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Signal transduction in Sjgren's syndrome T cells. Abnormalities associated with a newly described human A-type retrovirus

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I068
SIGNAL TRANSDUCTION IN
SJOGREN’S SYNDROME T CELLS
Abnormalities Associated with a Newly Described Human A-Type Retrovirus
ELIEZER FLESCHER, MICHAEL J. DAUPHINEE, DONNA FOSSUM,
JEFFREY LEDBETTER, and NORMAN TALAL
Objective. To study the effects of a novel A-type
retrovirus, detected in cocultures of lip biopsy specimens from Sjogren’s syndrome (SS) patients and a
human T cell line, on the infected T cells.
Methods. Interleukin-2 (IL-2) and IL-6 secretion
were measured by bioassay and enzyme-linked immunosorbent assay, respectively, in the infected and noninfected cell lines. Surface antigen expression was determined by flow cytometry, using monoclonal antibodies.
Protein kinase C (PKC) activity was measured using an
enzyme assay kit, and calcium mobilization was assessed
with a fluorescent probe.
Results. Infected cells expressed less CD4 and
IL-6 receptor, but more HLA-DR, compared with
From the Clinical Immunology Section, Audie L. Murphy
Memorial Veterans Hospital, and the Department of Medicine. The
University of Texas Health Science Center at San Antonio. San
Antonio, Texas, and the Bristol-Myers Squibb Pharmaceutical
Research Institute, Seattle. Washington.
Dr. Talal’s work was supported by USPHS grant ROIDEW3 I I .
Eliezer Flescher, PhD: Division of Clinical Immunology,
Department of Medicine, The University of Texas Health Science
Center; Michael J. Dauphinee, PhD: Clinical Immunology Section,
Audie L. Murphy Memorial Veterans Hospital. and Division of
Clinical Immunology. Department of Medicine, The University of
Texas Health Science Center (current address: Immunology Research Institute. Annandale, NJ): Donna Fossum. BSc: Division of
Clinical Immunology. Department of Medicine. The University of
Texas Health Science Center: Jeffrey Ledbetter. PhD: BristolMyers Squibb Pharmaceutical Research Institute: Norman Talal.
MD: Clinical Immunology Section, Audie L. Murphy Memorial
Veterans Hospital, and Division of Clinical Immunology, Depdrtment of Medicine, The University of Texas Health Science Center.
Address reprint requests to Eliezer Flescher. PhD, Division
of Clinical Immunology, Department of Medicine, The University of
Texas Health Science Center, 7703 Floyd Curl Drive. San Antonio.
TX 78284.
Submitted for publication September 17, 1991: accepted in
revised form April 29. 1992.
Arthritis and Rheumatism, Vol. 35, No. 9 (September 1992)
noninfected cells. Infected cells also produced less IL-2
and displayed reduced PKC activation and calcium
mobilization. A similar defect in calcium mobilization
was detected in T cells from SS patients.
Conclusion. These data suggest a possible involvement of the newly described retrovirus in T cell
abnormalities.
Sjogren’s syndrome (SS) is a chronic autoimmune and inflammatory disease in which lymphocytes
infiltrate and destroy exocrine glands and other parenchymal organs ( I ) . The peripheral blood of patients
with primary SS contains increased numbers of HLADR-bearing T lymphocytes. The ability of SS patient
peripheral blood T cells to produce interleukin-2 (IL-2)
is decreased (2,3). The predominant cell in the salivary
gland infiltrates of SS patients is a T cell bearing the
CD4 marker and activation markers such as HLA-DR
( I ) . We have suggested that T cells from SS patients
may have a defect in intracellular signaling, based on
our findings that these cells are impaired in their
proliferative response to phorbol myristate acetate
(PMA, a protein kinase C [PKC] activator) and ionomycin (a calcium ionophore) (4).
Antibodies to retroviral proteins can be detected in the serum of patients with SS (5). In addition,
an A-type retrovirus has been isolated from lymphoblastoid cells (the H9 cell line, which is a subclone of
Hut 78) exposed to lip biopsy material from SS patients (6,7). Since T cells from humans infected with
the human immunodeficiency virus (HIV) (8) and from
mice that exhibit acquired immunodeficiency syndrome (infected with LP-BM5 murine leukemia virus)
(9) have defects in signal transduction, we studied
signal transduction in a T cell line infected with the
A-TYPE RETROVIRUS A N D T CELL ABNORMALITIES IN SS
SS-associated A-type retrovirus, as well as in T cells
from SS patients. Our d a t a s h o w that early transmembrane signals (including calcium mobilization and PKC
activation), as well as IL-2 production, are significantly altered by infection with the SS-associated
A-type retrovirus.
PATIENTS AND METHODS
Patients. Fifteen patients who fulfilled diagnostic
criteria for primary SS ( I ) (14 women and I man) were
studied. The patients did not have associated rheumatoid
arthritis, nor were they taking corticosteroids or immunosuppressive drugs. Six healthy women served as normal
control subjects.
Mononuclear cells were obtained from heparinized
peripheral blood by centrifugation on Ficoll-Hypaque (Pharmacia, Piscataway, NJ); cells adhering to plastic were removed. Salivary gland tissue was obtained by performing lip
biopsies on 6 patients with SS.
The biopsy specimens were homogenized in a tissue
grinder, and the crude tissue homogenates added to cultures
of H9 cells. After 6-12 weeks, lysates of cells from 2 of these
cultures became positive for HIV-related gag antigens. A
characteristic intracisternal type A retroviral particle could
be seen by electron microscopic analysis of the infected
lines. The H9 cell line with (6,7), or without an A-type
retrovirus infection, was passed in complete medium (see
below). (These procedures were performed by Dr. R. F.
Carry, Tulane University, New Orleans, LA.)
Medium. Unless otherwise indicated, all cell cultures
were carried out in complete medium: RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mM
glutamine, 25 mM HEPES buffer, 100 units/ml penicillin,
and 100 pg/ml streptomycin. All reagents were from Gibco
(Grand Island, NY).
Assay for IL-2 secretion. H9 cells were suspended in
complete medium at 37°C in 95% air/5% CO,, at 2 x I06/ml
for 48 hours, with or without reagents as described in
Results. The cells were centrifuged and supernatants collected and assayed for the presence of IL-2, using a murine
IL-2-dependent cytotoxic T cell line (CTLL-2); the results
were calculated using probit analysis (10).
Assay for IL-6 secretion. H9 cells were incubated
under the same conditions as for the IL-2 secretion assay.
The cells were centrifuged and supernatants collected and
assayed for the presence of IL-6, using an enzyme-linked
immunosorbent assay (ELISA) for quantification of human
IL-6 (R & D Systems, Minneapolis, MN).
Flow cytometry using immunofluorescence staining.
H9 cells were analyzed for surface antigens (see Results)
using fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (MAb; Becton Dickinson, Mountain View,
CA). The analysis was performed as described elsewhere
(10,l I ) . The ability to bind IL-6 was measured using the
fluorokine kit (R & D Systems), in which biotinylated IL-6 is
allowed to bind to the cells and is detected with FITCconjugated avidin. This enables the determination of IL-6
receptor (IL-6R)-positive cells: The level of fluorescence is
1069
proportional to the number of receptors. The difference in
fluorescence intensity between 2 samples was calculated
using a formula recommended by Becton Dickinson, in
which the difference of the means of two logarithmic intensities =
where mean channeVl28 = log relative fluorescence (10).
Measurements of free intracellular calcium levels.
Nonadherent peripheral blood cells were loaded with the
calcium probe Indo-I and stimulated as indicated in Results.
The resting level of the cytoplasmic free calcium ions was
- 130 nM (12). Peaks of the mean fluorescence measured in
a flow cytometer are reported. H9 cells were loaded with the
calcium probe Fluo-3 (13) and stimulated as indicated in
Results. The resting level of the cytoplasmic free calcium
ions in noninfected cells was -90 nM; the measurement of
mean fluorescence in a flow cytometer was as described
elsewhere (14). Mean fluorescence in the range measured in
this study is in linear correlation with intracellular concentrations of free calcium ions ([Ca' +Ii).
Determination of PKC activation. Our method of
measuring PKC activation was a modification of procedures
described previously (15,16). Ten million H9 cells were
incubated with or without PMA (20 nM) for various periods
of time. Cells were washed and resuspended in isolation
buffer (20 mM HEPES, pH 7.4, 1 mM EGTA, I mM EDTA,
5 mM dithiothreitol) and lysed with a dounce homogenizer.
Cytosolic and membrane fractions were separated by centrifugation. The PKC in the membrane fraction was solubilized with 1 mM 3-[(3-chlamidopropyl) dimethylammonial-l
propane sulfonate (CHAPS) at 4°C for 20 minutes. PKC
activity in 10 pg protein of the cytosolic and solubilized
membrane fractions was assayed using the PKC enzyme
assay kit (Amersham, Buckinghamshire, UK). Any calciumand phospholipid-independent activity (which by definition
is not the result of PKC activity), as well as any nonspecific
effects of "P-ATP or its radiolytic decomposition products
binding to papers, were subtracted from the results obtained.
Reagents. All reagents were from Sigma (St. Louis,
MO) unless otherwise indicated. Phytohemagglutinin (PHA)
was purchased from Burroughs-Wellcome (Greenville, NC).
Anti-CD2 monoclonal antibodies (MAb 9.6) were prepared
according to the method of Martin et al (17). IgM anti-CD3
monoclonal antibodies (MAb 38.1) were prepared as described by Hansen et al (18). The fluorescent probes, Indo-I
and Fluo-3, were purchased from Molecular Probes (Eugene, OR). Ionomycin was from Boehring Diagnostics (La
Jolla, CA).
Statistical analysis. The statistical significance of the
results was calculated using Student's 1-tailed r-test.
RESULTS
Modulation of surface antigen expression on retrovirus-infectedT cells. T h e H9 cell line w a s used as a
T cell model t o study t h e possible effects of the
SS-associated retrovirus on lymphocytes. The per-
FLESCHER ET AL
1070
Table 1.
Expression of T cell surface proteins*
Surface protein
CD3
CD5
C D4
C D8
CD25
HLA-DR
IL-6R
Noninfected
cells
95.5 t 5.4
93.4 2 6.2
90.1 2 4.8
1.1 2 0.6
1.5 2 0.7
74.8 2 6.2
69.8 ? 5.9
Infected
cells
98.0
92.1
59.2
0.9
5
a-HLA-DR
:.:'.
2 6.1
4.4
4.9t
2 0.8
1 . 1 2 1.0
94.5 2 7.7$
47.0 2 5 . l t
2
2
* H9 cells (infected and noninfected) were stained with monoclonal
antibodies against the surface proteins indicated. The antibodies
were either biotinylated followed by fluorescein-avidin treatment
(interleukin-6 receptor [IL-6R]) or fluoresceinated (the rest of the
surface proteins). The cells were then analyzed in a flow cytometer.
Values are the mean ? SD percent stained cells ( n = 3).
t P < 0.005 versus noninfected cells.
$ P < 0.025 versus noninfected cells.
centage of retrovirus-infected cells expressing the T
cell activation marker HLA-DR was significantly
higher than in the noninfected cell line. In contrast, the
percentage of CD4 and ILdR-bearing cells was significantly lower in comparison with noninfected cells
(Table 1). The differences in HLA-DR and IL-6R
expression were also evident at the level of the mean
fluorescence of the cells (10): There was a 72.6% and a
20.8% difference in mean cellular expression of these
antigens, respectively, between noninfected and infected cells (Figure 1).
Similar differences in surface antigen expression between infected and noninfected cells were
observed at 3 different time points and after preincubation for up to 3 days in the presence of PHA, PMA, or
interferon-y. Stimulation with PHA and PMA did not
induce IL-2R expression on the surface of infected cells.
Lymphokine production by H9 cells. Levels of
IL-2 are decreased ( I 1). and levels of IL-6 increased
(191, in many chronic autoimmune diseases. Thus, we
studied production of these lymphokines in H9 T cells.
PHA (2 pg/ml) + PMA (20 ng/ml) stimulation of
infected or noninfected cells for 48 hours resulted in
the production of 15.5 ? 2.1 units/ml and 25.8 f 2.7
unitdm1 IL-2 (mean f SD; n = 3). respectively (P<
0.005), demonstrating decreased production in the
infected cell line. Neither the noninfected nor the
infected cells produced any IL-6 (even after PHA +
PMA stimulation), as assessed both by ELISA as well
as by a bioassay using B9 hybndoma cells as responders.
Calcium mobilization in H9 cells. Modulation of
[Ca"], is an essential early event in the transmission
0
1
2
3
4
0
1
2
3
4
Log Relative Fluorescence
Figure 1. Modulation of surface protein mean fluorescence in retrovirus-infected H9 cells. H9 cells were stained with monoclonal
antibodies against HLA-DR ((r-HLA-DR) and against interleukin-6
receptor (a-IL6R). The antibodies were either biotinylated, followed
by treatment with fluorescein-avidin ( I L d R ) , or fluoresceinated
(HLA-DR). The cells were then analyzed in a flow cytometer. The
distribution of the cell population according to the relative fluorescence of individual cells is shown. Dotted lines = control (unlabeled) cells; dashed lines = noninfected cells; solid lines = infected
cells. The results of I experiment are presented; 2 additional
experiments were performed, with similar results.
of an activation signal in T cells (20). Infected cells had
a significantly decreased response (response assayed
as a rise in [Ca"],) to a pharmacologic stimulus
delivered by ionomycin (Figure 2), as well as to a
mitogenic stimulus delivered by PHA (Figure 3). Basal
levels of [Ca"], in the infected cells were -25% lower
than in the noninfected cells.
3001
250
2
200
lonomycin
(10nM) -
$
150
ii
-100
1
I
I
I
I
I
I
I
-50
0
50
100
150
200
250
300
Time (seconds)
Figure 2. Decreased mobilization of calcium in retrovirus-infected
H9 cells following ionomycin treatment. The fluorescence of Fluo3-loaded H9 cells (representing [Ca"],), from I minute before
through 5 minutes after ionomycin stimulation, was determined
using flow cytometry. The results of 1 experiment are presented; 2
additional experiments were performed, with similar results.
A-TYPE RETROVIRUS AND T CELL ABNORMALITIES IN SS
30 1
0.7
m
250
Non-infected
Q,
2
$
Membrane
.-
E _ -
200-
0
Q,
I
PHA
150-
ii
g
1071
c
100-
\
Cytosol
50 -
O f
-100
1
I
1
I
I
I
I
-50
0
50
100
150
200
250
1
300
Time (seconds)
01
0
I
I
1
I
I
I
10
20
30
40
50
60
Time (minutes)
Figure 3. Decreased mobilization of calcium in retrovirus-infected
H9 cells following phytohemagglutinin (PHA) treatment. The fluorescence of Fluo-3-loaded H9 cells (representing [Ca+']J, from 1
minute before through 5 minutes after PHA stimulation, was determined using flow cytometry. The results of 1 experiment are
presented; 2 additional experiments were performed, with similar
results.
Figure 4. Kinetics of protein kinase C (PKC) translocation in H9
cells. PKC activity in the cytosol and membrane fractions of H9
cells incubated without (time 0) or with phorbol myristate acetate
(20 nM) for different periods of time was measured and expressed as
nmoles "P/mg protein/minute. The results of 1 experiment are
presented; 2 additional experiments were performed, with similar
results.
Calcium mobilization in SS peripheral blood T
cells. We assessed whether a defect in the ability to
mobilize calcium, identified in retrovirus-infected T
cells, also existed in T cells from the peripheral blood
of SS patients. In comparison with T cells from normal
peripheral blood, cells from SS patients were significantly deficient in their ability to respond with a rise in
[Ca"], following stimulation with MAb to CD2 (Table
2). The response of the latter cells to anti-CD3 anti-
bodies was also reduced, although the difference did
not reach statistical significance.
PKC activation in H9 cells. Translocation of
PKC from the cytosol to the membrane is essential for
human T cell activation (21-23). PKC activity in H9
cells was translocated from the cytosol to the membrane fraction following stimulation with 20 nM PMA;
this change reached a plateau after 30 minutes (Figure
4) and was kinetically similar to the response of PMA
+ ionomycin-stimulated resting human T cells (23). A
similar translocation occurred in infected cells after 30
minutes, but to a significantly lesser degree. PKC
activity in the membrane fraction of PMA-stimulated
infected cells was 0.35 nmoles "P/mg protein/minute,
while the activity in the same fraction from noninfected cells was 0.58 nmoles "P/mg proteitdminute ( P
< 0.0005) (Figure 5).
Table 2. Signal transduction by CD2 or CD3 receptors in peripheral blood T cells from control subjects (n = 4) and Sjogren's
syndrome ( S S ) patients (n = 15)*
Antibody, source of cells
CD2
Controls
S S patients
CD3
Controls
SS patients
Peak [Ca"],
response
1,510 2 241
981
5
364t
DISCUSSION
1,178 2 428
856 5 605
* Nonadherent cells were loaded with Indo-l dye and analyzed for
[Ca++J i response, using flow cytometry. CD2 receptor aggregation
was accomplished by treatment with biotin-conjugated monoclonal
antibody (MAb) 9.6 (10 pg/ml), followed 5 minutes later with avidin
(40 d r n l ) . CD3 receptor aggregation was accomplished by direct
addition of MAb 38. I (20 pg/ml). Values are the mean 5 SEM.
t P < 0.05 versus response of control cells.
We report that a human T cell line, containing
an A-type retrovirus which appears after coculture
with homogenates from SS patient lip biopsy specimens, has a different cell surface marker profile and
produces less IL-2 compared with noninfected cells.
In addition, the infected cells respond less well to
PMA, as measured by translocation of PKC activity
FLESCHER ET AL
1072
0.8
0.7
e
'E
e
.-
Q)
t
0.6
0.5
0.4
h
2 0.3
-I
0.2
c
0.1
0
30
0
Time (minutes)
Figure 5 Decreased protein kinase C (PKC) translocation in retrovirus-infected H9 cells. PKC activity in the cytosol fraction of
noninfected cells (W). the membrane fraction of noninfected cells
(0).
the cytosol fraction of infected cells (0).
and the membrane
fraction of infected cells ( H )was measured before and after stimulation with phorbol myristate acetate (20 nM) for 30 minutes. Values
are the mean 2 SD of 3 experiments.
from the cytosol to the membrane fraction. Finally,
these cells, as well as T cells from the peripheral blood
of SS patients, exhibit decreased calcium mobilization
following stimulation.
It is very important to confirm that the H9 cells
are infected with the A-type retrovirus. This accomplishment must await the development of a specific
probe for the virus, such as complementary DNA to be
used in in situ hybridization. Such a probe could also
be used to investigate for viruses in SS peripheral
blood T cells. A-particles are abundant in the infected
cell line, and our results reflect the virus effects at the
population level. On the other hand, primers from
conserved regions of HIV gug, pol, and m v genes, in
the polymerase chain reaction run under conditions of
low stringency, did not amplify nucleic acid sequences
from the infected cells. In addition, no C-particles
were detected in these cells by electron microscopy
(6,7). These findings indicate that the HPinfected cells
studied are not infected with HIV.
The infected H9 T cells, like HIV-l-infected T
cells, are deficient in the expression of surface CD4
(24). The percentage of HLA-DR+ cells, as well as
the density of this marker on the cell surface, are
increased in the infected cell population. The same is
true for T cells from the peripheral blood of SS
patients (2,3), suggesting enhanced ability to interact
at an intercellular level and function in the immune
response.
Since infection with HIV-I is associated with
enhanced 1L-6 production ( 2 3 , we assessed the potential of the infected cells in the present study to respond
by producing this cytokine. H9 cells did not produce
1L-6, as determined by both biologic and immunochemical means. IL-6R expression was reduced in
infected compared with noninfected cells. Thus, 1L-6
does not appear to be an autocrine factor for the
infected cells. Similar to induced transcription of IL-2
messenger R N A in human T lymphotropic virus type 1
(HTLV-Itinfected T cells, but unlike the findings in
HIV-l-infected cells (261, PHA + PMA-stimulated
cells in our system produced lower levels of IL-2,
compared with noninfected cells. Reduced IL-2 production is also characteristic of T cells from the
peripheral blood of SS patients (2,3).
H9-infected cells, as well as T cells from the
peripheral blood of SS patients, were defective in their
ability to generate a rise in [Ca"], following stimulation. Similar observations have been reported with
HIV-l-infected T cells (8,27), as well as with
HTLV- 1- and simian immunodeficiency virus ( S I V t
infected T cells (28,29). While H9-infected cells had
decreased basal concentrations of intracellular free
calcium, HIV-1 (8), HTLV-I (28), and SIV-infected
(28) cells had increased basal levels of intracellular
free calcium. The effect of an A-type retrovirus on T
cell basal [Ca"], may be different from the effects of
C-type retroviruses.
A synthetic peptide with sequence identity to
HIV-I gp41 inhibits PKC activity and anti-CD3induced C a + + influx in human T cells, without affecting levels of inositol triphosphate, the physiologic
second messenger that mobilizes Ca+ (30). Nevertheless, gp120 and gp160 of HIV-1 origin were recently
shown to inhibit CD3-mediated production of inositol
phosphate (3I ) . Since retrovirus-infected H9 cells
have an abnormally low response to the ionophore
ionomycin, suggesting that the cellular defect might be
distal to inositol phosphate production and directly
related to transmembrane calcium transport and/or its
accumulation intracellularly, it appears that the effect
of the A-type virus resembles the effect of HIV-1 gp41.
Using similar low concentrations of ionomycin (7-100
nM), Miller et al (32) were able to expose a defect in
the ability of T lymphocytes from old mice to respond
with an increase in [Ca"],.
As mentioned above, a peptide with sequence
identity to an HIV-1 glycoprotein has been shown to
+
A-TYPE RETROVIRUS AND T CELL ABNORMALITIES IN SS
inhibit PKC activity (30), and HIV-1 caused reductions in phospholipid synthesis and in levels of diacylglycerol, the physiologic second messenger that activates PKC (33,34). It thus appears that HIV-1 might
affect both levels of the PKC stimulator diacylglycerol, as well as PKC activity. Since we found a defect
in the ability of PMA, a direct activator of PKC, to
activate the enzyme in infected cells, a defect in PKC
activation may occur subsequent to the generation of
diacylglycerol in the virus-infected H9 cells.
Gerli et a1 (35) reported that T cells from SS
patients are defective in their ability to proliferate in
response to anti-CD2 antibodies. Although they did
not directly measure the generation of second messengers, their report suggests that the defect in the SS
cells is correlated with impaired PKC activation, and
not abnormal calcium mobilization. We found direct
evidence for defective calcium mobilization in antiCDZstimulated SS T cells. The difference between
our conclusions and those of Gerli et a1 (35)might arise
from different methodologic approaches.
Our findings suggest that the A-type retrovirus
infection is related to the observed abnormal signaling.
This does not preclude other possible contributing
factors such as another, concomitant, virus infection.
In summary, cells infected with an SS-associated
A-type retrovirus have abnormal characteristics which
they share both with human T cells infected with other
retroviruses as well as with T cells from the peripheral
blood of SS patients. This raises the possibility that the
A-type retrovirus isolated from SS lip biopsy material
might be involved in the immunologic abnormalities
characteristic of this autoimmune disease. Nevertheless, further investigation will be necessary in order to
establish a direct link between the A-type retrovirus
and signaling defects in T cells from the peripheral
blood of patients with SS.
ACKNOWLEDGMENTS
We thank Dr. I. R. Garrett for performing the IL-6
bioassay and Marian Langston for typing the manuscript.
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