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INTRODUCTION |
CD26 is a widely distributed lymphocyte activation antigen that
displays dipeptidyl peptidase IV activity in its extracellular domain.
The role of CD26 in HIV1
infection has been controversial. Its postulated role as coreceptor for
HIV entry (1) was rapidly contested (2, 3). However, it has been more
recently shown that the expression of CD26 may modulate HIV infection
by direct effects of dipeptidyl peptidase IV activity on the
antiviral activity of several chemokines or by unknown mechanisms
independent of peptidase activity (4-7).
In fact, CD26 can cleave out NH2-terminal dipeptides from a
protein with either L-proline or L-alanine at
the penultimate position and several chemokines can be processed by
CD26 (see Ref. 8 for review). For some chemokines proteolysis by CD26 does not affect the physiological activity. In contrast, truncation of
RANTES (regulated on activation normal T cell expressed and secreted)
by CD26 leads to higher receptor selectivity and anti-HIV activity (9,
10). Stromal cell-derived factor 1
(SDF-1
) also loses the
NH2-terminal Lys-Pro dipeptide by the action of CD26. This
processing significantly reduces the chemotactic and anti-HIV-1
activity of SDF-1
(4, 11).
Besides its enzymatic activity, CD26 interacts physically with
membrane-bound signal-transduction molecules. Thus, the group of
Morimoto has reported that CD26 associates to CD45, a membrane-linked protein tyrosine phosphatase (12) and adenosine deaminase (ADA; Ref.
13), which is a cytosolic globular protein that also appears on the
surface of lymphocytes (14). It has been shown that the binding of
iodinated ADA to CD26 is inhibited by HIV-1 particles and by the viral
envelope glycoprotein gp120, with IC50 values in the
nanomolar range. This inhibition is mediated by the C3 region of the
gp120 protein and occurs in human T and B cell lines by a mechanism
modulated by CD4 and CXCR4 expression (15, 16).
Given the functional relationship between dipeptidyl peptidase IV
activity of CD26 and CXCR4, and the role of CXCR4 in gp120-mediated inhibition of ADA binding to CD26, we have studied the distribution of
these proteins on the surface of human lymphocytes. In this paper we
present evidence of a colocalization, coimmunoprecipitation, and
comodulation of CD26 and CXCR4. On the other hand, there is a
redistribution of these cell surface proteins by the action of gp120 or
SDF-1
, which is the natural ligand of CXCR4. gp120-induced redistribution of CXCR4 and CD26 also affects cell surface ADA.
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EXPERIMENTAL PROCEDURES |
Materials--
Fluorescein isothiocyanate (FITC),
paraformaldehyde, and bovine serum albumin were purchased from
Sigma. Bovine ADA (type VIII, Sigma) was filtered through
Sephadex G-25. SDF-1
was purchased from R & D Systems
(Minneapolis, MN). Glycine and electrophoresis reagents were obtained
from Roche Molecular Biochemicals (Barcelona, Spain). The recombinant
HIV-1 envelope glycoprotein gp120 from viral isolate IIIB (construct
deposited by Dr. Jones) was produced in the baculovirus expression
system and was kindly provided by the Medical Research Council AIDS
Directed Program Reagent Project (Potters Bar, United Kingdom (UK)).
The recombinant gp120IIIB conjugated to FITC was from
Neosystem Isochem (Strasbourg, France). Sephadex G-25 fine grade
columns were obtained from Amersham Pharmacia Biotech (Uppsala,
Sweden). Deionized water further purified with a Millipore Milli-Q
system (Bedford, OH) was used throughout.
Cells and Transfections--
Peripheral blood mononuclear cells
from healthy donors were purified by Ficoll-Hypaque sedimentation and
used immediately after purification. The human B lymphoblast cell line
SKW6.4 (obtained from Dr. J. Moyano Laboratori d'Investigació,
MERCK Química, Barcelona, Spain) and the human T cell line
Jurkat J32 were grown in RPMI 1640 medium (Life Technologies, Inc.)
supplemented with 10% inactivated fetal bovine serum, 2 mM
L-glutamine, and antibiotics at 37 °C in a humid
atmosphere of 5% CO2. The human glioblastoma cell line
U373-MG, which does not express CXCR4 constitutively, was stably
transfected with either the cDNA for complete CXCR4 (U373-CXCR4
cells) or for a version of human CXCR4 lacking the COOH-terminal tail
(U373-CXCR4
cyt cells) as described elsewhere (17). Transfected U373
cells were grown in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) supplemented with 10% inactivated fetal bovine
serum and antibiotics (75 µg/ml hygromycin, 0.3 µg/ml puromycin,
and 0.5 mg/ml neomycin analog G-418).
Antibodies--
Rabbit anti-ADA antibody (Serotek, Oxford, UK)
has been developed in our laboratory (14). The anti-CD4 monoclonal
antibody Leu-3A was from Becton Dickinson (San Jose, CA). The UCHT-4
anti-CD8 monoclonal antibody was purchased from Sigma. Several
anti-CD26 monoclonal antibodies (mAb) were used: Ta1 was from Coulter
Clone (Izasa; Barcelona, Spain), 4H12 from Endogen Inc. (Cambridge, MA), 202.36p (purified from the antiserum kindly provided
by Dr. A. Gayà from the Servei d'Hematologia, Hospital
Clínic de Barcelona); TA5.9-CC1-4C8 (18) was kindly provided
by Dr. E. Bossman (Eurogenetics, Tessenderlo, Belgium); and 1F7 (19)
was kindly provided by Dr. C. Morimoto (Boston). The anti-CXCR4
monoclonal antibody 12G5 was purchased from PharMingen (San Diego, CA).
For Western blotting assays the goat polyclonal antibody sc-6190 raised
against the COOH-terminal region of CXCR4 (Santa Cruz Biotechnology,
Santa Cruz, CA) was used.
Protein Conjugation to Fluorochromes--
The antibodies
4H12, 202.36p, 12G5, Ta1, Leu-3A, and anti-ADA and the
enzyme ADA were conjugated to FITC or rhodamine isothiocyanate (TRITC)
as described elsewhere (20).
Immunostaining Assays--
Cells (4 × 106)
were washed with PBS; treated, if necessary, with several anti-CD26
monoclonal antibodies, ADA, gp120IIIB, SDF-1
P2G, or phorbol esters
at 37 °C; and washed with PBS or with PBS containing 10 mM glycine, pH 2, for the samples treated with SDF-1
or P2G (21). For cells other than U373, fixation, permeabilization, and
staining were done as described elsewhere (20). For fixation U273 cells
were washed in PBS and treated with 1% paraformaldehyde solution in
PBS at 20 °C for 5 min. For confocal microscopy analysis a
Leica TCS 4D confocal scanning laser microscope adapted to an inverted
Leitz DMIRBE microscope (Leica Lasertechnik GmbH, Heidelberg, Germany)
was used. The colocalization analysis was made by means of the
Multi Color software (version 2.0; Leica Lasertechnik GmbH). Flow
cytometry analysis was done with an EPICS Profile flow cytometer
(Coulter, Hialeah, FL). The parameters used to select cell populations
for analysis were forward and side light scatter.
Immunoprecipitation and Western Blots--
Cell extracts and
cell membranes were obtained as described by Ciruela et al.
(22). Cell membrane were solubilized (22) and immunoprecipitated with
anti-CD26 1F7 mAb or anti-CD8 mAb covalently coupled to a protein A
matrix as described elsewhere (23) and incubated overnight. Each sample
was washed and resuspended with 60 µl of SDS-polyacrylamide gel
electrophoresis nonreductor sample buffer (0.125 M
Tris-HCl, pH 6.8, 4% SDS, 20% v/v glycerol, 0.02% bromphenol blue).
The immune complexes were dissociated by heating to 37 °C for 15 min
and resolved by SDS-polyacrylamide gel electrophoresis in 7.5% gels
(24). Immunoblotting was performed using anti-CD26 1F7 antibody
(1/3,200) or anti-CXCR4 sc-6190 antibody (2 µg/ml) and anti-mouse
IgG-peroxidase (Dako A/S, Glostrup, Denmark) or anti-goat
IgG-peroxidase (Roche Molecular Biochemicals GmbH, Barcelona, Spain),
respectively. The polyvinyl difluoride membrane (Immobilon-P;
Millipore) was incubated in equal volumes of SuperSignal chemiluminescent substrates 1 and 2 (Pierce). The detection
reagent was drained off, and the filters were placed in contact with a film (Hyperfilm ECL), which was developed by chemiluminescence.
Protein Determination--
Protein was quantified by the
bicinchoninic acid method (Pierce) as described by Sorensen and
Brodbeck (25) and using bovine serum albumin as standard.
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RESULTS |
Colocalization of CXCR4 and CD26 in CD4+ and
CD4
Cell Lines--
To analyze the possible
codistribution of CXCR4, the coreceptor for T-tropic HIV-1, and CD26,
two cell lines were selected: the Jurkat J32 CD4+ T cell
line and the Epstein-Barr transformed B cell line SKW6.4, which does
not express the main HIV receptor CD4. To establish a possible
correlation between this codistribution and the well characterized
binding of the viral glycoprotein gp120 to the CXCR4 chemokine receptor
(26, 27), the degree of colocalization between CD26 and CXCR4 on the
surface of Jurkat J32 and SKW6.4 cells was determined in the absence
and in the presence of gp120. Jurkat J32 cells, fixed and labeled with
FITC-conjugated anti-CD26 Ta1 and TRITC-conjugated anti-CXCR4 12G5,
showed (Fig. 1A) a marked colocalization (in yellow) of the two proteins.
Preincubation of cells with gp120 for 30 min at 37 °C prior to
fixation and staining induces the formation of pseudopodia with a
redistribution on cell surface CXCR4 and CD26 that are coclustered in
these pseudopodia. This morphological change is reversible, with a
maximal formation of pseudopodia at 30 min of treatment with gp120, and
a disappearance at 2 h after the addition of the viral
glycoprotein.

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Fig. 1.
CD26 and CXCR4 colocalization.
Jurkat J32 (A) or SKW6.4 (B) cells (4 × 106) were incubated 30 min at 37 °C in PBS buffer in the
absence ( ) or in the presence (+gp120) of 15 µg/ml
gp120IIIB. Cells were fixed and stained with 100 µg/ml
FITC-conjugated Ta1 anti-CD26 (in green) and 15 µg/ml
TRITC-conjugated 12G5 anti-CXCR4 (in red), as indicated
under "Experimental Procedures." Bar, 10 µm.
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Staining of the CD4
SKW6.4 cell line with FITC-conjugated
anti-CD26 Ta1 and TRITC-conjugated anti-CXCR4 12G5 showed a high degree
of colocalization between CD26 and CXCR4 (Fig. 1B). However pretreatment with gp120 before fixation and labeling did not induce any
morphological redistribution of these molecules on the cell surface.
CXCR4 Coimmunoprecipitates with CD26--
The high degree of
colocalization between CD26 and CXCR4 on the Jurkat J32 and SKW6.4 cell
surface, and the coclustering of these proteins in the pseudopodia
induced by gp120 in T cells, suggest a molecular interaction between
both molecules. To test this possibility, coimmunoprecipitation
experiments were performed with solubilized Jurkat J32 and SKW6.4 cells
membranes. Immunoprecipitation was carried out with the 1F7 anti-CD26
mAb, and the Western blot of immunoprecipitates revealed a band of 110 kDa that corresponds to the CD26 when the Western blot was developed
with anti-CD26 1F7 antibody (Fig.
2A) and two bands of 90 and 50 kDa when the immunoblot was developed with anti-CXCR4 sc-6190 (Fig.
2B). These two bands correspond to the CXCR4 receptor as it
has been described previously (26, 27). These results and those shown
in Fig. 1 indicate that an interaction CD26/CXCR4 occurs on the surface of Jurkat J32 and SKW6.4 cells.

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Fig. 2.
Coimmunoprecipitation of CXCR4 and CD26.
Solubilized membranes from Jurkat J32 (J) or SKW6.4
(SK) cells were immunoprecipitated as indicated under
"Experimental Procedures" with 1F7 anti-CD26 mAb or with an
anti-CD8 mAb that is irrelevant for B and CD4+ T cells,
both covalently coupled to a protein A matrix. Immunoblotting of
immunoprecipitates was performed using 1F7 antibody to detect CD26 and
sc-6190 antibody to detect CXCR4.
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Modulation of the CD26/CXCR4 Module by SDF-1
, by an Antagonist
and by Pertussis Toxin--
It has been fully described that SDF-1
,
the physiological CXCR4 ligand, induces a rapid down-regulation of its
receptor (17, 28-31). The incubation of Jurkat J32 or SKW6.4 cells
with 400 nM SDF-1
induced the endocytosis of CXCR4 and
also of CD26 as stated by immunocytochemistry and confocal microscopy
analysis (Fig. 3). This process, which
was evident at 30 min, could be already detected 5 min after the
addition of the ligand (data not shown). These results show that the
modulation of the CD26·CXCR4 complex by chemokines takes place in
cells irrespective of CD4 expression or gp120-induced pseudopodia.

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Fig. 3.
SDF-1 -induced internalization of
CXCR4/CD26 in cell lines. 4 × 106 Jurkat
J32 (A) or SKW6.4 cells (B) were incubated in the
absence (Ctrl.) or presence of 400 nM SDF-1
for 10, 30, or 60 min at 37 °C. Subsequently, cells were fixed or
permeabilized and labeled with 100 µg/ml FITC-conjugated Ta1
anti-CD26 (in green) and 15 µg/ml TRITC-conjugated 12G5
anti-CXCR4 (in red) antibodies. For permeabilized
cells, colocalization (in yellow) is only shown.
Bar, 10 µm.
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Like gp120 (see Fig. 1), in Jurkat J32 cells, SDF-1
also induced the
formation of pseudopodia after 30 min of incubation (Fig.
3A: 30 min), although the number of cells presenting
pseudopodia was lower. In some cells the pseudopodia induced by the
SDF-1
were less labeled with fluorescent antibodies than the rest of the cell and, therefore, it seems that CXCR4 and CD26 are internalized with different kinetics in the pseudopodia than in the rest of the
cell. By analyzing permeabilized cells it is shown that CD26 and CXCR4
colocalize inside Jurkat J32 and SKW6.4 cells (Fig. 3) after 10 min of
SDF-1
-induced internalization. At a longer period of incubation with
SDF-1
(1 h), the two proteins do not codistribute inside the cell
(Fig. 3), since they are localized in different endocytic vesicles,
CXCR4 being more homogeneously distributed than CD26. In Jurkat J32
cells, cointernalization was also promoted by the phorbol ester PMA but
did not occur in the presence of the antagonist of CXCR4, P2G (Fig.
4).

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Fig. 4.
Cointernalization of CXCR4/CD26 in peripheral
blood lymphocytes and in Jurkat cells. 4 × 106 lymphocytes or Jurkat J32 cells were incubated in the
absence (Ctrl., 60 min) or presence of 100 nM
SDF-1 (60 min), 100 nM P2G (60 min), or 50 nM PMA (30 min) at 37 °C. Subsequently, cells were fixed
and labeled with 100 µg/ml FITC-conjugated Ta1 anti-CD26 and 15 µg/ml TRITC-conjugated 12G5 anti-CXCR4 antibodies. The images show
CD26/CXCR4 colocalization (in white). Bar, 10 µm.
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A colocalization between CXCR4 and CD26 was also evident on the cell
surface of peripheral blood lymphocytes (Fig. 4). The membrane
expression of both CD26 and CXCR4 in primary lymphocytes is
down-modulated by SDF-1
, but not modified by the antagonist P2G.
Therefore, CD26 and CXCR4 behave in primary cells as in the lymphocytic
cell lines. To confirm the specificity of the effect of SDF-1
upon
regulation of CD26 and CXCR4 expression, ligand-induced internalization
was assayed in human U373 cells stably transfected with cDNAs
encoding for full size CXCR4 (U373-CXCR4 cells) or for a version of the
receptor lacking the COOH-terminal tail (U373-CXRC4
cyt cells).
Parental U373 cells express endogenous CD26, and therefore, comodulation studies are possible using single transfectants. Again, in
cells expressing full size CXCR4, CD26/CXCR4 comodulation was found
when cells were treated with SDF-1
or PMA. In contrast, neither CD26
nor CXRC4 were internalized in cells expressing the short version of
the chemokine receptor (Fig. 5).
Cointernalization was inhibited by treatment with sucrose or acetic
acid, thus indicating that it is mediated by coated vesicles (Fig.
6), which is in agreement with the data
reported by Amara et al. (17). Interestingly, blockade of
Gi operation by pertussis toxin at either 150 ng/ml (Fig.
6) or 5 µg/ml (not shown) did not affect the SDF-1
-mediated cointernalization of CD26 and CXCR4.

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Fig. 5.
Comodulation of CXCR4/CD26 in transfected
U373 cells. Stably transfected U373 cells (see
"Experimental Procedures") were treated in the absence (black
line) or presence of 100 nM SDF-1 (green
line), 100 nM P2G (blue line), or 50 nM PMA (red line) for 30 min at 37 °C. For
flow cytometric analysis cells were fixed and labeled with 100 µg/ml
FITC-conjugated Ta1 anti-CD26 and 15 µg/ml TRITC-conjugated 12G5
anti-CXCR4 antibodies. Cells were then analyzed by flow
cytometry. Unspecific labeling is indicated by the gray shaded
area.
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Fig. 6.
Effect of acetic acid, sucrose, and pertussis
toxin upon SDF-1 -induced CXCR4 and CD26
internalization in Jurkat cells. For SDF1 -induced
internalization assays Jurkat cells were treated as described in the
legend of Fig. 3. For acetic acid treatment cells were pretreated with
the acid (5 mM, 5 min, 37 °C). For pertussis toxin
treatment cells were pretreated (6 h, 37°) with the toxin at a
concentration of 150 ng/ml. For sucrose treatment, the sugar (0.45 M final concentration) was added at the same time as
SDF-1 .
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Effect of gp120 upon the Redistribution of ADA on the Cell
Surface--
As it had been previously reported (15), gp120 inhibits
the binding of 125I-labeled ADA to CD26 in human
lymhocytes and T cell lines. To characterize better this effect,
experiments of confocal microscopy were performed using FITC-conjugated
ADA. As shown in Fig. 7, the interaction
of exogenous ADA with cells (Fig. 7A) decreases in the
presence of gp120 (Fig. 7B). The gp120-induced inhibition of
ADA binding to CD26 was also detected by flow cytometry (Fig. 7C). The displacement of ADA bound to CD26 by gp120 occurs
even for endogenous ADA. Thus, when nonfixed SKW6.4 cells were labeled with TRITC-conjugated anti-ADA (Fig. 7D) a decrease of
fluorescence was observed by the pretreatment with gp120 (Fig.
7E), indicating that gp120 is able to disrupt the ADA/CD26
interaction on the cell surface. Similar results were observed with
Jurkat J32 cells in the presence of an excess of anti-CD4 antibody
(result not shown). We also investigated whether in the gp120-induced
pseudopodia the CD26 would be free of cell surface ADA. In experiments
in which Jurkat J32 cells were labeled with FITC-conjugated anti-ADA and TRITC-conjugated 12G5, a good staining of cell surface ADA and
CXCR4 was observed (Fig. 8A)
with a notable degree of colocalization (in yellow). This is
consistent with the above described high degree of colocalization
between the CXCR4 and the cell surface ADA-binding protein, CD26. In
fact, the pretreatment of Jurkat J32 cells with gp120 induced the
formation of pseudopodia in which no labeling of ADA could be detected
by immunocytochemical assays (Fig. 8B). The inhibition of
exogenous ADA binding and the release of endogenous ADA bound to CD26
by gp120 suggests that gp120 recognizes the ADA binding site of CD26.
Interestingly enough, staining of fixed SKW6.4 cells with
FITC-conjugated gp120IIIB and with TRITC-conjugated anti-ADA led to a very high colocalization (in yellow) of
the two proteins (Fig. 9A).
However, when the same experiment was performed using cells that were
fixed after incubation with the reagents (see "Experimental
Procedures"), a poor colocalization was found (Fig.
9B). Thus, the interaction CD26/gp120 occurs through an
epitope different from the ADA binding site in CD26, and a subsequent conformational change in gp120 is required to block the ADA
binding site of CD26.

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Fig. 7.
Gp120-induced inhibition of ADA binding to
CD26. A and B, 4 × 106 SKW6.4 cells were incubated (45 min) with 2 units/ml
FITC-conjugated ADA in the absence (A) or presence
(B) of 15 µg/ml gp120IIIB. Cells were then
fixed and treated for confocal microscopy analysis as described under
"Experimental Procedures." C, flow cytometry analysis of
the samples: open peak, SKW6.4 cells labeled with
FITC-conjugated bovine ADA; dashed peak, cells incubated
with FITC-conjugated bovine ADA and gp120IIIB; and solid
peak, the nonspecific binding. D and E, SKW6.4
cells (4 × 106 cells) were labeled with 50 µg/ml
TRITC-conjugated anti-ADA antibody in the absence (D) or
presence (E) of 15 µg/ml gp120IIIB and then
fixed and analyzed by confocal microscopy as described under
"Experimental Procedures."
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Fig. 8.
Lack of ADA in gp120-induced
pseudopodia. Jurkat J32 cells (4 × 106)
were incubated (30 min, 37°) in PBS, in the absence (A) or
in the presence (B) of 15 µg/ml gp120IIIB.
Cells were fixed and stained with 50 µg/ml FITC-conjugated anti-ADA
(in green) and 15 µg/ml TRITC-conjugated 12G5 anti-CXCR4
(in red), as indicated under "Experimental Procedures."
Bar, 10 µm.
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Fig. 9.
Colocalization between gp120 and cell surface
ADA. Fixed (A) or nonfixed (B)
SKW6.4 cells (4 × 106) were incubated with 15 µg/ml
FITC-conjugated gp120IIIB and 50 µg/ml TRITC-conjugated
anti-ADA antibody as described under "Experimental Procedures."
Confocal microscopy analysis shows the gp120 (in green) and
the cell surface ADA (in red). The superposition of labels
and the confocal cytofluorogram show (in yellow) a high
degree of colocalization between both proteins in A but not
in B. Bar, 10 µm.
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gp120 Blocks the Binding of Specific Antibodies to CD26--
To
test the above described hypothesis, binding of FITC-conjugated
gp120IIIB was done with fixed SKW6.4 cells preincubated with several anti-CD26 mAbs raised against different epitopes. Preincubation was performed at 4 °C to prevent internalization of
cell surface molecules. Bound FITC-gp120IIIB was quantified by flow cytometry (Fig. 10). Two
anti-CD26 mAbs, 202.36p and 4H12, were able to decrease the
binding of gp120. This decrease could not be detected with other
anti-CD26 antibodies as Ta1 or TA5.9, which is directed against the ADA
binding site (not shown), or with anti-CD4 Leu-3a mAb used as an
irrelevant antibody for these CD4
cells. Moreover, the
preincubation of SKW6.4 cells with gp120IIIB (Fig.
11) diminished the staining obtained
using the anti-CD26 antibodies 202.36p and 4H12, but not
the staining obtained using Ta1. With the human T cell line Jurkat J32,
similar results were obtained when the gp120 binding to CD4 was blocked
with an anti-CD4 mAb (data not shown). All these results suggest that
there is a direct interaction between gp120 and CD26 and that the gp120
binding domain in CD26 is distinct from the ADA binding site.

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Fig. 10.
Anti-CD26 antibodies-induced inhibition of
gp120 binding. SKW6.4 cells (4 × 106) were
incubated in the absence (continuous line) or presence
(broken line) of 202.36p, 4H12, Ta1, or anti-CD4
Leu3A mAb in PBS buffer for 45 min at 4 °C. Subsequently, cells were
fixed and labeled with 15 µg/ml FITC-conjugated gp120IIIB
for flow cytometry analysis as described under "Experimental
Procedures." The solid peak represents unspecific
binding.
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Fig. 11.
gp120-induced inhibition of CD26
labeling. SKW6.4 (4 × 106) cells were untreated
(continuous line) or treated (broken line) with
15 µg/ml gp120IIIB for 45 min at 37 °C. Cells
were then fixed and stained with FITC-conjugated antibodies
202.36p, 4H12, Ta1, or anti-CD4 Leu3A mAb and analyzed by
flow cytometry (see "Experimental Procedures"). The solid
peak represents unspecific binding.
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DISCUSSION |
In this paper we demonstrate for the first time that CD26 and
CXCR4 interact in primary lymphocytes from human blood and also in T
and B cell lines. The CXCR4 natural ligand SDF-1
has the conserved
NH2-X-Pro sequence (where X is any
amino acid) at the NH2 terminus, which is a consensus
sequence for substrates of dipeptidyl peptidase IV activity of CD26. It
has been demonstrated that SDF-1
can be processed by CD26 (4, 9, 11,
32). Therefore the CD26·CXCR4 complex is likely a functional unit in which the expression of CD26 on the cell surface may modulate SDF-1
-induced chemotaxis. Since SDF-1
is a natural antiviral agent (33, 34), the expression of CD26 can also modulate the activity
of this substrate of CXCR4 against T-tropic strains of HIV.
The fact that SDF-1
induces the cointernalization of CXCR4 and CD26
in all cells tested (Figs. 3 and 4) is also relevant. The
SDF-1
-induced internalization of the CD26/CXCR4 module was not
CD4-dependent, as we could detect that the level of
expression of both molecules on the cell surface decreased with
SDF-1
incubation in T, but also in B, cells. The specificity and
physiological relevance of CD26/CXCR4 cointernalization was
demonstrated by the lack of internalization in the presence of the
antagonist P2G in primary lymphocytes and in cell lines. The cells
expressing a chemokine receptor lacking the COOH-terminal part were
also a suitable model to assess the relevance of the interaction. In fact, the lack of the cytoplasmic tail, which is known to prevent SDF-1
induced down-regulation of CXCR4, also prevents
internalization of CD26. These results indicate that treatment of
lymphocytes with chemokines or agents that activate protein kinase C
leads to the simultaneous internalization of CXCR4 and CD26, probably via the same endocytic pathway, as we have demonstrated for other interacting cell surface proteins (35). SDF-1
-induced
internalization of CXCR4 is mediated by coated vesicles (17). According
to this, the blockade of the cointernalization by sucrose and by acetic acid further suggests that endocytosis of CXCR4 and CD26 follows the
same endocytic pathway mediated by coated vesicles. Interestingly, after a long period of chemokine treatment, the codistribution of the
two proteins inside the cell is lost. At 1 h of treatment CXCR4 is
found homogeneously distributed near the plasma membrane (Fig. 3),
which would fit with the rapid recycling reported for this chemokine
receptor. In contrast, internalized CD26 is clustered in intracellular
vesicles, thus confirming that the traffic of the two proteins after
the cointernalization follows different routes. As reported previously,
ligand-induced internalization of CXCR4 is not mediated by
Gi proteins (17). The assays performed in the presence of
pertussis toxin indicate that blocking of G-protein-mediated signaling
does not prevent the cointernalization. This interesting finding is
evidence for a Gi protein-independent signaling pathway that regulates the SDF-1
-induced simultaneous internalization of the
two proteins. To our knowledge this is the second example of
ligand-induced cointernalization of a receptor and the enzyme that
inactivates the ligand for the receptor (35). In these models of
interacting proteins, the existence of membrane-bound and soluble forms
of degrading enzymes is relevant. Thus, in addition to
membrane-bound enzyme, soluble forms of CD26 are able to inactivate SDF-1
(4, 7). While the soluble enzyme could regulate circulating concentrations of SDF-1, the cell-bound enzyme should be responsible for the control of local changes in ligand concentration.
Cointernalization of CD26 with CXCR4 might represent a second mechanism
of control of CXCR4 signaling. Consistent with the results shown here,
it has been reported that peripheral blood lymphocytes migrating toward
SDF-1
show low expression of CXCR4 and CD26. This finding can be a
consequence of a higher response to SDF-1
from CD26low
cells, but it could reflect a SDF-1
-induced cointernalization of
CD26 and CXCR4 (see Fig. 3).
Iyengar et al. (36) have described that in peripheral blood
mononuclear cells the addition of gp120 cause cocapping of CD4 and
CXCR4 with subsequent pseudopodium formation. On the other hand, Feito
et al. (37) described that gp120 from the
syncytium-induced HIV-1 strain increased the cocapping of CD4
with some proteins, CD26 included. We here demonstrate that
CD26·CXCR4 complexes are coredistributed into gp120-induced
pseudopodia. Taken together, these results suggest that the viral
envelope glycoprotein gp120 induces the formation in T lymphocytes of
pseudopodia in which the main virus receptor, the CD4, the coreceptor
CXCR4, and the CD26 coexist.
Although the importance of CD4 and chemokine receptors in the process
of HIV entry has been extensively described, the role of CD26 in this
process is still not well understood. Previous results of our
laboratory (15) described the inhibition of the binding of
125I-labeled ADA to the CD26 by HIV-1 envelope gp120 and
viral particles, suggesting that gp120 recognizes the ADA binding site
of CD26. gp120 blocks binding of ADA to CD26 in a
CXCR4-dependent manner (16). It is possible that gp120
displaces ADA binding to CD26 in CD4-negative cells because of the
presence of CXCR4. These results support the hypothesis (see
Ref. 16) that gp120 has to bind to CXCR4 prior to its interaction with
CD26 and the disruption of the ADA/CD26 interaction. In this report we
confirm the gp120-induced inhibition of ADA binding to CD26, but we
also demonstrate that gp120 provokes the release of endogenous ADA
bound to CD26, and this happens without changes in the expression of
cell surface CD26 (Fig. 11). These data would suggest that gp120 and
ADA share a common binding site in CD26. As shown in Fig.
9B, when the FITC-conjugated gp120 incubation was done using
nonfixed cells, the viral glycoprotein could produce some membrane
protein rearrangements displacing cell surface ADA from CD26. However,
when cells were fixed before the incubation with gp120, a marked
colocalization between the viral glycoprotein and the cell surface ADA
was observed (Fig. 9A). It should be noted that
fixation avoids any cell membrane protein mobility or redistribution.
These results led to hypothesize the existence of a gp120 binding
domain in CD26 distinct from the ADA binding site. This indicates that
the gp120-mediated inability of ADA to bind to CD26 would be a
consequence of a first interaction of gp120 with a distinct epitope in
CD26. We detected two anti-CD26 mAb, 4H12 and 202.36p,
raised against a different epitope from ADA binding center that decrease the binding degree of gp120. Both antibodies are, also, less
effective in labeling CD26 when cells are incubated with the viral
glycoprotein gp120. These results correlate with the hypothesis of an
interaction of gp120 with CD26 at an epitope different from the ADA
binding domain, and they also indicate a recognition of cell
surface CD26 by the viral envelope protein gp120.
Comodulation of CXCR4 and CD26 by SDF-1
, gp120-induced formation of
pseudopodia in which ADA is not present, and the role of ADA in the
development and function of the immune system (38) suggest that the
ADA·CD26 and the CXCR4·CD26·ADA complex is important for the
functionality of human lymphocytes.