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CHEMOKINES
From the Department of Medicine, University of
Pennsylvania School of Medicine, and Department of Pathobiology,
University of Pennsylvania School of Veterinary Medicine, Philadelphia,
PA; Rega Institute for Medical Research, Katholieke Universiteit
Leuven, Leuven, Belgium; and Laboratory of Virology, Istituto Superiore
di Sanita', Rome, Italy.
Human immunodeficiency virus type 1 (HIV-1) uses the chemokine
receptors CCR5 and CXCR4 as coreceptors for entry. It was recently demonstrated that HIV-1 glycoprotein 120 (gp120) elevated calcium and
activated several ionic signaling responses in primary human macrophages, which are important targets for HIV-1 in vivo. This study
shows that chemokine receptor engagement by both CCR5-dependent (R5)
and CXCR4-dependent (X4) gp120 led to rapid phosphorylation of the
focal adhesion-related tyrosine kinase Pyk2 in macrophages. Pyk2
phosphorylation was also induced by macrophage inflammatory protein-1 Human immunodeficiency virus type 1 (HIV-1)
infection is initiated by the formation of a trimolecular complex on
the cell surface, consisting of the HIV-1 envelope (Env) glycoprotein
120 (gp120), the principal cellular receptor CD4, and a chemokine receptor that functions as a coreceptor (reviewed by
Berger1). Conformational changes induced in gp120 on
binding to CD4 increase its affinity for the coreceptor, leading to
fusion between the viral and cellular membranes. The chemokine
receptors that serve as HIV coreceptors are members of the 7 transmembrane G protein-coupled receptor (GPCR) family. CCR5 and CXCR4
play a dominant role in HIV-1 entry into primary cells, and are the
principal coreceptors used by macrophage (M)-tropic (R5) and T-cell
line (T)-tropic (X4) isolates, respectively.
Given their normal cell signaling function, the interaction of gp120
with the chemokine receptors may, in addition to facilitating viral
entry, also result in activation of cellular responses that could
modulate the activation status of the cells and affect postentry stages
of HIV replication (reviewed by Popik and Pitha2).
Although chemokine receptors and their ligands play central roles in
both HIV infection and in immune regulation, how signaling pathways mediated by CCR5 and CXCR4 contribute to the immunopathogenesis of HIV
infection is not well defined. In T cells, gp120 activation of the
chemokine receptors has been shown to elevate calcium3 and
activate the focal adhesion tyrosine kinases Pyk2 and
FAK4-6 as well as the mitogen-activated protein kinase
(MAPK) pathway.7,8
Macrophages are important targets for HIV-1 in vivo. Infected
macrophages may serve as a reservoir for viral persistence and participate in person-to-person transmission and are central to the
pathogenesis of brain, lung, and other end-organ disease in acquired
immunodeficiency syndrome (AIDS).9-12 In addition,
aberrant functions of both infected and uninfected macrophages have
been implicated in pathogenesis.13-15 Macrophages express
CCR5, which is used for entry by primary and prototype M-tropic R5
HIV-1 variants. Macrophages also express CXCR4, which mediates entry by
some X4 and R5X4 primary isolates, although laboratory-adapted X4
strains are restricted in their ability to use macrophage CXCR4 for
reasons that are not yet clear.16-18 However, relatively
little is known about how the chemokine receptors are coupled to
cellular signaling pathways in primary macrophages. Because the
cellular signaling pathways activated through 7-transmembrane GPCRs may
differ depending on the cell type,19 we have examined
whether gp120 engagement of the chemokine receptors leads to activation
signals in primary human macrophages. Recently we reported that Env
activates several ion channels and elevates calcium in macrophages
through CCR5 and CXCR4.20 In this study we examined the
ability of chemokine receptor stimulation by gp120 to activate protein
kinase pathways in primary macrophages, because protein phosphorylation
is an important molecular mechanism by which extracellular signals
produce biologic responses in cells. In addition, to address potential functional consequences of Env signaling in macrophages, we examined Primary human macrophages, Env glycoproteins, and
chemokines
Recombinant gp120 was produced as previously described23
in 293T cells infected with gp120-expressing recombinant vaccinia viruses vCB28 (JRFL) or vSC60 (IIIB, BH8 clone) and was a generous gift
of R. Doms (University of Pennsylvania). Supernatants were 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.23 Chemokines signaling was
investigated with macrophage inflammatory protein-1 Cell treatment
To test the effect of specific inhibitors, cells were treated prior to and during gp120 exposure with the CXCR4 antagonist AMD3100 (1 µg/mL); Bordetella pertussis toxin (PTX; 100 ng/mL; obtained from both Sigma, St Louis, MO, and Calbiochem, San Diego, CA); EGTA (2 mM; Sigma); and the calcium release-activated Ca++ (CRAC) channel blocker lanthanum chloride (100 nM; Sigma). Inhibitors were added to MDMs 20 to 60 minutes before gp120 exposure, except for PTX, which was added up to 18 hours before exposure. To ensure that inhibitory effects were not due to nonspecific toxicity, signaling antagonists were tested for cytotoxicity on human macrophages by vital staining using a methyl tetrazolium (MTT)-based assay (CellTiter AQ; Promega, Madison, WI). MDMs were plated at 5 × 104 cells/well in 96-well tissue culture. After 1 week in culture, medium was replaced with low serum medium (10% FBS) that was lacking phenol red. Test agents were added and cells were incubated for 18 hours. Tetrazolium reagent was then added and incubated for an additional 1 hour, and the optical density (OD; 492 nm) was determined using a plate spectrophotometer. Within each experiment a standard curve was established on the same plate by seeding MDMs at varying concentrations from 1 × 104 through 8 × 104 cells/well. The standard curve demonstrated a linear relationship between cell number and OD, and cytotoxicity was determined based on comparison with the standard curve. Five replicate wells were used to derive each data point. Immunoprecipitation and immunoblot analysis For Pyk2 immunoprecipitation, cell lysates containing identical amounts of protein (400 µg) from cells subjected to each treatment were clarified by incubation for 1 hour at 4°C with protein A+G-Sepharose (Santa Cruz Biotechnology; Santa Cruz, CA). Protein A+G-Sepharose was removed by centrifugation, and the supernatants were then incubated with 1 µg of the anti-Pyk2 monoclonal antibody (mAb) Pyk2/CAK (Transduction Laboratories; Lexington, KY) for 2 hours at
4°C. Antibody-antigen complexes were then immunoprecipitated by
incubation for 2 hours at 4°C with 50 µL protein A+G-Sepharose (50% suspension) and then washed 3 times with lysis buffer and once
with phosphate-buffered saline (PBS). Immunocomplexes were resuspended
in Laemmli buffer, boiled for 5 minutes, and subjected to
electrophoresis and immunoblotting.
Immunoblot analysis was carried out on immunoprecipitates generated as
described above or directly on cell lysates containing 40 µg protein
that were mixed with an equal volume of 2 × Laemmli buffer and boiled
for 5 minutes. Samples were subjected to SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes.
The membranes were blocked overnight with 5% nonfat milk protein in
PBS. They were then incubated sequentially with the primary specific
antibody for 3 hours at room temperature or overnight at 4°C, washed,
and then incubated with peroxidase-conjugated secondary antibody. The
immunoreactive bands were visualized using enhanced chemiluminescence
Western blotting system according to the manufacturer's instructions
(Amersham, Piscataway, NJ). Blots were then stripped (2% SDS, 62.5 mM
Tris, 100 mM Immunoblot detection was carried out using a 1:1000 dilution of
antiphosphotyrosine mAb 4G10 (Upstate Biotechnology, Lake Placid, NY)
in the case of Pyk2 immunoprecipitates. Whole cell extracts were
analyzed using 0.5 µg/mL of a polyclonal antibody Anti-Pyk2
[pY402], which is specific for the Tyr402 phosporylated form
of Pyk2 (Biosource International, Camarillo, CA), or 0.25 µg/mL mAb
Pyk2/CAK Measurement of chemokine secretion One-week-old macrophages were treated with gp120 (1 µg/mL) for 24 hours, in the presence or absence the MAPK-specific inhibitor SB-202190 (1 µg/mL; Calbiochem). Supernatant was clarified by centrifugation (5000 rpm for 5 minutes at 4°C) and stored at 80°C until analysis. Monocyte chemotactic protein-1 (MCP-1) and
MIP-1 production in supernatant was measured by enzyme-linked
immunoadsorbent assay (ELISA) as described by the manufacturer (R & D
Systems; Minneapolis, MN). The detection limit for MCP-1 was 5 pg/mL
and for MIP-1 4 pg/mL.
JRFL gp120 activates Pyk2 kinase in primary human macrophages Pyk2 is a nonreceptor tyrosine kinase related to focal adhesion kinase (FAK) that is expressed mainly in cells of hematopoietic and neuronal lineage.24-26 Pyk2 can be activated by several growth factors, chemokines, and GPCR ligands, and on activation is phosphorylated at 4 tyrosine phosphorylation sites (Tyr402, 579, 580, 881), with Tyr402 as the major autophosphorylation site.27-31To determine whether HIV-1 Env activates Pyk2 in primary human
macrophages, we treated MDMs with recombinant gp120 from the R5 isolate
JRFL and analyzed the levels of total and Tyr402-phosphorylated forms
by combined immunoprecipitation and immunoblot using specific antibodies (Figure 1A). This analysis
showed that MDMs express an abundance of total Pyk2 protein that had a
molecular mobility of approximately 110 kd, in accord with prior
studies of Pyk2 expression in monocytes.32 Exposure of
macrophages to gp120 resulted in rapid Pyk2 tyrosine phosphorylation.
We next determined the time course of Pyk2 activation using immunoblot
analysis of whole cell lysates with antibodies specific for the total
or activated forms of Pyk2. As shown in Figure 1B, MDMs responded to
JRFL Env in a time-dependent manner. Pyk2 tyrosine phosphorylation
generally peaked within 5 minutes and decreased to nearly background
levels by about 30 minutes. Analysis of earlier time points
demonstrated activation as early as 1 minute after stimulation and
showed that maximal activation occurred approximately 5 minutes after
treatment (data not shown).
Although MDMs from most donors had little or no basal Pyk2 phosphorylation and demonstrated a robust response to gp120 stimulation, there was some donor-to-donor variability in the level of constitutive Pyk2 phosphorylation under unstimulated conditions and the magnitude of phosphorylation response to gp120. In general, MDMs from donors that showed high levels of basal Pyk2 phosphorylation had relatively little up-regulation elicited by gp120 (data not shown). Interestingly, the higher levels of constitutive Pyk2 activation were seen primarily among MDMs from cigarette smokers, the significance of which is presently unknown, but for that reason MDMs from nonsmoker donors were used for further analysis. X4 gp120 as well as chemokines
MIP-1
We then tested whether macrophage chemokine receptor stimulation by the
natural ligands would elicit Pyk2 activation responses similar to that
seen with Env, using MIP-1 To address the concentration dependence of Pyk2 activation, we
tested the effects of varying gp120 and chemokine concentrations (Figure 2C,D). For gp120, Pyk2 activation was seen with concentrations ranging from 0.25 to 2.5 µg/mL (2-20 nM), but not at 0.06 µg/mL (0.5 nM) or below (Figure 2C and data not shown). Pyk2 was activated by
MIP-1 Macrophage Pyk2 activation by R5 and X4 gp120 is mediated by CCR5 and CXCR4 We next investigated whether the chemokine receptors were responsible for gp120-induced Pyk2 activation in macrophages (Figure 3). To probe CXCR4 we used the bicyclam antagonist AMD3100, which blocks both the HIV-1 coreceptor function and SDF-1 signaling capability of CXCR4.34 The role of CCR5
was assessed using CCR5-deficient macrophages, obtained from donors
homozygous for the CCR5  32 allele. This frameshift mutation in the
32 allele abrogates CCR5 surface expression and eliminates both its
chemokine signaling and coreceptor function.22,35
Immunoblot analysis of macrophage lysates using antibodies specific for
the activated or total forms of Pyk2 showed that in wild-type MDMs, the
CXCR4 antagonist AMD3100 completely inhibited IIIB gp120-induced Pyk2
activation but had no effect on the responses to JRFL gp120 (Figure
3A). In contrast, macrophages lacking CCR5 exhibited reduced activation
following JRFL gp120 exposure but responded to IIIB Env (Figure 3B).
AMD3100 also blocked Pyk2 activation by SDF-1 Pyk2 activation is calcium-dependent but insensitive to PTX We next wished to address the mechanisms involved in gp120-elicited chemokine receptor-mediated Pyk2 activation in macrophages. In some cell systems Pyk2 phosphorylation is regulated by calcium,25,26,30,31,36 and we and others recently found that gp120 induces intracellular calcium elevations in MDMs.20,37 We therefore determined whether Pyk2 activation by JRFL gp120 is regulated by [Ca++]i elevations. As shown in Figure 4A, immunoblot analysis of macrophage cell lysates showed that chelation of extracellular calcium with EGTA blocked gp120-elicited Pyk2 tyrosine phosphorylation (Figure 4A). We then tested the effect of lanthanum, a potent inhibitor of the CRAC channels38 that are known to be expressed in macrophages.39 Lanthanum also suppressed the Env-induced activation response (Figure 4B). Taken together, these results suggest that intracellular calcium elevations due to calcium influx, elicited by JRFL through CCR5, is responsible for Pyk2 phosphorylation.
We then tested the effect on gp120-induced Pyk2 phosphorylation of PTX,
which uncouples G JRFL gp120 and MIP-1
MDMs were stimulated with JRFL gp120 or MIP-1 Involvement of MAPK signaling pathway in gp120-mediated chemokine induction We next addressed the important question of whether gp120-induced chemokine receptor-mediated signaling in macrophages results in functional consequences with potential biologic significance. Macrophages are a major source for the production of-chemokines, which play a central role in inflammatory cell recruitment and activation.44 Changes in the production of specific
chemokines have been demonstrated during the course of HIV infection
and, particularly, in compartments where macrophages are believed to be
important in pathogenesis.45-47 In addition, several
groups have observed increased production of -chemokines by
macrophages after in vitro HIV infection or gp120
exposure.48-50 Therefore, we investigated gp120-induced
chemokine secretion by human macrophages and whether the MAPK pathway
might be involved.
The MDMs were exposed to JRFL gp120 for 24 hours, and MIP-1
Engagement of the chemokine receptor entry coreceptors by HIV-1 Env is essential for membrane fusion, but it may also initiate signaling events that alter cellular functions or even affect postentry stages of infection. Studies to date indicate that HIV-1 gp120 may participate in several signaling pathways through these receptors, including those involving G protein stimulation, ionic signaling, and protein phosphorylation.2 Macrophages are important targets for HIV-1 in vivo and express both CCR5 and CXCR4, the principal coreceptors for HIV-1 entry. However, relatively little is known about the pathways by which chemokine receptors signal in primary human macrophages in response to either gp120 or their natural chemokine ligands. We recently demonstrated that HIV-1 Env activates ionic signaling responses in macrophages through CCR5 and CXCR4.20 In the present study, we show that Env interaction with the coreceptors results in phosphorylation of the protein tyrosine kinase Pyk2, activation of the p38 and JNK/SAPK MAP kinases, and consequent induction of chemoattractant proinflammatory mediator secretion. These results begin to delineate the pathways by which the chemokine receptors are coupled in primary macrophages and, importantly, identify mechanisms by which HIV-1 may subvert the normal cellular signaling machinery. Protein kinase activation by gp120 had not previously been examined in
macrophages. We focused on the FAK-related protein Pyk2, also known as
related adhesion focal tyrosine kinase (RAFTK),24 calcium-dependent protein tyrosine kinase (CADTK),36 or
cell adhesion kinase We found that [Ca++]i elevation mediated by
CRAC channels is a principal link between CCR5 stimulation and Pyk2
activation in macrophages, because both EGTA and the CRAC channel
inhibitor lanthanum blocked gp120-induced Pyk2 phosphorylation. This
result is in accord with our previous finding that gp120 elevates
macrophage cytosolic calcium,20 and observations by others
that Pyk2 activation is linked to [Ca++]i in
various cell types.25,27,30,31,36 We also found that CCR5-mediated Pyk2 activation in macrophages was insensitive to PTX,
indicating that it is not coupled through G Because Pyk2 is implicated as an upstream regulator of the MAPK family
of proteins in a variety of cell types,28,29,31,36,42,43 we examined MAPK activity in macrophages and found that JRFL gp120 as
well as chemokines triggered activation of p38 and JNK/SAPK MAPK.
HIV-1- or gp120-induced MAP kinase activation has been reported in T
cells, where it may play a role in regulating viral
expression.7,8,60,61 Like Pyk2, activation of macrophage
MAPK was not blocked by PTX, indicating that it is independent of
G A central question regarding these data involves the biologic
significance of signaling cascades induced by gp120 in macrophages. MAPK activation plays an important role in regulating inflammatory responses, such as cytokine secretion in response to multiple stimuli.7,61,63-66 HIV-1 exposure or infection may induce
a variety of functional and secretory responses in primary
macrophages,13,48,50,67 including gp120-induced secretion
of Coreceptor signaling is dispensable for infection in cell lines,55,68 but indirect evidence has suggested that signaling might modulate infection in primary cells. Desensitization of CCR5 signaling has been associated with inhibition of lymphocyte infection,69 and chemokine receptor-mediated signaling was recently implicated in modulating productive versus nonproductive macrophage infection.37 Thus, whether signaling pathways elicited by Env in primary macrophages are involved in cell- and virus-specific mechanisms that modulate infection subsequent to entry is an important question that remains to be fully defined. In addition to Env on infectious virions, macrophages may be exposed to
gp120 on the surface of noninfectious virions or as free protein in
tissues or into the circulation of infected subjects.70,71 Concentrations of gp120 in circulation up to 92 ng/mL have been reported,71 which is close to the level seen to activate
Pyk2 here. Furthermore, we found chemokine secretion induced by as little as 50 ng/mL gp120 (data not shown). Importantly, however, macrophages are likely to be exposed to Env glycoprotein in vivo within
tissues, where higher concentrations of gp120 may be achieved locally
at sites of viral replication and contribute to induction of kinase
activation. Thus, the activation pathways identified in this report may
also contribute to pathogenesis through interactions with uninfected
cells. Various abnormalities of macrophage functions have been observed
in HIV-1 infection in vivo and in vitro that in many cases can be
induced by virions or soluble gp120 even in the absence of
infection.13,15,72 Also, although
We thank J. Cutilli, J. Riess and J. Moschella for technical assistance; R. Doms for Env proteins and valuable discussions; and blood donors who generously provided cells.
Submitted April 24, 2001; accepted July 16, 2001.
Supported in part by grants from the National Institutes of Health (MH 61139, AI 35502, and HL 58004) and from the Italian Ministry of Health (40D.7).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Ronald Collman, 522 Johnson Pavilion, 36th and Hamilton Walk, Philadelphia, PA 19104; e-mail: collmanr{at}mail.med.upenn.edu.
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Top
Abstract
Introduction
MDMs derived from donors homozygous
for the CCR5 
