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*
Pulmonary Center, Boston University School of Medicine, Boston, MA 02118;
Department of Pharmacology, Utrecht University, Utrecht, The Netherlands; and
Washington and Jefferson College, Washington, PA 15301
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(MIP-1) stimulation,
prompted us to investigate whether IL-16 could also alter CXCR4
signaling. These studies demonstrate that IL-16/CD4 signaling in T
lymphocytes also results in loss of stromal derived factor-1
(SDF-1)/CXCR4-induced chemotaxis; however, unlike MIP-1/CCR5, the
effects were not reciprocal. There was no effect on
eotaxin/CCR3-induced chemotaxis. Desensitization of CXCR4 by IL-16
required at least 10–15 min pretreatment; no modulation of CXCR4
expression was observed, nor was SDF-1 binding altered. Using murine
T cell hybridomas transfected to express native or mutated forms of
CD4, it was determined that IL-16/CD4 induces a
p56lck-dependent inhibitory signal for CXCR4,
which is independent of its tyrosine catalytic activity. By contrast,
IL-16/CD4 desensitization of MIP-1/CCR5 responses requires
p56lck enzymatic activity. IL-16/CD4 inhibition
of SDF-1/CXCR4 signals requires the presence of the Src homology 3
domain of p56lck and most likely involves
activation of phosphatidylinositol-3 kinase. These studies indicate the
mechanism of CXCR4 receptor desensitization induced by a natural ligand
for CD4, IL-16, is distinct from the inhibitory effects induced by
either gp120 or IL-16 on CCR5. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Functionally, binding of gp120 to CD4 results in subsequent
unresponsiveness to either macrophage-inflammatory protein-1
(MIP-1) (via CCR5) (12) or stromal derived factor-1
(SDF-1) (via CXCR4) chemokine stimulation (13). The
mechanism for this effect appears to be as a result of gp120-induced
down-modulation of surface-expressed CCR5 or CXCR4 proteins. Loss of
chemokine receptor expression and function following gp120 stimulation
is dependent on phosphorylation of the CD4-associated tyrosine kinase,
p56lck (13).
For many biological systems, the effects of gp120 stimulation mediated
through CD4 (8, 14, 15, 16, 17) are similar to the effects induced
following binding of a natural ligand for CD4, IL-16 (18, 19). A recent report has suggested that murine monocytes have an
alternative receptor for IL-16 (20), and therefore, we
have concentrated our current work on the interplay between IL-16/CD4
and chemokine receptors specifically in T cells. Along those lines, we
have recently demonstrated that IL-16 has the selective ability to
induce T cell unresponsiveness to MIP-1/CCR5 stimulation; however,
unlike gp120, does not affect signaling induced by monocyte
chemoattractant proteins (MCP) 1 (via CCR2), 2 (via CCR1, 2, 3, and 5),
and 4 (via CCR2 and 3) (21). This effect of IL-16 is not
mediated by loss of CCR5 membrane expression, nor by steric inhibition
of MIP-1 binding, but is mediated through the induction of
intracellular signals that block CCR5 signaling. Similar to gp120, the
inhibitory activity of IL-16/CD4 on CCR5 also requires enzymatic
activity of p56lck (21).
Interestingly, the inhibition is reciprocal, as pretreatment with
MIP-1 results in loss of responsiveness to IL-16 stimulation
(21).
In the present studies, we investigated whether IL-16 pretreatment
could also induce unresponsiveness to the other major chemokine
receptor utilized by HIV-1 binding, CXCR4 (22). Gp120
pretreatment, similar to its effects on CCR5, results in
p56lck enzymatic activity-dependent modulation
of CXCR4 associated with subsequent T cell unresponsiveness to SDF-1
stimulation (13). Our results indicate that IL-16
pretreatment does result in a transient unresponsiveness to
CXCR4/SDF-1 stimulation; however, unlike CCR5, the effect is not
reciprocal. In addition, we demonstrate that the presence and enzymatic
activity of the SH1 domain of p56lck are not
required, but that the inhibitory signal mediated through
p56lck is transduced by the Src homology (SH) 3
domain and most likely involves activation of phosphatidylinositol-3
(PI3) kinase. These studies identify a natural functional relationship
between CD4 and CXCR4, initiated by a natural ligand for CD4, which is
mechanistically different from the CD4/CCR5 relationship.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human and murine SDF-1, IL-16, and eotaxin were
purchased from Biosource International (Camarillo, CA).
Fluorescein-conjugated anti-CXCR4 Ab was purchased from PharMingen
(San Diego, CA). Unconjugated anti-IL-16 Ab (clone 14.1) was
isolated from hybridoma supernatants, purified using protein A affinity
chromatography, and used at a concentration of 5 µg/ml, which is
sufficient to neutralize 10-10 M rIL-16-induced
migration of human T cells (23). HIV-1
gp120IIIB was purchased from Intracel
(Issaquah, WA).
Cell culture
Normal human T lymphocytes were isolated from the blood of healthy volunteers using Hypaque-Ficoll separation of PBMCs, as previously described (19, 20). Preparations were enriched for T lymphocytes by nylon wool adherence. The nonadherent mononuclear cells were >95% T lymphocytes, as determined using flow cytometry to assess CD3+ cells. Enriched CD4+ T cells were generated as previously described (24). Briefly, the cells were then mixed with magnetic beads conjugated with anti-CD8 mAbs. Following incubation with the beads for 1 h at 37°C on a shaker, the CD4+ T cells were then isolated by negative selection using a strong magnet. The cells were cultured in medium 199 supplemented with 25 mM HEPES buffer, 100 U/ml penicillin, 100 µg/ml streptomycin, and containing 10% FBS for 18–24 h before use in the chemotaxis assay.
For the studies using herbimycin A, 2 x 106 cells/ml were incubated with herbimycin A (1 µM final concentration; Calbiochem, San Diego, CA) for 18 h before washing by centrifugation and resuspension with culture media. Following washing, rIL-16 (10-10 M) was added to the cells for 15 min, and the cells were washed again and resuspended at 8–10 x 106 cells/ml for use in the chemotaxis assay.
For the PI3 kinase inhibition experiments, cells were incubated with
either wortmannin (1 or 10 nM) for 18–24 h, followed by a 1-h
incubation period with IL-16, Ly294002 (10 or 40 µM; Santa Cruz
Biotechnology, Santa Cruz, CA) for 2 h, with IL-16 added for the
last hour of pretreatment. After washing, the cells were then subjected
to an SDF-1 dose response for induction of cell migration.
Murine T cell hybridoma cell lines
All CD4-expressing murine T cell hybridomas were generated as previously described (25, 26, 27) and were a generous gift from Steven J. Burakoff (Dana-Farber Cancer Institute, Boston, MA). To generate these cells, the L3T4-negative murine T cell hybridoma cell line By155.16 was infected with the mononuclear cell retroviral vector containing a neomycin resistance gene, a CMV promoter, and the gene for human CD4. Various constructs of CD4 were used to express either wild-type CD4 or point mutations corresponding to a Cys420 to Ser mutation, or a Cys430 to Ser mutation (25, 26). The mononuclear cell-CD4 transfectants were selected and assessed for CD4 surface expression. All cell lines were assessed for CD4 expression, as determined by anti-CD4 Ab binding and FACS analysis, and only those cell lines with comparable CD4 levels were used. In some studies, cells were infected to express a CD4/p56lck chimeric protein. Chimeric molecules containing the extracellular and transmembrane domains of CD4 directly ligated to different constructs of p56lck were expressed in the By155.16 cell line (27, 28) as either an intact p56lck molecule (full length); a truncated molecule containing only the N, SH2, and SH3 domains (N32); or a molecule comprised of only the N and SH3 domains (N3). Neomycin-resistant clones were screened for surface expression of CD4 by flow cytometric analysis using a FACScan (Becton Dickinson, Mountain View, CA), and only cells expressing comparable levels of CD4 were used. All cells were grown and maintained in RPMI 1640 medium (Sigma, St. Louis, MO) containing 200 U/ml penicillin, 200 µg/ml streptomycin, 2 mM glutamine, 20 mM HEPES (pH 7.4), and 10% FBS.
Lymphocyte chemotaxis assay
In vitro chemotaxis was performed as described previously (18, 29). Isolated CD4+ T cells were pretreated with rIL-16 (10-10 M) or control media for 0–1 h, depending on the experiment, at 37°C, then washed by centrifugation and resuspension in media twice before use in the chemotaxis assay. In the specificity experiments, the T cells were incubated under the same conditions with rIL-16 (10-10 M) and 10 µg/ml IL-16 mAb (14.1), an amount sufficient to neutralize 10-10 M rIL-16 activity (23). Migration was assessed using a 48-well microchemotaxis chamber with 8-µm nitrocellulose membrane filters used as the migration matrix (Neuroprobe, Cabin John, MD). Human CD4+ T lymphocytes (8–10 x 106/ml) or T cell hybridomas (5 x 106/ml), either with or without pretreatment with IL-16, were loaded into the upper well of the chamber, with 32 µl of various concentrations of chemoattractant or control buffer placed in the lower well. Chambers were incubated for 2 h for the human cells or 4 h for the T cell hybridomas (27), after which the filters were fixed in ethanol, stained with hematoxylin, and dehydrated by sequential washes in ethanol, propanol, and xylene. Cell migration was quantitated using light microscopy to visualize the number of cells that had migrated beyond a depth of 50 µm. All migration is expressed as percentage values of cell migration in control media (designated as 100%), and statistics were calculated using Student’s t test. As IL-16 is a chemoattractant factor, baseline migration was established as the migration of cells pretreated with IL-16, washed, and then stimulated with media alone. All samples were tested in duplicate, and four high-power fields were examined in each duplicate. Data are the mean value ± the SD of three or more experiments.
FACScan analysis
Surface expression of chemokine receptor was accomplished using a previously reported method (21). Briefly, CXCR4 expression was analyzed using fluorescein-conjugated anti-CXCR4 Abs (clone C-20; Santa Cruz Biotechnology). Human T cells (1 x 106 cells/ml) were either left untreated or pretreated with IL-16 (10-10 M) or with gp120IIIB (5 µg/ml) for 1, 4, 12, or 24 h, washed, and resuspended in staining buffer (PBS with 0.4% BSA and 0.1% sodium azide), and incubated with 0.25 µg of labeled Ab for 30 min at 4°C. Cells were then washed three times in cold PBS, resuspended and fixed with 10% Formalin, and analyzed with a FACScan (Becton Dickinson).
Binding assays
Binding assays were conducted, as previously described
(13), by stimulating 2 x 106
human T cells in 200 µl of culture media with either rIL-16 (10–1000
pg) or cold SDF-1 (10–1000 pg) for 30 min at room temperature
before the addition of labeled SDF-1. Radiolabeled
125I-SDF-1 (0.12 nM, 185 kBq; New England
Nuclear, Boston, MA) was added to each sample for 120 min at room
temperature. Following a 120-min incubation, the samples were quickly
aspirated through GF/C microfiber filters (Whatman, Maidstone, U.K.)
using a vacuum harvester. The filters were air dried and counted in a
gamma counter. Nonspecific radioactivity present with 10–1000-fold
excess of cold SDF-1 was subtracted from total bound counts for each
dose and used to calculate specifically bound counts. Percent
inhibition of SDF-1 binding induced by IL-16 or cold SDF-1 was
calculated by subtracting counts from IL-16- or SDF-1-treated cells
from specific counts, divided by specific binding counts, and
multiplied by 100%.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/CXCR4-induced chemotaxis mediated by rIL-16;
specificity and time course
Recent studies have identified that IL-16 pretreatment results in
selective desensitization of MIP-1/CCR5-induced chemotaxis
(21). We continued these observations by investigating
whether IL-16 stimulation could also desensitize another CD4-related
chemokine receptor, CXCR4. Human CD4+ T cells
were isolated and then incubated with IL-16
(10-10 M) for 1 h before washing and
stimulation by the CXCR4-specific ligand SDF-1. As shown in Fig. 1
A, SDF-1 induced a
migratory response in the T cells in a dose-dependent fashion. Cells
prestimulated with IL-16, however, did not respond to any concentration
of SDF-1 (Fig. 1A). This effect was selective, as
previous studies have identified no IL-16 effect on MCP-1-, MCP-2-, and
MCP-4 (21)-induced migration, and for these studies we
also demonstrate that migration to eotaxin, mediated through CCR3 was
not altered (Fig. 1A). Coincubation of IL-16 with
neutralizing concentrations of anti-IL-16 before pretreatment of
the T cells completely eliminated the inhibitory effect of IL-16 on
SDF-1 stimulation (data not shown).
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for 1 h before assessing for
IL-16-induced chemotaxis. Concentrations of SDF-1 used for
pretreatment ranged from 1 to 100 ng/ml; however, none of the
concentrations used were found to have any effect on IL-16 stimulation
(Fig. 1
B, only the 100 ng/ml concentration of SDF-1 is
shown).
A time course was then conducted to establish the minimal amount of
time required for IL-16/CD4 stimulation to cross-desensitize
CXCR4-induced migration. Human CD4+ T cells were
incubated with IL-16 (10-10 M) for a variety of
time points up to 1 h. The cells were then washed and assessed for
SDF-1-induced migration. As shown in Fig. 2A, receptor
cross-desensitization induced by IL-16 required between 10 and 15 min
of IL-16 stimulation, and was maximal by 60 min. To determine whether
the inhibitory effect was reversible, cells were incubated with IL-16
for 1 h, washed three times in media, and then incubated in
culture media alone for varying lengths of time up to 48 h before
subjected to SDF-1-induced chemotaxis. As shown in Fig. 2B, the cells required 18–24 h of incubation before they
demonstrated a normal dose response to SDF-1 stimulation, indicating
a transient effect by IL-16.
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A, CXCR4 expression was not
altered by IL-16 stimulation. CXCR4 expression was monitored for up to
24 h following IL-16 stimulation, with no observable changes (data
not shown). Similarly, there was no detectable induction of CXCR4
phosphorylation at either 1 or 24 h following IL-16 stimulation
(data not shown). The cells were responsive to gp120 stimulation,
however, as CXCR4 expression was significantly reduced following
exposure to 5 µg/ml gp120 for 1 h (Fig. 3A).
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binding, and/or that IL-16/CD4
signaling results in a conformational change in CXCR4, which then
reduces the avidity for SDF-1 binding. To address these
possibilities, binding studies were conducted with radiolabeled
SDF-1 in the presence of cold SDF-1 or IL-16. Human
CD4+ T cells were incubated with an increasing
concentration of either SDF-1 or IL-16 for 1 h. Without
washing, radiolabeled SDF-1 (0.1 nM) was then added for an
additional hour, and bound counts were analyzed. As shown in Fig. 3B, increasing concentrations of cold SDF-1 inhibited
bound counts by up to 80%, while IL-16, for all concentrations used,
was unable to alter SDF-1 binding. Taken together, these data
indicate that IL-16 stimulation results in loss of CXCR4
responsiveness, but that neither CXCR4 expression nor the ability of
SDF-1 to bind to CXCR4 is altered by the IL-16/CD4 interaction. In
addition, these studies indicate that while both gp120 and IL-16 bind
to CD4 and are capable of inhibiting SDF-1/CXCR4 signaling, the
mechanism for inhibition is different for the two CD4 ligands. CD4-p56lck association is required for IL-16-induced inhibition
The findings that IL-16 stimulation does not result in loss of
surface-expressed CXCR4 protein nor alters SDF-1 binding suggest
that IL-16/CD4 association generates a signal that results in the
inhibition of CXCR4-induced chemotaxis. To further define the mechanism
by which IL-16 inhibits CXCR4 signaling, murine T cell hybridomas were
retrovirally infected to express point-mutated human CD4 molecules. As
previously reported (25, 26), L3T4-negative T cells were
infected to express either wild-type CD4; a
Cys420 to Ser (CS420); or
Cys430 to Ser (CS430)
point-mutated CD4 molecules. Cysteine at position 420 is essential for
a CD4/p56lck association, and, therefore,
mutation of Cys420 to
Ser420 results in the disruption of this
association (25, 26). Cysteine at position 430 is outside
of the binding region, and therefore, the CS430
point mutation does not alter this association. We have previously used
these cells to demonstrate that a CD4/p56lck
association is required to confer IL-16-induced cell migration and
second messenger signaling (27). To determine the effects
of IL-16 on CXCR4-induced migration, it was first established that
murine SDF-1 could induce a migratory response in these cell lines
similar to what was observed for human CD4+ T
cells. As shown in Fig. 4, SDF-1
induced a migratory response in a dose-dependent fashion. Cells
expressing wild-type CD4, CS420, or
CS430 were then exposed to IL-16 for 1 h and
then stimulated with SDF-1. IL-16, as anticipated, blocked
SDF-1-induced migration in cells expressing the wild-type or
CS430 CD4 molecules; however, there was no IL-16
effect in the CS420-expressing cells (Fig. 4).
This finding was consistent with the hypothesis that an
IL-16/CD4-generated signal was inducing the observed inhibition, and,
furthermore, that a CD4/p56lck association was
required to transmit the inhibitory signal.
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We have previously reported that IL-16-initiated inhibition of
CCR5 signaling requires enzymatic activity of
p56lck (21). To determine whether
the inhibitory effects of IL-16 on CXCR4 signaling were also dependent
on p56lck enzymatic activity, we incubated
isolated human T cells with the specific src tyrosine kinase
inhibitor herbimycin A. These studies are feasible because cell
migration induced directly by SDF-1/CXCR4 is not herbimycin A
sensitive (data not shown). Human CD4+ T cells
were treated with herbimycin A (1 µM) for 1 h before the
addition of IL-16 for an additional hour. The cells were then assessed
for SDF-1-induced cell migration. Unlike CCR5 inhibition, herbimycin
A treatment did not reverse the desensitization effect of IL-16 (Fig. 5A). To demonstrate that
herbimycin A treatment was sufficient to eliminate enzymatic activity
of p56lck, herbimycin A-pretreated cells were
also pretreated with gp120 before SDF-1 stimulation. Under these
conditions, herbimycin A completely ablated the gp120-induced
inhibitory effect compared with cells treated with herbimycin alone
(Fig. 5B). We next assessed whether herbimycin A treatment
had an effect on gp120-induced modulation of CXCR4 expression. Flow
cytometric analysis of cell surface expression of CXCR4 indicated that
herbimycin A treatment did prevent gp120-induced loss of CXCR4 (Fig. 5C). Therefore, unlike the effects of gp120,
p56lck enzymatic activity does not appear to be
essential for transmission of the IL-16/CD4-mediated inhibitory
signal.
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(Fig. 6) or eotaxin (data not shown)
stimulation. The same dose response was repeated following a 1-h
incubation in the presence of IL-16 (10-10 M).
Similar to our findings with primary T cells, hybridoma cells
expressing the wild-type p56lck construct (full
length), exposed to IL-16, were subsequently unresponsive to SDF-1
stimulation (Fig. 6). Also similar to the data generated with primary T
cells, cellular response to eotaxin stimulation was unaffected by IL-16
pretreatment (data not shown). Cells expressing the SH1 deletional
mutation (N32) also demonstrated an IL-16-induced inhibitory effect.
This finding is consistent with the data generated on human T cells
following incubation with herbimycin A (Fig. 5). Interestingly, cells
expressing only the SH3 domain (N3) also demonstrated inhibitory
activity following IL-16 pretreatment (Fig. 6). This indicates that the
SH3 domain is at least in part capable of transmitting the inhibitory
signal. These data do not exclude the potential contribution by other
domains, such as SH2. The N domain is not involved in transmitting the
inhibitory effect of IL-16, as cells expressing only the N domain are
unresponsive to IL-16 pretreatment (Fig. 6).
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The ability of the SH3 domain to transmit the inhibitory signal is
similar to preliminary data identifying that the SH3 domain is involved
in transmission of an IL-16-induced migratory signal (T. Ryan,
unpublished observations). Those studies also suggest that the
migratory signal transduced by the SH3 domain is dependent on PI3
kinase activity. To determine whether PI3 kinase is also involved in
transmission of the CXCR4-inhibitory signal, human T cells were
incubated with the PI3 kinase-specific inhibitors, wortmannin or
Ly294002 (29). In these studies, T cells were incubated
with either wortmannin (1 nM, for 18–24 h) or Ly294002 (40 µM, for
1 h), followed by IL-16 pretreatment for 1 h before
conducting a SDF-1 dose response. As shown in Fig. 7, both wortmannin- and Ly294002-treated
cells demonstrated a partial reduction in the dose response as compared
with no inhibitor treatment, and support previous studies indicating
that SDF-1 signaling is in part PI3 kinase dependent (30, 31). When cells were pretreated with either inhibitor and IL-16,
response to SDF-1-induced chemotaxis was identical to the dose
response of cells pretreated with inhibitor alone, indicating that the
inhibitory effect of IL-16 was blocked (Fig. 7). These data suggest
that the IL-16/CD4/p56lck-transmitted inhibitory
signal of CXCR4 involves PI3 kinase activity. Cells pretreated with
higher concentrations of wortmannin (10 nM) or Ly294002 (80 µM) were
completely unresponsive to SDF-1 stimulation (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/CCR5 or SDF-1/CXCR4 signaling has
been well defined. Following binding by gp120, CCR5, CXCR4, as well as
CD4 are modulated from the cell surface, resulting in cellular
unresponsiveness to chemokine stimulation. This effect requires
enzymatic activity of the src kinase family member,
p56lck, mediated through the SH1 domain of
p56lck (13). The ability to
cross-desensitize chemokine signaling is not restricted to the CD4
ligand gp120, nor is it restricted mechanistically to the modulation of
chemokine receptors from the cell surface. We have previously reported
that another CD4 ligand, IL-16, could also desensitize CCR5 signaling
(21). Although IL-16, similar to gp120, requires enzymatic
activity of p56lck, unlike gp120, IL-16 does not
induce modulation of CCR5 from the cell surface. The CD4-CCR5
desensitization relationship is also reciprocal, whereby MIP-1
pretreatment results in cellular unresponsiveness to IL-16/CD4
stimulation (21). This indicates that the relationship
between CD4 and CCR5, first identified by gp120, exists also for the
natural ligands for CD4 and CCR5. The present studies were conducted to
investigate whether a similar relationship existed for CD4 and
CXCR4.
Our studies indicate that, like CCR5, a functional relationship does
exist for CD4 and CXCR4, which is facilitated by IL-16. Although there
are similarities between the effects of IL-16 on CXCR4 and CCR5
(inhibition of induced migration, time course required for inhibition,
and lack of an effect on chemokine receptor expression or chemokine
binding), there are some major differences. The effects of IL-16 on
SDF-1/CXCR4 signaling are not reciprocal. Pretreatment of primary T
cells or T cell hybridomas with SDF-1 had no effect on IL-16-induced
migration. This lack of reciprocity with CXCR4 may relate to the
findings that CD4 and CXCR4 are not constitutively associated, whereas
reciprocity is present for CCR5 and CD4, in which constitutive
association has been reported (11). Although gp120 is
capable of inducing CD4-CXCR4 receptor complex formation, it has not as
yet been determined whether IL-16 stimulation results in the induction
of a similar complex.
A second major difference between the interaction of CD4 with CXCR4 as
compared with that for CCR5 is that p56lck
enzymatic activity is not required to inhibit SDF-1/CXCR4 signaling.
Rather, based on the studies using p56lck
deletional mutant cell lines and on the PI3 kinase inhibitor
experiments, the inhibitory signal is transmitted, at least in part,
through the interaction of the SH3 domain and appears to require the
involvement of PI3 kinase. Interestingly, preliminary studies have
indicated that an IL-16/CD4-mediated migratory response in T cells is
also transmitted via the SH3 domain of p56lck,
which results in activation of PI3 kinase (T. Ryan, unpublished
observations). The preliminary observation that IL-16-induced migration
also involves PI3 kinase activation suggests common signaling pathways
for these two chemoattractant cytokines, despite SDF-1 using a
seven-membrane, G protein-associated receptor (CXCR4) and IL-16 using a
type I Ig family receptor (CD4). Without direct evidence for
IL-16-induced CD4-CXCR4 receptor complex formation, common use of
intracellular signaling pathways, such as depletion or occupancy of
intracellular pools of active kinases, is a potential mechanism for
receptor cross-desensitization.
The inhibitory effects of IL-16 are selective. We have previously demonstrated that IL-16 stimulation lacks an effect on MCP-1, MCP-2, or MCP-4 stimulation. In these studies, we identify that pretreatment of primary T cells by IL-16 did not effect the migratory response induced by eotaxin stimulation. Eotaxin binds exclusively to the chemokine receptor CCR3 and, similar to CCR5 and CXCR4, has been shown to facilitate HIV entry into CD4+ T cells (32). The lack of an effect on eotaxin/CCR3 stimulation by IL-16/CD4 indicates that the specificity of IL-16-inhibitory activity is not completely coincident with the subset of CD4-chemokine coreceptors, as defined by HIV-1 gp120 binding and internalization. Further studies are required to fully elucidate the spectrum of chemokine receptors that are affected by IL-16 signaling.
At present, we can only speculate as to the biological importance of
these findings. Clearly, the process of leukocyte migration from
circulation to sites of inflammation is complex and involves a variety
of stimuli acting either concomitantly or sequentially on selectively
recruited cells. As a result of exposure to multiple microenvironments,
proper interpretation of sequential stimuli is essential for directed
and efficient cellular recruitment. Previous reports have identified
both positive and inhibitory effects of one chemoattractant on
subsequent stimulation by a second chemoattractant
(33, 34, 35, 36). Foxman et al. (36) have reported on
the ability of neutrophils to maintain their ability to respond to
IL-8-induced chemotaxis even after preexposure to another neutrophil
chemoattractant leukotriene B4. There was also no
inhibitory effect when the chemoattractants were reversed. In contrast,
sequential stimulation of neutrophils by fMLP and IL-8 identifies a
different regulatory process (34). Pretreatment of
neutrophils with IL-8 has no effect on subsequent stimulation by fMLP;
however, fMLP stimulation totally abrogated the adhesive and
chemoattractant responses to subsequent IL-8 stimulation. Our data
indicate that this same paradigm exists for IL-16/CD4 and CXCR4,
whereby CD4+ T cells would be transiently unable
to respond to SDF-1 as a secondary stimulus following IL-16
stimulation.
In summary, our findings suggest that there is a functional
relationship between two chemoattractant cytokines of completely
different classes, IL-16 and SDF-1. A regulatory component exists
whereby pretreatment with IL-16 results in cross-desensitization of
SDF-1. The mechanism for the IL-16 effect on CXCR4 appears to be
quite distinct from the mechanism used by HIV-1 gp120, as inhibition
occurs as a result of a p56lck-independent
inhibitory signal, which is also in sharp contrast to the mechanism for
IL-16-induced inhibition of CCR5. As both of these cytokines have been
detected coincidently in association with inflammation, it is feasible
to hypothesize that this represents an adaptive process to restrict and
regulate recruitment of immune cells to sites of inflammation.
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Footnotes |
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2 C.V.D. and A.J. contributed equally to this manuscript.
3 Address correspondence and reprint requests to Dr. William Cruikshank, Pulmonary Center, R-304, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118.
4 Abbreviations used in this paper: CXCR, CXC chemokine receptor; gp, glycoprotein; MCP, monocyte chemoattractant protein; MIP, macrophage-inflammatory protein; PI3, phosphatidylinositol 3; SDF, stromal-derived factor; SH, Src homology.
Received for publication February 7, 2000. Accepted for publication August 31, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, MIP-1, as the major HIV-suppressive factors produced by CD8+ T cells. Science 270:1811., respectively. J. Immunol. 163:3123.-chemokine, stromal cell-derived factor-1, binds to the transmembrane G-protein-coupled CXCR-4 receptor and activates multiple signal transduction pathways. J. Biol. Chem. 273:23169.-induced lymphocyte polarization and chemotaxis. J. Immunol. 163:4001.-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 85:1135.[Medline]
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E. A. Lynch, C. A. W. Heijens, N. F. Horst, D. M. Center, and W. W. Cruikshank Cutting Edge: IL-16/CD4 Preferentially Induces Th1 Cell Migration: Requirement of CCR5 J. Immunol., November 15, 2003; 171(10): 4965 - 4968. [Abstract] [Full Text] [PDF]
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M. Inngjerdingen, B. Rolstad, and J. C. Ryan Activating and Inhibitory Ly49 Receptors Modulate NK Cell Chemotaxis to CXC Chemokine Ligand (CXCL) 10 and CXCL12 J. Immunol., September 15, 2003; 171(6): 2889 - 2895. [Abstract] [Full Text] [PDF]
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 F. F. Little, E. Lynch, G. Fine, D. M. Center, and W. W. Cruikshank Tumor Necrosis Factor-{alpha}-Induced Synthesis of Interleukin-16 in Airway Epithelial Cells: Priming for Serotonin Stimulation Am. J. Respir. Cell Mol. Biol., March 1, 2003; 28(3): 354 - 362. [Abstract] [Full Text] [PDF]
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 M. Inngjerdingen, K. M. Torgersen, and A. A. Maghazachi Lck is required for stromal cell-derived factor 1alpha (CXCL12)-induced lymphoid cell chemotaxis Blood, May 29, 2002; 99(12): 4318 - 4325. [Abstract] [Full Text] [PDF]
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 A. Z. Fernandis, R. P. Cherla, R. D. Chernock, and R. K. Ganju CXCR4/CCR5 Down-modulation and Chemotaxis Are Regulated by the Proteasome Pathway J. Biol. Chem., May 10, 2002; 277(20): 18111 - 18117. [Abstract] [Full Text] [PDF]
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C. Bandeira-Melo, K. Sugiyama, L. J. Woods, M. Phoofolo, D. M. Center, W. W. Cruikshank, and P. F. Weller IL-16 Promotes Leukotriene C4 and IL-4 Release from Human Eosinophils via CD4- and Autocrine CCR3-Chemokine-Mediated Signaling J. Immunol., May 1, 2002; 168(9): 4756 - 4763. [Abstract] [Full Text] [PDF]
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F. F. Little, W. W. Cruikshank, and D. M. Center IL-9 Stimulates Release of Chemotactic Factors from Human Bronchial Epithelial Cells Am. J. Respir. Cell Mol. Biol., September 1, 2001; 25(3): 347 - 352. [Abstract] [Full Text] [PDF]
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B.-Q. Li, M. A. Wetzel, J. A. Mikovits, E. E. Henderson, T. J. Rogers, W. Gong, Y. Le, F. W. Ruscetti, and J. M. Wang The synthetic peptide WKYMVm attenuates the function of the chemokine receptors CCR5 and CXCR4 through activation of formyl peptide receptor-like 1 Blood, May 15, 2001; 97(10): 2941 - 2947. [Abstract] [Full Text] [PDF]
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) or IL-16 (
) for 30 min before the
addition of radiolabeled SDF-1
) a 1-h pretreatment with IL-16 (10-10 M). The
data are expressed as percent control migration, in which control
migration is determined by the migration of unstimulated cells for each
CD4 construct. The asterisk denotes significantly different migration
in the treated cells as compared with migration seen in the untreated
cells, p < 0.05. The data represent the averages
of five separate experiments.
); or with IL-16 pretreatment only
(|og). This experiment was conducted five separate times, and the data
are expressed as the averages for those experiments. The asterisk
represents migration that was statistically different from the
migration observed for untreated cells for the same concentration of
SDF-1