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CHEMOKINES
From the Division of Experimental Medicine and
Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard
Institutes of Medicine, Boston, MA.
The mechanism whereby HIV-infected cells transit from
the bloodstream into tissues is not well defined. This phenomenon was addressed by studying the effects of HIV-1 Tat, a protein secreted by
infected cells, on human lung microvascular endothelial cells (HMVEC-Ls). It was found that monocyte chemoattractant
protein-1 (MCP-1) was released from HMVEC-Ls in a dose- and
time-dependent manner after Tat treatment. MCP-1 is a potent
Although both CD4+ T cells
and monocytes/macrophages are infected by HIV-1, it is generally
believed that monocytes/macrophages serve as reservoirs for virus
replication and act as the primary source of transmission of virus to
various tissues.1 Indeed, most of the virus found in the
extravascular tissues of AIDS patients has been shown to reside in
macrophages.2,3 In the lung, alveolar macrophages are the
predominant resident immune cells in the alveolar airspace and are
targets for HIV-1 infection.4 Direct infection of HIV-1
into these macrophages impairs monocyte/macrophage effector cell
functions, thereby inviting opportunistic infections. Thus,
HIV-1-infected monocytes/macrophages in peripheral tissues are an
important therapeutic target in AIDS treatment, particularly in
relation to restoring host defenses.
Localization of monocytes to the peripheral tissues begins
with interactions between the adhesion molecules LFA-1, -2, and -3, expressed on the monocyte surface, and the vascular
endothelium.5,6 The monocytes then traverse the
endothelial barrier by producing extracellular matrix-degrading
metalloproteinases, which allow passage through the vascular basement
membrane.7 In the process of monocytic infiltration, it is
believed that monocyte chemoattractant protein-1 (MCP-1), a member of
the beta (C-C) subfamily of chemokines, plays an important role. It is
known that mechanical shear stress increases MCP-1 secretion in
cultured human endothelial cells8-11 and in astrocytes,
the most abundant cell type in the brain,12,13 resulting
in increased monocyte adhesion to the endothelium. Further, MCP-1 is
the most potent of the various monocyte chemoattractants, which include
RANTES, macrophage inflammatory protein-1 HIV-1 Tat protein is released from HIV-1-infected T cells
and monocytes/macrophages15 as a protocytokine. It
interacts specifically with vascular endothelial growth factor (VEGF)
and the beta-integrin receptor molecules of endothelial
cells.16-19 Tat ligation of these specific surface
molecules activates several cellular protein kinases,18,20-22 including Flk-1/KDR, Flt-1, and MAP
kinase. HIV-1 Tat induces MCP-1 secretion from
astrocytes23 and is thought to enhance monocyte-macrophage
trafficking into the central nervous system. We postulated that MCP-1
could induce the transmigration of HIV-1-infected monocytes across an
endothelial monolayer. This transmigration would increase viral
transmission to extravascular compartments and play a role in
HIV-1-associated immune dysfunction. To address this hypothesis, we
assessed a model of Tat-mediated MCP-1 secretion from primary human
lung microvascular endothelial cells (HMVEC-Ls) and
characterized downstream signaling substrates involved in MCP-1
secretion and the transmigration of monocytes. We identified protein
kinase C (PKC) as a key signaling molecule that mediates Tat effects in
such microvascular endothelium.
Antibodies
Cell cultures
Tat protein HIV-1 IIIB Tat protein was purified, lyophylized, and reconstituted in Tat buffer (phosphate-buffered saline [PBS] containing 1 mg of bovine serum albumin [BSA] and 0.1 mM dithiothreitol [DTT]), as described.18 The purified protein was endotoxin free, as judged from the timed gel formation assay with the Limulus amoebocyte lysate reagent (Sigma). This protein was biologically active, as assessed by the rescuability of tat-defective provirus replication in HLM-1 cells with the purified Tat protein.18Tat treatment of cells At 80% confluence, cells were serum-starved overnight by replacing culture medium with the endothelial basal medium, EBM-2 (Clonetics), supplemented with 0.5% FBS. The cells were then treated with HIV-1 Tat protein (at the indicated concentrations) plus 10 U/mL heparin, or with 10 U/mL of heparin alone, in fresh EBM-2 medium containing 0.5% FBS for the indicated periods. Heparin was used in these experiments, as it is known to augment the biologic activities of Tat, such as the induction of endothelial cell growth, migration, and invasion in vitro.24 To examine the effect of inhibition of intracellular kinases on Tat-induced MCP-1 secretion, starved HMVEC-Ls were incubated with the indicated amounts of inhibitors or with diluent controls in EBM-2 medium 1 hour before treatment with Tat.Quantitation of MCP-1, MIP-1  , and MIP-1 levels in the clarified supernatants were measured by using a commercial MCP-1, MIP-1, or
MIP-1 antigen capture enzyme-linked immunosorbent assay (ELISA) kit
(R & D Systems) according to the manufacturer's instructions. All
results are the average of 2 or more experiments.
Flow cytometric analyses of CCR2 expression CCR2 is a major chemokine receptor for MCP-1. To investigate MCP-1-mediated chemotaxis, we first examined the expression of CCR2 on the surface of THP-1 cells and HMVEC-Ls by flow cytometric analysis. Briefly, THP-1 cells were washed once with PBS and incubated with antihuman CCR2 MAB150 (5 µg/mL) (R & D Systems) or with isotype IgG as a control in 1 mL of PBS with 10% normal goat serum for 1 hour on ice, followed by treatment with FITC-conjugated goat antimouse secondary antibody (1:500 dilution) (Boehringer-Mannheim, Indianapolis, IN) for 20 minutes on ice. Surface expression of each sample was then analyzed on an Epics Profile Flow Cytometer (Coulter Electronics, Hialeah, FL). To evaluate CCR2 expression in HMVEC-Ls, cells were detached, washed once, and then incubated with the primary antibody, as described previously.Chemotaxis assay Chemotaxis was assayed in 24-well plates (CoStar Corp, Cambridge, MA) carrying transwell inserts of 5 µm pore size. Briefly, CD14+ monocytes from human peripheral blood or THP-1 cells were washed twice, resuspended, and then loaded onto the inserts at 3 × 105 cells/200 µL for each assay. Chemotaxis medium containing the indicated chemoattractant was placed in the bottom chamber. After 3 hours of incubation at 37°C with 5% CO2, cells that migrated to the lower chamber were collected; the percentage of viable cells that migrated from the top to the bottom compartment was measured by counting cells with the trypan blue dye exclusion method. To investigate the effects of the inhibitors involved in the signal transduction or of the concentration gradients of the chemoattractants on the migration of CD14+ monocytes or THP-1 cells, the cells were incubated for 1 hour with the indicated inhibitors or chemoattractants before their loading onto transwell inserts.MAP kinase assay The in vitro MAP kinase assay was performed as described previously.18 Briefly, after the indicated treatments, cells were lysed by the addition of radioimmunoprecipitation assay (RIPA) buffer,18 and the lysates were immunoprecipitated with the indicated monoclonal antibodies. The precipitates were washed twice with deoxycholate-free RIPA buffer, followed by one wash with kinase assay buffer (20 mM HEPES, pH 7.4, 50 mM NaCl, 5 mM MgCl2, 5 mM MnCl2, 100 mM Na3VO4). The washed precipitates were then incubated at room temperature for 30 minutes in a kinase assay buffer containing 0.18 MBq (5 µCi) [ 32P]-ATP along
with: (1) myelin basic protein (MBP) (Upstate Biotechnology, Lake
Placid, NY) for the p44/42 MAP (Erk1/2) or p38 kinase assay, or (2)
c-Jun for the Jun kinase assay. The reaction was stopped by adding 4 times sodium dodecylsulfate (SDS) sample buffer. The kinase substrate
proteins were analyzed on 12% SDS-polyacrylamide gels (PAGE).
Protein kinase C assay PKC activity was assayed by quantification of 32P incorporation from [32P]-ATP into the substrate,
MARCKSPSD peptide (Calbiochem, Inc, La Jolla, CA). Assays were
performed at room temperature in a total volume of 20 µL containing
20 mM HEPES-NaOH (pH 7.5), 10 mM MgCl2, 10 mM DTT, 10 µg/mL leupeptin, 0.1 mM CaCl2, 0.1 mM [32P]-ATP, 0.03% Triton X-100, and 0.5 µg MARCKSPSD
peptide,25 5 µg cell lysate, 0.3 mg/mL
phosphatidylserine (PS), and 62 µg/mL 1,2-diolein. Reactions were
initiated by the addition of [32P]-ATP and terminated
after 15 minutes by adding 1 mL 25% trichloroacetic acid (TCA) and 10 µL of BSA (50 mg/mL). Samples were then precipitated by
centrifugation at 12 000 × g for 2 minutes, the pellet
rinsed twice with 0.5 mL of 25% TCA, and air-dried. Incorporation of 32P into the substrate was then quantitated by
Cherenkov counting.
Immunoprecipitation and Western blot analysis For the immunoprecipitation studies, an equivalent amount of protein from each sample was clarified by incubation with protein A-Sepharose for 1 hour at 4°C, and the antigen in the clarified solution was then immunoprecipitated with the indicated primary antibody, as described.18 The precipitates were next separated by SDS-PAGE, followed by transfer to nitrocellulose membranes. The membranes were blocked with 5% nonfat milk protein and probed with the indicated primary antibody at 4°C overnight. Immunoreactive bands were visualized by using horseradish peroxidase (HRP)-conjugated secondary antibody and the enhanced chemiluminescent (ECL) system (Amersham Pharmacia Biotech, Piscataway, NJ).
Tat effect on MCP-1 secretion To examine modulation of MCP-1 secretion from endothelial cells on HIV-1 Tat treatment, HMVEC-Ls at 80% confluence were stimulated with Tat protein plus heparin or with heparin alone in culture medium for the indicated periods. MCP-1 concentrations in the clarified culture supernatants were then measured by an ELISA.Treatment of HMVEC-L with Tat significantly induced MCP-1 secretion
into the culture supernatants. This induction of secretion peaked at 24 hours after Tat stimulation (Figure 1B).
We further assessed whether the Tat effect on MCP-1 release was
dose-dependent. HMVEC-Ls were stimulated over 24 hours with different
concentrations of Tat in culture media, and the amount of MCP-1 in the
supernatant was quantitated by ELISA. MCP-1 secretion was significantly
augmented by Tat, at concentrations as low as 10 ng/mL (Figure 1A). A
significant amount of MCP-1 was secreted from HMVEC-Ls treated with 6.4 ng/mL of Tat, whereas MCP-1 secretion was barely detectable at 1.6 ng/mL of Tat (data not shown). Because MIP-1
Activation of protein kinase C activity by Tat We then examined which signaling molecules were activated by Tat in HMVEC-Ls. Changes in PKC activity were sought. Cell lysates from Tat-treated HMVEC-Ls were incubated with the substrate, MARCKSPSD,25 and incorporations of 32P into the substrate were quantitated. Figure 2 shows that radioisotope incorporation increased steadily with the increasing incubation time of HMVEC-Ls with Tat. When the cells were treated for 20 minutes with the positive control phorbol 12-myristate 13 acetate (PMA), a potent activator of PKC, approximately 3 times higher PKC activity was observed than was seen with Tat treatment (data not shown). These results indicate that Tat activates PKC activity in HMVEC-Ls.
Activation of surface receptor molecules on HMVEC-Ls It is known that HIV-1 Tat protein interacts specifically with VEGF and the beta-integrin receptor molecules of endothelial cells.16-19 Tat ligation of these specific surface molecules activates several cellular protein kinases,18,20-22 including Flk-1 and Flt-1, which are important for endothelial cell proliferation and migration. Thus, we examined whether these surface molecules are activated by Tat protein. To this end, tyrosine phosphorylations of Flk-1 and Flt-1 in Tat-treated HMVEC-Ls were investigated by immunoprecipitation, followed by Western blot analysis. The data showed that both Flk-1 and Flt-1 were tyrosine-phosphorylated on Tat stimulation (Figure 3A,B, respectively). The tyrosine phosphorylation of Flk-1 was sustained for up to 20 minutes after stimulation, whereas that of Flt-1 was rapid and transient, with decreased amounts of phosphorylated Flt-1 observed beginning 2 minutes after stimulation. The amount of Flt-1 or Flk-1 protein loaded at the different time points was equivalent, indicating that the observed differences in the amount of phosphorylated protein did not reflect the quantitative variance of each protein. Taken together, these data suggest that the signaling cascade leading to the Tat-mediated modulation of chemokine secretion might be initiated on the ligation of Tat by Flk-1 and/or Flt-1.
Activation of MAP kinase activity The MAP kinase pathway is known to be a major conduit for the transmission of downstream signals regulating cell proliferation,26-28 cell cycle control,26,29,30 and chemotaxis31-33 in different cell types. To investigate whether MAP kinase is involved in Tat-induced MCP-1 release, 200 µg of lysates generated from Tat-treated HMVEC-Ls were precipitated with Erk 1/2 antibodies, and then the kinase activity in the precipitates was determined by using MBP as a substrate. As shown in Figure 3C, strong Erk 1/2 kinase activity was observed; this activity increased with time, peaking at 20 minutes. By contrast, activity of the other major MAP kinases, p38 and c-Jun kinase, was not enhanced in the presence of Tat (data not shown). These results indicate that Erk 1/2 was activated by Tat in HMVEC-Ls and suggest that signals for the modulation of MCP-1 release could be mediated through the MAP kinase and/or PKC pathway.Effect of protein kinase inhibitors on MCP-1 secretion We further tested whether p44/42 MAP (Erk 1/2) and/or PKC were necessary for the induction of MCP-1 release by Tat protein. Serum-starved HMVEC-Ls were treated with various concentrations of protein kinase inhibitors 1 hour before Tat stimulation. The MAP kinase specific inhibitor, PD98059, had no significant effect on MCP-1 secretion (Figure 4). By contrast, the PKC inhibitor, GF109203X, significantly decreased MCP-1 secretion induced by Tat treatment (Figure 4). These data indicate that the signaling cascade for the secretion of MCP-1 acts primarily through PKC, but not through the MAP kinase.
MCP-1-dependent transwell migration of CD14+ monocytes and THP-1 cells Because MCP-1 is one of the most potent chemoattractants for monocytes/macrophages,14 we assessed the levels of MCP-1-dependent transwell migration of CD14+ monocytes and model THP-1 monocytic cells. To this end, we first examined whether THP-1 cells and HMVEC-Ls express CCR2, the major chemokine receptor for MCP-1, using flow cytometric analysis. The data show that significant amounts of CCR2 were expressed on the surface of THP-1 cells but not on HMVEC-Ls (data not shown), suggesting that MCP-1 secreted from HMVEC-Ls could function to chemoattract THP-1 cells. Expression of CCR2 on the surface of peripheral blood monocytes has already been reported.34,35 We then assayed for possible MCP-1-dependent transwell migration of THP-1 cells by quantitating cell migration from the upper to lower compartment, which contained various concentrations of MCP-1. Migration of THP-1 cells was concentration dependent, peaking at 50 ng/mL and was abrogated by the removal of the concentration gradient (Figure 5A). In contrast, in controls treated with anti-CCR2 antibody alone, there was no chemoattraction for THP-1, even at high concentrations of antibody (100 ng/mL) (data not shown). Transmigration of CD14+ monocytes was also concentration dependent up to 12.5 ng/mL of MCP-1 and was saturated beyond that concentration (Figure 5B). Similar to the migration of THP-1 cells, the removal of concentration gradient completely abrogated the migration of CD14+ monocytes (Figure 5B). These data indicate that the observed transwell migrations of CD14+ monocytes and THP-1 cells were specifically chemoattractive for MCP-1, rather than the result of a simple MCP-1 receptor/ligand interaction.
Chemotaxic properties of culture supernatants from Tat-treated HMVEC-Ls The above data indicate that treatment of HMVEC-Ls with HIV-1 Tat induces MCP-1 secretion and that MCP-1 is a strong chemoattractant for CD14+ monocytes and THP-1 cells. Thus, we investigated whether culture supernatants of HMVEC-Ls treated with Tat were chemotactic. Consistent with the above data, MCP-1 (50 ng/mL) showed strong chemotactic activity (Figure 6A, lane 2). When Tat-treated culture supernatants (containing 1 ng of MCP-1 as determined by an ELISA) were placed in the lower chamber, more than 20% of the THP-1 cells migrated (Figure 6A, lane 4), which is a higher percentage of migration compared with a gradient of 6.25 ng/mL of MCP-1 (Figure 5A). This result indicated that culture supernatants from Tat-treated HMVEC-Ls were chemotactic and implied that these supernatants contain other chemoattractants for the migration of monocytes in addition to MCP-1. As previously addressed, the small amount of MIP-1 secretion observed in our earlier experiment might
partially account for the THP-1 cell migration. The resultant
chemotactic activity was abrogated by pretreatment of the THP-1 cells
with either MCP-1 (20 ng/mL) (Figure 6A, lane 5) or supernatants from
Tat-treated HMVEC-Ls (Figure 6A, lane 6). Similar data were obtained
with CD14+ monocytes (Figure 6B), even though significant
portions of the monocytes were nonspecifically transmigrated; that is,
approximately 15% of the monocytes migrated in the absence of
chemokines (Figure 6B, lane 1). These data demonstrate that the culture
supernatants induced the migration of CD14+ monocytes and
THP-1 cells, and that this migration was dependent on a
concentration gradient.
HIV-1 infection of monocytes increases their interaction with human microvascular endothelial cells, thereby disrupting the integrity of the endothelial cell monolayer.36-38 This perturbation of the endothelium may promote extravasation of HIV-1-infected cells into peripheral tissues. Our results demonstrate that HIV-1 Tat up-regulates MCP-1 secretion and transmigration of monocytic THP-1 cells, an effect that would enhance the passage of virus and virus-infected cells through the endothelium. This induction of MCP-1 secretion and the migration of THP-1 cells was dependent on PKC activation in HMVEC-Ls. MCP-1, a member of the beta subfamily of chemokines, attracts blood monocytes and T cells and facilitates their transendothelial migration.39,40 MCP-1 messenger RNA (mRNA) or protein has been detected at high levels in several diseases, including pulmonary fibrosis,41,42 arthritis,43 and various tumors,44,45 strongly suggesting that the chemokine recruits monocytes in these settings. Our data suggest that Tat modulates MCP-1 secretion from lung microvascular endothelium. This would enhance the infiltration of virus-infected cells from the pulmonary microcirculation into the extravascular spaces, ie, the alveoli. The MCP-1 secretion pathway may be initiated by activation of the
surface molecules Flk-1 and/or Flt-1. We found that although HIV-1 Tat
treatment activated several signaling molecules, including p44/42 MAP
kinases and PKC, only inhibition of PKC resulted in significantly
diminished MCP-1 secretion and THP-1 monocyte chemotaxis. This result
indicated that regulation of MCP-1 secretion is primarily mediated
through this specific protein kinase signaling cascade. These data are
consistent with a previous report, wherein a general protein kinase
inhibitor blocked tumor necrosis factor alpha (TNF Culture supernatants from Tat-treated HMVEC-Ls displayed chemotactic activity for THP-1 cells. When the THP-1 cells and culture supernatants containing 1 ng of MCP-1 were placed onto upper and lower compartments, respectively, more than 20% of the THP-1 cells migrated from the top to the bottom chamber (Figure 6). This is a higher proportion of cells migrating compared with a gradient of 6.25 ng/mL of MCP-1 (Figure 5). The percentage inhibition of MCP-1 secretion by the PKC inhibitor did not parallel that of the THP-1 migration; that is, the reduced level of migration induced by the PKC inhibitor (25% decrease with 9 µM of GF109203X compared with the control) was less than the level of inhibition of MCP-1 secretion (80% decrease with the same concentration of inhibitor). These data imply that the culture supernatants from the Tat-treated HMVEC-Ls contain several chemoattractants, including MCP-1, for the migration of monocytes. We observed higher random transmigration of the CD14+ monocytes compared with the THP-1 cells. However, transmigration of the CD14+ monocytes was significantly increased in response to either MCP-1 or Tat-treated culture supernatants, and this increased transmigration was eliminated on removal of the concentration gradient. These data indicate that the observed transmigration was specific to MCP-1. Taken together, our data demonstrate that HIV-1 Tat induces secretion of MCP-1 and other chemoattractants that facilitates the transendothelial migration of monocytes from HMVEC-Ls; this occurs via a PKC-dependent pathway. The other chemoattractants released from HMVEC-Ls in the Tat-mediated PKC signaling pathway remain to be elucidated. Additional studies to characterize this signaling pathway should provide insight into the passage of HIV-1-infected monocytes across the microvascular endothelium to the tissue spaces, a signature event in the pathogenesis of reduced host defense in AIDS.
We thank Janet Delahanty for editing this manuscript and Nancy DesRosiers for preparation of the figures.
Submitted April 12, 2000; accepted September 13, 2000.
Supported by NIH grants HL61940 and HL53745.
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: Jerome E. Groopman, Division of Experimental Medicine, Harvard Institutes of Medicine/BIDMC, 4 Blackfan Circle, Rm 351, Boston, MA 02115; e-mail: jgroopma{at}caregroup.harvard.edu.
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J. Sun, T. Soos, V. N. KewalRamani, K. Osiecki, J. H. Zheng, L. Falkin, L. Santambrogio, D. R. Littman, and H. Goldstein CD4-Specific Transgenic Expression of Human Cyclin T1 Markedly Increases Human Immunodeficiency Virus Type 1 (HIV-1) Production by CD4+ T Lymphocytes and Myeloid Cells in Mice Transgenic for a Provirus Encoding a Monocyte-Tropic HIV-1 Isolate J. Virol., February 15, 2006; 80(4): 1850 - 1862. [Abstract] [Full Text] [PDF]
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J. Li, Y. Liu, B. O. Kim, and J. J. He Direct Participation of Sam68, the 68-Kilodalton Src-Associated Protein in Mitosis, in the CRM1-Mediated Rev Nuclear Export Pathway J. Virol., July 17, 2002; 76(16): 8374 - 8382. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
CXC and CC chemokines.
Adv Immunol.
1994;55:97-179