| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Interaction between T cell-mounted LFA-1 and its ligands, ICAM-1, -2, and -3, is important for T cell surveillance and migration, T cell-APC interaction, and cytotoxic T cell function (7, 8). Also, a number of reports suggest a role for signaling through LFA-1 in costimulation of T cells (reviewed in Ref. 9). As examples, engagement of cell surface LFA-1 with plate-bound ICAM-1 enhances T cell activation following cross-linking of the TCR-CD3 complex (10), and stimulator cells transfected with MHC and ICAM-1, but not those transfected with MHC alone, induce proliferation and IL-2 secretion in naive T cells (11). In support of a role in second signal generation, LFA-1 coengagement induces transmembrane signals distinct from those delivered through the TCR-CD3 complex alone (12).
In contrast to the considerable body of work dealing with signaling into T cells through LFA-1, the effect of signaling into T cells through its counterreceptors the ICAMs remains relatively unstudied (reviewed in Ref. 13). ICAM-1 (CD54) is expressed on endothelial cells, epithelial cells, fibroblasts, keratinocytes, astrocytes, and leukocytes as well as conventional APCs (reviewed in Ref. 14). Thus, many cell types express ICAM-1 and can use ICAM-1 to deliver signals into T cells through LFA-1 on the T cell surface. In contrast, only leukocytes express LFA-1 and thus only comparatively few cell types can potentially deliver signals into T cells through ICAM-1 expressed on the T cell surface. Also, unlike the case with LFA-1, ICAM-1 on T cells is not thought to participate in migration, so signals delivered into the T cell through ICAM-1 are more likely to be involved in cell recognition or activation. Thus, the differential expression of LFA-1 and its counterreceptor ICAM-1 on different cell types raises interesting questions about T cell regulation.
A few reports exist that investigate the signaling events induced by stimulation through ICAM-1 and the effect that these events have on cellular function. Cross-linking ICAM-1 with mAbs in rat brain endothelial cells causes phosphorylation on tyrosine of the cytoskeleton-associated proteins FAK, paxillin, and cortactin (15). Stimulation through ICAM-1 in human B cell lines results in increased phosphorylation of the Src family kinases p53/p56Lyn and pp60Src (16, 17). Work from our laboratory has begun to define signaling events that occur following ICAM ligation on T cells. We observed that cross-linking of ICAM-1 leads to the transient phosphorylation on tyrosine and concomitant inactivation of the cell cycle regulator cdc2 kinase (18). Although a unifying hypothesis is not apparent, the data collected to date suggest that stimulation of various cells through ICAM-1 might play a direct role in cell function. The importance of ICAM-1 to the immune response is underscored by diminished responses following immune challenge in ICAM-1-deficient mice (19).
In the present study, we examined the ability of stimulation through
ICAM-1 to deliver a costimulatory signal for T cell activation. We
observed that in combination with stimulation through CD3,
costimulation through ICAM-1 induced proliferation of human T cells,
resulted in increased expression of the activation marker CD69, and
promoted sustained cell division in naive CD4+ T
cells. In addition, we observed that phosphatidylinositol 3-kinase
(PI3K)5 was
activated only by the double stimulus of anti-CD3 plus
anti-ICAM-1 and not by either used alone. Finally, we found that
this costimulatory signal is capable of driving IL-2 transcription and
leads to secretion of IL-2 and IFN-
, but not IL-5. Thus, it seems
that signaling of resting T cells through ICAM-1 delivers a distinct
costimulatory signal resulting in T cell activation and
proliferation.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Anti-CD3 (OKT3), anti-CD11a (HB202), and anti-CD54 (R6.5D6) were purchased from American Type Culture Collection (Manassas, VA) and purified from serum-free hybridoma culture medium using protein G-Sepharose. Results were verified with a second anti-ICAM-1 Ab, clone HA58 (BD PharMingen, San Diego, CA). Anti-CD28 (clone 28.2) was purchased from BD PharMingen. Anti-CD69-FITC, anti-CD4-PE, and anti-CD8-PE Abs were purchased from Caltag Laboratories (Burlingame, CA). Anti-PI3K Ab (p85 subunit) was from obtained BD Transduction Laboratories (Lexington, KY). PMSF, phorbol 12,13-dibutyrate (PDB), ionomycin, and PHA were obtained from Sigma-Aldrich (St. Louis, MO), as were all chemicals unless otherwise noted. CFSE was purchased from Molecular Probes (Eugene, OR).
Cell purification and culture
T cells were isolated from fresh tonsils or peripheral blood from multiple healthy donors as previously described (20). Briefly, cell suspensions from minced tonsils or peripheral blood were layered over Ficoll-Hypaque (Pharmacia, Piscataway, NJ) and spun for 30 min at 670 x g to isolate mononuclear cells. T cells were purified by E-rosetting. Typically, >98% of the purified cells were positive for CD3, as assessed by flow cytometry. Where indicated, purified T cells were negatively separated into CD4+ and CD8+ subpopulations using a MACS LS column (Miltenyi Biotec, Auburn, CA) by using the appropriate isolation kit according to the manufacturer’s directions. For purification of naive CD4+ T cells, PBMC were subjected to negative selection using a naive T cell enrichment kit (StemCell Technologies, Vancouver, British Columbia, Canada). All magnetically selected T cell subsets were of >99% purity, as assessed by flow cytometry. The leukemic T cell line Jurkat E6.1 was purchased from American Type Culture Collection (TIB-152). Culture medium for all cell types was RPMI 1640 (Mediatech, Herndon, VA), containing 10% FBS (Atlanta Biologicals, Norcross, GA), 50 U/ml each of penicillin and streptomycin (Life Technologies, Grand Island, NY), and 20 mM glutamine (Life Technologies).
Proliferation assay
Abs in PBS were attached to 96-well tissue culture plates (Midwest Scientific, St. Louis, MO) by incubation at 37°C for 2 h, and the wells were washed three times with PBS. Tonsil T cells or peripheral blood T cells (4 x 105 cells) were added to each well. Stimulations were performed in triplicate on plates coated with anti-CD3 (1 µg/ml), anti-LFA-1 (10 µg/ml), anti-CD28 (2 µg/ml), or anti-ICAM-1 (10 µg/ml) in PBS. After 66 h, [3H]thymidine was added (1 µCi/well, 67 Ci/mmol; New England Nuclear, Boston, MA) for 6 h, and samples were harvested using a PHD Cell Harvester (Cambridge Technology, Watertown, MA). Incorporated [3H]thymidine was assessed by liquid scintillation counting (Packard Instrument, Downers Grove, IL). Controls for proliferation were various, PHA (1 µg/ml) or the combination of PDB (10 nM) plus ionomycin (0.5 µM).
Activation marker expression
Cells were stimulated for 6 h as indicated above for cell proliferation, removed from the plate, and washed in ice-cold PBS. Cells were then fixed in 2% paraformaldehyde (Sigma-Aldrich), and samples were washed twice in PBS and resuspended in 100 µl of blocking buffer (Dulbecco’s PBS plus 0.5% BSA) for 30 min on ice. After blocking, cells were washed twice and conjugated Abs were added at dilutions recommended by the manufacturers, and the samples were incubated for 30 min in the dark on ice. Cells were washed twice with blocking buffer before analysis. Flow cytometry was performed on a FACScan (BD Biosciences, San Jose, CA) and data were analyzed with CellQuest or WinMDI software.
CFSE dilution assay
Analysis of cell division was performed as previously described by others (21). Naive CD4+ T cells were labeled with 1.5 µM CFSE for 10 min at 37°C in serum-free RPMI 1640. Cells were washed twice in RPMI 1640 containing 10% FBS and stimulated as indicated. Cell division was assessed at day 7 by determining the pattern of CFSE dilution using flow cytometry. Propidium iodide (Sigma-Aldrich) was included for acquisition to discriminate dead cells.
PI3K assay
PI3K activity was measured essentially as we previously
described (22). Tonsil T cells were stimulated as
described in the text. Lysates (500 µg) were precleared with 15 µl
of Omnisorb (Calbiochem, La Jolla, CA) for 30 min at 4°C. Precleared
lysates were then immunoprecipitated overnight at 4°C with 0.5 µg
of anti-p85 PI3K Ab plus 25 µl of Omnisorb. Immune complexes were
washed sequentially once in 100 mM Tris-HCl and 0.5 M LiCl, (pH 7.5);
once in 10 mM Tris-HCl, 100 mM NaCl, and 0.1 mM EDTA (pH 7.5); and once
in 20 mM HEPES, 50 mM NaCl, 5 mM EDTA, 0.03% Triton X-100, 200 µM
Na3VO4, 10 µg/ml
pepstatin, and 1 mM PMSF. Immune complexes were resuspended in 40 µl
of reaction buffer (25 mM Tris-HCl, 94 mM NaCl, 12.5 mM
MgCl2, 25 mM HEPES, and 250 µM adenosine, pH
7.5) plus sonicated phosphatidylinositol (5 µg; Sigma-Aldrich),
followed by incubation on ice for 10 min. The kinase reaction was
initiated by adding 5 µCi of [-32P]ATP
(800 Ci/mmol; New England Nuclear) and 1 µl of 5 mM nonlabeled ATP,
incubated at room temperature for 20 min, and stopped by addition of
100 µl of CHCl3:MeOH:HCl (100:200:2), 100 µl
of CHCl3, and 100 µl of distilled water. The
organic phase was harvested and dried under vacuum. Samples were
resuspended in 25 µl of chloroform:methanol (1:1) and separated by
TLC on Silica gel G plates (Analtech, Newark, DE) with a
CHCL3:acetone:MeOH:acetic
acid:H2O (80:30:26:24:14) mobile phase. Plates
were dried and exposed to film for autoradiography, after which the
migration marker (phosphatidylinositol phosphate; Avanti
Polarlipids, Alabaster, AL) was visualized using iodine vapor.
Plasmids and transient transfections
IL-2:lucif vector (23) was a generous gift
from Dr. C. Hughes (University of California, Irvine, CA). IL-2:lucif
contains the IL-2 regulatory region (600 bp) inserted into the multiple
cloning site of the pGL2 vector (Promega, Madison, WI). pGL2 is an
enhancer vector that contains the gene for firefly luciferase. pRL-TK
(Promega) is a renilla luciferase vector that is driven by the HSV
thymidine kinase promoter. For transient transfections, 250 µl of
culture medium containing 20 x 106 Jurkat T
cells/ml, 15 µg of IL-2:lucif plasmid, and 0.5 µg of pRL-TK plasmid
were electroporated (ElectroPorator; Invitrogen, Carlsbad, CA) at 960
µF, 
, and 250 V, and allowed to stand at room temperature for
10 min. Cells were centrifuged at 1600 rpm for 5 min, resuspended at
5 x 106 cells/ml in culture medium, and
stimulated as described below.
Luciferase assays
Transiently transfected cells (7.5 x 106/well, 2 wells/sample) were stimulated on plates coated at 37°C for 2 h with 50 µl of anti-CD3 (2 µg/ml), anti-LFA-1 (10 µg/ml), anti-CD28 (2 µg/ml), or anti-ICAM-1 (10 µg/ml) in PBS (20). Before the addition of cells, plates were washed three times with PBS. Following incubation for 12 h, cells were lysed and assayed for luciferase activity using the dual-luciferase assay kit (Promega) according to the manufacturer’s instructions. Luciferase activity was measured in a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA) and was recorded as relative light units (RLU). Data are represented as a ratio of firefly luciferase RLU:Renilla luciferase RLU.
Cytokine measurements
Peripheral blood T cells were stimulated as described for the
proliferation assay. Culture supernatants were collected at 24 h
and analyzed for IL-2, IL-5, and IFN- production using Quantikine
kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s
instructions.
RT-PCR
Postnuclear RNA was extracted from T cells as previously
described (24). One microgram of RNA was combined with 0.5
µg of oligo(dT16) and 5 U of RNasin (Promega),
heated at 65°C for 5 min, and incubated on ice for 5 min. The
reaction mixture was completed by the addition of 4 U of avian
myeloblastosis virus-RT (Promega), dNTP (0.2 mM final), and RT
buffer (Promega), for a final volume of 20 µl. The mixture was
incubated for 30 min at 50°C, heated at 95°C for 5 min, and cooled
on ice before the addition of 30 µl of H2O. The
PCR mixture was prepared with 5 µl of cDNA, 10 mM Tris (pH 8.4), 50
mM KCl, 0.2 mM dNTP, 1.5 mM MgCl2, 1 U of
Taq polymerase, 5 mM DTT, and 0.5 µM of each primer.
Primer sequences used were as follows: IL-2
(5'-ATGTACAGGATGCAACTCCTGAAAC-3' and
5'-GTCAGTGTTGAGATGATGCTTTGAC-3'),
IFN-(5'-TTTAATGCAGGTCATTCAGATG-3' and
5'-CTGGGATGCTCTTTCGTCCTCGAAAC-3'), and GAPDH
(5'-GAATCTACTGGCGTCTTCACC-3' and 5'-GTCATGAGCCCTTCCACGATGC-3').
Primers for IL-2R
were purchased from CLONTECH Laboratories (5432-1;
Palo Alto, CA). The cDNA was amplified in a GeneAmp 2400 thermocycler
(PerkinElmer/Cetus, Norwalk, CT) at 94°C for 30 s, 55°C for
30 s, 72°C for 1 min for 25 cycles, followed by an extension at
72°C for 7 min using Taq polymerase (generous gift from
Visible Genetics, Toronto, Canada). PCR products were resolved on a
1.5% gel and visualized by ethidium bromide (0.2 µg/ml).
Densitometric units for each gel were normalized to GAPDH values as
follows. The GAPDH densitometric values exceeded those of the cytokine
RNAs. Thus, each lane of GAPDH was first assigned a numerical value
relative to lane 1 by setting the pixel number for
lane 1 equal to 1.0 and dividing each GAPDH pixel number by
the lane 1 pixel number to assign a relative value for each
lane. To normalize the cytokine values, the pixel number for each
cytokine lane was divided by the relative GAPDH value for that
lane.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To determine whether ICAM-1 could meet the basic criterion as a
costimulatory signal for TCR/CD3-induced T cell proliferation, we
stimulated fresh human tonsil or PBL-T cells with a combination of Abs
to CD3 and to ICAM-1 and compared this to proliferation driven by Abs
to the known costimulatory molecules CD28 and LFA-1. Cells were
incubated on a tissue culture plate and treated as indicated for
72 h, with [3H]thymidine added for the
final 6 h. As seen in Fig. 1
A, nonstimulated cells showed
minimal incorporation of thymidine, and anti-CD3 used alone did not
induce significant T cell proliferation. As expected, T cells
costimulated through CD28 or LFA-1 in conjunction with anti-CD3
showed similar increases in proliferation. Interestingly, costimulation
with anti-ICAM-1 Abs plus anti-CD3 also resulted in a
significant increase in proliferation. Anti-ICAM-1 had no effect on
proliferation when used alone (data not shown). Similar results were
obtained with CD4+ or CD8+
T cell subsets (data not shown). Thus, costimulation through ICAM-1
induced T cell proliferation.
|
B). Moreover, when
approximately equal amounts of total Ab were used (10 µg of
anti-CD3 compared with 1 µg of anti-CD3 plus 10 µg of
anti-ICAM-1) a significant increase was observed in proliferation
as the result of costimulation but not stimulation through CD3 alone.
These data suggest that the ICAM-1-coinduced proliferation was not
merely a result of enhanced signaling through the TCR.
In addition to proliferation, we examined the ability of costimulation
through ICAM-1 to increase surface expression of the activation marker
CD69. CD69 expression has been shown to increase dramatically following
lymphocyte activation (25). Peripheral blood T cells were
stimulated with anti-CD3 alone, or in conjunction with
anti-ICAM-1 or anti-LFA-1, and CD69 expression was measured by
flow cytometry in both CD4+ and
CD8+ T cell subsets at 6 h (Fig. 2). Anti-CD3 resulted in a moderate
increase in CD69 expression on both CD4+ (Fig. 2A, 20%) and CD8+ (Fig. 2B, 34%) cells. Anti-ICAM-1 used in combination with
anti-CD3 (Fig. 2, third column) led to a marked increase in CD69
expression in CD4+ cells (49%) and
CD8+ cells (61%) compared with anti-CD3 used
alone. Anti-LFA-1 was used in conjunction with anti-CD3 as a
control and, as expected, costimulation through LFA-1 caused increased
expression of CD69 (CD4+ cells, 65%;
CD8+ cells, 73%). Stimulation through either
ICAM-1 alone or LFA-1 alone had no effect on expression of CD69 (data
not shown). Thus, costimulation through ICAM-1 caused increased
expression of CD69 that was comparable to that induced by costimulation
through LFA-1.
|
Clonal expansion of naive T cells is essential to mounting a
successful adaptive immune response against Ags that have not been
encountered previously. The ability of naive T cells to divide and
acquire effector functions is dependent upon receiving a costimulatory
signal, and stimulation through TCR (CD3) alone is not able to activate
naive T cells to proliferate (26, 27). To investigate
whether costimulation through ICAM-1 could result in substantial cell
division, we stimulated naive CD4+ T cells and
assessed cell division by CFSE dilution at 7 days (Fig. 3). Naive T cells left not stimulated or
stimulated through the TCR alone were unable to divide, whereas naive T
cells that were costimulated through either ICAM-1, LFA-1, or CD28
underwent multiple divisions by 7 days. In several experiments, CD28
costimulation induced approximately five to six divisions; LFA-1
costimulation induced approximately four cell divisions; and ICAM-1
costimulation induced approximately six cell divisions. Since
costimulation is required for expansion of naive T cells, these data
support the ability of ICAM-1 to provide a legitimate costimulatory
signal.
|
Thus far, the data formed a convincing argument that ICAM-1 could
signal T cells in a costimulatory manner. Ligation of costimulatory
molecules is believed to generate signals distinct from those delivered
through the TCR. To address costimulatory signaling in our system, we
examined PI3K activation, which is thought to be a reasonable index of
a true costimulatory process. It was shown previously that stimulation
through CD3 alone was unable to activate total cellular PI3K
(28). However, PI3K was activated when both CD3 and LFA-1
were stimulated simultaneously (12). We attempted to
replicate this using anti-ICAM-1 as a costimulus. In Fig. 4, tonsil T cells were stimulated with
anti-CD3, anti-ICAM-1, or anti-LFA-1 alone, or with
anti-CD3 in combination with either anti-ICAM-1 or
anti-LFA-1. Used alone, none of the stimuli activated PI3K (Fig. 4, lanes 2–4), but PI3K was activated when T cells were
treated with anti-CD3 plus anti-ICAM-1 in combination (Fig. 4, lane 5) and achieved a level of activation comparable to
anti-CD3 plus anti-LFA-1 (Fig. 4, lane 6). As a
further control, we used a 10-fold greater amount (10 µg) of
anti-CD3 alone in four separate experiments and this treatment did
not activate PI3K (data not shown). This is in contrast to a
considerable amount of the early work involving PI3K in T cells that
demonstrated that stimulation through the TCR alone resulted in
activation of PI3K and an accumulation of phosphatidylinositides
(29, 30). However, these studies were performed in the
leukemic T cell line Jurkat, and recent evidence has shown that PI3K is
constitutively active in these cells due to defects in the negative
regulators PTEN and SHIP (Ref. 31 , reviewed in Ref.
32). More recent work involving primary cell cultures has
demonstrated that stimulation through the TCR alone did not result in
an increase in total cellular PI3K activation (28).
Variation in intensity of origins is a common manifestation in
occasional lanes, and replicate experiments gave similar results in
that neither anti-ICAM-1 nor anti-LFA-1 was able to activate
PI3K alone. Thus, ICAM-1 seems to be a true costimulatory molecule that
is capable of synergizing with CD3 to deliver signals that are distinct
from either CD3 or ICAM-1 alone.
|
The ability to promote production of IL-2 is one of the criteria
usually applied to established costimulatory molecules such as CD28 and
LFA-1. To determine whether costimulation through ICAM-1 was capable of
modulating IL-2 gene expression, we transiently transfected
Jurkat T cells with a construct of the IL-2 regulatory region driving
the firefly luciferase gene. Transfection efficiency was normalized by
cotransfection with the pRL-TK vector, which expresses a basal level of
Renilla luciferase. In Fig. 5, transfected cells were left not stimulated or stimulated for 12 h
with anti-CD3 or anti-CD3 plus anti-CD28, anti-LFA-1,
or anti-ICAM-1. Nonstimulated cells exhibited a low luciferase
expression ratio (luciferase RLU:Renilla RLU), and
expression from the IL-2 vector (
), was slightly induced with
anti-CD3 alone (0.6-fold increase). In contrast, when cells were
costimulated with anti-CD28, anti-LFA-1, or anti-ICAM-1,
the luciferase expression ratio increased by 3.4-, 3.5-, and 4.7-fold,
respectively. The empty vector, pGL2 (
) was used as a negative
control and showed no response to any of the stimuli. The positive
control of PHA resulted in a 7.0-fold increase in luciferase activity.
Thus, costimulation through ICAM-1 induced expression through the IL-2
regulatory region in human T cells that was comparable to that observed
with costimulation through LFA-1 or CD28.
|
Since our transfection experiments indicated that costimulation
through ICAM-1 could increase transcription from the IL-2 promoter, we
investigated the ability of costimulation through ICAM-1 to increase
RNA levels and secretion of the cytokines IL-2, IL-5, and IFN-.
Recent evidence suggests that the costimulatory pathway that is
utilized can affect whether the T cell becomes polarized toward a Th1
or Th2 response (33). We examined whether ICAM-1
costimulation resulted in differential secretion of either type 1 or
type 2 cytokines. Peripheral blood T cells were stimulated with
anti-CD3 alone or in conjunction with anti-CD28,
anti-LFA-1, or anti-ICAM-1, and culture supernatants were
examined for secretion of IL-2, IFN-, or IL-5. Although T cells left
not stimulated or stimulated with anti-CD3 alone secreted low
amounts of the measured cytokines, all three costimulatory signals
significantly increased secretion of the Th1 cytokines IL-2 (Fig. 6A) and IFN- (Fig. 6B). The levels were comparable to those induced by the
positive control PHA. However, secretion of the Th2 cytokine IL-5 (Fig. 6C) was markedly lower in response to costimulation through
CD3 plus either LFA-1 or ICAM-1 when compared with CD28 and the
positive control PHA.
|
following costimulation (Fig. 7). T cells that received only signal 1
(anti-CD3, Fig. 7, lane 2) did not exhibit an increase
in either IL-2 or IFN- mRNA, but when costimulation was delivered
through CD28, LFA-1, or ICAM-1 (Fig. 7, lanes 3–5) there
was a marked increase in the levels of RNA encoding both cytokines. As
a control, IL-2R mRNA was measured and, as expected, was
up-regulated by all four of the stimuli. The corresponding graphs
represent densitometric values normalized to GAPDH from the
representative experiment shown here. Taken together, these results
suggest that ICAM-1 acts similarly to LFA-1 in that this mechanism of
costimulation results in the secretion of type 1 but not type 2
cytokines.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
It is accepted that CD28 is the most prominent costimulatory receptor. However, CD28 gene-targeted mice can still mount an effective immune response, suggesting that other molecules can substitute for CD28 (34). The number of surface molecules recognized as being capable of delivering a costimulatory signal to T cells has grown markedly in the last several years. Presently, the list of surface proteins thought to deliver such a signal includes, but is not limited to, CD28 (2), LFA-1 (35), SLAM (36), 4-1BB (6), OX-40 (37), CD27 (38), and ICOS (5). As the knowledge base and theoretical support have increased, it has become apparent that assessment of increased cell proliferation is, by itself, no longer a sufficient barometer of whether a particular stimulus can serve as a second signal. In the present work, we have attempted to support with several additional parameters our observation that costimulation through ICAM-1 caused increased proliferation in a manner similar to LFA-1 and CD28. We demonstrated that costimulation through ICAM-1 1) induced expression of an early activation marker and caused an increase in proliferation regardless of the strength of signal through the TCR; 2) increased activity of PI3K differently from stimulation through either signal alone; 3) induced a level of cell division by naive CD4+ T cells that was comparable to that induced by CD28, whereas ICAM-1 promoted a greater number of divisions than LFA-1; and 4) caused differential regulation of various cytokines. We feel that these additional parameters provide strong support for our contention that ICAM-1 can provide a legitimate second signal.
The ability of T cells to secrete the proper cytokines in response to
antigenic stimulation is important for allowing the immune system to
respond effectively. Many factors, such as the cytokine environment
(39), strength of TCR-mediated signals (40),
and specific costimulatory molecules (41, 42), can
regulate Th1/Th2 polarization. We have observed that costimulation
through ICAM-1 increased transcription from the IL-2 promoter and
increased secretion of the Th1 cytokines IL-2 and IFN-, but did not
induce secretion of the Th2 cytokine IL-5. This was of particular
interest considering that costimulation through LFA-1 results in
secretion of primarily Th1 cytokines (12, 43). Thus, the
costimulatory signals delivered through both LFA-1 and ICAM-1 are, at
least in some ways, similar. However, it will be interesting to
investigate differences that may exist between the costimulatory
signals delivered through LFA-1 and ICAM-1.
During this work, it was at least possible that the costimulatory effect of ICAM-1 in our system was not the result of an independent signal delivered by ICAM-1. Instead, it could have occurred because anti-ICAM-1 Ab caused increased signaling through the TCR-CD3 complex by causing the T cells to bind more strongly to the plate, allowing increased time of association with the anti-CD3 Ab. To address this possibility, we examined the ability of costimulation through ICAM-1 to activate the PI3K pathway. The costimulatory receptor CD28 functions in part by generating signals required for T cell activation that are distinct from those activated by the TCR, such as activation of PI3K (44) and sphingomyelinase (45). Additional work has demonstrated that LFA-1 may function as a costimulatory receptor in a similar manner (12). Thus, activation of these TCR-independent signals is considered a hallmark of costimulatory signaling. We observed that PI3K activation occurred only when cells were stimulated through both CD3 and ICAM-1 simultaneously. PI3K was not activated by either signal alone or by an amount of anti-CD3 equivalent to a 10-fold increase in the CD3 signal (data not shown). In addition, we attempted to induce proliferation by increasing the amount of anti-CD3 to 10 µg/ml, which is equivalent to the amount of ICAM-1 added in other cultures. Little difference was observed until we added anti-ICAM-1, at which time significant proliferation was induced. Thus, the role of ICAM-1 in costimulation seems to be more than simple physical modulation of TCR signaling, a supposition that is supported by the observations that signaling through ICAM-1 alone can induce signaling events in several cell types (15, 16, 17, 18).
ICAM-1 is known to be at relatively low density on the surface of resting T cells (46). We demonstrate that, despite this low surface expression, stimulation through ICAM-1 can still deliver a costimulatory signal. Since ICAM-1 expression is up-regulated by inflammatory cytokines (reviewed in Ref. 47), the importance of costimulation through ICAM-1 may be even more pronounced following an initial inflammatory response. Alternatively, stimulation through ICAM-1 at later times after activation may play an entirely different role than that presented here. The role that costimulation through ICAM-1 plays at different phases during T cell-dependent immune response is under investigation. It is important to note that LFA-1 is not the only physiologically important ligand for ICAM-1. Mac-1 (48), rhinovirus (49), Plasmodium falciparum-infected erythrocytes (50), and fibrinogen (51) have all been demonstrated to bind to ICAM-1. The capacity to signal into T cells through ICAM-1 by these ligands may play a role in determining the efficacy of the immune response.
In the case of costimulation through ICAM-1 in concert with Ag presentation through MHC, it is likely that T cells would only receive the signal through ICAM-1 when they interact with APCs that express LFA-1. In contrast, costimulatory signals could be delivered through LFA-1 expressed on the T cell surface by interaction with any cell expressing ICAM-1. Such cells include endothelial cells, epithelial cells, fibroblasts, keratinocytes, astrocytes, and leukocytes as well as conventional APCs (reviewed in Ref. 14). Thus, it is at least formally possible that, in addition to their roles as adhesion molecules, the LFA-1:ICAM-1 dual array of surface molecules may serve as a form of sensor by which T cells sample the cellular environment. For example, a T cell might use only a part of the array to receive signals through the TCR plus LFA-1 on its surface delivered by MHC plus ICAM-1 expressed on nonlymphoid cells, e.g., endothelial cells. That T cell would perceive a different environment than a T cell receiving signals through a different part of the array, TCR plus ICAM-1 or even through all three, TCR plus LFA-1 plus ICAM-1. These two sets of signals could be delivered only by leukocytes capable of expressing both counterreceptors, ICAM-1 and LFA-1. Presumably, the different potential arrays of signals would engender differential but perhaps subtle responses in the T cell. Because only APCs express LFA-1 capable of stimulating ICAM-1 on the T cell, then ICAM-1, resident on a T cell, may provide a means by which T cells can distinguish between leukocytes and nonlymphoid cells and respond appropriately.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: Faculty of Medicine, Chulalongkorn University, Payathai Road, Pathumwan, Bangkok 10330, Thailand.
3 Current address: Washington University School of Medicine, St. Louis, MO 63110.
4 Address correspondence and reprint requests to Dr. Stephen H. Benedict, Department of Molecular Biosciences, University of Kansas, 1200 Sunnyside Avenue, Lawrence, KS 66045. E-mail address: sbene{at}ku.edu
5 Abbreviations used in this paper: PI3K, phosphatidylinositol 3-kinase; PDB, phorbol 12,13-dibutyrate; RLU, relative light unit.
Received for publication April 12, 2001. Accepted for publication March 26, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
induces activation of phosphatidylinositol-3 kinase that associates with Pyk-2 and is necessary for B-cell migration. Exp. Cell Res. 268:77.[Medline]
-phosphatidylinositol 3-kinase becomes activated by T cell receptor cross-linking and requires pp56lck. J. Biol. Chem. 269:19435. production. J. Immunol. 158:4036.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  K.M. Mitchell, A.L. Dotson, K.M. Cool, A. Chakrabarty, S.H. Benedict, and S.M. LeVine Deferiprone, an orally deliverable iron chelator, ameliorates experimental autoimmune encephalomyelitis Multiple Sclerosis, November 1, 2007; 13(9): 1118 - 1126. [Abstract] [PDF]
|
|
|
|
 |
|
 B. Graf, T. Bushnell, and J. Miller LFA-1-Mediated T Cell Costimulation through Increased Localization of TCR/Class II Complexes to the Central Supramolecular Activation Cluster and Exclusion of CD45 from the Immunological Synapse J. Immunol., August 1, 2007; 179(3): 1616 - 1624. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Owaki, M. Asakawa, N. Morishima, K. Hata, F. Fukai, M. Matsui, J. Mizuguchi, and T. Yoshimoto A Role for IL-27 in Early Regulation of Th1 Differentiation J. Immunol., August 15, 2005; 175(4): 2191 - 2200. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Parameswaran, R. Suresh, V. Bal, S. Rath, and A. George Lack of ICAM-1 on APCs during T Cell Priming Leads to Poor Generation of Central Memory Cells J. Immunol., August 15, 2005; 175(4): 2201 - 2211. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. L. Barnard, T. Igakura, Y. Tanaka, G. P. Taylor, and C. R.M. Bangham Engagement of specific T-cell surface molecules regulates cytoskeletal polarization in HTLV-1-infected lymphocytes Blood, August 1, 2005; 106(3): 988 - 995. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Li, A. L. Carr, E. J. Donald, J. J. Skitzki, R. Okuyama, L. M. Stoolman, and A. E. Chang Synergistic Effects of IL-12 and IL-18 in Skewing Tumor-Reactive T-Cell Responses Towards a Type 1 Pattern Cancer Res., February 1, 2005; 65(3): 1063 - 1070. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. C. Barlow, H. Xu, C. T. Weaver, J. R. Lindsey, T. R. Schoeb, and D. C. Bullard Development of dermatitis in CD18-deficient PL/J mice is not dependent on bacterial flora, and requires both CD4+ and CD8+ T lymphocytes Int. Immunol., February 1, 2004; 16(2): 345 - 351. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. H. Qureshi, J. Cook-Mills, D. E. Doherty, and B. A. Garvy TNF-{alpha}-Dependent ICAM-1- and VCAM-1-Mediated Inflammatory Responses Are Delayed in Neonatal Mice Infected with Pneumocystis carinii J. Immunol., November 1, 2003; 171(9): 4700 - 4707. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Gregori, N. Giarratana, S. Smiroldo, and L. Adorini Dynamics of Pathogenic and Suppressor T Cells in Autoimmune Diabetes Development J. Immunol., October 15, 2003; 171(8): 4040 - 4047. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Admyre, J. Grunewald, J. Thyberg, S. Gripenback, G. Tornling, A. Eklund, A. Scheynius, and S. Gabrielsson Exosomes with major histocompatibility complex class II and co-stimulatory molecules are present in human BAL fluid Eur. Respir. J., October 1, 2003; 22(4): 578 - 583. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Burne-Taney, J. Kofler, N. Yokota, M. Weisfeldt, R. J. Traystman, and H. Rabb Acute renal failure after whole body ischemia is characterized by inflammation and T cell-mediated injury Am J Physiol Renal Physiol, July 1, 2003; 285(1): F87 - F94. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Marwali, J. Rey-Ladino, L. Dreolini, D. Shaw, and F. Takei Membrane cholesterol regulates LFA-1 function and lipid raft heterogeneity Blood, July 1, 2003; 102(1): 215 - 222. |
). After
66 h in culture, [3H]thymidine was added for 6
h and proliferation was measured by liquid scintillation counting. Data
are presented as cpm ± SEM and an asterisk designates
statistically relevant values (*, p 
0.02;
**, p 
