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IFN-γ-Inducible T Cell α Chemoattractant Is
a Potent Stimulator of Normal Human Blood
T Lymphocyte Transendothelial Migration:
Differential Regulation by IFN- γ and TNF-α
Karkada Mohan, Ziqiang Ding, John Hanly and Thomas B.
Issekutz
J Immunol 2002; 168:6420-6428; ;
doi: 10.4049/jimmunol.168.12.6420
http://www.jimmunol.org/content/168/12/6420
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Copyright © 2002 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
The Journal of Immunology
IFN-␥-Inducible T Cell ␣ Chemoattractant Is a Potent
Stimulator of Normal Human Blood T Lymphocyte
Transendothelial Migration: Differential Regulation by IFN-␥
and TNF-␣1
Karkada Mohan,* Ziqiang Ding,* John Hanly,†‡ and Thomas B. Issekutz2*†‡
E
xtravasation and accumulation of leukocytes in the tissues
is a hallmark of chronic inflammatory conditions. Lymphocyte trafficking, both during normal circulation and
recruitment to sites of inflammation involves a multistep process,
in which lymphocyte surface integrins, endothelial adhesion molecules, and chemokines produced in the local microenvironment
play an essential role (1–3). There are ⬃50 human chemokines
identified to date (4 –7), which, based on the number and arrangement of the first two cysteine residues in their structure, are subdivided into four groups, CXC (␣), CC (␤), C (␥), and CX3C (␦).
Chemokines up-regulated at the site of inflammation by proinflammatory cytokines, such as IFN-␥ and TNF-␣, are thought to directly recruit lymphocytes by interacting with their chemokine receptors (4 – 6). CXC chemokines which have been shown to act on
T lymphocytes include IL-8, stromal cell-derived factor (SDF-1),3
Departments of *Pediatrics, †Medicine, ‡Microbiology/Immunology, and Pathology,
Dalhousie University, Halifax, Nova Scotia, Canada
Received for publication July 25, 2001. Accepted for publication April 8, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by The Arthritis Society (Grant no. TAS89/0002) and a
grant from the Canadian Institutes of Health Research (Grant no. MOP-42379). K.M.
was supported by a Cancer Research and Education Nova Scotia trainee award with
funding from Cancer Care Nova Scotia.
2
Address correspondence and reprint requests to Dr. Thomas B. Issekutz at the current address: Division of Immunology, Rheumatology and Infectious Diseases,
Department of Pediatrics, Izaak Walton Killam Grace Health Center, 5850 University Avenue, Halifax, Nova Scotia, Canada, B3J 3G9. E-mail address:
[email protected]
3
Abbreviations used in this paper: SDF-1, stromal cell-derived factor-1; TEM, transendothelial migration; EC, endothelial cell; I-TAC, IFN-␥-inducible T cell ␣ chemoattractant; IP-10, IFN-␥-inducible protein-10; Mig, monokine induced by IFN-␥;
VLA-4, very late Ag 4; MCP-1, monocyte chemoattractant protein-1; MIP, macrophage inflammatory protein; PPP, platelet-poor plasma; HSA, human serum albumin.
Copyright © 2002 by The American Association of Immunologists, Inc.
IFN-␥-inducible protein-10 (IP-10), monokine induced by IFN-␥
(Mig) and IFN-␥-inducible T cell ␣ chemoattractant (I-TAC).
Among these chemokines, SDF-1 shows broad specificity for different subtypes of T cells, while IP-10, Mig, and I-TAC are reported to be selectively chemotactic for memory or IL-2-activated
T cells (8, 9). Hence, these latter three chemokines are thought to
be important in the process of T cell recruitment in inflammation.
CXCR3 is the only receptor on T cells for IP-10, Mig, and
I-TAC, and the binding affinity for I-TAC is reported to be much
higher than for IP-10 and Mig (9). Nevertheless, all three of these
chemokines are known ligands for CXCR3⫹ activated/memory T
lymphocytes (8 –10). I-TAC mRNA has been found to be upregulated in IFN-␥-treated astrocytes, monocytes (9), bronchial
epithelial cells (11), neutrophils (12), and keratinocytes (13). In
addition, I-TAC was recently shown to be up-regulated in IFN␥-stimulated human endothelial cells (ECs), suggesting a role for
this chemokine in T lymphocyte recruitment to sites of inflammation (14). The pathophysiological role for I-TAC is not fully understood, although its expression by ECs in atherosclerotic lesions
has been shown to correlate with CXCR3⫹ T cell accumulation
within the lesions (15). Moreover, marked enrichment of CXCR3⫹
cells in the synovium, over blood, of patients with rheumatoid
arthritis (16), and increased levels of circulating as well as intralesional CXCR3⫹ cells in pateints with multiple sclerosis (17, 18)
suggest a possible chemotactic role for CXC chemokines in inflammation. In addition, enhanced expression of I-TAC mRNA by
IFN-␥-stimulated bronchial epithelial cells (11) and in allergic skin
reactions (19) suggest that this chemokine may play an important
role in the recruitment of activated T cells to lesions in the tuberculoid lung and to delayed type hypersensitivity reactions in
the skin. Recently, prolonged cardiac allograft survival has been
reported in IP-10⫺/⫺ mice, also suggesting a role for CXC chemokines in graft rejection (20).
0022-1767/02/$02.00
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Previous studies have shown that the CXC chemokine, IFN-␥-inducible T cell ␣ chemoattractant (I-TAC), was chemotactic for
IL-2-activated human T lymphocytes, which express abundant CXCR3. However, because most memory T lymphocytes are also
CXCR3ⴙ, the ability of I-TAC to promote the migration of normal human blood T cells across HUVEC monolayers in Transwell
chambers was examined. I-TAC induced a marked (4- to 6-fold) increase in transendothelial migration (TEM) of T cells across
unstimulated HUVEC from 5.6 to 28% of input T cells and was substantially more active than IFN-␥-inducible protein-10, another
CXCR3 ligand. I-TAC significantly enhanced TEM of T cells across TNF-␣, but not across IFN-␥ or IFN-␥ plus TNF-␣-activated
HUVEC. IFN-␥ or IFN-␥ plus TNF-␣-activated HUVEC produced substantial amounts of I-TAC, in contrast to TNF-␣-treated
EC. Both CD4ⴙ and CD8ⴙ T cells migrated in response to I-TAC to a similar extent, while memory T cells migrated several fold
better than naive T cells. Blockade of LFA-1 strongly inhibited I-TAC-induced T cell TEM across unstimulated HUVEC, and
⬃50 – 60% of the TEM across cytokine-activated HUVEC. However, blocking both LFA-1 and very late Ag-4 abolished I-TAC
induced T cell TEM. In vivo significant levels of I-TAC were detected in arthritic synovial fluid. Thus, I-TAC is one of the most
potent chemoattractants of normal human blood CD4 and CD8 T cell TEM and is likely a major mediator of blood memory T
lymphocyte migration to inflammation. The Journal of Immunology, 2002, 168: 6420 – 6428.
The Journal of Immunology
Previously, it was shown that T cells activated for 7–15 days in the
presence of IL-2 and/or PHA, migrated in chemotaxis assays in response to CXC chemokines, IP-10, Mig, and I-TAC (9, 10). However,
it is not clear whether blood T lymphocytes, which are not stimulated
with IL-2 or PHA, can chemotax to these CXC chemokines. Because
most circulating memory T cells express CXCR3, it was important to
determine whether these lymphocytes would migrate without longterm activation to CXC chemokines and especially to I-TAC, which
has the highest binding affinity of these chemokines (9). Moreover,
transendothelial migration (TEM) of T cells in response to I-TAC,
especially across cytokine-stimulated EC, as would occur during inflammation, has not been previously examined.
In the present study, chemotaxis and TEM of freshly isolated
human peripheral blood T cells, without IL-2 preactivation, was
investigated in response to I-TAC and IP-10. These freshly isolated T cells demonstrated substantial chemotaxis and TEM in response to I-TAC. TEM was further enhanced by TNF-␣, but in
contrast to other chemokines, not IFN-␥ stimulation of ECs.
Abs and reagents
Recombinant human I-TAC and IP-10 were obtained from PeproTech
(Rocky Hill, NJ), TNF-␣ (specific activity ⫽ 5 ⫻ 107 U/mg) was from
R&D Systems (Minneapolis, MN), and IFN-␥ (107 U/mg) was purchased
from Genentech (South San Francisco, CA). Anti-CXCR3 (1C6) was a gift
from Dr. P. Ponath (Leukocyte, Cambridge, MA). Affinity purified, rabbit
polyclonal anti-I-TAC Ab, with or without biotin label, were purchased
from PeproTech. Anti-CD45 mAb (4B2) was obtained from the American
Type Culture Collection (ATCC, Manassas, VA). Anti-CD45RA (G1–15)
was a kind gift of Dr. J. Ledbetter (Bristol-Meyers-Squibb, Seattle, WA).
Anti-CD45RO (UCHL-1) was obtained from Immunotech (Westbrook,
ME). Affinity isolated goat-anti-mouse Ig was purchased from DAKO
(Glostrup, Denmark). Anti-CD4 (OKT4), anti-CD8 (OKT8), and antiLFA-1␤ (IB4) Abs were obtained from ATCC. Anti-very late Ag (VLA)-4
(HP2/1) was a generous gift from Dr. F. Sanchez-Madrid (Universidad
Autonoma de Madrid, Madrid, Spain).
Preparation of lymphocytes
Lymphocytes were isolated from human peripheral blood from healthy
donors by gradient centrifugation and passage over a nylon wool column.
In brief, acid citrate dextrose-heparin anticoagulated blood was gently
mixed with an equal volume of warm PBS, layered onto Ficoll-Paque
(Pharmacia, Uppsala, Sweden), and centrifuged at 900 ⫻ g for 20 min. The
PBMC on top of the Ficoll-Paque were collected and washed three times
with Ca2⫹Mg2⫹ free Tyrode’s solution. The PBMC were resuspended in
RPMI 1640 (RPMI) medium with 10% human platelet-poor plasma (PPP)
and were applied onto a nylon wool column. After 60 min of incubation,
the unbound T lymphocytes were eluted, washed, resuspended in fresh
RPMI medium plus 10% PPP at 2 ⫻ 106 cells/ml, and cultured overnight
in tissue culture flasks. The nonadherent cells contained ⬎96% T cells,
⬍3% B cells, and ⬍0.1% monocytes by immunofluorescence staining, and
were ⬎98% viable by trypan blue dye exclusion. In some experiments, to
study the effect of IL-2 activation, T cells were cultured in RPMI ⫹ 10%
PPP, with or without 400 U/ml recombinant human IL-2 for 8 days. Cells
were cultured at a concentration range of 1.5–2 ⫻ 106 cells/ml medium;
and to maintain the cell numbers at this concentration, IL-2-supplemented
cultures were diluted at 2- to 3-day intervals using fresh IL-2 containing
medium.
In some studies, naive (CD45RA⫹), memory (CD45RA⫺), CD4⫹- or
CD8⫹-enriched T cells were purified by using MACS (Miltenyi Biotec,
Bergisch Gladbach, Germany). Briefly, T cells were incubated with mAb
to either CD45RA (biotinylated G1–15), or CD4 (OKT4) or CD8 (OKT8)
at 50 ␮g/108 cells/ml in RPMI medium plus 10% FCS at 4°C for 30 min.
The cells were then washed twice and resuspended in HEPES-buffered
HBSS containing 10% FCS at 107 cells/90 ␮l medium. Streptavidin-conjugated- or goat anti-mouse Ig-conjugated magnetic beads as appropriate
were added and incubated at 6 –12°C for 15 min, mixing every 5 min.
Finally, cells were washed once and passed through a column in a magnetic
field and the flow through cells were collected as CD45RA⫺, CD4⫺ cells,
or CD8⫺ cells. The purity was ⬎97% for the CD45RA⫺ cell population,
and ⬎99% for CD4⫹ and CD8⫹ T cells by immunofluorescence staining.
Unfractionated, memory, naive, CD4⫹, CD4⫺ or CD8⫹, CD8⫺ T
cells were labeled by incubating 5 ⫻ 107 cells/ml in RPMI ⫹ 15 mM
HEPES ⫹ 10% FCS with 50 ␮Ci/ml Na251CrO4 (Amersham, Oakville,
Canada) at 37°C for 45 min. Cells were washed three times with RPMI and
resuspended in RPMI ⫹ 5 mg/ml human serum albumin (HSA) for chemotaxis and TEM assays. Depending on the assay, cells were left untreated
or pretreated with 20 ␮g/ml of anti-CD45, anti-LFA-1, anti-VLA-4, or
anti-LFA-1 plus anti-VLA-4 mAbs for 20 min at room temperature, and
then added on top of the EC monolayers in the TEM assay without removing the mAbs. In some assays, T cells were also pretreated with 10
␮g/ml anti-CXCR3 mAb (1C6) before being tested for TEM to I-TAC.
Isolation and culture of ECs
HUVEC were isolated by collagenase digestion as described (21). Briefly,
human umbilical veins were flushed with Ringer’s Lactate, then incubated
with 0.5 mg/ml collagenase type II (Sigma-Aldrich, St. Louis, MO) at 37°C
for 30 min. Detached EC were collected, washed, and then cultured in
gelatin-coated flasks (Nunc, Naperville, IL) in RPMI containing 20% FCS
(HyClone Laboratories, Logan, UT), 25 ␮g/ml EC growth supplement (BD
Labware, Bedford, MA), 45 ␮g/ml heparin, 2 mM L-glutamine, 50 ␮M
2-ME, 100 U/ml penicillin, and 100 ␮g/ml streptomycin. Confluent
HUVEC in the flasks were gently trypsinized and seeded onto polycarbonate Transwell filters of 6.5-mm diameter and 5-␮m pore size (Costar Corning, Cambridge, MA). The Transwell filters were prepared by coating with
0.01% gelatin at 37°C overnight followed by 3 ␮g of human fibronectin
(Life Technologies, Grand Island, NY) at 37°C for 3 h. Then, 1.2 ⫻ 104
HUVEC in 100 ␮l HUVEC medium were seeded onto each filter and 0.6
ml of the culture medium was added to each lower chamber beneath the
filter. After 6 days of culture, the integrity of confluent HUVEC monolayers was assessed by microscopic observation and by measuring the permeability of the monolayer using 125I-albumin diffusion.
Measurement of lymphocyte chemotaxis to I-TAC
Percentage of T cells migrating in response to I-TAC across 5 ␮m pore size
polycarbonate filters (6.5-mm diameter) was investigated in 24-well Transwell chambers (Costar Corning). 51Cr-labeled T lymphocytes were suspended at 2 ⫻ 106 cells/ml in RPMI plus 5 mg/ml HSA and 100 ␮l of the
cell suspension was added to the upper chamber of each Transwell precoated with gelatin and fibronectin. Chemokines were added to the lower
chamber at the indicated concentrations. The plates were incubated for 60
min at 37°C in 5% CO2. The migrated T cells in the lower chambers were
collected and the extent of T cell migration was measured by gamma
counting. Percentage of migration was calculated by dividing the radioactivity of cells in the lower chamber by the total input radioactivity of the
labeled cells added to the upper chamber.
Measurement of lymphocyte TEM
HUVEC were left untreated or were stimulated by adding TNF-␣ (200
U/ml), IFN-␥ (200 U/ml), or IFN-␥ plus TNF-␣ (200 U/ml each) to the
lower chamber of the Transwells for 18 h. The endothelial monolayers in
the Transwell inserts were rinsed with RPMI, 100 ␮l labeled cells at 2 ⫻
106 cells/ml in RPMI plus HSA were added to the HUVEC monolayers,
and the inserts were transferred to new wells (lower chambers) of a 24-well
plate containing 0.6 ml of fresh RPMI plus HSA and the indicated chemokine. The Transwells were then incubated at 37°C in 5% CO2. After 4 h,
T cells which had migrated through the HUVEC monolayer into the lower
chambers were recovered. The radioactivity in these samples was determined by gamma counting. The percentage of migrated cells was calculated as above. Spontaneous release of 51Cr from the labeled cells during
the 4-h migration assay was ⬍2%.
Immunofluorescence staining
Briefly, T cells were washed, resuspended in PBS plus 0.5% BSA, and
incubated with 10 ␮g/ml mAb to CD4, CD8, CD45RA, CD45RO, and
CXCR3 at 4°C for 30 min. Cells were washed twice and incubated with
FITC-conjugated sheep anti-mouse IgG (Sigma-Aldrich). Finally, cells
were washed, fixed in 1% paraformaldehyde in PBS, and analyzed by flow
cytometry.
Measurement of lymphocyte adhesion
HUVEC were grown to confluence in gelatin-coated 96-well tissue culture
plates. 51Cr-labeled T lymphocytes (2 ⫻ 105 cells in 100 ␮l RPMI plus
10% FCS) were added to triplicate wells with or without I-TAC (200
ng/ml). In some experiments, HUVEC was pretreated with 200 ng/ml ITAC for various times before adding labeled T cells. The cells were allowed to adhere at 37°C for 60 min. Nonadherent cells were removed by
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Materials and Methods
6421
6422
I-TAC-INDUCED HUMAN LYMPHOCYTE TEM
four washes with warm RPMI. The bound T cells were lysed with 0.1 N
NaOH, collected into tubes, and the radioactivity was measured by gamma
counting. Percentage of cell adhesion was calculated by dividing the radioactivity of bound cells by the radioactivity of total input cells.
Measurement of I-TAC
Statistical analysis
Data were expressed as either the mean ⫾ SEM of multiple assays, or as
the mean ⫾ SD of replicate from a representative experiment. ANOVA and
Student’s unpaired t test were used to compare the differences between
means.
FIGURE 1. Dose-dependant effect of I-TAC and IP-10 on T cell chemotaxis (A) and TEM (B). 51Cr-labeled blood T cells were added to the
upper chamber of Transwells having either gelatin and fibronectin-coated
membranes (A) or membranes with confluent HUVEC monolayers (B).
Chemokines were added in the lower chamber at the indicated concentrations and migration was allowed to proceed for either 1 h (chemotaxis) or
for 4 h (TEM) at 37°C. C, The effect of I-TAC induced TEM in the presence or absence of a chemokine gradient was tested by adding 200 ng/ml
I-TAC to either lower, upper, or both Transwell chambers. D, The effect of
anti-CXCR3 mAb (1C6) treatment of T cells on chemotaxis and TEM is
shown. The percentage of migration was calculated by dividing the radioactivity of migrated cells by the total radioactivity of the cells added to the
upper chamber. Data are expressed as means ⫾ SD of triplicate wells of
four to six similar experiments.
Results
Effect of I-TAC on T cell chemotaxis and TEM
The migration of unstimulated blood T cells, either across gelatin
and fibronectin-coated microporous Transwell membranes or
across HUVEC monolayers grown on Transwell membranes to
various concentrations of I-TAC in the lower chamber of the Transwells was tested. For comparison, migration to IP-10, which also
binds to CXCR3, was also measured. As shown in Fig. 1A, I-TAC
at 10 ng/ml had little effect on T cell migration, but increased T cell
chemotaxis by 5-fold from a background of 4 –20% migration at a
concentration of 100 –300 ng/ml. In contrast, IP-10 at 100 ng/ml
induced only a small increase in T cell migration, and at 300 ng/ml
induced only a 3-fold increase in T cell chemotaxis, which was still
less than that with I-TAC.
I-TAC also induced the migration of T cells across HUVEC
monolayers. As shown in Fig. 1B, I-TAC at 100 – 400 ng/ml induced a 4- to 5-fold increase in T cell TEM from 6% to 22–28%,
while IP-10 induced only a small increase in T cell TEM, and even
at 400 ng/ml, only induced a 4% increased migration above medium control. Because there seems to be a plateau in the TEM to
I-TAC between 200 and 400 ng/ml, in subsequent studies 200
ng/ml was used. Blood T cells from ⬎10 donors showed a range
of migration from 15 to 28% in response to this concentration of
I-TAC with backgrounds of 2– 6%. This variation is likely to be
related to donor differences in I-TAC responsive T cells in the
circulation, but in all experiments a strong TEM to I-TAC was
observed and a much greater response to I-TAC than to IP-10.
To determine whether the T cell TEM to I-TAC was chemotactic or due to increased chemokinesis, the effect of adding I-TAC in
the upper chamber with the T cells, or in both upper and lower
chambers was tested. As shown in Fig. 1C, T cell migration across
the HUVEC to the lower chamber was completely lost when ITAC was added to either the upper or both upper and lower cham-
bers, which indicated that I-TAC induced TEM was chemotactic
not chemokinetic for the T cells in this system. This also suggested
that the effect of I-TAC was on the T cells and not the HUVEC.
I-TAC specifically binds to CXCR3 on T lymphocytes; therefore, the effect of treating T cells with anti-CXCR3 on T cell
chemotaxis and TEM was determined. I-TAC-induced T cell
chemotaxis was inhibited by 90% and TEM by 84% following antiCXCR3 mAb treatment (Fig. 1D), suggesting that I-TAC induced
migration of the blood lymphocyte was mediated through CXCR3.
Effect of I-TAC on chemotaxis and TEM of T cells cultured with
or without exogenous IL-2
Previous reports had shown that T cells cultured with IL-2 for 8 or
more days to induce T cell proliferation and activation chemotaxed
to I-TAC. Therefore, the effect of culturing T cells with and without IL-2 for 8 days on T cell chemotaxis and TEM to I-TAC was
examined. Up to 90% of the T cells cultured without IL-2 were
recovered and there was a 3-fold increase in the T cells in the
presence of IL-2. Both groups of T cells demonstrated higher spontaneous migration to medium alone than did freshly isolated T
cells (Table I vs Fig. 1A). However, both sets of cultured T cells
showed similar and significant ( p ⬍ 0.05) levels of chemotaxis to
I-TAC whether they were cultured with or without IL-2.
The migration of T cells cultured in the presence or absence of
IL-2 across HUVEC was quite different (Table I). T cells cultured
without IL-2 had a relatively low spontaneous TEM and a marked
increase in migration to I-TAC similar to fresh T cells. Although
T cells cultured with IL-2 demonstrated chemotaxis to I-TAC in
the absence of HUVEC (Table I); in the presence of HUVEC,
these T cells spontaneously migrated across the EC in large numbers and the addition of I-TAC did not increase this further. Interestingly, these IL-2-activated T cells also had an increased
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Synovial fluids were obtained from the rheumatology clinic of the Queen
Elizabeth II Hospital (Dalhousie University), and the cell-free supernatants
were stored at ⫺80°C until tested for I-TAC. To obtain endothelial-conditioned medium, HUVEC was grown to confluency in 35-mm tissue culture wells in HUVEC medium and the growth medium was replaced with
3 ml/well RPMI plus 5 mg/ml HSA just before stimulating cells using 200
U/ml IFN-␥ and/or TNF-␣ for 18 h. HUVEC supernatant was collected and
tested for I-TAC secretion using a sandwich ELISA with the ELISA amplification system (Life Technologies). Briefly, 50 ␮l/well of 0.5 ␮g/ml
rabbit polyclonal anti-human I-TAC Ab in carbonate buffer (pH 9.6) was
coated overnight at 4°C to 96-well Maxi-Sorp plates (Nunc Immuno Plates,
Roskilde, Denmark), washed twice with 0.01% Tween 20/TBS, and
blocked with 2% BSA at 37°C for 2 h. After three washes, samples and
known concentrations of recombinant human I-TAC were added and incubated overnight at 4°C. Biotin-labeled polyclonal anti-human I-TAC Ab
at 0.5 ␮g/ml (50 ␮l/well) was added and incubated for 2 h at 37° C,
followed by streptavidin-alkaline phosphotase for 30 min at room temperature. Each step was separated by four washes. Then, 50 ␮l of NADPH
substrate was added for 45 min, followed by 50 ␮l of the amplifier solution
containing alcohol dehydrogenase and diaphorase according to manufacturer’s instructions. OD was read at 490 nm after stopping the reaction with
0.3 N H2SO4 solution. The detection limit of this ELISA amplification
system was ⬃4 pg/ml.
The Journal of Immunology
6423
Table I. Effect of culturing blood T cells with IL-2 on chemotaxis and TEM to I-TACa
Treatment
Chemotaxis (% migration)
TEM (% migration)
Extp.
IL-2
Medium
I-TAC
1
⫺
⫹
⫺
⫹
13.3 ⫾ 3.8
15.1 ⫾ 0.5
12.6 ⫾ 0.1
9.1 ⫾ 1.5
30.1 ⫾ 8.0
32.1 ⫾ 5.0†
24.0 ⫾ 1.8†
25.4 ⫾ 2.1†
2
†
Medium
I-TAC
8.4 ⫾ 0.3
32.0 ⫾ 6.8
1.7 ⫾ 0.9
31.0 ⫾ 0.5
39.6 ⫾ 3.0*
38.2 ⫾ 3.4
15.7 ⫾ 0.5*
39.2 ⫾ 1.8
a
Human blood T cells were cultured in the presence or absence of 400 U/ml recombinant human IL-2 for 8 days, labeled
with 51Cr, and their migration in response to 200 ng/ml I-TAC was tested as in Fig. 1. Migration is expressed as the percentage
of total input radiolabeled cells, and represent the mean ⫾ SD of triplicate Transwells studied in each experiment.
†
p ⬍ 0.05.
* p ⬍ 0.01; I-TAC vs medium.
I-TAC-induced chemotaxis of T cell subpopulations
To identify the phenotype of T cells migrating in response to ITAC, T cells were separated into CD4, CD8, CD45RA⫹ (naive),
and CD45RA⫺ (memory) fractions, and their chemotaxis to I-TAC
across bare membranes was determined (Fig. 2). CD4⫹ T cells
demonstrated lower spontaneous migration than CD4⫺ T cells, but
both CD4⫹ and CD4⫺ T cells had a strong chemotactic response
to I-TAC (Fig. 2A). Similarly, when T cells were separated into
CD8⫹ and CD8⫺ populations, their migration to I-TAC was comparable (data not shown), not only between these two populations,
but also to that seen with CD4⫺ and CD4⫹ cells, respectively.
Fractionation of T cells into naive and memory T cells demonstrated that memory-enriched T cells had a slightly greater spontaneous migration than naive T cells, and only memory but not
naive T cell chemotaxis was markedly increased to I-TAC (Fig.
2B). Memory-enriched T cells migrated ⬃4-fold (5–19%) more to
I-TAC than to the medium control, while there was no significant
response by naive T cells. Thus, I-TAC induced the chemotaxis of
both CD4 and CD8 memory, but not naive T cells.
ulated EC from 15 to 30% of input T cells. The TEM to I-TAC
across TNF-␣-activated HUVEC was also significantly greater
than observed across unstimulated EC in response to I-TAC, and
appeared to be additive to that of either I-TAC alone or TNF-␣
activation alone. In striking contrast to TEM across TNF-␣-treated
EC, T cell migration to I-TAC across IFN-␥ or IFN-␥ plus TNF␣-stimulated EC was similar to the migration seen in the absence
of I-TAC across HUVEC treated with these cytokines. Similarly, migration to I-TAC across IFN-␥ or IFN-␥ plus TNF-␣-stimulated EC
was not significantly higher than that to I-TAC alone across unstimulated endothelium. Thus, IFN-␥ treatment of the HUVEC reduced the
response to added I-TAC and also prevented the increase in TEM to
I-TAC across TNF-␣-stimulated HUVEC. These findings show that T
cell TEM to I-TAC is differentially regulated by IFN-␥ and TNF-␣
activation of the endothelium. Moreover, this effect was not caused by
change in T cell adhesion to cytokine-stimulated HUVEC, as we did
not observe any effect of I-TAC on T cell adhesion to IFN-␥ and/or
TNF-␣-stimulated endothelium (data not shown).
Migration of CD4 and CD8 T cells to I-TAC across normal and
cytokine-activated endothelium
The effect of I-TAC on TEM of CD4 and CD8 T cells across
normal and cytokine-stimulated EC was investigated. I-TAC significantly increased TEM of both CD4⫹ and CD4⫺ T lymphocytes
across unstimulated HUVEC (Fig. 4). Both CD4⫹ and CD4⫺ T
Effect of cytokine activation of the endothelium on T cell
migration to I-TAC
In inflammation, T cells migrate across cytokine-activated EC in
response to chemokines. Therefore, the effect of treating the
HUVEC with TNF-␣, IFN-␥, and both cytokines for 18 h on ITAC-induced T cell TEM was determined. As shown in Fig. 3, T
cell migration across HUVEC treated with TNF-␣, IFN-␥, or both
cytokines was increased 4- to 5-fold from 4% to 15–23%. I-TAC
significantly increased lymphocyte TEM across the TNF-␣-stimTable II. CXCR3 expression and phenotype of human T cells cultured
with or without IL-2a
Percent Positive Cells
T Cells
CD45RA⫹
CD45RO⫹
CXCR3⫹
Freshly isolated
8-day culture without IL-2
8-day culture with IL-2
54.3
36.7
33.2
43.2
63.3
66.8
29.1
31.1
56.4
a
Human blood T cells, freshly isolated or cultured for 8 days with or without 400
U/ml IL-2 were stained with anti-CD45RA (G1–15), anti-CD45RO (UCHL-1), or
anti-CXCR3 (1C6) as described in Materials and Methods. Values shown are representative of three similar experiments using different donors.
FIGURE 2. Chemotaxis of blood T cell subpopulations in response to
I-TAC. CD4⫹, CD4⫺, CD45RA⫹ naive, and CD45RA⫺ memory T cell
subsets were isolated as described in Materials and Methods. Cells were
51
Cr-labeled and 2 ⫻ 105 cells were allowed to migrate for 1 h to 200 ng/ml
I-TAC. Values shown are percentage of total input radiolabeled cells and
represent means ⫾ SD of one of two similar experiments done in triplicate.
Two additional experiments using CD8⫹ and CD8⫺ T cells showed similar
results to that of CD4⫹, CD4⫺ T cells shown here. ⫹, p ⬍ 0.05 vs medium
controls; #, p ⬍ 0.02 naive vs memory T cells.
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spontaneous adherence to the HUVEC (data not shown). Immunofluorescence staining of fresh and cultured T cells showed
that there was an increase in the proportion of T cells with a
memory phenotype after culture, and culturing the cells did not
increase CXCR3 expression, but IL-2 treatment nearly doubled
the percentage of cells expressing CXCR3 (Table II).
6424
FIGURE 3. Effect of endothelial activation by cytokines on T cell TEM
in response to I-TAC. ECs were unstimulated or stimulated with 200 U/ml
of the indicated cytokines for 18 h and labeled T cells were allowed to
migrate to either medium control or in response to 200 ng/ml I-TAC in a
4-h TEM as outlined in Fig. 1. Values shown are percentage of total input
radiolabeled cells. Pooled data from 10 –18 independent experiments, using
T cells and HUVEC from different donors, are expressed as means ⫾ SEM.
ⴱ, p ⬍ 0.001 vs medium control; #, p ⬍ 0.0001, migration to I-TAC across
unstimulated vs TNF-␣-stimulated HUVEC.
Migration of memory and naive T cells to I-TAC across normal
and cytokine-activated endothelium
I-TAC induced a marked increase in CD45RA⫺ memory-enriched
T cell TEM across unstimulated HUVEC, but had a relatively meager effect on CD45RA⫹ naive-enriched T cells (Fig. 5) similar to
the chemotactic response to I-TAC. Cytokine treatment of
HUVEC increased memory, but not naive T cell TEM. I-TAC
further enhanced memory-enriched lymphocyte TEM across
TNF-␣, but not IFN-␥ or IFN-␣ plus TNF-␣-treated HUVEC. Naive enriched T cell migration was only slightly increased across
TNF-␣-activated EC, and was much less than that of memory T
FIGURE 5. TEM of CD45RA⫺ memory (A and C) and CD45RA⫹ naive (B and D) T cells in response to I-TAC. HUVEC monolayers were
either unstimulated or stimulated with 200 U/ml of the indicated cytokines
for 18 h. CD45RA⫹ or CD45RA⫺ T cells were labeled with 51Cr and used
for TEM as outlined in Fig. 1. Migration is expressed as the percentage of
total input radiolabeled cells. Data from two independent experiments performed in triplicate are expressed as means ⫾ SD. ⌾, p ⬍ 0.01 vs medium
control; #, p ⬍ 0.01 naive vs memory T cells.
cells to all stimuli. Thus, memory T cells, but not naive T cells,
were responsible for the increased TEM to I-TAC across unstimulated HUVEC, and for the increased response across TNF-␣-stimulated endothelium. Supporting this observation, freshly isolated
memory T cells had nearly 10-fold higher proportion of cells expressing CXCR3 (33%) compared with naive T cells (3.5%).
Effect of cytokine stimulation of HUVEC on I-TAC secretion
Because cytokine stimulation of the HUVEC differentially regulated T cell TEM to I-TAC, the effect of cytokine treatment on the
production of I-TAC by HUVEC was determined in EC-conditioned media. As shown in Table III, unstimulated and TNF-␣stimulated HUVEC produced no detectable amounts of I-TAC.
However, treatment of the HUVEC with IFN-␥ induced a large
amount of I-TAC to be produced by the EC and released into the
medium. Stimulation of the HUVEC with both IFN-␥ and TNF-␣
also induced I-TAC secretion and caused an even greater (⬎60%)
increase than IFN-␥ activation alone. Thus, IFN-␥, but not TNF-␣,
induced I-TAC production by HUVEC, and TNF-␣ acted synergistically with IFN-␥ to augment I-TAC secretion.
The role of LFA-1 and VLA-4 in I-TAC-induced T cell TEM
To determine the cell adhesion molecules involved in T cell migration to I-TAC, the effect of function blocking mAbs to LFA-1
Table III. Effect of cytokine stimulation on I-TAC production by
HUVECa
FIGURE 4. TEM of CD4⫹ (A) and CD4⫺ (B) T cells in response to
I-TAC. HUVEC monolayers were either unstimulated or stimulated with
200 U/ml of the indicated cytokines for 18 h. CD4⫹ or CD4⫺ T cells were
labeled with 51Cr and used for TEM as outlined in Fig. 1. The migrated
cells were collected and the values are expressed as percentage of total
input radiolabeled cells. Data from one of two independent experiments,
performed in triplicate, were expressed as means ⫾ SD. Two more experiments using positively selected CD8 T cells (CD8⫹ and CD8⫺) showed
similar results to that of CD4⫹, CD4⫺ T cells shown here. ⴱ, p ⬍ 0.001 and
⌾, p ⬍ 0.01, I-TAC vs medium control.
Endothelial Stimulation
—b
TNF-␣
IFN-␥
IFN-␥ ⫹ TNF-␣
I-TAC Concentration
(ng/ml)
⬍ 0.004
⬍ 0.004
22.4 ⫾ 1.6
36.3 ⫾ 4.3
a
Confluent monolayers of HUVEC were stimulated with TNF-␣, IFN-␥, or both
cytokines, and the concentration of I-TAC in the conditioned medium was determined
18 h later by ELISA as outlined in Materials and Methods. Values shown are mean ⫾
SD from two independent experiments, each with duplicate determinations.
b
—, no stimulation.
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cells demonstrated an increase in TEM across IFN-␥ and/or TNF␣-treated HUVEC. I-TAC-stimulated TEM was enhanced across
TNF-␣ but not IFN-␥ and IFN-␣ plus TNF-␣-treated endothelium
for both CD4⫹ and CD4⫺ T cells. Similar results were obtained
using CD8⫹ and CD8⫺ T cells (data not shown).
I-TAC-INDUCED HUMAN LYMPHOCYTE TEM
The Journal of Immunology
and VLA-4 on T lymphocyte TEM across unstimulated and cytokine-treated HUVEC was examined. I-TAC-induced TEM of
⬎20% of the T cells across unstimulated, IFN-␥, and IFN-␥ plus
TNF-␣-treated HUVEC, and ⬎30% T cell migration across TNF␣-stimulated HUVEC (Fig. 6). Anti-LFA-1 mAb blocked ⬃80%
of the migration across unstimulated HUVEC, anti-VLA-4 mAb
had little effect, while combined blockade of both LFA-1 and
VLA-4 inhibited ⬃90% of the lymphocyte migration to I-TAC.
Migration across IFN-␥-stimulated HUVEC was completely abolished by anti-LFA-1 mAb, and unaffected by VLA-4 blockade. T
cell TEM to I-TAC across TNF-␣ and IFN-␥ plus TNF-␣ stimulated HUVEC was significantly, but only partially (50 – 60%), inhibited by anti-LFA-1 mAb. Blocking VLA-4 inhibited this migration by ⬃10%, and the combination of LFA-1 and VLA-4
blockade completely abolished T cell TEM to I-TAC across these
activated endothelia. These findings suggested that I-TAC-induced
TEM is completely dependent on LFA-1 and VLA-4.
6425
FIGURE 7. Measurement of the concentration of I-TAC in the synovial
fluid of patients with arthritis. Synovial fluid was obtained from patients
with osteoarthritis (n ⫽ 12) and rheumatoid arthritis (n ⫽ 15) and the
concentration of I-TAC was determined by ELISA as described in Materials and Methods. Each data point represents the I-TAC concentration in
an individual synovial fluid sample and the lines represent the mean ⫾
SEM I-TAC in the rheumatoid arthritis samples.
Presence of I-TAC in synovial fluid of arthritic joints
in all of the rheumatoid joint synovial fluids. The mean concentration of I-TAC was 735 pg/ml. In contrast, no I-TAC was detected (⬍4 pg/ml) in the synovial fluid of patients with osteoarthritis. This suggests that large amounts of I-TAC are produced
during joint inflammation in rheumatoid arthritis; and that osteoarthritis is not associated with production of this chemokine. Moreover, I-TAC is present at a site of chronic inflammation in vivo.
Discussion
FIGURE 6. Effect of blocking LFA-1 and VLA-4 on I-TAC-induced T
cell TEM. HUVEC monolayers were either left unstimulated (A), or stimulated with 200 U/ml of the indicated cytokines (B–D) for 18 h. Blood T
cells were labeled with 51Cr and either left untreated or treated with mAb
to either CD45, LFA-1, VLA-4, or both LFA-1 plus VLA-4, and their TEM
was measured as in Fig. 1. Values shown are means ⫾ SD from triplicate
wells, and are representative of three to four similar experiments. ⴱ, p ⬍
0.001 untreated vs mAb treated T cells.
The importance of chemokines in lymphocyte extravasation has
been established in recent years; however, the relative contribution
of many chemokines, even chemokines binding to the same receptors, in lymphocyte migration is inadequately understood (4, 6, 7,
22, 23). The CXC chemokines, I-TAC, IP-10, and Mig all bind to
CXCR3, and these chemokines have been shown to act in vitro on
IL-2-activated T cells, on allogeneically activated T cells in MLR,
and on thymocytes during lymphopoiesis (8 –10, 16, 24, 25). Although exogenous IL-2 can enhance CXCR3 expression on T cells
in culture, we and others have also found CXCR3 expressed on
35– 40% of normal blood T cells (Table II) (10, 16, 26). Therefore,
we investigated the chemotaxis and TEM of freshly isolated blood
T cells in response to I-TAC, which is the most potent of the
CXCR3 ligands, and compared it to IP-10 (9).
This is the first study to demonstrate that I-TAC is a chemoattractant for freshly isolated normal human blood T cells. I-TAC
was effective at inducing both chemotaxis and TEM at 100 –300
ng/ml, which is similar to the concentrations found to be effective
with several other inflammation-associated chemokines, including
RANTES, macrophage-inflammatory protein (MIP)-1␣, and
monocyte chemoattractant protein-1 (MCP-1) (27, 28). In addition
to stimulating T lymphocyte chemotaxis across bare membranes,
this study also demonstrates that I-TAC is a very potent inducer of
T cell TEM. Moreover, with the exception of SDF-1, I-TAC appeared to be among the strongest stimulators of human T cell
TEM, which we have observed (28). Concentrations as low as 25
ng/ml stimulated significant TEM, suggesting that in vivo I-TAC
may be an effective recruiter of normal blood T lymphocytes.
Cole et al. (9) were unable to show that I-TAC induced chemotaxis of unstimulated human T cells. This may be the result of the
differences in the techniques used to measure and enumarate the T
cells. Cole et al. (9) coated filters with collagen on the bottom
surface and the cells that migrated to the lower surface of the filter
were scored microscopically. In contrast, in our experiments the
upper surface of Transwell membranes were coated with gelatin
and fibronectin and radiolabeled T cells, which had migrated
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To evaluate the potential relevance of I-TAC to inflammation in
vivo, the concentration of I-TAC in the synovial fluid of arthritic
joints from patients with rheumatoid and osteoarthritis was determined. The synovial fluid samples from rheumatoid patients had
significantly higher leukocyte counts (11.7 ⫾ 0.9 ⫻ 106/ml) than
those of patients with osteoarthritis (0.6 ⫾ 0.2 ⫻ 106/ml). As
shown in Fig. 7, significant concentrations of I-TAC were present
6426
CD4⫹ and CD8⫹ T cells, and induces the migration of only memory (CD45RA⫺) and not naive (CD45RA⫹) T lymphocytes. This
demonstrates that CXCR3 is functional on both memory CD4⫹
and CD8⫹ T cells in the blood. In this respect, I-TAC resembles
MCP-1, RANTES, and MIP-1␣ in inducing human blood memory
T cell TEM (28).
During inflammation, EC are stimulated by cytokines such as
TNF-␣ and IFN-␥ to up-regulate adhesion molecule expression,
which promotes TEM and lymphocyte recruitment into the inflammatory site (3, 32–35). Migration of T cells, monocytes, and neutrophils in response to chemokines has also been shown to be
augmented across cytokine-activated endothelium (28, 36, 37). ITAC-induced T cell TEM was markedly increased by TNF-␣ activation of HUVEC (Fig. 3). This is similar to the previous report
with the chemokines, RANTES, MIP-1␣, and SDF-1 (28). TEM to
these latter chemokines was also augmented by IFN-␥ activation
of HUVEC, while TEM to I-TAC did not increase further across
IFN-␥ or IFN-␥ plus TNF-␣-activated endothelium. Furthermore,
the increase in TEM across TNF-␣, but not across IFN-␥ or IFN-␥
plus TNF-␣-stimulated HUVEC was observed for both CD4⫹ and
CD8⫹ memory T cells. Therefore, I-TAC appears to be unusual
among these inflammation-associated chemokines in that I-TACinduced TEM is differentially regulated by IFN-␥ and TNF-␣ treatment of the endothelium.
ECs stimulated with cytokines can produce chemokines which
can affect T cell TEM (28, 38 – 40). As previously shown, T cell
TEM across IFN-␥- and TNF-␣-activated endothelium has been
shown to be pertussis toxin sensitive (28). Chemokines present at
the luminal (apical) surface of EC enhance leukocyte adhesion but
not migration, while chemokines on the abluminal (basal) surface
of EC induce TEM (41). This suggests that there is a requirement
for a chemokine gradient for the T cell to transmigrate the endothelium. Our results demonstrate that treatment of the HUVEC
with IFN-␥ and IFN-␥ plus TNF-␣, but not TNF-␣ alone induces
large amounts of I-TAC to be produced (Table III). In addition,
IFN-␥-activated EC can also produce IP-10 and Mig (14, 15, 42,
43). Because I-TAC-stimulated TEM is highly dependent on a
chemokine gradient (Fig. 1C), one explanation for the lack of any
increase in cell migration to I-TAC across IFN-␥ or IFN-␥ plus
TNF-␣ activated HUVEC may be the disruption of the required
chemotactic gradient.
Another explanation for the decreased migration to I-TAC
across IFN-␥-treated EC may relate to the proteoglycans on the
surface of the EC, which can bind chemokines (42, 44 – 46). These
proteoglycans are thought to produce a high local concentration of
chemokines in vivo to induce rapid integrin activation on rolling
leukocytes (47). Thus, I-TAC, IP-10, and Mig induced by IFN-␥
and IFN-␥ plus TNF-␣ treatment of the HUVEC is likely immobilized on the EC. These immobilized CXCR3⫹ ligands may also
interfere with the chemotactic gradient or may contribute to desensitization and internalization of CXCR3⫹, as has been shown
for some other chemokines (48, 49). It was also shown recently
that surface-bound I-TAC, IP-10, and Mig on IFN-␥ activated, but
not on resting EC, upon coculture, could rapidly induce T cell
CXCR3 internalization (50). Experiments to be reported elsewhere
by us also indicate that CXCR3 on T cells contributes together
with other chemokine receptors to TEM across IFN-␥-stimulated
endothelium, further suggesting that IFN-␥ activated EC-derived
I-TAC might play a role in desensitizing CXCR3⫹ T cells.
In the case of monocytes, MCP-1 produced by IL-1-activated
EC has been shown to inhibit monocyte TEM in response to exogenous MCP-1 (36). Taken together, it appears that the chemokines produced by the endothelium in response to TNF-␣ and/or
IFN-␥ may play an important role in differential regulation of T
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through the membrane in response to I-TAC, were quantified by
gamma counting. Chemokines can enhance binding to fibronectin
and collagen, so the coating on the Transwells may have facilitated
chemotaxis to I-TAC. Unactivated T cells are less adhesive than
IL-2-activated T cells to extracellular matrix proteins, such as the
collagen on the underside of the filters. This may have resulted in
better adherence and detection of the IL-2-activated T cells than
the unstimulated lymphocytes. In any case, it appears that I-TAC
under appropriate circumstances is a potent stimulator of unstimulated T cell chemotaxis.
Our results also indicated that I-TAC was having a specific chemotactic effect. I-TAC induced a concentration-dependent increase
in lymphocyte TEM, and I-TAC-stimulated TEM was abolished in
the absence of a concentration gradient for the chemokine. Blockade of CXCR3 also prevented I-TAC-stimulated chemotaxis and
TEM, demonstrating that the effect of I-TAC on blood T cells was
mediated through binding to CXCR3. This suggests that CXCR3
on blood lymphocytes is functional without prolonged lymphocyte
activation.
Previous studies had shown that the percentage of T cells expressing CXCR3 increased after culture of the lymphocyte in the
presence of exogenous IL-2 and/or PHA for 8 –21 days (10, 16).
Moreover, T cells cultured with IL-2 chemotaxed in response to
I-TAC (9, 10). In the present study, lymphocytes cultured for 8
days with exogenous IL-2 also increased their expression of
CXCR3 (Table II) and I-TAC induced both chemotaxis as well as
TEM of these cells (Table I). In addition, our results show that T
cells maintained in culture without exogenous IL-2 migrated to
I-TAC to a similar extent as lymphocytes incubated with IL-2, and
the chemotactic response to I-TAC of the T cells cultured for 8
days was comparable to that observed with T cells freshly isolated
from the blood. Thus, our studies suggest that I-TAC-stimulated
migration of T cells does not require prolonged IL-2 preactivation
of the T cells and demonstrate that there are significant numbers of
I-TAC responsive T cells in blood. This finding suggests a role for
I-TAC in the recruitment of unactivated circulating blood
CXCR3⫹ T cells to inflammatory sites.
The T cells used in this study are considered “resting” T cells
because they were freshly isolated from healthy donors, and the
cells were not activated in vitro using cytokines or mitogens. These
freshly isolated T cells lack activation markers and are CD25low
and ⬍3% CD69⫹ similar to those previously reported by others
and found to express CXCR3 (16, 24). Because we found that
I-TAC induced 12–25% of T cells to migrate almost all of the
migration observed was by T cells lacking activation markers. Activation with IL-2 did not substantially increase the already considerable chemotaxis induced by I-TAC (Table I) under the conditions used in this study. Thus, blood T cells in the absence of
cytokine or mitogen stimulation appear to be highly responsive to
I-TAC.
Our studies demonstrate that I-TAC can stimulate blood T lymphocyte chemotaxis, and suggest that it acts on T cells to induce
transmigration of endothelium in vitro. Although the chemotactic
effect of I-TAC on blood T cells suggests that I-TAC-induced
TEM is mediated by its actions on T cells, ECs have also been
shown to express functional CXCR3 (29 –31). However, I-TACinduced T cell TEM does not appear to be due to an effect of
I-TAC on the HUVEC. Addition of I-TAC in the upper chamber
of the Transwell containing the EC monolayer did not result in T
cell TEM, and a chemotactic gradient was required for TEM.
Previous studies have shown that CXCR3 is expressed on both
CD4⫹ and CD8⫹ T lymphocytes of the memory phenotype, and
on some naive CD8⫹ cells (16, 26). Our results show that I-TAC
stimulates the chemotaxis and TEM of both freshly isolated blood
I-TAC-INDUCED HUMAN LYMPHOCYTE TEM
The Journal of Immunology
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cell TEM induced by I-TAC. One thing which is not clear is why
this effect is observed with I-TAC, but not with other chemokines,
such as RANTES. Cytokine activation of HUVEC can induce substantial secretion of RANTES (our unpublished observation), but
RANTES-induced TEM is enhanced across TNF-␣- and IFN-␥treated EC (28). Further studies on EC chemokine production and
T cell chemokine receptor modulation will be needed to understand these processes.
Our studies also examined the role of the major T lymphocyte
integrins in I-TAC-induced TEM, because previous studies had
shown that both LFA-1 and VLA-4 could mediate TEM, but the
contribution of these integrins varied based on the cytokines and
chemokines used to induce lymphocyte migration (51). I-TACinduced TEM of T cells across unstimulated and IFN-␥-stimulated
EC was completely or almost completely abolished by blockade of
LFA-1 (Fig. 6). In contrast, I-TAC stimulated T cell TEM across
TNF-␣ and TNF-␣ plus IFN-␥ was partially inhibited by antiLFA-1 and abolished when both VLA-4 and LFA-1 were blocked.
Previous studies had shown that TEM induced by RANTES, MIP1␣, and SDF-1 across unstimulated and IFN-␥-treated HUVEC
was mediated by LFA-1, while migration across TNF-␣-activated
HUVEC stimulated by these chemokines, but not in the absence of
the chemokines, was dependent on both LFA-1 and VLA-4 (51).
TEM induced by I-TAC across unstimulated and TNF-␣-activated
HUVEC similarly appears to depend on these two integrins, suggesting that I-TAC increases not only LFA-1 but also VLA-4 function during TEM. It is also interesting to note that TEM across EC
treated with both TNF-␣ and IFN-␥ was also mediated by these
two integrins, even though this migration was not enhanced by
I-TAC, suggesting that TEM across this activated endothelium is
induced by endothelial-derived chemokines, probably in part
CXCR3 ligands.
Our findings that I-TAC induces normal blood T lymphocyte
TEM suggests that T cell recruitment to inflamed tissues may be
stimulated by the action of I-TAC on resting blood T cells. To
determine whether I-TAC may be contributing to T cell migration
in vivo, the concentration of I-TAC in arthritic synovial fluid was
examined. I-TAC was abundant in synovial fluid from joints of
patients with rheumatoid arthritis, but not osteoarthritis (Fig. 7).
The finding of substantial levels of I-TAC in RA synovial fluid
further supports an in vivo role for I-TAC in recruiting T cells to
inflammation.
A recent report has shown increased levels of IP-10 in synovial
fluid (52), and suggests that CXCR3 ligands likely play an important role in RA. The studies here indicate that I-TAC may play a
particularly important role in this T cell infiltration, because unlike
IP-10, I-TAC can stimulate TEM by resting blood lymphocytes.
Increased expression of CXCR3 on T cells in inflammatory reactions mediated by type 1 cytokines has been previously reported
(15, 16, 18, 23, 43, 52–55). Our results suggest that the CXCR3 on
T cells in these inflammatory sites likely mediates TEM to the site,
and may not be the result of the up-regulation of CXCR3 by T cells
in response to the cytokines in the inflamed tissue. Future studies
of the in vivo migration of CXCR3⫹ T cells are needed to determine the full role of this receptor in recruitment of T lymphocytes
to inflammation.
6427
6428
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I-TAC-INDUCED HUMAN LYMPHOCYTE TEM