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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 6, 2000 as doi:10.1096/fj.00-0492fje. |
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Departments of Medicine and Microbiology-Immunology, University of California Medical Center, San Francisco, California 94143-0711, USA
2Correspondence: University of California, UB8B, Box 0711, 533 Parnassus at 4th, San Francisco, CA 94143-0711, USA. E-mail: egoetzl{at}itsa.ucsf.edu
SPECIFIC AIMS
To determine the mechanisms for different responses of unstimulated and activated human blood T cells to lysophosphatidic acid (LPA) growth factor, studies were conducted to elucidate effects of mitogens on patterns of expression and immune functions of the predominant types 2 and 4 endothelial differentiation gene-encoded, G-protein-coupled LPA receptors (Edg-2 and Edg-4 Rs) of T cells.
PRINCIPAL FINDINGS
1. Mitogen activation of human T cells down-regulates Edg-4 R
and up-regulates Edg-2 R for LPA
Newly isolated human blood CD4+ T
cells express predominantly Edg-4 LPA Rs, much lower levels of Edg-2
LPA Rs, and no detectable Edg-7 LPA Rs (Table 1
). Incubation of these T cells with a mitogenic stimulus in
vitro alters substantially the pattern of expression of mRNA
encoding Edg Rs. After exposure to phytohemagglutinin (PHA) for 24 h, Edg-2 LPA R-specific mRNA in the CD4+ T cells
of two healthy subjects increased to a level of approximately 50% of
that of Edg-4 Rs, which decreased concurrently (Table 1)
. Reverse
transcription-polymerase chain reaction semiquantification of Edg-2 R
mRNA in a pool from PHA-activated CD4+ T cells of
20 healthy subjects showed a much higher signal than that detected
previously in unstimulated CD4+ T cells, with a
ratio of intensity to Edg-4 R mRNA similar to that observed in
CD4+ T cells of individual subjects (Table 1)
.
The prevalence of Edg-2 Rs relative to Edg-4 Rs in PHA-activated
CD4+ T cells also was apparent from Western blot
analyses, where again it was approximately 50%.
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The very low levels of mRNA and antigenic protein of both Edg-3
and -5 sphingosine 1-phosphate Rs decreased to an only marginally
detectable level or were absent after PHA activation of
CD4+ T cells (Table 1)
. In summary, unstimulated
CD4+ T cells express Edg-4 >> Edg-3 > Edg-2,
whereas PHA-activated CD4+ T cells express Edg-4
Edg-2, without Edg-3 or Edg-5 Rs (Table 1)
. Unstimulated
CD8+ T cells did not express any Edg Rs
prominently, with only traces of Edg-2 or -5 in those from some
individuals, and this was not changed by incubation with mitogen. The
trace of Edg-2 R mRNA observed in mitogen-activated
CD8+ T cells of some individuals was the only Edg
R detected and could not be seen on most Western blots. B cells
expressed a high level of Edg-4 R alone both without stimulation and
after incubation with mitogen for 24 h.
2. Edg-4 R transduces LPA suppression and Edg-2 R LPA enhancement
of interleukin 2 (IL-2) production/secretion
CD4+ T cell functional consequences of
the activation-induced shift from nearly exclusive expression of Edg-4
LPA Rs to codominant expression of Edg-2 and Edg-4 LPA Rs were examined
in relation to T cell receptor-evoked generation of IL-2. Human blood
purified CD4+ T cells were preincubated with PHA
or complete medium alone for 24 h to establish respective Edg-2 +
Edg-4 R-codominant and Edg-4 R-predominant sets prior to incubation in
serum-free medium for 6 h, addition to wells bearing adherent
anti-CD3 plus anti-CD28 antibodies, and introduction of LPA or
functional anti-Edg-2 or anti-Edg-4 R monoclonal antibodies. LPA
signaling through Edg-4 Rs alone in serum-free, medium-conditioned
CD4+ T cells suppressed T cell receptor-evoked
IL-2 secretion (Fig. 1
). Suppression was significant at 10-10 M LPA,
increased in an LPA concentration-dependent relationship, and reached
an apparent mean maximum of over 60% at 10-6 M
LPA (Fig. 1)
. In contrast, LPA signaling concurrently through both
Edg-2 and Edg-4 Rs in PHA-activated CD4+ T cells
enhanced T cell receptor-evoked IL-2 secretion. Augmentation of IL-2
secretion was significant at 10-10 M LPA, and
rose progressively with increases in LPA concentration up to a mean
of nearly 200% of control at 10-6 M LPA
(Fig. 1)
. The control phospholipids
lysophosphatidyl-ethanolamine and dioleoyl-phosphatidic acid had
no effect on any set of T cells.
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As the effect of LPA on IL-2 secretion by CD4+ T cells preincubated with PHA reflected signals through both Edg-2 and Edg-4 Rs, stimulatory monoclonal antibodies specific for the extracellular amino terminus of each Edg R were applied separately to resolve their individual contributions experimentally. Anti-Edg-4 R antibody suppressed IL-2 generation by Edg-4 R-dominant CD4+ T cells, as well as Edg-2 and Edg-4 R codominant CD4+ T cells. The suppressive effects of equal concentrations of anti-Edg-4 R antibody were greater in the Edg-4 R-dominant set of CD4+ T cells, presumably due to their higher level of Edg-4 R expression. For the Edg-4 R-dominant set, IL-2 secretion was inhibited significantly by 100 ng/ml of anti-Edg-4 R antibody and reached a mean maximum of nearly 60% at 3 µg/ml. At the same concentrations, anti-Edg-2 R antibody increased IL-2 generation by PHA-activated Edg-2 and Edg-4 R-codominant CD4+ T cells, with significant enhancement at 300 ng/ml of anti-Edg-2 R antibody and mean maximum enhancement similar to that for LPA in the same T cells at 1 µg/ml. These findings are consistent with LPA mediation of suppression of T cell receptor-evoked IL-2 generation through Edg-4 Rs and of enhancement of T cell receptor-evoked IL-2 generation through Edg-2 Rs.
3. Edg-2 and Edg-4 LPA Rs expressed separately in Jurkat T cell
transfectants transduce opposite effects on IL-2 production/secretion
Jurkat model T cells, designed respectively to have Edg-2 R-
and Edg-4 R-predominance, were treated with LPA just prior to exposure
to anti-CD3 antibody and PMA for assessment of Edg R specificity of
regulation of IL-2 secretion. In Jurkat-Edg-4 cells, LPA inhibited IL-2
generation significantly at 10-8 M to
10-6 M; in Jurkat-Edg-2 cells, LPA enhanced IL-2
generation significantly at 10-7 M and
10-6 M. For Jurkat-Edg-4 R cells, 0.1 to 3
µg/ml of anti-Edg-4 R antibody significantly suppressed IL-2
generation, whereas 0.1 to 3 µg/ml of anti-Edg-2 R antibody enhanced
significantly the generation of IL-2 by Jurkat-Edg-2 cells, without any
effect on IL-2 generation by Jurkat-Edg-4 cells. Neither LPA nor
anti-Edg R antibody at the concentrations studied had any effects alone
on IL-2 generation in Jurkat-Edg-2 or Jurkat-Edg-4 cells.
CONCLUSIONS AND SIGNIFICANCE
The specific responses of T cells to antigens and other activating stimuli are regulated by many different immune cytokines, as well as growth factors and cellular mediators from nonimmune sources. That such regulatory factors exert effects that differ adaptively with the state of differentiation and activation of the target T cells is attributable to one or more types of cellular mechanisms. The first type is increased production of a factor by T cells or other immune cells only at one stage of differentiation or state of activation. The second type is differentiation- and/or activation-dependent increases in expression or signaling properties of receptors specific for a regulatory factor. The third type of mechanism is alteration in generation of another factor or in expression or functions of its receptor(s), which indirectly augments or suppresses T cell responses to the primary factor.
The human blood CD4+ T cell and Jurkat T
cell transfectant systems studied for LPA effects do not produce LPA in
functionally relevant quantities under any known circumstances.
Preliminary data suggest that LPA acts directly on T cell secretion of
IL-2, without major involvement of second or intermediate cytokines or
other factors. Thus, the most relevant alteration in the T cells
resulting from mitogenic activation is the shift from predominance of
Edg-4 Rs to codominance of Edg-4 and Edg-2 Rs (Table 1)
. This increase
in ratio of Edg-2 to Edg-4 Rs shifted the effect of LPA from
suppression to enhancement of T cell receptor-evoked generation of IL-2
(Fig. 1)
. To confirm the signaling activities of Edg-2 R in the absence
of Edg-4 R, Jurkat T cells were developed that expressed predominantly
Edg-2 R and these were analyzed in parallel with Edg-4 R-dominant
Jurkat T cells. As for Edg-4 R-predominant unactivated
CD4+ T cells from human blood, Jurkat-Edg-4 cells
generated IL-2 after stimulation with anti-CD3 antibody and PMA, which
was suppressed by LPA and anti-Edg-4 R antibody. As for Edg-2 and -4
codominant mitogen-activated CD4+ T cells,
Jurkat-Edg-2 cells generated IL-2 after T cell receptor-mediated
stimulation, which was enhanced by LPA and anti-Edg-2 R antibody more
strikingly than that of the mitogen-activated set of
CD4+ T cells, in part due to the lack of
opposition from Edg-4 R signals.
The differences between Edg-2 and Edg-4 Rs in mechanisms of
regulation of expression and of signal transduction have not been fully
elucidated nor is it clear what determines distinctive functional
profiles of these receptors in any cell type. Current evidence suggests
that Edg-4 R couples most effectively to G
q and thereby directly
activates phospholipase C (PLC) and inositol phosphate signaling of
increases in intracellular concentration of calcium
([Ca2+]i). In contrast,
Edg-2 R is more closely coupled to G i and thus elicits increases in
[Ca2+]i through Gß/
enhancement of PLC activity both directly and by augmenting a
concurrent lower level of activation of G q by Edg-2 R. Differences
in the time course, magnitude, and susceptibility to inhibition of
[Ca2+]i responses
mediated by Edg-2 and Edg-4 Rs have been demonstrated in Edg R-null
HTC4 rat hepatoma cells and Jurkat T cells transfected with the
respective Edg Rs. The Edg-4 R-mediated increases in
[Ca2+]i were greater,
decreased more rapidly, had a lesser contribution from late increases
in calcium influx, were preceded by lower rises in cellular
concentration of inositol trisphosphate, and were more sensitive to
inhibition by Pertussis toxin. As some forms of phosphoinositol
3-kinase (PI3 kinase) also are activated by both Gß/ and G 13,
it is expected that differences in coupling of Edg-2 and -4 Rs to these
G-proteins will lead to distinctive capabilities in their recruitment
of PI3 kinase and its many downstream effectors. Some of these
signaling pathways, such as focal adhesion kinase, are likely to be
involved in T cell responses to LPA.
Activation-induced alterations in T cell patterns of relative
expression of the Edg-2 and -4 LPA Rs result in major changes in the
net effects of LPA on T cell receptor-induced generation of IL-2 (Fig. 1)
. Of great recent interest is the finding that Edg-4 R transduces
LPA-evoked increases in T cell trans-basement membrane migration,
whereas Edg-2 R does not mediate direct stimulation, but suppresses
chemokine-elicited chemotaxis of T cells in the same model system.
Thus, Edg-2 and –4 LPA receptors may participate in orchestration of
the phenotypic changes characteristic of the transition from mobile
unstimulated T helper cells to activated regionally committed helper T
cells (Fig. 2
). In resting human blood T helper cells, Edg-4 Rs thus may mediate or
enhance migration while inhibiting cytokine responses. After T cell
activation elicits expression of Edg-2 Rs, the net effects of LPA may
be to prevent migration and augment cytokine generation. The changes in
profile of T cell regulatory effects of LPA with activation appear to
be determined principally by the ratio of Edg-2 to Edg-4
Rs.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0492fje To cite this article, use (October 6, 2000) FASEB J. 10.1096/fj.00-0492fje 
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