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Published online before print February 24, 2004
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receptor signaling in primary human microglia: differential roles of PI-3K and Ras/ERK MAPK pathways in phagocytosis and chemokine induction
Departments of
* Pathology and
Anatomy, Albert Einstein College of Medicine, Bronx, New York; and
Department of Orthopedic Surgery, University of Tokyo, Japan
1Correspondence: Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail: slee{at}aecom.yu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R). In this report, we investigated microglial FcR signal-transduction pathways by using adenoviral-mediated gene transfer and specific inhibitors of cell-signaling pathways. We found that Src inhibitor PP2 and Syk inhibitor piceatannol inhibited phagocytosis, macrophage-inflammatory protein-1
(MIP-1) release, as well as phosphorylation of extracellular-regulated kinase (ERK) and Akt, consistent with Src/Syk involvement early in FcR signaling. Constitutively active mitogen-activated protein kinase kinase (MEK) induced MIP-1, and Ras dominant-negative (DN) inhibited IC-induced ERK phosphorylation and MIP-1 production. These results suggest that the Ras/MEK/ERK pathway is necessary and sufficient in IC-induced MIP-1 expression. Neither Ras DN nor the MEK inhibitor U0126 inhibited phagocytosis. In contrast, phosphatidylinositol-3 kinase (PI-3K) inhibitors Wortmannin and LY294002 inhibited phagocytosis without affecting ERK phosphorylation or MIP-1 production. Conversely, Ras DN or U0126 did not affect Akt phosphorylation. Together, these results demonstrate distinct roles played by the PI-3K and Ras/MEK/ERK pathways in phagocytosis and MIP-1 induction, respectively. Our results demonstrating activation of functionally distinct pathways following microglial FcR engagement may have implications for human central nervous system diseases.
Key Words: macrophage-inflammatory protein-1 • immune complex • MEK • Src kinase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rs) are expressed in myeloid cells in species ranging from lower vertebrates to humans. Cross-linking FcRs triggers essential cellular events, including phagocytosis, inflammatory mediators release, as well as immune complex (IC) clearance and regulation of antibody production. In this way, FcRs provide a critical feedback between the humoral and cellular immune response [1
, 2
].
Phagocytosis of IgG-opsonized particles by macrophages is initiated by clustering FcRs, followed by phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) within the FcR subunit. Tyrosine kinases of the Src family are thought to be responsible for the initial phosphorylation of ITAMs, which then serve to recruit and activate Syk, a Src homology 2 (SH2) domain containing tyrosine kinase. Evidence supports that Src and Syk kinases play important roles in FcR-mediated signaling leading to phagocytosis. For instance, overexpression of C-terminal Src kinase (Csk), a negative regulator of Src, in mouse macrophages abolishes FcR-mediated phagocytosis, and the introduction of Lyn and Hck Src kinases restores the phagocytic signaling in these cells [3
]. Csk overexpression also abolishes Syk phosphorylation, in keeping with an earlier study that found defective phagocytosis in Syk–/– macrophages [4
]. Collectively, these data indicate that Syk kinase plays an indispensable role in FcR-mediated phagocytosis. Conversely, macrophages deficient in triple Src kinase (Hck, Fgr, and Lyn) show only moderate reduction of phagocytosis (although they are defective in actin polymerization and activation of Syk), suggesting that the Src family kinases are dispensable for phagocytosis [5
]. These observations indicate that the Src and Syk kinases have distinct functions in the propagation of a phagocytic signal through the FcR. Rather than a linear flow of information from the receptor to Src, Syk, and to downstream events, FcR signals may diverge at some point after receptor activation. Events downstream of Syk activation in FcR signaling have also been studied extensively; these include activation of phospholipase C, paxillin, phosphatidylinositol 3-kinase (PI-3K), and extracellular signal-regulated (ERK) mitogen-activated protein kinase (MAPK) [6
7
8
]. These studies report a profound reduction in FcR-triggered protein tyrosine phosphorylation including PI-3K and MAPK in Syk-deficient macrophages [4
]. The importance of PI-3K in FcR signaling is particularly well studied with respect to its role in phagocytosis, which requires activation of multiple transmembrane signaling pathways that culminate in cytoskeletal assembly and particle ingestion. The requirement for specific signal components in the different stages of phagocytosis has been defined: Particle ingestion via FcR requires actin assembly, pseudopod extension, and phagosomal closure, and PI-3K has been implicated these processes [9
, 10
].
ERK MAPKs function in a protein kinase cascade that plays a critical role in the regulation of cell growth and differentiation. ERK involvement in FcR signaling has been suggested in various cell types. Studies have shown that activation of MAPK is necessary for the FcR-dependent induction of tumor necrosis factor (TNF-) mRNA expression in monocytes and natural killer cells [11
]. Using rabbit IC as the stimuli, Garcia-Garcia et al. [6
, 12
] have shown that FcR cross-linking in THP-1 cells leads to secretion of interleukin (IL)-1 through a mechanism involving ERK kinase induction. These results underscore the role of MAPK as signal-transducing molecules controlling the gene expression on FcR stimulation. Whether MAPK participates in phagocytic signaling is less clear: ERK activation is shown during IgG-mediated phagocytosis by human neutrophils and monocytes, but conflicting data exist regarding its role in phagocytosis [13
14
15
]. In primary human microglia, we demonstrated the involvement of ERK kinase in FcR-mediated macrophage-inflammatory protein-1 (MIP-1) production [16
, 17
]. The upstream signaling involved in ERK activation, its role in microglial phagocytosis, and its relationship to other FcR-activated signaling molecules have not been studied.
Microglia are the resident macrophages of the brain, which is responsible for the clearance of the pathogens from the central nervous system (CNS). Although microglial cells represent the major endogenous brain phagocytes, little is known about the consequences of the activation of their phagocytic receptors such as FcRs and complement receptors. We have previously reported that Cryptococcus neoformans (CN) can induce chemokine [MIP-1, MIP-1ß, and regulated on activation, normal T expressed and secreted (RANTES)] production and phagocytosis in primary human microglia in the presence of specific antibodies [16
]. Further studies using FcR-blocking antibodies and FcR-deficient murine microglia demonstrated that FcRs, specifically FcRI and RIII, were involved in these processes [17
]. In this report, we investigated the signaling components involved in FcR activation and subsequent biological outcomes, namely, microglial chemokine gene production and phagocytosis. We found that PI-3K and ERK MAPK have distinct and nonoverlapping roles in the FcR signaling, leading to phagocytosis and microglial chemokine expression, respectively. Using adenoviral-mediated gene transfer in primary microglial cells, we also identified that Ras is crucial in the activation of MAPK kinase (MEK)/ERK, leading to chemokine expression following FcR cross-linking.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Organism
CN (American Type Culture Collection, Manassas, VA, strain 24067), a D-serotype strain, was used in this study. This strain was selected for study, as it has been studied extensively and was used in our prior investigations. Cells were grown in Sabouraud’s dextrose broth in a rotary shaker at 30°C until stationary phase. Cells were then washed three times in sterile, phosphate-buffered saline and counted with a hemocytometer.
Inoculation of microglia with CN
CN were added to microglia cultures at 4–6 x 105 per well to yield a CN-to-microglia ratio of 10:1 in the presence or absence of monoclonal antibody (mAb). Protein G-purified murine monoclonal (mAb) IgG1 from hybridomas (18B7), which binds glucuronoxylomannan, was used in this study. Cultures incubated at 37°C. After 16 h or indicated time, microglial culture supernatants were collected for determination of chemokines with enzyme-linked immunosorbent assay (ELISA), and cells were fixed with methanol and then stained with Giemsa for phagocytosis assay, as described [16
, 17
].
Chemokine ELISAs
The chemokine concentration (MIP-1 and RANTES) in human microglial culture supernatants was determined by ELISA using kits from R&D Systems (Minneapolis, MN). For some experiments, ELISA was performed using capture and detection antibody pairs from R&D Systems. The sensitivity of detection was similar in both ELISA systems. Microglial culture supernatants were diluted 1:5–1:20 before ELISA.
Western blot analysis and immunocytochemistry
Microglia at 0.5 x 106 cells per 60-mm Petri dish were incubated with CN + mAb for the indicated time intervals, and then cells were lysed in 8 M urea. Cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with rabbit polyclonal antibodies to ERK, phospho-ERK (pERK), Akt, or phospho-Akt (pAkt) from Cell Signaling (Beverly, MA). Antibodies were diluted at 1:1000 for ERK, pERK, and Akt and 1:500 for pAkt. In some experiments, blots were incubated with two antibodies simultaneously (pERK/pAkt or pAkt/total ERK). Secondary antibody was goat–anti-rabbit–horseradish peroxidase conjugate at 1:2000. Signals were detected by enhanced chemiluminescence (Amersham, Little Chalfont, UK). For immunostaining, microglia were treated with CN + 18B7 for 30 min or indicated time and were then fixed with methanol. Fixed cells were incubated with anti-pERK or nuclear factor (NF)-
B p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:200 for 16 h and then with peroxidase-conjugated goat–anti-rabbit antibody at 1:300 for 2 h. Color was developed by diaminobenzidine.
Drug treatment of microglial culture
A specific inhibitor of MEK, U0126, was purchased from Promega (Madison, WI). Syk inhibitor piceatannol (PIC) was purchased from Sigma Chemical Co. Src inhibitor PP2 and PI-3K inhibitors LY294002 and Wortmannin were purchased from Calbiochem (San Diego, CA). All inhibitors were dissolved in dimethyl sulfoxide. Microglial cultures were treated with drugs for 1 h before exposure to CN + mAb. Culture supernatants were tested for lactate dehydrogenase (LDH) efflux using a commercially available kit (Promega), according to the manufacturer’s instructions. Cells grown in triplicates in medium containing 5% FCS with or without drug treatment were compared for the LDH content (Student’s t-test). The baseline optical density values in fresh culture medium were subtracted from all values.
Adenovirus infection
The replication-deficient adenovirus vectors carrying dominant-negative Ras (Rase DN; Ser17 to Asn) or constitutively active MEK1 (MEK CA; Ser218 and Ser222 to Glu) were obtained from Dr. S. Tanaka. Adenovirus encoding the proteolysis-resistant (S32, 36E) super-repressor inhibitor of B (SR IB) or adenovirus carrying the cytomegalovirus (CMV) promoter (Ad-CMV: control virus) were obtained from Dr. Richard Pestell (Albert Einstein College of Medicine). Microglial cells were plated at 40,000 cells per well in 96-well tissue-culture plates and were fed with medium (DMEM+5% FCS) containing the recombinant adenovirus at a concentration of 10–100 x 103 plaque-forming units (pfu) per cell for Ad-IB or Ad-CMV. For Ad-MEK CA or Ad-Ras DN, infection was performed according to the method described previously [19
]. Briefly, microglia were incubated with the recombinant adenoviruses at 10–1000 pfu per cell for 1 h. Then, cells were washed twice and further incubated. All infection proceeded for 24 h, and then medium was replaced with fresh, complete medium containing CN + 18B7. Chemokine levels in the culture supernatants were determined by ELISA 16 h later.
Statistics
Means of chemokine levels in different groups were compared using ANOVA. Relevant groups were then compared by the Student’s t-test. P values of <0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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production and phagocytosis in microglia as well as phagocytosis in the presence of specific antibody (mAb: 3E5 or 18B7) [16
]. Subsequent studies using FcR-deficient murine microglia determined that FcR signaling plays an essential role in these activities [17
]. It is believed that Src family tyrosine kinases and Syk tyrosine kinase are the earliest detectable kinases to be activated following FcR cross-linking. Thus, we used PP2, a Src inhibitor, and PIC, a Syk inhibitor, to determine the role of Src/Syk in microglial FcR signaling. As shown in Figure 1
, PP2 and PIC treatment inhibited MIP-1 and phagocytosis. Both inhibitors produced consistent inhibition at 10 µM and 100 µM, respectively, and lower concentrations showed no or variable effects (Fig. 1)
. The inhibitions were not a result of microglial cell toxicity, as shown by the viable microglial cells during phagocytosis assay (Fig. 1B
and 1D)
as well as by LDH efflux assays (data not shown). Neither PP2 nor PIC alone changed the basal level MIP-1 production (P>0.05 by Student’s t-test). These results indicate that the Src and Syk kinases are involved in the early FcR signaling, leading to chemokine production and phagocytosis.
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R cross-linkingR-activated THP-1 cells found this not to be the case. Thus, we asked if Ras were upstream of ERK in microglial FcR signaling. We tested this by adenoviral transduction of primary human microglia with Ras DN or MEK CA. As shown in Figure 2A
, transduction with MEK CA alone induced MIP-1 production, and in microglia treated with IC, transduction with Ras DN completely inhibited MIP-1 production. Control vector (Ad-CMV) had little activity by itself but synergized with IC to produce more MIP-1, consistent with the known, immunogenic effects of adenoviral vectors [20
].
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production in microglia and that Ras is upstream of ERK MAPK in FcR-mediated MIP-1 induction in microglia.
Role of the Ras/MEK/ERK pathway in FcR-mediated CN phagocytosis
Whether FcR-activated Ras/ERK pathway is also involved in phagocytosis is unknown. We determined this by use of Ras DN and the specific pharmacological inhibitor of MEK/ERK, U0126. We show that in contrast to chemokine production, IC-mediated phagocytosis was not affected by Ras DN (Fig. 2C)
or by U0126 (Fig. 2E)
at the same concentrations that were effective in inhibiting chemokine production (Fig. 2D)
. U0126 alone did not change the basal level of MIP-1 production (P>0.05 by Student’s t-test). These results show that although the Ras/MEK/ERK pathway is critical to FcR-mediated inflammatory gene expression in microglia, it is not involved in phagocytosis.
Role of PI-3K–Akt pathway in MIP-1 expression and phagocytosis
The PI-3K pathway has been shown to be crucial for pseudopod extension, an essential step in phagocytosis. Whether it is also involved in phagocytic receptor-mediated macrophage gene expression is unknown. We tested PI-3K inhibitors for their effects in IC-treated microglia. LY294002 and Wortmannin inhibited phagocytosis with IC50 at 
30 µM and 20 nM, respectively (Fig. 3B
and 3D
, and data not shown). However, neither agent inhibited MIP-1 production, basal or IC-induced (Fig. 3A
and 3C) . These results suggest that in microglia, FcR cross-linking activates the PI-3K pathway and that PI-3K is required for particle ingestion. To determine PI-3K activation more directly, immunoblot analysis was performed in IC-challenged microglia for pAkt, a downstream kinase activated by PI-3K. Figure 3E
shows phosphorylation of Akt in microglial cultures challenged with IC, demonstrating PI-3K pathway activation by FcR engagement. Akt phosphorylation was apparent at 10 min and was sustained through 150 min after IC challenge. It is interesting that cytochalasin D, an agent that inhibits IC phagocytosis without affecting MIP-1 production [16
] had no effect on pAkt expression. This demonstrates that actin assembly is not required for PI-3K activation.
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R transduction, we further analyzed their relationship by Western blot analysis. We first determined whether the PI-3K pathway was involved in ERK activation in IC-challenged microglia by use of LY294002 or Wortmannin. As shown, LY294002 or Wortmannin did not inhibit IC-induced ERK phosphorylation at concentrations that effectively inhibited Akt phosphorylation (Fig. 4A
and 4B
). U0126 treatment abolished pERK without affecting pAkt, and PP2 and PIC inhibited ERK and Akt phosphorylation (Fig. 4A
and 4B)
. Furthermore, microglia transduced with Ras DN did not inhibit Akt phosphorylation, and it completely inhibited ERK phosphorylation (Fig. 4C)
. The relationship between PI-3K and ERK was again examined by immunostaining for pERK, which showed failure of Wortmannin to inhibit IC-induced pERK expression (data not shown). These results confirm the relationship among various signaling pathways (Src, Syk, Ras, ERK, and PI-3K) in IC-challenged microglia as proposed in Figures 1
2
3
and demonstrate that PI-3K and Ras/MEK/ERK pathways do not intersect in these cells.
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B by IC and role in microglial activation and phagocytosisB has been implicated in the induction of human MIP-1 gene expression [21
, 22
]. We have previously shown that the NF-B inhibitor pyrrolidinedithiocarbamate (PDTC) moderately inhibits IC-induced MIP-1 expression in microglia [17
]. To what extent NF-B contributes to FcR-mediated macrophage gene expression and whether it is involved in phagocytosis are unclear. In this report, we used a proteolysis-resistant SR IB to induce specific inhibition of NF-B activation. As shown in Figure 5
, adenoviral-mediated transduction of SR IB induced inhibition of chemokine expression, consistent with our previous data with PDTC. However, SR IB differentially affected MIP-1 and RANTES. Whereas MIP-1 was only modestly inhibited at both concentrations of the virus (10,000 and 100,000 pfu), near-complete inhibition was achieved in the expression of RANTES (Fig. 5B)
. Control adenovirus produced moderate levels of chemokines, which agrees with previous reports [20
].
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B had no effect on phagocytosis used at the same concentrations (Fig. 5C) , demonstrating that NF-B is not required for FcR-mediated phagocytosis.
To determine the relationship between NF-B activation and Src, Syk, and ERK pathways, we used p65 immunocytochemistry. At 30 min after challenge with IC, most cells displayed nuclear translocation of p65 (Fig. 5D)
. Stimulation with LPS for 30 min was used as a positive control. The Src inhibitor PP2 inhibited NF-B nuclear translocation, and the Syk inhibitor or the MEK inhibitor failed to affect p65 nuclear translocation (Fig. 5E)
, suggesting that Src family kinase but not Syk or ERK kinases is upstream of NF-B. Together, these results demonstrate that NF-B is activated in microglia by IC challenge via a mechanism that involves Src kinases. NF-B contributes to the chemokine expression but not phagocytosis.
Summary of our results and hypothesis
Our hypothesis linking FcR activation and the following signaling events leading to phagocytosis and microglial chemokine induction is presented in Figure 6
. We identified that following IC-mediated FcR activation, Ras/MEK/ERK and PI-3K pathways are activated, which contributes to MIP-1 expression and phagocytosis, respectively. It is interesting that no cross-talk between the two pathways could be demonstrated. NF-B activation was not necessary for phagocytosis and had a variable role in chemokine induction depending on the types of chemokines examined.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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production, which suggested their involvement early in FcR signaling. This was further supported by the inhibition of pAkt and pERK by PP2 and PIC. Although the inhibition was very dramatic, PP2 still failed to abrogate phagocytosis completely, consistent with the notion that Src kinases are not absolutely required for phagocytosis [5
]. Although the Src and Syk kinases are known to play a role in phagocytosis and the generation of reactive oxygen species [23
], their involvement in macrophage gene expression has not been demonstrated until now.
We also show for the first time that PI-3K and ERK have differential roles in FcR signaling. We have confirmed our previous results that ERK MAPKs were required for chemokine induction. Furthermore, using adenovirus-mediated gene transfer, we show that Ras is upstream of ERK in FcR signaling and that activation of MEK/ERK is sufficient for MIP-1 induction in microglia. These results are intriguing, as MAPKs have been shown to activate NF-B by activating upstream kinases in certain systems [24
]. In THP-1 cells stimulated with sheep red blood cell (RBC) IC, NF-B is activated in a manner dependent on ERK and PI-3K [6
]. These results suggest that MAPKs and NF-B can be induced in an interdependent manner, resulting in proinflammatory gene expression in macrophages.
However, several lines of evidence suggest that this is an unlikely scenario in microglia. First, NF-B activation occurred independent of ERK, as the MEK inhibitor U0126 did not inhibit NF-B nuclear translocation (Fig. 5)
. Second, transduction of microglia with DN NF-B moderately reduced MIP-1, and it abolished RANTES, suggesting that NF-B was not absolutely required for the induction of MIP-1 [21
, 22
, 25
]. Third, the amounts of RANTES induced by IC are small compared with MIP-1 and MIP-1ß [16
]. Fourth, IL-1 and TNF-, cytokines whose expression is NF-B-dependent, are not inducible in microglia by IC (D. L. Goldman and S. C. Lee, unpublished). From these data, we conclude that ERK is the dominant pathway induced by FcR engagement in microglia, whereas NF-B activation is variable. The molecular basis for the low-to-variable NF-B activation is unclear but suggests the presence of negative regulatory signals that may target NF-B. For instance, FcR-mediated NF-B activation has been shown to be inhibited by the SH2 domain-containing inositol polyphosphate phosphatase [26
, 27
]. Further studies will determine the precise molecular mechanisms underlying the positive and negative FcR signaling in macrophages and microglia.
In our study, although ERK was sufficient for the induction of MIP-1, not all stimuli that induced ERK led to chemokine induction. For instance, we showed previously that soluble IC (CN capsular polysaccharide) induced ERK phosphorylation but failed to induce MIP-1 [16
]. Furthermore, although CN IC induced phosphorylation of ERK in a subpopulation of microglia, adenoviral transduction of MEK induced pERK in virtually all cells (Fig. 2B) . These results suggest the presence of threshold amounts of kinases necessary for induction of MIP-1. Soluble IC may be unable to induce sufficient amounts of ERK and/or fails to produce other necessary signals. Transduction of all microglial cells with MEK CA probably represents a super-physiological condition, which may or may not occur in vivo.
Our results with microglia are at variance with those obtained with another macrophage-lineage cell, THP-1. In THP-1 cells, ERK activation occurred independent of Ras, and PI-3K and ERK were required for phagocytosis upon differentiation [12
]. Furthermore, PI-3K was required for gene activation after FcR engagement [6
]. These results suggest that FcR signaling requirements differ among various macrophage populations. The differences may originate from the fact that microglia are terminally differentiated tissue macrophages adapted to the CNS environment. The differences may also lie in the phagocytic stimuli applied, as phagocytosis of CN by microglia dramatically increased in the presence of specific antibody, and phagocytosis of sheep RBC in THP-1 cells increased only marginally [6
]. Furthermore, as IC can also transmit signals through the complement receptors such as CD18 [28
], complement components and receptors may also play a role in the divergent findings. Further studies are needed to determine the physiological relevance of the observed FcR signaling among various macrophage populations.
In microglia, we demonstrate that the PI-3K pathway was important in FcR-mediated phagocytosis and was not involved in MIP-1 expression. PI-3Ks are a family of enzymes that phosphorylate the membrane phosphoinositides, which serve as membrane-bound, second messengers that control cellular responses to stimuli leading to cell growth, survival, and cellular movement [29
]. Based on the sequence homology and substrate preference, three classes of PI-3Ks have been recognized. Of these, class I PI-3Ks are heterodimeric enzymes composed of a p110 catalytic subunit and a p85 regulatory subunit, which appear to be essential for the pseudopod extension and/or fusion around the ingested particles [30
, 31
]. Akt, also termed protein kinase B, is a serine/threonine kinase that binds to phosphorylated lipids at the membrane in response to the activation of PI-3K and is the best-characterized, downstream effector of PI-3K [32
]. PI-3K and the MAPK are activated by many of the same ligands. Several groups have reported involvement of PI-3K in the activation of ERK, whereas others found that activation of ERK is not sensitive to Wortmannin [33
].
The specific role of PI-3K during FcR-mediated phagocytosis has been well delineated. PI-3K does not appear to be involved in actin assembly but rather involved in the membrane trafficking required for pseudopod extension. PI-3K involvement depends on the particle size, and ingestion of a large particle and not a small particle is inhibitable by PI-3K inhibitors [9
]. These results suggest that PI-3K contributes to membrane remodeling during phagocytosis. In microglia, we also have evidence that PI-3K activation occurs independent of actin assembly, as cytochalasin D failed to inhibit PI-3K activation (Fig. 3)
, and internalization of soluble (small) IC failed to activate PI-3K (data not shown).
In summary, we have identified the signaling transduction following FcR cross-linking in microglia cells, and Ras is critical in the activation of MEK/ERK, leading to chemokine expression, and the PI-3K pathway, leading to phagocytosis. As the mAb 18B7 is now in a phase-II clinical trial, unraveling the mechanisms that enhance host defense against invading CN could provide clinically relevant information useful in the refinement of therapy. These studies also have implications for autoimmune-inflammatory CNS diseases such as multiple sclerosis, as well as conditions such as Alzheimer’s disease for which therapeutic vaccines are being tested.
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ACKNOWLEDGEMENTS |
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B. This study was supported by AI44641 and MH55477 to S. C. L. Received April 1, 2003; revised December 15, 2003; accepted January 29, 2004.
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