Mònica Comalada

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.

Protein kinase C epsilon is required for the induction of mitogen-activated protein kinase phosphatase-1 in lipopolysaccharide-stimulated macrophages.

LPS induces in bone marrow macrophages the transient expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1). Because MKP-1 plays a crucial role in the attenuation of different MAPK cascades, we were interested in the characterization of the signaling mechanisms involved in the control of MKP-1 expression in LPS-stimulated macrophages. The induction of MKP-1 was blocked by genistein, a tyrosine kinase inhibitor, and by two different protein kinase C (PKC) inhibitors (GF109203X and calphostin C). We had previously shown that bone marrow macrophages express the isoforms PKCβI, ε, and ζ. Of all these, only PKCβI and ε are inhibited by GF109203X. The following arguments suggest that PKCε is required selectively for the induction of MKP-1 by LPS. First, in macrophages exposed to prolonged treatment with PMA, MKP-1 induction by LPS correlates with the levels of expression of PKCε but not with that of PKCβI. Second, Gö6976, an inhibitor selective for conventional PKCs, including PKCβI, does not alter MKP-1 induction by LPS. Last, antisense oligonucleotides that block the expression of PKCε, but not those selective for PKCβI or PKCζ, inhibit MKP-1 induction and lead to an increase of extracellular-signal regulated kinase activity during the macrophage response to LPS. Finally, in macrophages stimulated with LPS we observed significant activation of PKCε. In conclusion, our results demonstrate an important role for PKCε in the induction of MKP-1 and the subsequent negative control of MAPK activity in macrophages.

Molecular Mechanisms Involved in Macrophage Survival, Proliferation, Activation or Apoptosis

Macrophages play a critical role during the immune response. Like other cells of the immune system, macrophages are produced in large amounts and most of them die through apoptosis. Macrophages survive in the presence of soluble factors, such as IFN-gamma, or extracellular matrix proteins like decorin. The mechanism toward survival requires the blocking of proliferation at the G1/S boundary of the cell cycle that is mediated by the cyclin-dependent kinase (cdk) inhibitor, p27kip and the induction of a cdk inhibitor, p21waf1. At the inflammatory loci, macrophages need to proliferate or become activated in order to perform their specialized activities. Although the stimuli inducing proliferation and activation follow different intracellular pathways, both require the activation of extracellular signal-regulated kinases (ERKs) 1 and 2. However, the kinetics of ERK-1/2 activation is different and is determined by the induction of the MAP-kinase phosphatase-1 (MKP-1) that dephosphorilates ERK-1/2. This phosphatase plays a critical role in the process of proliferation versus activation of the macrophages.

Immunosenescence of macrophages: reduced MHC class II gene expression

In order to determine the effect of aging on macrophages, we produced bone marrow-derived macrophages in vitro from young and aged mice. We analyzed the effect of aging on the genomic expression of macrophages in these conditions, without the influence of other cell types that may be affected by aging. Macrophages from young and aged mice were present in similar numbers and showed an identical degree of differentiation, cell size, DNA content and cell surface markers. After incubation with interferon-gamma (IFN-gamma), the expression at the cell surface of the MHC class II gene IA complex product and the levels of intracellular IAbeta protein and mRNA were lower in aged macrophages. Moreover, the transcription of IAbeta gene was impaired in aged macrophages. The amount of transcription factors that bound to the W and X boxes, but not to the Y box of the IAbeta promoter gene were lower in aged macrophages. Similar levels of CIITA mRNA were found after IFN-gamma treatment of both young and aged macrophages. This shows that neither the initial cascade that starts after the interaction of IFN-gamma with the receptor, nor the second signals involved in the expression of CIITA, are impaired in aged macrophages. These data could explain, at least in part, the impaired immune response associated to senescence.

Macrophage proinflammatory activation and deactivation: a question of balance.

Macrophages play key roles in inflammation. During the onset of the inflammatory process, these phagocytic cells become activated and have destructive effects. Macrophage activation, which involves the induction of more than 400 genes, results in an increased capacity to eliminate bacteria and to regulate many other cells through the release of cytokines and chemokines. However, excessive activation has damaging effects, such as septic shock, which can lead to multiple organ dysfunction syndrome and death. In other situations, persistence of proinflammatory activity results in the development of chronic inflammation, such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease. To prevent undesirable effects, several mechanisms have evolved to control the excess of activation, thereby leading to macrophage deactivation and the resolution of inflammation. In this review, we discuss several mechanisms that mediate macrophage deactivation.

JNK1 Is required for the induction of Mkp1 expression in macrophages during proliferation and lipopolysaccharide-dependent activation

Macrophages proliferate in the presence of their growth factor, macrophage colony-stimulating factor (M-CSF), in a process that is dependent on early and short ERK activation. Lipopolysaccharide (LPS) induces macrophage activation, stops proliferation, and delays ERK phosphorylation, thereby triggering an inflammatory response. Proliferating or activating responses are balanced by the kinetics of ERK phosphorylation, the inactivation of which correlates with Mkp1 induction. Here we show that the transcriptional induction of this phosphatase by M-CSF or LPS depends on JNK but not on the other MAPKs, ERK and p38. The lack of Mkp1 induction caused by JNK inhibition prolonged ERK-1/2 and p38 phosphorylation. The two JNK genes, jnk1 and jnk2, are constitutively expressed in macrophages. However, only the JNK1 isoform was phosphorylated and, as determined in single knock-out mice, was necessary for Mkp1 induction by M-CSF or LPS. JNK1 was also required for pro-inflammatory cytokine biosynthesis (tumor necrosis factor-α, interleukin-1β, and interleukin-6) and LPS-induced NO production. This requirement is independent of Mkp1 expression, as shown in Mkp1 knock-out mice. Our results demonstrate a critical role for JNK1 in the regulation of Mkp1 induction and in LPS-dependent macrophage activation.

Macrophage colony‐stimulating factor‐, granulocyte‐macrophage colony‐stimulating factor‐, or IL‐3‐dependent survival of macrophages, but not proliferation, requires the expression of p21Waf1 through the phosphatidylinositol 3‐kinase/Akt pathway

Mouse bone marrow‐derived macrophages proliferate in the presence of macrophage colony‐stimulating factor (M‐CSF), granulocyte‐macrophage colony‐stimulating factor, or IL‐3, but undergo apoptosis in their absence. Inhibition of extracellular signal‐regulated kinases (ERK)‐1/2 blocks growth factor‐dependent proliferation but not survival, indicating that the two processes require independent signaling pathways. Although M‐CSF induces the activation of other kinase pathways, such as c‐Jun N‐terminal kinase, p38, and phosphatidylinositol 3‐kinase (PI‐3K), these pathways are not required for proliferation. However, PI‐3K is the only one necessary for the induction of survival, as demonstrated using the inhibitors LY294002 and Wortmannin. Growth factors also activate Akt kinase and a transient expression of the cdk inhibitor p21Waf1, which inhibits apoptosis but is not required for proliferation. PI‐3K inhibitors also block growth factor‐dependent expression of p21Waf1 and the activation of Akt. Moreover, the survival induced by cyclosporinu2004A or decorin is also dependent on the PI‐3K/Akt kinases and p21Waf1. These findings demonstrate that the induction of p21Waf1 through the PI‐3K/Akt pathway is a general survival response of macrophages. Our results show that growth factors in macrophages use two pathways: one for proliferation, mediated by ERK, and the other for survival, which requires the PI‐3K/Akt kinases and p21Waf1.

Macrophage-Colony-Stimulating Factor-Induced Proliferation and Lipopolysaccharide-Dependent Activation of Macrophages Requires Raf-1 Phosphorylation to Induce Mitogen Kinase Phosphatase-1 Expression

Macrophages are key regulators of immune responses. In the absence of an activating signal, murine bone marrow-derived macrophages undergo proliferation in response to their specific growth factor, namely M-CSF. The addition of bacterial LPS results in macrophage growth arrest and their engagement in a proinflammatory response. Although participation of ERKs is required for both macrophage proliferation and activation, ERK phosphorylation follows a more delayed pattern in response to activating agents. In primary macrophages, mitogen kinase phosphatase-1 (MKP-1) is a key regulator of the time course of MAPK activity. Here we showed that MKP-1 expression is dependent on Raf-1 activation. The time course of Raf-1 activation correlated with that of ERK-1/2. However, whereas ERK phosphorylation in response to M-CSF is Raf-1 dependent, in response to LPS, an alternative pathway directs the activation of these kinases. Inhibition of Raf-1 activity increased the expression of cyclin-dependent kinase inhibitors and growth arrest. In contrast, no effect was observed in the expression of proinflammatory cytokines and inducible NO synthase following LPS stimulation. The data reported here reveal new insights into how signaling determines opposing macrophage functions.

Macrophage colony-stimulating factor-dependent macrophage proliferation is mediated through a calcineurin-independent but immunophilin-dependent mechanism that mediates the activation of external regulated kinases

Calcineurin is constitutively expressed in bone marrow‐derived macrophages. However, macrophage response to macrophage colony‐stimulating factor (M‐CSF) was not impaired by the use of either calcineurin inhibitors (W‐13, chlorpromazine and trifluoperazine), calcium chelators (BAPTA‐AM) or Ca2+ channel antagonists (verapamil, nifedipine and diltiazem). Inhibition of calcineurin expression by inhibitory antisense RNA treatment did not result in an inhibition of M‐CSF‐dependent proliferation. Only very high doses of cyclosporinu2004A and FK506 inhibited macrophage proliferation induced by growth factors, such as M‐CSF, granulocyte‐macrophage (GM)‐CSF or IL‐3. This inhibitory action is mediated by the peptidylprolyl isomerase activity of the immunophilins, as demonstrated bythe use of specific inhibitors (rapamycin and sanglifehrinu2004A). These isomerase inhibitors exerted a negative effect on a key element involved in macrophage proliferation, namely the M‐CSF‐dependent activation of the extracellular signal‐regulated kinases (ERK). In summary, the data presented here provide new insights in the mechanism of macrophage proliferation, which may have relevant consequences. First, we showed that in M‐CSF‐dependent proliferation calcineurin is not involved, and second, that immunophilins play a key role and their activation blocks ERK activation.

Cyclophilin A is required for M-CSF-dependent macrophage proliferation.

The immunosuppressor sanglifehrin A (SfA) is a member of a family of immunophilin cyclophilin A‐binding molecules and does not inhibit calcineurin activity. Sanglifehrin A inhibits M‐CSF‐dependent macrophage proliferation by arresting the G1 phase of the cell cycle but does not affect cell viability. This immunosuppressor exerts its action on proliferation by inactivating cyclin‐dependent kinase 2 (Cdk2) activity. Moreover, c‐myc expression is also repressed. In the early steps of M‐CSF signaling, SfA inhibits the phosphorylation of Raf‐1 and the external regulated kinases (ERK)1/2 and mitogen‐activated protein kinase phosphatase‐1, which are required for proliferation. The effects of SfA are not related to a block of the proteosome activity. These data show that immunophilin contributes to M‐CSF‐dependent proliferation through activation of the Raf‐1/MEK/ERK pathway and the regulation of Cdk activities, which is required for cell cycle progression.