Published online before print August 4, 2005
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production by primary human macrophages is mediated by phosphatidylinositol-3 (PI-3) kinase and mitogen-activated protein (MAP) kinase pathways
* Departments of Medicine, University of Pennsylvania School of Medicine, and
Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia
1Correspondence: University of Pennsylvania School of Medicine, 522 Johnson Pavilion, 36th & Hamilton Walk, Philadelphia, PA 19104. E-mail: collmanr{at}mail.med.upenn.edu
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(TNF-), but the mechanisms have not been determined. We sought to define the pathways responsible for TNF- secretion by monocyte-derived macrophages (MDM) following HIV-1 gp120 stimulation. MDM exposure to recombinant macrophage-tropic (R5) gp120 led to dose- and donor-dependent release of TNF-, which was cyclohexamide-sensitive and associated with up-regulated message. Pretreatment with specific inhibitors of the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase 1/2 (ERK-1/2; PD98059, U0126) and p38 (SB202190, PD169316) inhibited the secretion of TNF-. gp120-elicited TNF- production was also blocked by phosphatidylinositol-3 kinase (PI-3K) inhibitors (wortmannin, LY294002). Moreover, PI-3K inhibition ablated gp120-induced phosphorylation of p38 and ERK-1/2. The response was inhibited by a CC chemokine receptor 5 (CCR5)-specific antagonist, indicating that CCR5 was in large part responsible. These results indicate that gp120-elicited TNF- production by macrophages involves chemokine receptor-mediated PI-3K and MAPK activation, that PI-3K is an upstream regulator of MAPK in this pathway, and that p38 and ERK-1/2 independently regulate TNF- production. These gp120-triggered signaling pathways may be responsible for inappropriate production of proinflammatory cytokines by macrophages, which are believed to play a role in immunopathogenesis and in neurological sequelae of AIDS.
Key Words: monocyte • AIDS • signal transduction • HIV-associated dementia
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-associated protein-70 (ZAP-70), and mitogen-activated protein kinases [MAPK; extracellular signal-regulated kinase (ERK), Jun N-terminal kinase, and p38], can be activated by Env interactions with chemokine receptors in several cell types [8
9
10
11
], and functional consequences include calcium mobilization, chemotaxis, or the induction of cytokine and chemokine secretion [6
, 12
13
14
15
].
Several cytokines are reported to be produced by human macrophages in vitro upon infection with HIV-1 or exposure to virions or Env glycoprotein [15
16
17
18
19
20
]. Macrophage activation and proinflammatory cytokine production play a particularly important role in vivo in HIV-associated dementia (HAD). Tumor necrosis factor (TNF-) is among the most consistently observed and markedly elevated of these cytokines in HAD and is produced by uninfected brain macrophages and microglia (M/M) as well as infected cells [21
22
23
24
25
26
27
28
]. TNF- is also functionally important, as it is believed to contribute to neuronal injury through direct and indirect mechanisms [29
30
31
32
]. In addition to its role in neuropathogenesis, elevated TNF- may contribute to immunopathogenesis and can up-regulate viral replication [33
, 34
]. However, the mechanism by which HIV-1 Env elicits TNF- secretion by macrophages has not been defined.
We previously reported that Env glycoprotein triggers ion channels, intracellular calcium mobilization, calcium-dependent Pyk2 phosphorylation, and MAPK activation and induces the secretion of macrophage-inflammatory protein-1ß (MIP-1ß) and monocyte chemoattractant protein-1 in primary human macrophages [14
, 15
]. As macrophage production of TNF- appears to play such an important role in HIV-1 pathogenesis, our aim in this study was to define the intracellular signaling pathways activated by HIV-1 gp120, which result in TNF- secretion by primary macrophages in the absence of infection. Our findings indicate that R5 gp120 signals through phosphatidylinositol-3 kinase (PI-3K), which results in the phosphorylation of the MAPK p38 and ERK-1/2, and that this pathway leads to up-regulated TNF- message and protein synthesis.
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32 deletion allele by polymerase chain reaction (PCR) [36
], and only donors homozygous for the wild-type allele were used. Unless otherwise indicated, all data shown are representative of results carried out in at least four experiments using different donors.
Reagents
Recombinant JRFL gp120 (kindly provided by Bridget Puffer and Robert W. Doms, University of Pennsylvania, Philadelphia) was produced in 293T cells infected with gp120-expressing recombinant vaccinia virus as described previously [37
]. Supernatant was clarified by centrifugation and filtered (0.45 µm pore size). Virus was inactivated (0.1% Triton X-100), and gp120 was purified using Galanthus nivalis lectin-coupled agarose beads (Vector Labs, Burlingame, CA) followed by protein concentration and buffer exchange. Env integrity was confirmed by Western blot with a rabbit polyclonal antibody as described [37
]. To confirm that responses could not be a result of potential endotoxin contamination, in selected experiments, polymyxin B (10 µg/ml) was added to gp120 and control wells, and heat-inactivated gp120 protein (95°C for 30 min, which would inactivate protein but not endotoxin) without polymyxin B was included as a control. Wortmannin and LY294002 (PI-3K inhibitors), PD98059 and U0126 (ERK-1/2 inhibitors), and SB202190 and PD169316 (p38 MAPK inhibitors) were purchased from Calbiochem (San Diego, CA). The CCR5 blocker M657 [38
, 39
] was kindly provided by Michael Miller (Merck and Co., Whitehorse Station, NJ).
Determination of TNF- production
Monocytes were plated at 5 x 104 cells/well in 96-well tissue-culture plates and maintained for 1 week prior to stimulation. Triplicate wells of MDM were exposed to gp120 for 4 h, and TNF- levels in culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA; Quantikine, R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. For blocking studies, kinase inhibitors were added 1 h before and maintained during the period of stimulation.
Reverse transcriptase (RT)-PCR analysis of TNF- mRNA
Cells were plated at 2 x 105 cells/well in 24-well plates, allowed to differentiate into MDM, and exposed to gp120, and total RNA was extracted 2 h later using Trizol reagent (Gibco Life Technologies, Gaithersburg, MD). cDNA synthesis was performed with 0.1 µg total RNA using oligo-dT priming and the ThermoScript RT-PCR system (Invitrogen, Carlsbad, CA) according to the manufacturer’s specifications. PCR amplification was done in a 25-ul reaction mixture containing 5 µl cDNA, Tris-HCl (pH 8.5), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 200 uM each deoxy-unspecified nucleoside 5'-triphosphate, 10 pmol each primer, and 2.5 units EXPAND high-fidelity DNA polymerase II (Roche Diagnostic Corp., Indianapolis, IN). After denaturation for 3 min at 95°C, they were subjected to 35 cycles of 95°C for 30 s, 57°C for 45 s, and 72°C for 30 s, followed by a final extension step of 72°C for 10 min. The primers for PCR were as follows: TNF-, 5'-CAG AGG GAA GAG TTC CCC AG (forward), 5'-CCT TGG TCT GGT AGG AGA CG (reverse); actin, 5'-ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG (forward), 5'-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC (reverse). PCR products were analyzed on 1.5% agarose gels containing ethidium bromide and visualized with ultraviolet light.
Western blot analysis
Week-old cultures of MDM (5–10x105 cells/well in 24-well plates) were exposed to gp120 for the indicated time periods, washed once using ice-cold phosphate-buffered saline, and then used for Western blot analysis as described [15
]. Cells were lysed using 100 µl lysis buffer containing 50 mM Tris-HCl, pH 7.4, 1% Nonidet-40, 0.5% sodium deoxycholate, 150 mM NaCl, and 0.1% sodium dodecyl sulfate (SDS), supplemented with protease and phosphatase inhibitors (5 mM phenylmethylsulfonyl fluoride, 5 µg/ml pepstatin, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM sodium vanadate, 1 mM sodium fluoride, 1 mM sodium pyrophosphate). Cell lysates were clarified (1000 g at 4°C for 10 min), subjected to 10% SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to nitrocellulose membranes. Immunoblotting was done using rabbit polyclonal antibody specific for phospho-ERK (p44/42; Thr 202/Tyr 204), phospho-p38 MAPK (Thr 180/Tyr 182), or phospho-Akt (Ser 473/Thr 308; Cell Signal Technology, Beverly, MA) and then visualized using a chemiluminescent substrate (Pierce, Rockford, IL). The same membranes were then stripped and reprobed with rabbit polyclonal antibodies, which detect total ERK (p44/42), total p38 MAPK, or total Akt protein (Cell Signal Technology). In the indicated experiments, MDM were pretreated with kinase inhibitors for 1 h prior to and during stimulation.
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secretion in primary human macrophages [17
18
19
20
], which may have important implications for several aspects of pathogenesis. Toward this end, we addressed the ability of recombinant gp120 from an R5 HIV-1 isolate to induce TNF- production by human macrophages. MDM were induced to a quiescent state, stimulated with gp120 from the R5 prototype strain JRFL, and cell supernatants were assayed for TNF- by ELISA.
In the absence of stimulation, most donors’ macrophages produced low concentrations of TNF- (<5 pg/ml), although some produced levels up to 30 pg/ml (Fig. 1
). Consistent with previous reports, gp120 elicited TNF- secretion in these MDM cultures. Env stimulation of TNF- production was dose-dependent, whereas heat-inactivated gp120 failed to induce TNF- (Fig. 1A)
. We then compared the secretory response among cells from different donors. The TNF- response to gp120 was consistent, although donors showed a range in the absolute levels elicited (Fig. 1B) , and peak levels ranged from 30 to 3000 pg/ml. Thus, MDM produce TNF- in response to gp120 stimulation in a manner that is dose-dependent as well as donor-dependent.
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Figure 1. R5 gp120 activates primary MDM to produce TNF-. Monocytes from healthy donors were allowed to differentiate into MDM for 7 days and were then stimulated with gp120. (A) Cells were treated with R5 gp120 (strain JRFL) for 4 h, and culture supernatants were assayed for TNF- by ELISA. Heat-inactivated JRFL gp120 ( HI) was used as a negative control. (B) Cells from six different normal donors were treated with 20 nM JRFL gp120 for 4 h, and supernatant TNF- was measured by ELISA. Data represent means ± SD of triplicate wells.
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production is regulated at the level of mRNA and de novo protein synthesis could reflect up-regulated protein production or release from cellular or membrane-bound stores. Therefore, we assessed TNF--specific mRNA by RT-PCR and determined whether the secretory response was sensitive to the protein synthesis inhibitor cyclohexamide (CHX). As shown in Figure 2A
, unstimulated MDM had minimal or no detectable TNF- mRNA, and stimulation with 20 nM gp120 for 2 h resulted in clearly up-regulated message levels. Similar results were observed following stimulation with LPS. In contrast, heat-inactivated gp120 had no effect on TNF- mRNA expression. We then tested if CHX would block gp120-mediated TNF- release. As shown in Figure 2B
, preincubation of MDM with 10 µM CHX completely blocked TNF- release. These results demonstrate that JRFL gp120 stimulation of MDM increases the secretion of TNF- via up-regulation of mRNA expression and de novo protein synthesis, rather than release of preformed cytokine from intracellular stores or cleavage of membrane-bound TNF-.
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Figure 2. RNA up-regulation and protein synthesis dependence of gp120-induced TNF- secretion. (A) MDM were stimulated with gp120 (20 nM) or LPS (1 µg/ml) for 2 h, at which point total RNA was extracted and subjected to cDNA synthesis using oligo-dT priming followed by PCR amplification using primers specific for TNF- or ß-actin. LPS, Lipopolysaccharide. (B) MDM were pretreated for 1 h with or without CHX (10 uM) and then exposed to JRFL gp120 (20 nM) for 4 h, following which, supernatants were assayed for TNF- by ELISA. Data represent means ± SD for triplicate wells.
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production is MAPK-dependent secretion, we focused first on the MAPK signaling pathways, as MAPK are linked to TNF- regulation in several models [40
], and we previously found MAPK activation in response to gp120 [15
]. Pretreatment of cells with inhibitors of p38 MAPK (SB202190 and PD169316) abolished the TNF- response to gp120 (Fig. 3A
). Similar inhibition was observed when inhibitors of the ERK-1/2 MAPK pathway (PD98059 and U0126) were used (Fig. 3A)
. For both MAPK, the reduction in TNF- secretion was dose-dependent.
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Figure 3. R5 gp120-induced TNF- production is ERK-1/2- and p38 MAPK-dependent. MDM were incubated with or without protein kinase inhibitors for 1 h prior to and during exposure to 20 nM JRFL gp120 for 4 h, following which supernatants were assayed for TNF- by ELISA. Data represent means ± SD for triplicate wells. (A) Two different inhibitors of p38 (SB202190, PD169316) and ERK-1/2 (PD98059, U0126) MAPK were tested. (B) Cells were incubated with the p38 inhibitor SB202190, the ERK-1/2 inhibitor PD98059, or both inhibitors together.
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response to gp120, we then addressed whether blocking both pathways together would be additive. To test this, MDM were pretreated with the p38 inhibitor SB209021 and ERK inhibitor PD98059 in combination for 1 h before gp120 stimulation, using concentrations that independently had modest effects (Fig. 3B)
. We found that concentrations of inhibitors that individually had limited effects (0.1 ug/ml SB209021 and PD98059) markedly suppressed TNF- production compared with either inhibitor alone (Fig. 3B)
. This result suggests that ERK and p38 MAPK pathways contribute independently to gp120-elicited TNF- production in MDM.
PI-3K is an upstream regulator of MAPK activation in response to gp120
PI-3K is an important modulator of extracellular signals in many cell types and is often involved in MAPK activation by growth factors and other stimuli. Therefore, we tested the role of PI-3K in the response under study here by treating MDM with two different PI-3K inhibitors, LY294002 and wortmannin, before JRFL gp120 stimulation. As shown in Figure 4
, LY294002 and wortmannin blocked gp120-induced TNF- production in a dose-dependent manner, indicating that PI-3K is also involved in TNF- secretion in response to gp120.
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Figure 4. Activation of PI-3K is responsible for TNF- production. MDM were incubated with or without the PI-3K inhibitors LY294002 or wortmannin at the indicated concentrations for 1 h prior to and during exposure to 20 nM JRFL gp120. Supernatants were collected after 4 h and assayed for TNF- by ELISA. Data represent means ± SD for triplicate wells.
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Figure 5. PI-3K is an upstream regulator of MAPK activation by gp120. Macrophages were stimulated for 5 min with 20 nM JRFL gp120. Cell lysates were separated by SDS-PAGE and then analyzed by Western blot for kinase activation using antibody-specific phosphorylated forms of the proteins. Membranes were then stripped and reprobed using antibodies that detect total forms of the proteins. (A) As an indicator of PI-3K activation, MDM were analyzed by Western blot for phosphorylation of Akt (P-Akt), a downstream target of PI-3K. (B) MDM were incubated in the presence or absence of the PI-3K inhibitor LY294002 at the indicated concentration for 1 h prior to and during gp120 stimulation, followed by Western blot analysis of ERK and p38 MAPK activation. Data shown are representative of two independent experiments using cells from different donors.
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secretion, PI-3K inhibitors also blocked gp120-induced MAPK phosphorylation. Taken together, these results suggest that gp120 stimulation of MDM results in the secretion of TNF- through pathways that are dependent on PI-3K and the MAPK ERK-1/2 and p38 and that PI-3K is an upstream regulator of the MAPK in this cascade.
Induction of macrophage TNF- by R5 gp120 involves CCR5
We next addressed whether chemokine receptor signaling was responsible for gp120-elicited TNF- production by blocking CCR5 with the specific inhibitor M657 [38
, 39
]. Pretreatment of MDM with M657 decreased the level of TNF- production in response to R5 (JRFL) gp120 (Fig. 6
). This effect was dose-dependent, and maximal inhibition was observed at 10 µM M657. In contrast, the CXCR4 blocker AMD3100 had no effect on JRFL-triggered production, nor did M657 block secretion triggered by LPS or by X4 gp120 (data not shown). Thus, R5 gp120 signaling, which leads to TNF- production, involves CCR5, consistent with our prior observation that R5 gp120 signals through the chemokine receptors in macrophages [14
, 15
]. It is interesting that TNF- secretion was not completely blocked, even at high concentrations of M657 (Fig. 6
and data not shown), raising the possibility that although CCR5 is involved in gp120 activation, other pathways may contribute to the TNF- response as well. We then asked if gp120 activation of CCR5 signaling was linked to Gi, a G protein subunit often associated with CCR5, using the Gi-specific inhibitor pertussis toxin. Pertassis toxin did not block TNF- production in response to gp120 (data not shown), indicating that gp120-triggered CCR5 activation and consequent TNF- secretion are linked to mediators other than Gi.
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Figure 6. gp120 induction of macrophage TNF- involves signaling through CCR5. Macrophages were incubated for 1 h in the presence or absence of the CCR5 antagonist M657, exposed for 4 h to JRFL (R5) gp120 (20 nM) in the continued presence of the inhibitor, and supernatants were assayed for TNF- production by ELISA. Data represent means ± SD for triplicate wells.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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production by primary human macrophages. Many previous studies have shown that HIV-1 virions or recombinant Env can trigger TNF- release by macrophages, and ample evidence supports a role for macrophage TNF- production in HIV-1 pathogenesis. However, the specific mechanism and intracellular signals responsible have not been well defined. Here, we show that macrophage stimulation with recombinant R5 Env results in up-regulated TNF- mRNA and new protein production. We further show that gp120 induces TNF- through a pathway that involves PI-3K and two members of the MAPK family, ERK-1/2 and p38. Finally, we demonstrate a role for the chemokine receptor CCR5 in this response. Taken together, our results indicate that HIV-1 gp120 elicits TNF- production in macrophages through a chemokine receptor—PI-3K—MAPK pathway, leading to up-regulation of mRNA levels and de novo protein synthesis. Recently, Francois and Klotman [42 ] reported that gp120 induced PI-3K activation in primary macrophages and that it was necessary for viral replication. Others have shown that PI-3K activation by HIV-1 Env, Nef, or Tat is necessary for optimal viral replication, MHC class I down-regulation, and regulation of apoptosis in infected cells [43 44 45 46 47 ]. In contrast, we sought to investigate the effects of gp120 apart from viral infection, as cells may be exposed to Env on noninfectious virions or shed from virus or infected cells, and evidence in vivo suggests that uninfected macrophages, as well as infected cells, are activated and produce proinflammatory cytokines. The pathways identified here, therefore, have implications for the mechanism by which HIV-1 can affect the function of uninfected cells and how soluble Env or gp120 on noninfectious virions may contribute to immune cell dysfunction.
We found that ERK-1/2 and p38 MAPK were required for TNF- production in response to gp120. These results are consistent with data in several cell models using other stimuli in which multiple MAPK may be necessary for TNF- induction [48
, 49
]. Cooperative regulation by multiple MAPK is a common mechanism of macrophage TNF- regulation, which depending on the stimulus, may involve cooperative control of transcription or distinct transcriptional and post-transcriptional effects on mRNA stability [40
, 50
51
52
]. Whether ERK-1/2 and p38 MAPK regulate macrophage TNF- production in response to gp120 through up-regulated transcription, post-transcriptionally, or both will require further investigation. Soluble TNF- release can also result from cleavage and release of the membrane-bound form of the protein [53
, 54
], but this does not appear to be involved in gp120-induced TNF-, as it was CHX-sensitive and associated with increased mRNA levels.
The CCR5 antagonist M657 markedly reduced the levels of secreted TNF- following exposure to JRFL R5 Env, indicating that CCR5 was largely responsible for this response. This result is consistent with our previous findings that several intracellular signals elicited by gp120 are chemokine receptor-mediated [14
, 15
]. However, the secretory response was not completely abrogated by CCR5 blocking, raising the possibility that another receptor might be involved as well. As chemokine receptor antagonists would not block the ability of gp120 to engage CD4, studies are under way to determine if stimulation of CD4 may contribute as well. Direct engagement of CD4 alone using specific antibody or of CCR5 alone using MIP-1ß leads to only modest TNF- release (data not shown), and so, it remains to be determined whether CD4 is involved directly or might augment the response to chemokine receptor stimulation. Synergistic cross-talk between CD4 and CCR5 was described recently in T cells, where CD4 markedly enhanced the response to CCR5 stimulation by MIP-1ß [55
]. However, macrophages lack p56lck, the principal CD4-associated intracellular signaling molecule in T cells, and whether and how CD4 signals in macrophages are not known [56
]. Of note, X4 gp120 also triggers TNF- secretion by macrophages (refs. [18
19
20
] and data not shown), but whether the pathways linked to X4 gp120 and CXCR4 activation are the same as those detailed here for R5 gp120-triggered CCR5 stimulation remains to be determined.
Depletion of CD4+ T cells during later stages of disease is the major pathogenic consequence of HIV-1 infection. However, inappropriate immune activation and cytokine secretion and immunological dysfunction independent of direct infection are believed to be important as well, and so dysregulated TNF- production as a result of gp120-macrophage interactions may contribute to immunopathogenesis. A recent study of lymphoid tissue histocultures exposed to HIV-1 did not observe TNF- up-regulation [57
], which implies that these responses might not operate in lymphoid tissue sites of virus replication, although that study used low-level virus exposure and bulk tissue analysis and thus, might not detect important differences within specific cell subsets or that occur in response to higher local concentrations. In HAD, inappropriate activation of uninfected as well as infected brain M/M leads to release of soluble products that injure neurons [58
]. TNF- is prominent among these products and functions as a direct neurotoxin and indirectly, by inhibiting astrocyte uptake of the excitotoxic neurotransmitter glutamate, thereby potentiating glutamate neurotoxicity [29
30
31
32
, 59
]. Despite numerous reports that virion or gp120 exposure can trigger macrophages to release TNF- [18
19
20
], how HIV-1 up-regulates TNF- production by macrophages in the absence of infection has been obscure. Our findings here begin to define the molecular pathways and receptors responsible for this process. As donors varied considerably in the levels of TNF- produced following gp120 stimulation, further studies will be warranted to test whether this results from host genetic polymorphisms or other determinants underlying differences in levels of CCR5 expression [60
, 61
], genetic polymorphisms that affect TNF- responsiveness [62
], or other factors. In addition, it will be important to address whether the degree of macrophage responsiveness to gp120 is a host determinant that regulates why only a subset of infected individuals develops HAD.
Received January 31, 2005; revised May 4, 2005; accepted May 5, 2005.
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