with [␣-32 P]dCTP (ICN Pharmaceuticals, Costa Mesa, CA). After incubating in hybridization solution (20% formamide, 5ϫ Denhardt's solution, 5ϫ SSC, 10 mM EDTA, 1% SDS, 25 mM Na 2 HPO 4 , 25 mM NaH 2 PO 4 , and 0.2 mg/ml salmon sperm DNA) at 65°C, membranes were exposed to Kodak X-AR films (Eastman Kodak Co.). Bands of interest were quantified with a Molecular Analyst System (Bio-Rad).
Determination of ERK Activity by in-gel Kinase Assay-The cells were washed twice in cold phosphate-buffered saline and lysed on ice with lysis solution (1% Triton X-100, 10% glycerol, 50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM sodium orthovanadate, 1 g/ml aprotinin, 1 g/ml leupeptin, 1 g/ml iodacetamide, 1 mM phenylmethylsulfonyl fluoride). The analysis of ERK activity was performed as described (24). Briefly, 50 g of total protein were separated by 12.5% SDS-PAGE containing 0.1 mg/ml of myelin basic protein (Sigma) co-polymerized in the gel. After electrophoresis, SDS was removed by washing the gel with two changes of 20% 2-propanol in 50 mM Tris-HCl (pH 8.0) for 1 h at room temperature. The gel was then incubated with 50 mM Tris-HCl (pH 8.0) containing 5 mM ␤-mercaptoethanol (Buffer A) for 1 h at room temperature. The proteins were denatured by incubating the gel with two changes of 6 M guanidine-HCl for 1 h at room temperature and then renatured by incubating it with five changes of Buffer A containing 0.04% Tween-20 for 16 h at 4°C. To perform the phosphorylation assay, the gel was first equilibrated in 40 mM Hepes-NaOH (pH 7.4) containing 2 mM dithiothreitol, 0.1 mM EGTA, 15 mM MgCl 2 , 300 M sodium orthovanadate for 30 min at 25°C and then incubated in the same solution but also containing 50 M ATP and 100 Ci of [␥-32 P]ATP (ICN). The reaction was terminated by washing the gel with 5% trichloroacetic acid containing 10 mM sodium pyrophosphate to inhibit phosphatase activity. The gel was dried, exposed to x-ray films (Kodak), and quantitated with a Molecular Analyst (Bio-Rad).
Determination of JNK Activity-This assay was performed as described (25) with minor modifications. Briefly, the cells were washed with phosphate-buffered saline and lysed in cold lysis buffer (1% Nonidet P-40, 20 mM Hepes-Na, pH 7.5, 10 mM EGTA, 40 mM ␤-glycerophosphate, 25 mM MgCl 2 , 2 mM sodium orthovanadate, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 g/ml aprotinin, 1 g/ml leupeptin, 1 g/ml iodacetamide). 150 g of total protein were mixed with 75 l of 20% protein A-Sepharose and 1 l of anti-JNK1 antibody (sc-474, Santa Cruz Biotechnology, Santa Cruz, CA) in a total volume of 500 l. The samples were rotated for 2 h at 4°C. The immune complexes were washed three times with cold phosphate-buffered saline supplemented with 1% Nonidet P-40 and 2 mM sodium orthovanadate, once with cold JNK buffer (20 mM Hepes-Na, pH 7.5, 20 mM ␤-glycerophosphate, 20 mM MgCl 2 , 0.1 mM sodium orthovanadate, 2 mM dithiothreitol), and resuspended in JNK reaction buffer (JNK buffer supplemented with 1 g of GST-c-Jun (1-169) (Calbiochem) as a sub-strate, 20 M ATP, and 1 Ci of [␥-32 P]ATP). The reaction was allowed to proceed for 30 min at 30°C and was then stopped by adding 12 l of 5ϫ Laemmli buffer. The samples were incubated for 3 min at 100°C and separated by 10% SDS-PAGE. After the electrophoresis, the gels were fixed in isopropanol:water:acetic acid (25:65:10), dried and exposed to Kodak X-AR films.

RESULTS
In this work, we have used bone marrow-derived macrophages because they represent a homogeneous population of primary macrophages that can either proliferate efficiently in response to M-CSF or become activated in response to LPS. Both agents induce the activity of ERK kinases. To determine the involvement of ERK activation in the response to M-CSF or LPS, we used PD98059, an inhibitor specific for the ERK kinase (MEK-1) (26). The preincubation with PD98059 inhibited in a dose-dependent manner the activation of ERK 1 and 2 induced by M-CSF (Fig. 1A). The percentage of inhibition of ERK activity correlated with the capability of PD98059 to block the proliferation of quiescent bone marrow macrophages as measured by [ 3 H]thymidine incorporation (Fig. 1B). The inhibition of macrophage proliferation was also observed by cell counting (Fig. 1C). The maximal inhibition of both ERK activation and macrophage proliferation was obtained at a PD98059 concentration of 50 M. We have previously observed that macrophages do not undergo a process of apoptosis upon exposure to this concentration of the inhibitor (29). In the same studies, we found that PD98059 did not alter certain cellular processes, such as the induction of the phosphatase MAPK phosphatase-1. Taken together, our results indicate that the effect of PD98059 on macrophage proliferation is specifically mediated by the inhibition of ERK activation rather than by a general toxic effect. For subsequent experiments, we used 50 M PD98059 to fully inhibit ERK activation in macrophages.
We further extended our investigations to analyze the involvement of ERK 1 and 2 activity in the macrophage activation by LPS. Preincubation of macrophages with PD98059 also abolished the activation of ERK 1 and 2 by LPS (Fig. 1D). The treatment with this compound inhibited the LPS-induced expression of IL-1␤ and IL-6 ( Fig. 1, E and F). In addition, the blockage of ERK activation had also an inhibitory effect on the late induction of TNF-␣ by LPS. These results suggest that activation of ERK 1 and 2 is required for the correct induction of cytokines during the macrophagic response to LPS.
We next determined the time-course of ERK activation in response to M-CSF or LPS. Using an in-gel kinase assay, we detected ERK 1 and 2 activity as early as 2 min after the stimulation with M-CSF; it peaked at 5 min and decreased progressively thereafter ( Fig. 2A). By contrast to the M-CSF signal transduction, we did not detect ERK activation within the first 5 min of stimulation with LPS (Fig. 2B). Instead, ERK 1 and 2 activity started to be detected at 10 min and peaked at 15 min of LPS treatment. Therefore, important differences were observed between the time-course of ERK 1 and 2 activity induced by M-CSF and LPS; the former was more rapidly induced by the proliferating factor than by the activating agent. These results were confirmed by mobility shift assays (data not shown).
In order to determine whether differences in the time-course of ERK activation had any specific relationship with the macrophagic response toward proliferation versus activation, we tested the effect of other growth factors. Although M-CSF is the major and specific growth factor for macrophages, these cells are also able to proliferate in response to GM-CSF and IL-3 (Fig. 3, A and B). However, the signaling pathways induced after the binding of these growth factors to their specific receptors (27,28) is different from that induced by the M-CSF receptor (5). As shown above for the M-CSF-induced response, the macrophage proliferation induced by GM-CSF or IL-3 was also blocked by the use of the specific MEK inhibitor PD98059 (Fig. 3, A-D). This indicates that ERK activation is required for this process. The inhibition of macrophage proliferation is not due to a general toxic effect of PD98059, because in previous experiments, this compound did not reduce macrophage viability or induce macrophage apoptosis (data not shown). In addition, a number of cellular responses, such as the induction of MAPK phosphatase-1, were not modified by this inhibitor (29).
In order to determine any correlation between the earliest peak of ERK activity and macrophage proliferation, we studied the pattern of ERK activation in response to GM-CSF and IL-3. The treatment of macrophages with either one of these two growth factors induced the activation of ERK 1 and 2 within the first 5 min of stimulation (Fig. 3, E and F). This activation was extended up for 10 min more and thereafter decayed to basal levels.
Moreover, we studied the effect of the phorbol ester TPA. This agent induced the proliferation of quiescent bone marrow macrophages (Fig. 4A). Interestingly, macrophage proliferation induced by TPA was inhibited by the pretreatment with PD98059 (Fig. 4, A and B). We also compared the effect of TPA with that of LPS on macrophage activation and found that in bone marrow macrophages, TPA was a much weaker inducer of the expression of the proinflammatory cytokines TNF-␣ and IL-1␤ (Fig. 4C). Our results suggest that in macrophages, TPA plays a more important role as an inducer of proliferation than as an activating factor. When the time-course of ERK activation was analyzed, a peak of ERK 1 and 2 activity was detected within the first 5 min of stimulation with TPA (Fig. 4D). Taken together, our results suggest that in macrophages, the earliest peak of ERK activation is a common feature induced by mitogenic factors.
Next, we were interested in assessing whether other agents that mimic the effect of LPS also induced a similar pattern of ERK activation. Although IFN-␥ is the major macrophage activating factor, it induces different functional activities than LPS. In fact, no ERK activity was detected in macrophages stimulated with IFN-␥ (data not shown). Interestingly, the incubation of macrophages with exogenous PC-PLC from B. cereus induced potently and very quickly the mRNA expression of the cytokines TNF-␣ and IL-1␤ (Fig. 5A). This induction was partially inhibited by the use of PD98059 (Fig. 5A), thus suggesting that ERK activation was involved in this event. The effect of PC-PLC on macrophage proliferation was also analyzed. The treatment with PC-PLC alone did not induce proliferation on quiescent macrophages (Fig. 5B). Because macrophage activation is linked to a loss of proliferation, as demonstrated by the treatment of macrophages with M-CSF and LPS simultaneously (Fig. 5C), the effect of exogenous PC-PLC on the M-CSF-induced proliferation of macrophages was also studied. In fact, PC-PLC significantly inhibited macrophage proliferation in response to M-CSF (Fig. 5C). Taken together, these results indicate that exogenous PC-PLC acts as a macrophage activating agent. This effect was not due to contaminating LPS in the PC-PLC samples because autoclaved PC-PLC aliquots lost their capability to induce macrophage activation (data not shown). Therefore, we analyzed the timecourse of ERK activation induced by this agent and compared it with that triggered by LPS. PC-PLC mimicked the time-course of ERK activity induced by LPS, with maximal activation after 15 min of stimulation (Fig. 5D). These results further supported our hypothesis that the pattern of ERK activation helped to define the macrophage response toward proliferation or activation.
We were also interested in analyzing whether the timecourse of activation of the JNK pathway could also play a role in determining the macrophage dichotomy between proliferation and activation. Interestingly, both M-CSF and LPS induced JNK activity, as assessed by an in vitro kinase assay (Fig. 6). However, the time-course of this activation was virtually identical. JNK1 activity was not detected during the first 5 min of stimulation, peaked at 15 min, remained elevated at 30 min, and thereafter decayed to basal levels. PC-PLC was also observed to induce JNK activity in a similar way. In contrast, TPA did not induce detectable JNK activity in macrophages. Therefore, we found no tight correlation between the capability of a certain agent to induce JNK activity and its effect on macrophage biology.

DISCUSSION
Lymphocytes undergo a clonal expansion when they are activated either after interaction with a peptide presented by the major histocompatibility complex (T cells) or after the direct recognition of an antigen (B cells) (30). In contrast, macrophages cannot simultaneously proliferate and become activated. In fact, macrophage activation is linked to a growth arrest and an enhancement of their ability to perform specialized functions in the immune system. Our goal is to determine the signaling mechanisms that induce macrophages to either proliferate or become activated.
In this report, we have used bone marrow-derived macrophages because they constitute a homogeneous population of primary macrophages. An advantage over macrophagic cell Cell counting after trypan blue staining was performed, and the mean of three independent experiments is represented. C, the TPA-induced expression of proinflammatory cytokines was compared with that induced by LPS in macrophages. The cells were either left untreated or incubated with LPS (100 ng/ml) or TPA (100 ng/ml) for 90 min. The expression of TNF-␣ and IL-1␤ was studied by Northern blotting (15 g of total RNA per lane). The expression of the L32 transcript was analyzed to check for differences in RNA loading and transfer. D, TPA induces ERK activation within the first 5 min of stimulation. The cells were incubated with TPA (100 ng/ml) for the indicated periods of time. ERK activity was determined by an in-gel kinase assay. The experiments shown in this figure were performed three independent times with identical results. lines is that bone marrow macrophages can be rendered quiescent by removing M-CSF from the medium and then induced to proliferate efficiently in response to growth factors. In addition, these cells become activated after exposure to activating agents, such as LPS. One of the main properties of the activation of macrophages by LPS is the production of proinflammatory cytokines that help resolve the immune response against microorganisms. However, although representing a suitable model for studying several aspects of the regulation of macrophage proliferation versus activation, at present, transfection of these primary cultures is very inefficient (21). For this reason, we need to use chemical inhibitors to assay the involve-ment of specific molecules in macrophage biology.
In this report, we have shown that in macrophages, activation of ERK 1 and 2 is necessary for the proliferative processes induced by different growth factors, including M-CSF, GM-CSF, and IL-3, and by the tumor promoting agent TPA. The activation of these kinases is also necessary for the proliferation of other cell types in response to specific growth factors or serum (31,32). The inhibition of this pathway led to a growth arrest of macrophages at the G 1 phase of the cell cycle, without inducing apoptosis (29). In addition, we have demonstrated that activation of ERK 1 and 2 by LPS is required for the correct induction of the cytokines IL-1␤, IL-6, and, to a lesser extent, TNF-␣. These results complement recent observations in human monocytes, in which blockage of the ERK pathway inhibited the LPS-induced secretion of IL-1␤ and TNF-␣ (33,34). We have also found that the treatment with exogenous PC-PLC mimics some of the aspects of the macrophage activation by LPS, including the growth arrest and the production of proinflammatory cytokines. Again, ERK activity was required for the normal expression of TNF-␣ and IL-1␤ induced by PC-PLC. These observations contrast with the mitogenic effect attributed to the treatment with exogenous PC-PLC in some other cellular system (35). Although our data do not allow us to conclude that endogenous PC-PLC plays a major role in the context of macrophage activation, the results shown here are in agreement with the findings that mouse septic shock during Gram-negative bacterial infections can be down-modulated by the blockage of PC-PLC activation (36). The fact that both proliferating and activating agents induced ERK activity in macrophages does not reduce the importance of these kinases in the regulation of these two processes. In fact, we have shown that the time-course of ERK activity was markedly different in one case or the other. Our results confirm previous observations that LPS and M-CSF induced two distinct patterns of ERK activation in macrophages (7). For the first time, we have demonstrated that GM-CSF, IL-3, and TPA, which induce macrophage proliferation, trigger a pattern of ERK activation similar to that induced by M-CSF, with an early peak of ERK 1 and 2 activity within the first 5 min of stimulation. In contrast, LPS and PC-PLC, which inhibited macrophage proliferation and induced the expression of cytokines, triggered a more delayed peak of ERK activation, which was maximal after 15 min of stimulation.
The mechanisms by which the proliferating agents induce an earlier peak of ERK activation in comparison to that induced by the activating agents may be a consequence of the usage of different pathways to activate the ERK cascade. In BAC1.2F5 macrophages, ERK activation by M-CSF was postulated to be mediated by both Ras-dependent and Ras-independent mechanisms, whereas the activation by LPS partially involved the action of a PLC isoform specific for phosphatidylcholine (6). These observations are in agreement with our finding that the treatment of macrophages with exogenous PC-PLC induces a time-course of ERK activation similar to that induced during the macrophage response to LPS. Our results also indicate that although M-CSF, GM-CSF, and IL-3 have been shown to activate different signaling molecules, e.g. signal transducer and activator of transcription-1 in response to M-CSF (5, 38) versus signal transducer and activator of transcription-5 in response to GM-CSF (39) or IL-3 (40), convergence at the level of ERK activation exists between the different macrophage mitogenic networks.
We are currently investigating the mechanisms downstream or in parallel to ERK 1 and 2 that may help define a certain cellular response, depending on the time-course of ERK activation. The fact that a particular pattern of ERK activity correlates with a specific macrophage response does not necessarily mean that ERK 1 and 2 are the unique regulator of that process. In fact, the blockage of ERK activation did not result in a complete inhibition of the expression of proinflammatory cytokines in any of the systems tested. Our results point to an involvement of ERK activity in the determination of the fate of the macrophage response rather than on the extent of the final response itself. We should also think on the existence of finely regulated interactions between active ERK 1 and 2 and other signaling molecules, most probably transiently switched on, in order to regulate gene transcription leading to a specific macrophage response. In this regard, other members of the MAPK group of kinases, such as JNK/stress-activated protein kinase, have been also implicated in the control of proliferation (41,42) and the production of cytokines in different cell types (37,43,44). Unfortunately, we have not been able to demonstrate any tight correlation between the activation of the JNK pathway and the macrophage response toward proliferation or activation. However, we are currently assessing whether these kinases play a role in synergy to ERK 1 and 2 in the control of cytokine expression in response to LPS or PC-PLC.
Taken together, our results allow us to reach two important conclusions. First, the ERK pathway is required both for macrophage proliferation and for the correct production of cytokines during macrophage activation. And second, a clear correlation exists between the time-course of ERK activation and the decision of macrophages to either proliferate or become activated; this decision is the initial peak of ERK activation common to all the proliferative signals and the more delayed peak induced by activating agents. Our results suggest a crucial role for the ERK pathway in the control of this dichotomy.