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Deficient phytohemagglutinin-induced interleukin-2 activity in patients with inactive systemic lupus erythematosus is correctable by the addition of phorbol myristate acetate.

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826
DEFICIENT PHYTOHEMAGGLUTININ-INDUCED
INTERLEUKIN-:! ACTIVITY IN PATIENTS WITH
INACTIVE SYSTEMIC LUPUS ERYTHEMATOSUS
IS CORRECTABLE BY THE ADDITION O F
PHORBOL MYRISTATE ACETATE
YOHKO MURAKAWA and TSUYOSHI SAKANE
In systemic lupus erythematosus (SLE) patients,
the production of interleukin-2 (IL-2) by blood T lymphocytes in response to stimulation with phytohemagglutinin (PHA) either alone or with phorbol myristate acetate (PMA) or ionomycin, a Ca2+ ionophore, was
examined. Deficiency in PHA-stimulatedIL-2 production
by cells from SLE patients was repaired by the addition
of PMA, but not ionomycin. PMA alone did not stimulate
IL-2 production but, in concert with PHA, induced IL-2
synthesis. Moreover, PMA was effective in the repair of
the deficiency of PHA-induced IL-2 production by both
T4+ and TS+ subsets. Thus, for effective IL-2 production, SLE T cells required signals either distinct from or
in addition to those supplied by PHA.
Interleukin-2 (IL-2) has the ability to interact with
activated T cells and to promote their proliferation and
differentiation (1-3). Thus, deficient IL-2 activity could
disrupt T cell differentiation to effector cells (4). Our
previous studies (9,as well as those of other investigators (6-9), have demonstrated that lymphocytes from
patients with systemic lupus erythematosus (SLE) have
From the Third Division, Department of Internal Medicine,
Shimane Medical University, Shimane, Japan.
Supported in part by a 1986/1987 grant-in-aid for scientific
research (project no. 61570312) from the Ministry of Education,
Science, and Culture of Japan; by a 1987 research grant from the
Autoimmune Disease Research Committee of Japan, Ministry of
Welfare; and by a 1986 research grant from the Tokyo Biochemical
Research Foundation, Tokyo, Japan.
Yohko Murakawa, MD, PhD: Medical Staff, Tsuyoshi
Sakane, MD, PhD: Senior Associate Professor.
Address reprint requests to Tsuyoshi Sakane, MD, PhD,
Third Division, Department of Internal Medicine, Shimane Medical
University, Izumo, Shimane 693, Japan.
Submitted for publication May 18, 1987; accepted in revised
form December 22, 1987.
Arthritis and Rheumatism, Vol. 31, No. 7 (July 1988)
a profound deficiency in phytohemagglutinin (PHA)induced IL-2 production that is due largely to a T cell
defect, and not to a monocyte defect alone (5,7,9).
Recently, investigations have focused on signals that are required to activate T cells (10-14).
Several investigators have demonstrated that an increase in the concentration of intracellular free Ca2+
([Ca2']i) and the activation of protein kinase C (PKC)
play critical roles in the full activation of T cells
(10-14); both signals are required for the secretion of
IL-2 by human peripheral blood T cells following
immune stimulation (1 1,13). Thus, it is conceivable
that T cells from SLE patients could be fully activated
by delivering signals either distinct from or in addition
to those supplied by PHA to induce the production of
IL-2. This possibility was investigated by supplementing PHA-stimulated cultures either with phorbol myristate acetate (PMA), a direct activator of PKC, or
with ionomycin, a Ca2+ ionophore. The apparent
deficiency in PHA-stimulated IL-2 production manifested by SLE T cells was completely eliminated by
the addition of PMA, but not by the addition of
ionomycin. Our results indicate that while the ability
to produce IL-2 activity is intact in SLE T cells, these
patients may have a defect in intracellular signal
transduction, especially in the activation of PKC, for T
cell activation. This abnormality may explain many of
the T cell defects observed in SLE patients, including
reduced IL-2 production.
PATIENTS AND METHODS
Patients. Fifteen patients who fulfilled the American
Rheumatism Association 1982 revised criteria for the classification of SLE (15) were entered into the study. The mean
age of the SLE patients was 33 years, and all were women.
PMA AND IL-2 ACTIVITY
827
Patients taking immunosuppressive drugs or high doses of
corticosteroids (215 mg of prednisolone per day) were
excluded. Seventeen healthy blood donors served as control
subjects; 11 were women and 6 were men. Their mean age
was 29 years.
At the time the blood samples were drawn, disease
activity was assessed on the basis of clinical and laboratory
findings, as follows: fever, active arthralgia, active rash, oral
ulcers or alopecia, elevated erythrocyte sedimentation rate
(>30 mndhour), leukopenia (<4,OOO/pl), hypoalbuminemia
(<3.5 gnddl), h ypocomplementemia (CH50 <20 unitdml),
positive LE cell preparation. Patients who fulfilled 3 or more
criteria were categorized as having active disease. If patients
fulfilled less than 2 criteria, they were included in the inactive
disease groups. Based on the above criteria, all 15 patients in
this study were classified as having inactive disease.
Isolation of T cells and monocytes. Peripheral blood
mononuclear cells were isolated, and T and non-T cells were
separated by the sheep erythrocyte rosetting technique
described previously (16). Twice-purified rosetting cells are
referred to as T cells. These T cell preparations consisted of
more than 95% cells that were reactive with OKT3 monoclonal antibody (Ortho Pharmaceutical, Raritan, NJ). Nonrosetting cells were incubated in petri dishes for 1 hour at
37°C in a 5% CO,/95% air humidified environment. Monocytes were then obtained by collecting the cells that were
firmly adhered to the dishes. Ninety-five percent of the cells
in the adherent cell preparations were identified as monocytes after staining with Giemsa.
Isolation of T cell subsets. The rosetting T cells
obtained were further separated into T cell subsets (T4 and
T8 + ) by complement-mediated cell lysis with monoclonal
antibodies OKT8 and OKT4 (Ortho), respectively (16,17).
Briefly, T cells (1 x 10’) were incubated on ice for 1 hour
with either 10 pg of OKT8 or 5 pg of OKT4 in 100 p l of
phosphate buffered saline. Thereafter, 25 pl of rabbit complement (Behringwerke AG, Marburg, FRG) was added, and
the cells were incubated for 1 hour at 37°C. This procedure
was repeated twice. The OKT8-treated population contained
more than 94% T4+ cells and less than 1% T8+ cells,
whereas the OKTCtreated population contained less than
2% T4+ cells and more than 96% T8+ cells. These cell
populations depleted of T8 and T4 + cells will be referred
to as T4+ and T 8 + , respectively.
Culture medium. The culture medium consisted of
RPMI 1640 (Flow Laboratories, Rockville, MD) supplemented with 10% Nu-serum (Collaborative Research, Lexington, MA), 2 mM L-glutamine (Gibco, Grand Island, NY),
1% HEPES buffer solution (Gibco), 100 units/ml penicillin,
and 100 pg/ml streptomycin (5).
Cultures for preparation of IL-kontaining supernatants. T cells, either unfractionated or fractionated, were
incubated at 1 x lo6 cells/ml in culture medium, together
with autologous monocytes (5 x 104/ml) that had been
previously treated with mitomycin (Sigma, St. Louis, MO).
They were stimulated for 3 hours with 1 of the following: no
mitogen at all, 1 pg/ml of PHA (Wellcome Research Laboratories, Beckenham, UK), 5 ng/ml of PMA (Sigma), 1 nM
ionomycin (Calbiochem-Behring, La Jolla, CA), 1 pg/ml of
PHA + 5 ng/ml of PMA, 1 pg/ml of PHA + 1 nM ionomy1 nM ionomycin. Thereafter, the
cin, or 5 ng/ml of PMA
+
cells were washed 6 times to remove mitogens, resuspended
in an original volume of culture medium, and further incubated for 24 hours in 5% CO, at 37°C in a humidified
atmosphere. Supernatants were then collected, filtered
through 0.45-pm Millex filters (Millipore, Bedford, MA),
aliquoted, and stored at -70°C (5,18).
In some studies, the supernatants were tested with
cell lysates prepared by freeze-thawing. Cell mixing studies,
in which purified T cell populations were pretreated for 3
hours with either PHA or PMA and then washed 6 times
before culture with other cells, either untreated or similarly
exposed to PHA or PMA, were also performed. These cell
mixtures were incubated for another 24 hours, and the
supernatants were collected.
Determination of IL-2 activity of the supernatants. To
measure IL-2 activity in culture supernatants, IL-2-dependent PHA blasts were prepared as previously described
(5,6). Briefly, normal human peripheral blood mononuclear
cells were stimulated with PHA (1 pg/ml) and were kept in
culture for at least 20 days in RPMI 1640 that was supplemented every 3 days (subsequent to the first 4 days) with
both fetal bovine serum (Gibco) and partially purified IL-2
(Electro-Nucleonics, Silver Spring, MD). Before being used
as indicator cells in an IL-2 assay system, they were washed
and incubated for 3 hours in IL-2-free culture medium.
Indicator cells (100 pl, 1 X lo4)were placed in 96-well
round-bottom microtiter plates (Costar Data Packaging, Cambridge, MA) with serial dilutions (100 pl) of either the supernatants being tested for IL-2 activity or recombinant IL-2
(Takeda Chemical, Osaka, Japan). All cultures were incubated for 72 hours at 37°C in a 5% C0,/95% air humidified
environment. Twenty hours before the termination of the
incubation period, 1 pCi of (methyL3H)-thymidine (2 Wmmole; New England Nuclear, Boston, MA) was added to each
culture well. Cells were then processed by using a microharvester, and the proliferative response for each was evaluated
by measuring the incorporation of thymidine. IL-2 activity
was analyzed by probit analysis and expressed as unitdm1 as
previously described by Farrar et a1 (1).
Statistical analysis. Differences in IL-2 activity were
analyzed by Student’s t-test.
+
+
RESULTS
Effect of PHA and PMA on IL-2 production.
PMA, by itself, did not induce significant IL-2 production in cells from either the healthy controls or from
the SLE patients (results not shown). The stimulation
of T cells by PHA alone produced considerable IL-2
activity in healthy controls. Conversely, as has been
previously reported (5-9), a profound deficiency in
PHA-induced IL-2 production was present in patients
with SLE (Figure 1). This was corrected by culturing
SLE T cells with PHA PMA. That is, when normal
T cells were stimulated by PHA
PMA, the 1L-2
activity in the culture supernatants was only slightly
increased compared with stimulation by PHA alone.
+
+
MURAKAWA AND SAKANE
828
NS
P<0.005
m
r-----l
I
I
I
PHA
PHA+PMA
PHA
PHA+PMA
Figure 1. Interleukin-2 (IL-2) activity in culture supernatants derived from phytohemagglutinin (PHAtpulsed or PHA + phorbol
myristate acetate (PMAtpulsed T cells from normal controls (0)
and from patients with systemic lupus erythematosus (0).Shaded
areas designate the mean 5 SEM IL-2 activity. NS = not significant.
When SLE T cells were stimulated by PHA + PMA,
IL-2 levels rose substantially and equalled those produced by normal cells (Figure 1). These results indicated that the deficiency in PHA-induced production
of IL-2 by SLE T cells can be reversed by the addition
of PMA.
source
of
SuDernatant
Stimulant for
Monocytes
PHA
PMA
Stimulant for
T cells
PHA PMA I
In parallel studies, the addition of cell lysates to
the supernatants did not increase IL-2 activity in any
of the experimental groups (data not shown). This
suggests that the 1L-2 production defect in SLE patients does not reflect an inability to release IL-2
accumulated within the cells and that PMA does not
correct this defect by promoting IL-2 release from
PHA-stimulated cells.
Restoration mechanisms of the deficient PHAinduced production of 1L-2 by the addition of PMA.
PMA has been reported to activate macrophages directly (19) and to promote the release of cytokines,
such as IL-1, from macrophages (19). Furthermore,
dysfunction of monocytes has been demonstrated to
contribute, at least in part, to the defective IL-2
production observed in patients with SLE (5,7).
Therefore, the mechanism for reversal of the IL-2
defects in SLE could have been IL-1-related or monocyte-related. However, the addition of varying concentrations of recombinant human 1L-la (1-100
units/ml; Dai-Nippon Pharmaceutical, Osaka, Japan)
had no effect on PHA-induced production of IL-2 (data
not shown).
To address the role of monocytes in the IL-2
defects in SLE, monocytes were preincubated for 3
hours with 1 pg/mI of PHA alone or I pg/rnl of PHA +
5 ng/ml of PMA, washed 6 times, and treated with
mitomycin. These preincubated monocytes were then
added to the culture of autologous T cells (1 x 106/ml)
at a final concentration of 5%. These cell mixtures
were further cultured for 3 hours with 1 pg/ml of PHA
or 1 pglml of PHA + 5 ng/ml of PMA, washed 6 times,
and incubated in culture medium for another 24 hours.
As shown in Figure 2, IL-2 activity remained low in
EXP. 1
EXD.2
+
-
+
7
+
+
+
+
+
I+
-
+
-
+
+
+
+
Normal
+
SLE
-
+ +
+ + -
5
5x2
5
~
5X23
2 ~
5x24
5x25
5x26
5
5x2
5x22
5x23
5X24
5x25
5x26
IL-2 A c t w t y W m l )
Figure 2. Lack of contribution of monocytes pretreated with phorbol myristate acetate (PMA) and/or phytohemagglutinin (PHA) to the
correction of deficient interleukin-2 (IL-2) activity in patients with systemic lupus erythematosus (SLE).
PMA AND IL-2 ACTIVITY
829
the culture supernatants derived from PHA-stimulated
SLE T cells with autologous monocytes pretreated
with PHA + PMA. It was equal to the IL-2 activity in
the culture supernatants derived from PHA-stimulated
SLE T cells with autologous monocytes either pretreated with PHA alone or untreated. When the combination of SLE T cells + 5% monocytes was stimulated
simultaneously by PHA + PMA, normal production
of IL-2 activity was observed, regardless of pretreatment of monocytes (Figure 2). This indicated that the
correction of deficient IL-2 production by the addition
of PMA did not occur through enhancement of monocyte function.
Since stimulation with either PHA (Figure 1) or
PMA (results not shown) failed to induce normal
amounts of IL-2 activity in SLE T cells, and only a
combination of PHA and PMA could correct the IL-2
deficiency, it seemed likely that the same IL-2producing cells were responding to signals provided by
both mitogens. Indeed, the IL-2 activity present in
supernatants derived from PHA + PMA-pulsed SLE
cells was far higher than the IL-2 activity present in
the supernatants derived from either PHA-pulsed or
PMA-pulsed SLE T cells.
To clarify this, SLE T cells were pulsed for 3
hours with PHA and/or PMA, washed, then mixed
together, cultured for another 24 hours, and the supernatants assayed for IL-2 activity. The results of 3 such
experiments are shown in Figure 3. SLE cells pulsed
with either PHA or PMA produced little IL-2, but
when the SLE cells were pulsed with PHA + PMA,
PHA
PMA
-
+
0
+
+
0
+
I
41
0 ‘5x20
I
5x2’
I
5x22 5x23
I
5X24
I
5x25
IL-2 ACTIVITY (U/rnl)
Figure 3. Increased interleukin-2 (IL-2) production by a single cell
population responding to signals provided by both phytohemagglu-
tinin (PHA) and phorbol myristate acetate (PMA) in patients with
systemic lupus erythernatosus. There was no synergy between a
PHA-pulsed single cell population and a PMA-pulsed single cell
population. Results shown are the mean and SEM of 3 experiments.
IL-2 production by SLE T cells was substantially
increased. However, there was minimal 1L-2 activity
in the supernatants that were obtained by 24-hour
incubation of the cell mixtures of SLE T cells that had
been previously pulsed for 3 hours with PHA alone
and SLE T cells that had been previously exposed to
PMA alone (Figure 3). These data indicated that
effective production of 1L-2 required activation of the
cell by both PHA and PMA. PHA and PMA appear to
act synergistically on the same IL-2-producing cells.
T cell subsets involved in the correction of deficient IL-2 production by addition of PMA. We have
previously found that normal T4+ and T8+ T cell
subsets are able to produce equal amounts of IL-2
activity when stimulated with PHA, and the defect in
cells from patients with SLE resides in both T4+ and
T8+ subsets (5). This raises the possibility that the
addition of PMA corrects the deficiency in PHAinduced IL-2 production by T4 + cells or by T8 + cells,
or both. To address this question, negatively selected
(by complement-mediated cell lysis) T4 + or T8 + cells
were pulsed simultaneously with PHA + PMA for 3
hours, washed, and incubated in culture medium for 24
hours. As shown in Figure 4, T4 + and T8 + cells from
SLE patients produced normal levels of IL-2 activity in
response to simultaneous stimulation with PHA and
PMA. The results indicated that PMA is equally effective in the repair of the deficiency in PHA-induced IL-2
production by both T cell subsets.
In contrast with previous observations in murine and human systems that it is the L3T4+ or T4+
cells that predominantly produce IL-2 (3,9,20-23), we
and other investigators (5,24-28) have demonstrated
that Lyt-2+ or T8+ cells produce equal amounts of
IL-2 as L3T4 + or T4 + cells. To exclude the possibility that IL-2 is produced by the few T4+ cells that
remain in the T8 + cell preparations after the complement lysis procedures, T4 + or T8 + cells were also
obtained by “panning,” as described elsewhere (29).
This procedure yielded populations of at least 98%
purity based on analysis by indirect immunofluorescence. In normal individuals, these positively selected
T8 + cells could indeed produce as much IL-2 as either
positively or negatively selected T4+ cells on stimulation with PHA or PHA + PMA. Mean (+SEM)
levels of PHA-induced or PHA + PMA-induced 1L-2
activity (unitdml) by positively isolated T4 + and T8 +
cells (by panning) in 3 normal individuals were 46.4 &
3.0 or 70.5 2 11.3 and 40.2 & 6.1 or 58.1 f 7.3,
respectively. Mean ( ? SEM) levels of PHA-induced or
PHA + PMA-induced IL-2 activity by negatively
830
NS
NS
1
1
I
P<0.05
'
-
-
P <b.05
[
NS
1
-
;<0.005
I
I
I
1
Ti
I
I
T4
+
+
-
1
+
- +
MURAKAWA AND SAKANE
Effect of ionomycin on PHA-induced IL-2 production by SLE T cells. When T cells are stimulated by
external ligands, 2 signals are required to induce IL-2
production: an increase in [Ca2+]iand the activation
of PKC (11,13). To study whether deficiency in PHAinduced 1L-2 production by SLE T cells is correctable
by the increase in [Ca*+]i, SLE T cells were exposed
for 3 hours to PHA + the Ca2.+ionophore, ionomycin.
These pulsed T cells were washed, incubated for
another 24 hours in culture medium, and the supernatants were assayed for IL-2 activity. Ionomycin, by
itself, did not induce detectable IL-2 activity (results
not shown); IL-2 activity present in the supernatants
from SLE T cells pulsed with ionomycin + PHA was
not different from the activity present in the supernatants from SLE T cells pulsed with PHA alone (Figure
5). Thus, ionomycin did not correct the deficiency in
IL-2 production. However, the combination of ionomycin + PMA could induce SLE T cells to produce
normal levels of IL-2. The IL-2 activity in SLE T cell
supernatants pulsed with ionomycin + PMA was
NS
T8'
Figure 4. The effect of the addition of phorbol myristate acetate
(PMA) on deficient interleukin-2 (IL-2) production in T cell subsets
from 4 normal controls ( D ) and 4 systemic lupus erythematosus
patients (~3).
T cell subsets plus 5% autologous monocytes were
pulsed for 3 hours with phytohemagglutinin (PHA) ( - ) or with PHA
+ PMA (+), washed, and the supernatants prepared by incubating
the pulsed cells for another 24 hours in cultlire medium. Results
shown are the mean and SEM IL-2 activity. N S = not significant.
isolated T4 + and T8 + cells (by complement-mediated
cell lysis) were 50.6 k 4.9 or 68.2 ? 10.4, and 44.6 ?
8.6 or 59.4 & 9.3, respectively. Moreover, the addition
of PMA corrected the deficiency in PHA-induced 1L-2
secretion by positively selected SLE T4+ and T8+
cells (data not shown).
It should be further noted that the additioii of
anti-Tac ascites (kindly provided by Dr. Takashi Uchiyama, Kyoto University School of Medicine, Kyoto,
Japan) at a final dilution of 1:1,000 to the IL-2 assay
cultures that contained IL-2-dependent PHA blasts +
culture supernatants derived from T cell subsets completely inhibited the proliferation of IL-2-dependent
PHA blasts. This suggested that the proliferation of
the IL-2-dependent PHA blasts used as indicator cells
in the present study could be solely due to IL-2 in the
supernatants, but not due to cytokines other than IL-2.
5XZ'O
s1
'
NS
n
v)
I
J
-
5X2P
PHA
pMA
l o n o k ~ c mP M A
PHA
PhA
londrn~crP M A
Figure 5. The effect of phorbol myristate acetate (PMA) or ionomycin on phytohemagglutinin (PHA)-induced interleukin-2 (IL-2) production in 5 normal controls (0)and 5 systemic lupus erythemat o w s (SLE) patients (a). Deficient 1L-2 production in PHAstimulated SLE T cells was increased by the addition of PMA, but
not by the addition of ionornycin. Results shown are the mean and
SEM 1L-2 activity. NS = not significant.
PMA AND IL-2 ACTIVITY
almost the same as the 1L-2 activity present in SLE T
cell supernatants pulsed with PHA + PMA (Figure 5).
These findings suggested that when SLE T cells
are stimulated by external ligands, the concentration
of [Ca2+]irises to a level sufficient for the stimulation
of IL-2 production. Thus, the defect observed in cells
from patients with SLE appears to be in the signal
transduction pathway that activates PKC.
Although there is considerable evidence that a
3-hour incubation is sufficient time for stimulation with
PHA or PMA, it has not been established whether the
effects of ionomycin last for 24 hours. Nevertheless,
the finding that 3-hour stimulation with ionomycin and
PMA could induce normal levels of 1L-2 in SLE T cells
(Figure 5) was highly suggestive that this incubation
time could also be sufficient for stimulation with the
Ca2+ ionophore. Therefore, further studies were performed with the Ca2+ ionophore and/or PHA in the
cultures for the entire 24-hour incubation period. In 3
patients with inactive SLE, there were no significant
amounts of IL-2 activity in either of the supernatants
derived from cells stimulated for the entire period with
PHA alone, from cells stimulated with ionomycin
alone, and from cells stimulated with PHA + ionomycin (mean t SEM IL-2 activity 4.7 -C 2.2 units/ml, not
detectable, and 4.5 2 2.2 units/ml, respectively).
Taken together, these data provide strong evidence
that the 1L-2 defect in SLE patients is not attributable
to the defect in the level of [Ca2+]i, but rather to the
defect in the activation of PKC.
DISCUSSION
We have previously found that SLE T cells fail
to produce normal 1L-2 activity when stimulated with
PHA, regardless of the disease activity or the dosage
of corticosteroids (5). Moreover, we have observed a
deficiency of IL-2 production in approximately 40% of
the healthy family members of SLE patients (unpublished observation). This suggests that the deficiency
may contribute to the pathogenesis of the disease, and
that the defect may be inherited. A similar abnormality
in IL-2 production has been observed in each of 3
strains of inbred mice with SLE-like disease (30-32);
this defect, moreover, precedes the development of
the disease, suggesting that in SLE-prone mice, the
deficiency in IL-2 production might contribute to the
disease (33-35). Thus, decreased IL-2 production in
human SLE, as well as in murine SLE, could contribute to abnormal immune regulation and a predisposition to autoimmune disease.
83 1
It remains to be determined, however, whether
deficient PHA-stimulated IL-2 production by cells
from SLE patients is an intrinsic or an acquired defect,
and whether the cells will respond to signals either
distinct from or in addition to those supplied by PHA
for effective IL-2 production (36-38). Wofsy et al (35)
found that, in SLE-prone MRL-lpr/lpr mice, the defect
in concanavalin A (Con A)-induced IL-2 production is
not corrected by the presence of IL-I, a macrophage
product, or by PMA. Santoro et al (38) also noted that
the addition of IL-1 failed to correct the defect but, in
contrast to the findings of Wofsy and colleagues,
reported correction of the deficiency by Con A +
PMA. From these results, they concluded that PMA
may render the T cells more receptive to Con A by
extending the G, phase of the cell cycle and by
reducing IL-2 absorption by S phase retardation
(32,323). In view of the 2-signal model for T cell
activation (10-14), their findings could indicate that
the defect(s) in the signal transduction pathway may
be present in lymphocytes in MRL-lpr/lpr mice, since
PMA, a direct activator of PKC, has been known to
deliver 1 of the 2 signals required for IL-2 gene
activation (1 1,13).
A similar controversy exists in human SLE. In
our studies of human SLE, the addition of PMA
corrected the deficient PHA-induced IL-2 production.
Moreover, we determined that (a) this restoration was
not mediated by augmenting monocyte function; (b)
PMA did not promote the release of intracellular IL-2;
(c) PMA, by itself, did not induce IL-2 production at
the concentration used in the study; and (d) the defect
could not be corrected by mixing cells exposed separately to PHA and to PMA. This indicated that these 2
agents act in synergy. PMA is likely to provide a
signal(s) different from those of PHA for the same
1L-2- producing cells, and thus, fix the impaired signal
transduction process in PHA-stimulated SLE T cells.
Moreover, we have also demonstrated that the addition of PMA corrects the deficiency in PHA-induced
IL-2 production by both T4 and T8 + subsets.
Our findings differ from the data obtained by
Huang et al (39), who reported that the response of
SLE T cells to PHA is not restored by PMA unless the
cells are cultured in vitro for 2 days. The differences
between our findings and those of Huang et al can be
explained in part by differences in the patients' disease
activity, since almost all of theirs had active disease
and all of ours had inactive disease. We previously
demonstrated that although many of the activated
lymphocytes in the peripheral blood of SLE patients
+
MURAKAWA AND SAKANE
with active disease are B cells, their peripheral blood T
cells are also activated (40). Many abnormal T cells in
the blood of SLE patients with active disease could
have already been activated in vivo. These abnormal T
cells would no longer respond to additional in vitro
stimuli; rather, they would be preferentially eliminated
during the 2-3-day culture period. Most cells precultured in vitro for 2-3 days would thus consist of the
remaining normal T cell populations. In contrast, most
abnormal T cell populations in the blood of patients
with inactive S L E could be found to be in the resting
phase. These abnormal T cells may fail to respond to
PHA alone; however, when provided with an additional
signal delivered by PMA, the inability of these cells to
respond to PHA could be reversed. Therefore, we
believe that the different T cell populations that were
investigated explain the major differences between our
present findings and those of previous studies.
In this study, it was not possible to document
whether the defect in cells from SLE patients might be
reversed by increasing the concentration of rnitogen or
by taking the supernatants at different times. Therefore, it is possible that under different conditions,
greater o r lesser impairment in IL-2 activity of SLE T
cells might be observed.
It has recently been established that turnover of
membrane inositol phospholipids is an important signal in the initiation of T cell activation and proliferation (41). When cells are stimulated by an external
ligand, the increase in [Ca2+]i and the activation of
PKC are set in motion synergistically (41). To study
these 2 synergistically activated pathways, it is suitable to use agents that activate only one of the
pathways. Tumor promotors, such as PMA, activate
PKC without inducing an increase in [Ca2+]i (41).
Calcium ionophores, such as ionomycin, can increase
[Ca2']i without activating PKC (41). 1L-2 production
has been reported to occur through simultaneous
activation of these 2 pathways (1 1,13). In the present
study, we used PMA and/or ionomycin to examine the
role of these 2 pathways in the stimulation of SLE T
cells. When ionomycin was added to PHA-stimulated
cultures, SLE T cells produced small amounts of IL-2;
however, by the addition of PMA, PHA-stimulated
SLE T cells could be induced to generate normal
levels of 1L-2 activity. If the effect of PMA is limited to
the activation of PKC, these data indicate that in SLE
patients, the physiologic signal(s) that activates PKC
may be defective following PHA stimulation, while the
pathway that induces increased [Ca2+]i is intact.
Thus, the primary lymphocyte defect in SLE may be a
disorder of signal transduction. If that is the case, SLE
might be treatable by providing additional lymphocyte
activation signal( s).
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inactive, myristate, patients, induced, systemic, phytohemagglutinin, erythematosus, phorbol, correctable, lupus, deficiency, additional, activity, interleukin, acetate
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