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*
UCLA AIDS Institute, Department of Medicine, and
Department of Microbiology and Molecular Genetics, University of California at Los Angeles School of Medicine, Los Angeles, CA 90095
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemokine receptor expression has received a great deal of attention following the discovery that certain chemokine receptors function in conjunction with CD4 to mediate HIV infection of human cells (9). CXCR4 was initially identified as a coreceptor for T cell-tropic strains of HIV-1 (10), and CCR5 and several other CC chemokine receptors were subsequently identified as coreceptors for macrophage-tropic strains of HIV-1 (11, 12, 13). CXCR4 can also mediate CD4-independent infection of human cells by HIV-2 (14, 15). Varying levels of chemokine receptor expression can influence cellular susceptibility to HIV infection (16, 17), and chemokines and anti-receptor Abs can block HIV infection under certain circumstances (18, 19, 20, 21, 22, 23).
Because chemokine receptors play a critical role in both normal immune system function and HIV pathogenesis, understanding the factors that regulate their expression may provide options for therapeutic intervention in a variety of pathophysiologic settings (1, 2, 3, 4). In addition to differential expression across distinct leukocyte subpopulations, chemokine receptor density is also altered in the presence of its natural ligand (24), over the course of mitosis (5, 24, 25), and during cellular maturation (26). Cytokines can alter the expression of some CC chemokine receptors (27, 28) although their effects on lymphocyte CXC chemokine receptors remain poorly defined. Chemokine receptors are members of a diverse family of cell surface receptors characterized by a seven-transmembrane serpentine structure and signal transduction via heterotrimeric guanine nucleotide binding proteins (4, 29, 30). Cell surface expression of G-protein-linked receptors is regulated both by gene expression and by continual recirculation of receptors between the cell surface and endosomal compartments (24, 31). Distinct signaling pathways govern these two modes of receptor regulation. For example, whereas certain mitogenic stimuli suppress CXCR4 expression by increasing receptor internalization via the protein kinase C (PKC) signaling cascade, this pathway does not appear to mediate ligand-induced suppression of CXCR4 (24).
With the exception of mitogenic stimuli and ligand-induced
down-regulation, little is known about the role of extracellular
factors in regulating lymphocyte expression of CXC chemokine receptors.
However, the pronounced effects of cytokines, growth factors, and
hormones on lymphocyte localization (3, 32, 33, 34) suggest that
extracellular factors may well play an important role in chemokine
receptor expression. One major mode of extracellular influence on
lymphocyte function comes from a class of secreted molecules that
activate the cellular cAMP-dependent protein kinase A (PKA) (35). The
cAMP-PKA signaling pathway represents a common second messenger system
for a variety of distinct receptors that bind a diverse array of
hormones, neurotransmitters, and peptide-signaling molecules (e.g., E
series PGs, histamine, catecholamines, neurohypophyseal hormones (such
as corticotropin-releasing factor and vasopressin), and
pro-opiomelanocortin-derived peptides (such as adrenocorticotropic
hormone)) (30). As a result of cAMP signaling, PKA phosphorylates
multiple intracellular substrates, including elements of other
signaling pathways (e.g., phospholipase C
1) and
regulators of gene transcription (e.g., cAMP response element binding
protein (CREB)). The cAMP-PKA signaling pathway plays a critical
"switching" role in a variety of physiologic settings ranging from
glycolysis to ontogenic differentiation. In the immune system, cAMP
signaling modulates cellular activation (35, 36) and alters cytokine
production profiles (37, 38, 39, 40, 41). cAMP-inducing stimuli can also alter
leukocyte traffic and localization (33, 34, 42), although the molecular
basis for such effects is not fully understood.
In the present study, we explore the role of the cAMP-PKA signaling pathway in modulating lymphocyte expression of CXCR4, the receptor for the chemokine SDF-1 (19, 23). In contrast to most other chemokine systems, which recruit cells to sites of inflammation, the SDF-1/CXCR4 system is believed to localize cells to lymphoid organs (43, 44, 45). Consistent with this function, CXCR4 is richly expressed on hemopoietic progenitor cells and immature thymocytes, on fully differentiated but antigenically naive B and T lymphocytes, and on monocytes and cultured dendritic cells (5, 26, 46). Lymphocyte CXCR4 expression is down-regulated during cellular activation (24), and CXCR4 is virtually absent from NK cells, neutrophils, eosinophils, and freshly isolated Langerhans cells (5, 21, 47). Here, we examine the role of cAMP in regulating CXCR4 expression on both activated and unstimulated PBMC. CD4+ cells represent a special focus of attention due to their potential vulnerability to HIV infection. In addition, we consider the functional implications of cAMP regulation of CXCR4 for ligand-induced internalization, SDF-1-induced chemotaxis, and vulnerability to infection with CXCR4-tropic HIV-1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Healthy donor PBMC were isolated by Ficoll density gradient and cultured at 3 x 105/ml in RPMI 1640 supplemented with 10% (v) human AB serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine, at 37°C in an atmosphere of 5% CO2. PKA-activating ligands were added once, at the beginning of culture, and included the membrane-permeable cAMP analogue, N6,2'-O-dibutyryl adenosine-3',5'-cyclic monophosphate (dbcAMP), the adenylyl cyclase activator Forskolin, and the physiologic cAMP inducers PGE2, histamine (H), adrenocorticotropic hormone (ACTH), epinephrine (E), and norepinephrine (NE) (all from Sigma, St. Louis, MO). Activated cells were costimulated with Abs to CD3 (0.1 µg/ml adhered to flask by goat anti-mouse Ab; Southern Biotechnology, Birmingham, AL) and CD28 (0.1 µg/ml soluble; Biodesign, Kennebunkport, ME) at the beginning of culture. CD4+ T cell cultures were established by isolating cells on negative selection columns (R&D Systems, Minneapolis, MN) and culturing them under conditions described above.
CXCR4 cell surface expression
Cell surface expression of CXCR4 was quantified by flow cytometry using the phycoerythrin-conjugated 12G5 mAb (PharMingen, San Diego, CA). CD4+, CD8+, CD14+, and CD19+ subsets were identified by FITC- and PerCP-conjugated mAb binding (Becton Dickinson Immunocytometry, Mountain View, CA). Ab staining was conducted according to the manufacturer’s protocol, and all flow cytometric data were acquired using a FACScan instrument (Becton Dickinson Immunocytometry). Data were analyzed using CELLQuest software (Becton Dickinson Immunocytometry), with gating to exclude dead cells and debris on the basis of forward- vs side-scatter profiles. Linear regression was used to assess the statistical significance of dose-response relationships between dbcAMP concentrations and CXCR4 mean fluorescence intensity.
CXCR4 gene transcription
CXCR4 mRNA was quantified by RT-PCR. Total cellular RNA from 107 PBMC was extracted (RNeasy; Qiagen, Chatsworth, CA), subject to DNase-1 treatment (Promega, Madison, WI), and reverse transcribed using murine leukemia virus reverse transcriptase (Perkin-Elmer Cetus, Norwalk, CT) and a 20-thymidine oligonucleotide primer. One-tenth of the resultant cDNA was amplified by PCR using the CXCR4-specific primers 5'-TCA TCT ACA CAg TCA ACC TCT ACA-3' and 5'-gAA CAC AAC CAC CCA CAA gTC ATT-3' (47). To verify equivalent RNA loading, ß-actin cDNA was amplified in parallel using commercial primers (R&D Systems). Primers were radio end-labeled with 32P, and samples were amplified using Taq DNA polymerase (Perkin-Elmer Cetus) with 34 cycles of denaturing at 94°C for 1 min, annealing at 54°C for 1 min, and extension at 72°C for 1 min. Standards consisting of serial 10-fold dilutions of cDNA from total cellular RNA extracted from 107 PBMC were amplified in parallel. Radiolabeled amplified products were resolved on a 6% polyacrylamide gel and quantified by radioanalytic image analysis in comparison with standard curves. All amplifications were RNA specific as demonstrated by no-reverse-transcriptase negative controls.
CXCR4 internalization
Receptor internalization was measured by retention of anti-CXCR4 Ab binding following elimination of cell surface-bound Ab via acid wash (24). Total cell-associated Ab binding reflects both receptors expressed at the cell surface and those subsequently internalized into endosomal compartments. When surface-bound Ab is removed by acid wash, residual Ab binding reflects internalized receptors only (24). Removal of surface-bound Ab was confirmed by establishing that acid washing abrogated both anti-CD4 Ab binding at 37°C (CD4 does not internalize under normal circumstances; Ref (48) and anti-CXCR4 Ab binding on cells maintained at 4°C to inhibit internalization (see Results). Receptor internalization was quantified as the fraction of total anti-CXCR4 fluorescence intensity (pH 7 wash) retained following acid wash (pH 3). Fluorescence intensity was measured by FACS analysis gated to exclude dead cells and debris on the basis of forward- vs side-scatter profiles. We used t tests to assess the statistical significance of differences in receptor internalization.
CXCR4 compartmentalization
The distribution of CXCR4 in intracellular vs cell surface compartments was assessed by flow cytometric quantitation of anti-CXCR4 Ab binding following permeabilization of fixed cells (measuring both intracellular and extracellular binding) vs fixation in the absence of permeabilization (measuring extracellular binding only). PBMC (106) were surface stained with anti-CD4 Ab according to the manufacturer’s protocol, fixed by suspension in 1 ml 4% paraformaldehyde, pelleted, and resuspended in 1 ml of saponin buffer (0.1% w/v saponin plus 0.05% azide in HBSS) for permeabilization. Unpermeabilized cells were suspended in 1 ml HBSS. All cells were then pelleted, 800 µl of suspension solution was aspirated, and 10 µl of anti-CXCR4 Ab was added. Following 30 min of incubation at room temperature, cells were washed once in 1.5 ml of either saponin buffer (permeabilized cells) or HBSS (unpermeabilized cells), pelleted, and resuspended in PBS for flow cytometry. Fluorescence intensity was measured by FACS analysis gated to exclude dead cells and debris on the basis of forward- vs side-scatter profiles. Intracellular CXCR4 compartmentalization was quantified by subtracting the mean fluorescence intensity of anti-CXCR4 Ab binding on unpermeabilized cells (extracellular CXCR4) from that of permeabilized cells (intracellular plus extracellular CXCR4). Statistical significance of differences in intracellular compartmentalization was assessed by t test. Nonspecific Ab binding was assessed by parallel staining with an isotype-matched phycoerythrin-conjugated anti-keyhole limpet hemocyanin (KLH) Ab and did not differ across cAMP-treated and -untreated cells. Analyses subtracting nonspecific fluorescence intensity from anti-CXCR4 fluorescence intensity produced similar results.
Ligand-induced down-regulation
CXCR4 down-regulation in response to exogenous ligand was
assessed by flow cytometric quantification of cell surface CXCR4
expression on costimulated PBMC (as described above) at 5 and 30 min
after addition of 100 ng/ml SDF-1
(R&D Systems). Effects of
ligand-induced internalization were quantified by expressing CXCR4 mean
fluorescence intensity on SDF-1-treated cells as a fraction of CXCR4
mean fluorescence intensity on untreated cells.
Recirculation of CXCR4 to the cell surface following SDF-1-induced
internalization was assessed by flow cytometric quantification of CXCR4
expression on costimulated PBMC at 0, 20 and 60 min after washing
SDF-1-treated cells twice in PBS and resuspending them in fresh
culture medium. Surface expression recovery rates were estimated by
linear regression of CXCR4 mean fluorescence intensity vs time since
SDF-1 washout, and recovery rates on costimulated cells and cells
costimulated in the presence of dbcAMP were compared by t
test.
Chemotaxis
Chemotactic response to SDF-1 was measured by a membrane
transmigration assay in which 5 x 105 PBMC in 100
µl medium (RPMI + 0.25% BSA) were loaded into the upper chamber of a
Transwell insert with a 5-µm pore size polycarbonate membrane
separating cells from a lower chamber containing graded concentrations
of SDF-1. The number of cells transmigrating during 2 h of
incubation was quantified by FACS (gating out dead cells and debris on
the basis of forward- and side-scatter profiles), and SDF-1-induced
chemotaxis was calculated by subtracting background transmigration (0
ng/ml SDF-1) and standardizing relative to a 500-µl sample
containing 10% of the input cell number. SDF-1-induced chemokinesis
(ambidirectional motility) was assessed by transmigration rates in the
presence of 300 ng/ml SDF-1 in both upper and lower chambers (45).
Statistical significance of differential chemotaxis rates was
assessed by t test and comparison of dose-response
curves by linear regression.
HIV-1 infection and expression
Healthy donor PBMC were infected with CXCR4-tropic
HIV-1NL4–3 (49) (0.05 infectious units per cell) for 30
min in the presence of 10 µg/ml polybrene. Following infection, cells
were washed twice and costimulated as described above. To prevent viral
spread, postinfection medium included the HIV protease inhibitor,
Indinavir (100 nM; Merck, Rahway, NJ). To determine whether
effects of cAMP were mediated specifically by altered CXCR4 expression,
cells were preincubated for 1 h in the presence of 1 mM
recombinant SDF-1 to block interactions between HIV-1 and CXCR4
(18). Equivalent preincubation in the presence of 1 mM recombinant
MIP-1 (R&D Systems) served as a nonblocking control.
The number of infected cells was quantified by PCR analysis of proviral DNA at 14 h post infection (AA55 and M667 primer pair specific for the R/U5 region of the viral long terminal repeat, quantified relative to input cell number as indicated by ß-globin primers LA1 and LA2) (50). Briefly, one primer of each pair was radio end labeled, and amplification was conducted for 25 cycles in parallel with control standards consisting of linearized cloned HIV-1 DNA and known quantities of cellular DNA. Radiolabeled amplified products were resolved on a 6% polyacrylamide gel and quantified by radioanalytic image analysis in comparison with standard curves. To ensure that viral spread did not occur, proviral DNA was also quantified at 48 h postinfection. In no case did the fraction of provirus-bearing cells at 48 h exceed that at 14 h postinfection.
HIV-1 gene expression was quantified by flow cytometric detection of a reporter gene product from a genetically altered HIV-1NL4–3. HIV-1NL-r-HSAS was created by cloning the murine heat-stable Ag (HSA) gene into a vacancy created by deleting nucleotides 5625 through 5742 in the vpr gene of HIV-1NL4–3 (51). Upon transcription and translation of HIV-1NL-r-HSAS, murine HSA is expressed on the cell surface and can be quantified by flow cytometric assessment of anti-murine CD24 Ab binding (FITC-conjugated M1/69; PharMingen). This virus is pathogenic in vivo in SCIDhu mice, and HSA reporter gene expression correlates closely with other means of verifying HIV infection, including PCR assessment of proviral DNA and flow cytometric quantitation of intracellular p24 (51).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activation of the lymphocyte cAMP-PKA signaling pathway alters
lymphocyte traffic and localization (33, 34, 42) and increases HIV
replication (52, 53, 54), although the mechanisms underlying such effects
remain incompletely understood. To determine whether altered expression
of chemokine receptors might play a role, we examined the effect of
cAMP in regulating cell surface expression of CXCR4. On unstimulated
PBMC, CXCR4 expression spontaneously increased over the first 12 h
of culture and subsequently remained stable for up to 3 days (Fig. 1
). Equivalent culture in the presence of
100–300 µM dbcAMP significantly increased CXCR4 cell surface
expression by 12 h, with CXCR4 levels declining gradually
thereafter but remaining significantly elevated above those of
untreated cells (Fig. 1). dbcAMP treatment did not significantly affect
CXCR4 expression levels during the first 6 h of culture as
receptor expression spontaneously increased on both untreated and
dbcAMP-treated cells (data not shown). Pronounced effects of
dbcAMP on CXCR4 expression were observed only once CXCR4 levels
reached a steady state (between 6 and 12 h of culture), suggesting
that cAMP regulates the constitutive receptor set point rather than the
receptor generation process itself. cAMP up-regulation of CXCR4 was
dose dependent (Fig. 1, Table I), and
cAMP-inducing ligands produced similar effects, with 100 µM Forskolin
increasing CXCR4 cell surface expression by up to sixfold at 24 h
(Table I). CXCR4 expression was also increased by a variety of
physiologic cAMP-inducing ligands, including PGE2,
histamine, ACTH, and the catecholamines epinephrine and NE (Table I).
Similar dynamics were observed in CD4+, CD8+,
and CD19+ subsets (Fig. 1) although with differing kinetics
in each subset. On CD4+ cells, cAMP-induced CXCR4
expression peaked at 12 h and declined gradually thereafter,
whereas peak expression on CD8+ and CD19+ cells
occurred at 24 h, followed by a rapid decline toward baseline
levels (Fig. 1). At all time points observed, CD19+ cells
showed significantly greater levels of CXCR4 expression than did
CD4+ or CD8+ cells (all p <
0.0001). In contrast to cAMP effects on lymphocyte CXCR4, cAMP
significantly suppressed CXCR4 expression on CD14+ cells
(Fig. 1). cAMP did not significantly alter cell surface CD4 expression
at any time point examined (data not shown).
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) (p < 0.001 at each time point).
Equivalent costimulation in the presence of dbcAMP significantly
up-regulated CXCR4, with peak expression levels significantly exceeding
those of both costimulated PBMC and unstimulated PBMC in the absence of
cAMP (p < 0.0001) (Fig. 1). Elevated CXCR4
cell surface expression was observed by 12 h of costimulation,
peaked at 24 h, and declined gradually back toward unstimulated
levels at 48 and 72 h (Fig. 1). As with unstimulated cells,
cAMP-inducing ligands also up-regulated CXCR4 expression on
costimulated PBMC (Table I), with 100 µM Forskolin increasing CXCR4
levels by 10-fold. Similar CXCR4 expression kinetics were observed for
CD4+, CD8+, and CD19+ cells, with
CXCR4 levels on CD19+ cells significantly exceeding those
on CD4+ and CD8+ cells (Fig. 1). In the absence
of cAMP, CXCR4 expression on CD19+ cells from costimulated
cultures was significantly suppressed relative to CD19+
cells from unstimulated cultures, implying some degree of cross
regulation by T cell activation (since CD19+ cells should
not be directly activated by CD3/CD28 costimulation). As with
unstimulated cells, cAMP significantly suppressed CXCR4 expression on
CD14+ cells (Fig. 1) and did not significantly alter CD4
expression at any time point examined (data not shown). cAMP does not alter CXCR4 gene expression
To determine whether cAMP-induced up-regulation of cell surface
CXCR4 was mediated by increased gene expression, receptor mRNA levels
were quantified by RT-PCR following 20 h of culture (preceding the
period of peak difference in receptor expression). dbcAMP failed to
significantly increase CXCR4 mRNA levels in either resting or
costimulated PBMC (Fig. 2). Across five
experiments, the average CXCR4 mRNA level in cAMP-treated cells
differed from that of untreated cells by less than 5%. Moreover,
dbcAMP-induced up-regulation of CXCR4 was not abrogated by the protein
synthesis inhibitor cyclohexamide (data not shown), confirming that
cAMP up-regulation of CXCR4 cell surface expression is not mediated
by increased CXCR4 gene expression.
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To determine whether altered receptor trafficking might play a
role in cAMP up-regulation of CXCR4 cell surface expression, receptor
internalization rates were quantified by flow cytometric measurement of
internalized anti-CXCR4 Ab following removal of surface-bound Ab by
acid washing. Residual Ab binding reflects internalized receptors not
exposed to extracellular low pH (24). Low pH washing efficiently
removed cell surface-bound Ab, as demonstrated by the abrogation of
anti-CD4 Ab binding (Fig. 3A) and the abrogation of
anti-CXCR4 Ab binding in cells incubated at 4°C to prevent
internalization (Fig. 3B). Under normal incubation
conditions (37°C), acid-resistant anti-CXCR4 Ab binding averaged
20% of total CXCR4 fluorescence intensity at 60 min (Fig. 3, C and D). Parallel incubation in the presence of
dbcAMP decreased acid-resistant anti-CXCR4 Ab binding by 39%, to
an average of 12% of total fluorescence intensity at 60 min
(p < 0.001). In contrast, dbcAMP did not
significantly alter acid-resistant anti-CD4 binding (Fig. 3E), indicating a specific effect of cAMP on CXCR4
internalization. Similar effects were observed in isolated
CD4+ T cell cultures, with dbcAMP decreasing acid stable
anti-CXCR4 binding by an average of 44% (data not shown). Thus
cAMP appears to up-regulate cell surface expression of CXCR4 in part by
reducing receptor internalization rates.
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cAMP-induced CXCR4 up-regulation counteracts ligand-induced down-regulation
CXCR4 undergoes rapid internalization after binding its natural
ligand, SDF-1 (18, 31). To determine whether cAMP-induced up-regulation
of CXCR4 cell surface expression can offset ligand-induced
down-regulation, 100 ng/ml SDF-1 was added to PBMC costimulated for
24 h in either the presence or absence of dbcAMP. As shown in
Table II, 100 ng/ml SDF-1
suppressed CXCR4 cell surface expression within 5 min, and this
suppression persisted for at least 30 min (p =
0.007). dbcAMP (10 µM) (Table II) offset the effects of SDF-1
and restored cell surface CXCR4 expression to levels that did not
differ significantly from those observed on cells costimulated in the
absence of SDF-1 (p = 0.277). dbcAMP (100 µM)
(Table II) counteracted ligand-induced internalization effects
and elevated CXCR4 expression to levels significantly greater than
those observed on costimulated PBMC (untreated with either SDF-1 or
dbcAMP; p = 0.020). Thus cAMP up-regulation of CXCR4
counteracts ligand-induced CXCR4 suppression.
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for 1 h and then washed
twice and resuspended in fresh medium. Following suppression to 57% of
pre-SDF-1 levels, CXCR4 expression levels on costimulated PBMC
recovered by an average of 0.34% per minute during the first 60 min
following SDF-1 removal. In cells costimulated in the presence of
dbcAMP, CXCR4 recovery rates increased to 0.67% per minute
(p = 0.029). Thus cAMP up-regulation of cell
surface CXCR4 can influence both ligand-induced receptor
internalization and reexternalization following ligand removal.
cAMP-induced up-regulation of CXCR4 increases SDF-1-induced
chemotaxis
To explore the functional consequences of cAMP-induced
up-regulation of CXCR4, lymphocyte chemotactic response to SDF-1 was
assessed in membrane transmigration assays. In PBMC costimulated for
24 h, SDF-1 produced dose-dependent increases in lymphocyte
transmigration, with the fraction of transmigrating cells increasing by
5.8% with each 100 ng/ml increase in SDF-1 concentration (Fig. 4). Costimulation in the presence
of 100 µM dbcAMP increased transmigration rates by approximately
threefold (to 15.8% per 100 ng/ml, p < 0.0001) (Fig. 4). More pronounced effects were observed using the adenylyl cyclase
activator Forskolin (100 µM), which increased transmigration rates by
fivefold (28.6% per 100 ng/ml, p < 0.0001; data not
shown). Physiologic cAMP-inducing ligands also increased
SDF-1-induced chemotaxis, with 10 µM NE or PGE2 each
approximately doubling transmigration rates (p
= 0.004).
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on both sides of the membrane. PBMC showed minimal
chemokinetic response to SDF-1 (transmigration rates differed from
those of no-SDF controls by less than 5%), and dbcAMP did not
significantly alter these effects (data not shown). Thus cAMP
up-regulation of SDF-1-induced chemotaxis was not mediated by
increased random mobility. cAMP-induced up-regulation of CXCR4 increases vulnerability of CD4+ cells to HIV-1 infection
PCR was used to quantify reverse-transcribed proviral DNA at
14 h following infection by CXCR4-tropic HIV-1NL4–3.
When PBMC were infected following 24 h of costimulation, an
average of 4% bore proviral DNA 14 h later (Fig. 5A). Costimulation in the
presence of dbcAMP increased proviral penetrance by approximately
eightfold (Fig. 5A). These cAMP-induced increases in HIV-1
infectivity were mediated specifically by altered CXCR4 expression, as
demonstrated by the fact that cAMP effects on proviral penetrance could
be blocked by preincubating cells in the presence of the CXCR4-specific
ligand SDF-1 (1 mM), but not by parallel preincubation in the
presence of the CCR5 ligand MIP-1 (1 mM; see Fig. 5A).
Thus cAMP-induced up-regulation of CXCR4 expression increases
cellular vulnerability to infection by CXCR4-tropic HIV-1.
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B). Similar effects
were observed using physiologic inducers of cAMP (e.g., 10 µM NE
increased HIV-1 gene expression to 230% of costimulated control
levels, p = 0.022). To ensure that cAMP-induced
increases in HIV-1 gene expression did not reflect enhanced viral
spread, cells were cultured in the presence of an HIV-1 protease
inhibitor (100 nM Indinavir). PCR analysis of proviral load at 48
h postinfection confirmed that antiretroviral treatment prevented
spread of provirus beyond cells infected by 14 h (data not shown).
Thus cAMP-induced up-regulation of cell surface CXCR4 expression
renders T lymphocytes more vulnerable to productive infection by
CXCR-4-tropic HIV-1.
Kinetic analysis of HSA expression as a function of CXCR4 density
indicated that HSA initially appeared on cells expressing high
levels of CXCR4 (mean fluorescence intensity > 1000; data not
shown). As HSA intensity increased over time, CXCR4 levels declined,
with average expression dropping below 100 fluorescence intensity units
on cells showing maximal HSA expression (Fig. 5B). Such
results may arise from several possible mechanisms, including
down-regulation of CXCR4 expression by HIV gene expression and
differential survival of cells expressing high vs low levels of CXCR4.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and increased
vulnerability to infection by CXCR4-tropic HIV-1. cAMP up-regulates
CXCR4 expression on both CD4+ and CD8+ T
lymphocytes as well as on CD19+ B cells. In contrast to
these effects on lymphocytes, cAMP suppresses CXCR4 expression on
CD14+ cells by promoting CXCR4 internalization. These data
imply that the diverse family of extracellular factors that activate
the cAMP-PKA signaling pathway may function as natural modulators of
leukocyte traffic and localization and incidentally render
CD4+ T cells more vulnerable to infection with CXCR4-tropic
HIV-1. Because SDF-1 also regulates aspects of lymphopoiesis and tissue
development (55, 56), cAMP regulation of CXCR4 expression may have
additional implications for cellular maturation and morphogenesis. The present results suggest that cAMP may modulate lymphocyte circulation patterns by altering expression of chemokine receptors. Such results are consistent with evidence that cAMP-inducing ligands can selectively mobilize specific lymphocyte subsets into circulation while retaining other subsets in lymphoid organs (33, 34, 42). cAMP-inducing ligands are distributed both systematically via the endocrine system and locally via paracrine secretion from cells of the nervous and immune systems. Potential paracrine activators of the lymphocyte cAMP pathway in vivo include PGs and histamine secreted by myeloid cells at sites of inflammation (57, 58), catecholamines secreted by sympathetic nervous system neurons terminating in lymphoid organ parenchymal tissues (59), and ACTH secreted by activated lymphocytes (60).
Signaling via the cAMP-PKA pathway may interact with other
CXCR4-modulators to modify cellular localization in response to
antigenic signals. For example, CXCR4 expression on T lymphocytes
typically declines following cellular activation (Refs. 24 and 31, and
Fig. 1), and this effect is particularly pronounced for
CD45RA+ (naive) cells. This regulatory pathway may
facilitate migration of activated cells out of lymphoid organs and into
peripheral tissue sites (5). However, cAMP-inducing ligands may
counteract this effect and thus promote retention of activated
lymphocytes in areas of high SDF-1 concentration.
CXCR4 plays an important role in HIV pathogenesis by functioning in conjunction with CD4 as a coreceptor for virulent syncytium-inducing viral strains (9). Activation of the lymphocyte cAMP-PKA signaling pathway can accelerate HIV-1 replication (52, 53, 54). The present data suggest that cAMP effects on HIV replication may be mediated in part by increased cellular vulnerability to HIV infection as a function of up-regulated CXCR4 expression. Such effects may have significant clinical implications since the emergence of CXCR4-tropic HIV strains is associated with progression from chronic infection to the development of life-threatening illness (61, 62). Several cAMP-inducing mediators are increased during HIV infection (e.g., PGE; 63 , and exogenous HIV-1 proteins can increase intracellular cAMP levels (64, 65). Such effects could conceivably promote disease progression by increasing T lymphocyte vulnerability to infection and thereby facilitating replication of CXCR4-tropic viral strains. Such a dynamic may undermine the use of cAMP-inducing agents to suppress chemokine receptors on myeloid cells (as demonstrated above) as an antiviral strategy (66).
The present data indicate that cAMP up-regulates lymphocyte CXCR4 expression primarily by altering receptor compartmentalization. The negligible effects of cAMP on total CXCR4 pool size and receptor gene expression are consistent with the absence of any known cAMP response element in the CXCR4 promoter (67, 68). Although cAMP up-regulation of cell surface CXCR4 is mediated by altered receptor trafficking, the molecular mechanism of these effects remains to be clarified. Activated PKA may directly phosphorylate the receptor system itself (as in other receptor families; 30 , or it may interact with other signaling pathways that influence CXCR4 compartmentalization (69). Another outstanding question regards the teleologic rationale for receptor regulation by cAMP-inducing ligands. Many cAMP-inducing factors exert significant effects over lymphocyte function (e.g., PGs, catecholamines, ACTH), but their physiologic roles remain poorly understood. A more comprehensive understanding of both the sources and function of CXCR4 ligands may be helpful in elucidating the role of cAMP in the immune response more generally (1).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Steve W. Cole, Division of Hematology-Oncology, Department of Medicine, Factor 11-934, UCLA School of Medicine, Los Angeles, CA 90095-1678. E-mail address:
3 Abbreviations used in this paper: MIP-1, macrophage inflammatory protein-1; SDF-1, stromal cell-derived factor-1; PKA, protein kinase A; dbcAMP, N6,2'-O-dibutyryl adenosine-3',5'-cyclic monophosphate; ACTH, adrenocorticotropic hormone; HSA, heat-stable Ag; NE, norepinephrine.
Received for publication July 13, 1998. Accepted for publication October 26, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dependent internalization of the chemokine receptor CXCR4 contributes to inhibition of HIV replication. J. Exp. Med. 186:139., and MIP-1ß as the major HIV suppressive factors produced by CD8+ T cells. Science 270:1811.. Virology 194:345.[Medline]
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 J. B. Sundstrom, G. A. Hair, A. A. Ansari, W. E. Secor, A. M. Gilfillan, D. D. Metcalfe, and A. S. Kirshenbaum IgE-Fc{epsilon}RI Interactions Determine HIV Coreceptor Usage and Susceptibility to Infection during Ontogeny of Mast Cells J. Immunol., May 15, 2009; 182(10): 6401 - 6409. [Abstract] [Full Text] [PDF]
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 G. R. Campbell and S. A. Spector CCL2 Increases X4-tropic HIV-1 Entry into Resting CD4+ T Cells J. Biol. Chem., November 7, 2008; 283(45): 30745 - 30753. [Abstract] [Full Text] [PDF]
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 S. W. Cole Psychosocial Influences on HIV-1 Disease Progression: Neural, Endocrine, and Virologic Mechanisms Psychosom Med, June 1, 2008; 70(5): 562 - 568. [Abstract] [Full Text] [PDF]
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 E. K. Sloan, J. P. Capitanio, R. P. Tarara, S. P. Mendoza, W. A. Mason, and S. W. Cole Social Stress Enhances Sympathetic Innervation of Primate Lymph Nodes: Mechanisms and Implications for Viral Pathogenesis J. Neurosci., August 15, 2007; 27(33): 8857 - 8865. [Abstract] [Full Text] [PDF]
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V. Mariani, S. Gilles, T. Jakob, M. Thiel, M. J. Mueller, J. Ring, H. Behrendt, and C. Traidl-Hoffmann Immunomodulatory Mediators from Pollen Enhance the Migratory Capacity of Dendritic Cells and License Them for Th2 Attraction J. Immunol., June 15, 2007; 178(12): 7623 - 7631. [Abstract] [Full Text] [PDF]
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 E. K. Sloan, R. P. Tarara, J. P. Capitanio, and S. W. Cole Enhanced Replication of Simian Immunodeficiency Virus Adjacent to Catecholaminergic Varicosities in Primate Lymph Nodes J. Virol., May 1, 2006; 80(9): 4326 - 4335. [Abstract] [Full Text] [PDF]
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 P. Goichberg, A. Kalinkovich, N. Borodovsky, M. Tesio, I. Petit, A. Nagler, I. Hardan, and T. Lapidot cAMP-induced PKC{zeta} activation increases functional CXCR4 expression on human CD34+ hematopoietic progenitors Blood, February 1, 2006; 107(3): 870 - 879. [Abstract] [Full Text] [PDF]
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M. Chang, H. J. Brown, A. Collado-Hidalgo, J. M. Arevalo, Z. Galic, T. L. Symensma, L. Tanaka, H. Deng, J. A. Zack, R. Sun, et al. {beta}-Adrenoreceptors Reactivate Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication via PKA-Dependent Control of Viral RTA J. Virol., November 1, 2005; 79(21): 13538 - 13547. [Abstract] [Full Text] [PDF]
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B. E. Hurwitz, K. A. Brownley, S. J. Motivala, J. R. Milanovich, J. L. Kibler, L. Fillion, W. G. LeBlanc, M. Kumar, N. G. Klimas, M. A. Fletcher, et al. Sympathoimmune Anomalies Underlying the Response to Stressful Challenge in Human Immunodeficiency Virus Spectrum Disease Psychosom Med, September 1, 2005; 67(5): 798 - 806. [Abstract] [Full Text] [PDF]
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C.-A. Amella, B. Sherry, D. H. Shepp, and H. Schmidtmayerova Macrophage Inflammatory Protein 1{alpha} Inhibits Postentry Steps of Human Immunodeficiency Virus Type 1 Infection via Suppression of Intracellular Cyclic AMP J. Virol., May 1, 2005; 79(9): 5625 - 5631. [Abstract] [Full Text] [PDF]
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W. Wang, Z. Jobbagy, T. H. Bird, M. V. Eiden, and W. B. Anderson Cell Signaling through the Protein Kinases cAMP-dependent Protein Kinase, Protein Kinase C{epsilon}, and RAF-1 Regulates Amphotropic Murine Leukemia Virus Envelope Protein-induced Syncytium Formation J. Biol. Chem., April 29, 2005; 280(17): 16772 - 16783. [Abstract] [Full Text] [PDF]
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S. J. Curnow, K. Wloka, J. M. Faint, N. Amft, C. M. G. Cheung, V. Savant, J. Lord, A. N. Akbar, C. D. Buckley, P. I. Murray, et al. Topical Glucocorticoid Therapy Directly Induces Up-Regulation of Functional CXCR4 on Primed T Lymphocytes in the Aqueous Humor of Patients with Uveitis J. Immunol., June 1, 2004; 172(11): 7154 - 7161. [Abstract] [Full Text] [PDF]
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 O. Rohr, C. Marban, D. Aunis, and E. Schaeffer Regulation of HIV-1 gene transcription: from lymphocytes to microglial cells J. Leukoc. Biol., November 1, 2003; 74(5): 736 - 749. [Abstract] [Full Text] [PDF]
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Z. Ding, T. B. Issekutz, G. P. Downey, and T. K. Waddell L-selectin stimulation enhances functional expression of surface CXCR4 in lymphocytes: implications for cellular activation during adhesion and migration Blood, June 1, 2003; 101(11): 4245 - 4252. [Abstract] [Full Text] [PDF]
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V. Odemis, B. Moepps, P. Gierschik, and J. Engele Interleukin-6 and cAMP Induce Stromal Cell-derived Factor-1 Chemotaxis in Astroglia by Up-regulating CXCR4 Cell Surface Expression. IMPLICATIONS FOR BRAIN INFLAMMATION J. Biol. Chem., October 11, 2002; 277(42): 39801 - 39808. [Abstract] [Full Text] [PDF]
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 A. D. CRISTILLO, H. C. HIGHBARGER, R. L. DEWAR, D. S. DIMITROV, H. GOLDING, and B. E. BIERER Up-regulation of HIV coreceptor CXCR4 expression in human T lymphocytes is mediated in part by a cAMP-responsive element FASEB J, March 1, 2002; 16(3): 354 - 364. [Abstract] [Full Text] [PDF]
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A. la Sala, S. Sebastiani, D. Ferrari, F. Di Virgilio, M. Idzko, J. Norgauer, and G. Girolomoni Dendritic cells exposed to extracellular adenosine triphosphate acquire the migratory properties of mature cells and show a reduced capacity to attract type 1 T lymphocytes Blood, March 1, 2002; 99(5): 1715 - 1722. [Abstract] [Full Text] [PDF]
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 J. C. Boldrick, A. A. Alizadeh, M. Diehn, S. Dudoit, C. L. Liu, C. E. Belcher, D. Botstein, L. M. Staudt, P. O. Brown, and D. A. Relman Stereotyped and specific gene expression programs in human innate immune responses to bacteria PNAS, January 22, 2002; 99(2): 972 - 977. [Abstract] [Full Text] [PDF]
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 A. P. Kohm and V. M. Sanders Norepinephrine and beta 2-Adrenergic Receptor Stimulation Regulate CD4+ T and B Lymphocyte Function in Vitro and in Vivo Pharmacol. Rev., December 1, 2001; 53(4): 487 - 525. [Abstract] [Full Text] [PDF]
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S. W. Cole, B. D. Naliboff, M. E. Kemeny, M. P. Griswold, J. L. Fahey, and J. A. Zack Impaired response to HAART in HIV-infected individuals with high autonomic nervous system activity PNAS, October 23, 2001; 98(22): 12695 - 12700. [Abstract] [Full Text] [PDF]
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L. M. Ebert and S. R. McColl Coregulation of CXC Chemokine Receptor and CD4 Expression on T Lymphocytes During Allogeneic Activation J. Immunol., April 15, 2001; 166(8): 4870 - 4878. [Abstract] [Full Text] [PDF]
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 P. S. Mattila, A. Nykanen, M. Eloranta, and J. Tarkkanen Adenoids provide a microenvironment for the generation of CD4+, CD45RO+, L-selectin-, CXCR4+, CCR5+ T lymphocytes, a lymphocyte phenotype found in the middle ear effusion Int. Immunol., September 1, 2000; 12(9): 1235 - 1243. [Abstract] [Full Text] [PDF]
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H. Nagase, M. Miyamasu, M. Yamaguchi, T. Fujisawa, K. Ohta, K. Yamamoto, Y. Morita, and K. Hirai Expression of CXCR4 in Eosinophils: Functional Analyses and Cytokine-Mediated Regulation J. Immunol., June 1, 2000; 164(11): 5935 - 5943. [Abstract] [Full Text] [PDF]
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P. Secchiero, D. Zella, S. Curreli, P. Mirandola, S. Capitani, R. C. Gallo, and G. Zauli Engagement of CD28 Modulates CXC Chemokine Receptor 4 Surface Expression in Both Resting and CD3-Stimulated CD4+ T Cells J. Immunol., April 15, 2000; 164(8): 4018 - 4024. [Abstract] [Full Text] [PDF]
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M. J. Orsini, J.-L. Parent, S. J. Mundell, and J. L. Benovic Trafficking of the HIV Coreceptor CXCR4. ROLE OF ARRESTINS AND IDENTIFICATION OF RESIDUES IN THE C-TERMINAL TAIL THAT MEDIATE RECEPTOR INTERNALIZATION J. Biol. Chem., October 22, 1999; 274(43): 31076 - 31086. [Abstract] [Full Text] [PDF]
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