By
From the * Divisions of Immunobiology and Rheumatology, Department of Medicine, The University
of Vermont College of Medicine, Burlington, Vermont 05405-0068; Immunex Corporation, Seattle,
Washington 98101; § Institute of Biochemistry, University of Lausanne, Swiss Institute for Cancer
Research, Epalinges, Switzerland;
Division of Rheumatology and Connective Tissue Research,
Department of Medicine, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson
Medical School, New Brunswick, New Jersey 08903
The function of the minor subset of T lymphocytes bearing the T cell antigen receptor is
uncertain. Although some
T cells react to microbial products, responsiveness has only rarely been demonstrated toward a bacterial antigen from a naturally occurring human infection. Synovial fluid lymphocytes from patients with Lyme arthritis contain a large proportion of
cells that proliferate in response to the causative spirochete, Borrelia burgdorferi. Furthermore,
synovial
T cell clones express elevated and sustained levels of the ligand for Fas (APO-1,
CD95) compared to
T cells, and induce apoptosis of Fashigh CD4+ synovial lymphocytes.
The findings suggest that
T cells contribute to defense in human infections, as well as manifest an immunoregulatory function at inflammatory sites by a Fas-dependent process.
While most T lymphocytes express a TCR composed
of Patients.
Lyme arthritis patients came from areas endemic for
Lyme disease and were followed at the Lyme Disease Clinic at
the University of Medicine and Dentistry of New Jersey, Robert
Wood Johnson Medical School. All patients had histories, exams,
and serologies consistent with Lyme arthritis, including Borreliaspecific antibody titers that were higher in synovial fluid relative
to serum. Synovial fluid lymphocytes were examined from seven
patients with Lyme arthritis of 6-mo to 3.2-yr duration.
Flow Cytometry.
Lymphocytes were isolated from peripheral
blood or synovial fluid by Ficoll-Hypaque centrifugation. Cells
were stained with the indicated fluorochrome-conjugated antibody at 4°C for 30 min. Antibodies were specific for TCR- Proliferation Assays and Derivation of Lyme Synovial PCR Analysis of Synovial Fluid T Lymphocyte V Assay of Cytotolytic Activity.
Faslow variants of the wild-type
Jurkat T cell line, H7 (3% normal surface Fas levels) and B4 (1%
normal Fas levels), were derived through irradiation mutagenesis
using five doses of 200 Rads each, delivered at 5-d intervals. After
each irradiation, cells were cultured in wells coated with lytic
anti-Fas antibody (M2, 3 µg/ml)(24). The Faslow variants and
wild-type Jurkat cells were incubated with 51Chromium (51Cr)
for 1 h, washed, and then mixed at various effector/target ratios
with cloned V and
chains, a subpopulation of T cells bearing alternate
and
chains exists as a minor subset of peripheral blood lymphocytes (PBL)1 (1). While the function
of
T cells is uncertain, a clue may lie in their increased
proportion at epithelial barriers, during certain infections,
and at sites of chronic inflammation such as synovial tissue
in rheumatoid arthritis (2). Some
T cells respond to
bacterial products and can be identified after infection of
mice with particular bacteria (8). However, in humans,
leprosy is the only infectious disease to date in which
cells from affected individuals have been shown to respond to the causative organism (9).
T cells frequently manifest cytolytic activity toward
a broad array of target cells (2, 16). Such a spectrum of cytolysis might occur when a target molecule is widely expressed, such as the Fas antigen (APO-1, CD95) (17). Fas
is a 45-kD cell surface molecule that mediates apoptosis
and is a member of a family of molecules that includes the
type I receptor for TNF. Fas is one of the principle components responsible for T cell-mediated cytotoxicity (18).
Expression of mRNA for the Fas ligand (FasL) was originally described as being transiently expressed by activated
T cells, although higher mRNA levels were noted in
T cells (21). More recent findings have noted constitutive expression of FasL by nonlymphoid cells, including
Sertoli cells of the testis (22) and certain components of the
eye (23). FasL expression by these tissues parallels their ability
to suppress immune-mediated inflammation. These collective observations suggested that
T cells in Lyme arthritis
might respond to Borrelia burgdorferi as well as contribute to
regulation of the synovial inflammatory infiltrate.
(JOVI-1; Ancell Corp., Bayport, MN), TCR-
(5A6.E9; T Cell
Sciences, Inc., Cambridge, MA), TCR-V
1 and TCR-V
2 (AB
and BB3, respectively, courtesy of Dr. Alessandro Moretta, University of Genoa, Genoa, Italy), CD4 (SFCI12T4D11; Coulter
Corp., Hialeah, FL), CD8 (SFCI21Thy2D3; Coulter Corp.), Fas
(M38) (24) and FasL (polyclonal C-20; Santa Cruz Biotechnology, Santa Cruz, CA; or monoclonal A11 [25]). Surface staining
for FasL was performed by one of three methods. The first approach used a fusion protein composed of the extracellular domain of murine Fas linked to the human Ig Fc portion (Fas-Fc)
(26). This was followed by goat anti-human Fc-biotin and then
avidin-phycoerythrin. Control staining was accomplished by
staining for surface IL4 using an IL4 receptor-Fc fusion protein.
Alternatively, surface FasL was measured using either a rabbit antiserum to the extracellular carboxyl-terminal portion of human
FasL and purified on a FasL sepharose column (C-20), or monoclonal antibody A11 that recognizes both mouse and human Fas
(25). To measure FasL induction, cells were examined 3 h after
stimulation with PMA (10 ng/ml) and ionomycin (250 ng/ml),
in the absence or presence of metalloprotease inhibition using 5 mM EDTA (27). Samples were analyzed on a Coulter Elite flow
cytometer (Coulter Corp.) and at least 2 × 104 events were accumulated for analysis.
T Cell
Clones.
Synovial fluid lymphocytes were cultured in AIM-V serum-free medium (GIBCO BRL, Gaithersburg, MD) in either
bulk cultures (106/ml) for phenotyping, or in round-bottomed
microtiter wells (105/well) for proliferation assays. Cells were
stimulated with 3 µg/ml of a sonicate of B. burgdorferi grown in
BSK II medium as previously described (28). Triplicate cultures
were pulsed with 3H-TdR during the last 18 h of a 6-d culture,
harvested, and counted. From parallel cultures, responding cells
were cloned at 0.3 cells/well in AIM-V with 5% FCS in the presence of irradiated PBL (3 × 105/well), human recombinant IL2
(10 U/ml), and 3 µg/ml of B. burgdorferi sonicate. Responding
wells were phenotyped and the
cells expanded by restimulation at 10-d intervals.
Repertoire.
Semi-quantitative PCR was performed on samples using cDNA
prepared from oligo-dT-primed RNA and reverse transcriptase
(GIBCO BRL) as previously described (29). The 5
V
- and
C
-specific primers are modifications of published sequences (30)
as follows: V
1: 5
-AGCAACTTCCCAGCAAAGAG-3
; V
2:
5
-AGGAAGACCCAAGGTAACACAA-3
; V
3: 5
-CACTGTATATTCAAATCCAGA-3
; V
4: 5
-TGACACCAGTGATCCAAGTTA-3
; V
5: 5
-CTGTGACTATACTAACAGCATGT-3
; V
6: 5
-TATCATGGATTCCCAGCC-3
; 5
C
:
5
-CTTGTCTGGTGCAG-3
; 3
C
: 5
-CTTCACCAGACAAGCGACAT-3
. A PCR reaction master mix that was common
to all samples contained 100 mM Tris HCl, pH 8.3, 500 mM
KCl, 2 mM MgCl2, 200 µM dNTPs, with 25 pmoles of 3
C
primer, 2.2 µCi
-32P-dCTP, and 2.5 U Taq polymerase
(GIBCO BRL) per tube. The final volume was 100 µl and
contained 10 ng cDNA, and 25 pmoles of individual V
primer.
Samples were run on a thermocycler (model 9600; Perkin-Elmer Corp., Norwalk, CT) for 24 cycles using the parameters: cycle 1:
94°C × 3 min, 50°C × 45 s, 72°C × 1 min; cycles 2-23: 94°C × 30 s, 50°C × 45 s, 72°C × 1 min; cycle 24: 94°C × 30 s, 50°C × 45 s, 72°C × 7 min. Samples were resolved on a 29 cm 10%
polyacrylamide gel containing 7 M urea in TBE buffer and electrophoresed at 80 V for 18 h. The gel was dried and developed
on an analyzer (Betascope 603; Betagen, Waltham, MA). The
percentage expression of each V
was assigned by dividing the
actual cpm for a specific V
by the total cpm for V
1-V
6 after
correction for the total C
message in each sample.
1 cells in a total volume of 200 µl. After a 4-h incubation at 37°C, 100 µl of supernatant were removed and counted for
emission. Spontaneous release was determined
from labeled targets in the absence of effector cells. Maximum release was determined by lysing target cells with 1.0 N HCl. The
percentage of specific 51Cr release was calculated as:
.
TUNEL Assay for Apoptosis.
Cells were initially stained for
expression of surface , CD4, or CD8 and then fixed for 15 min
in 1% paraformaldehyde. Cell membranes were then permeabilized for 15 min using 70% ethanol at 4°C. Samples were incubated at 37°C for 1 h in 100 µl containing 10 U terminal
deoxyribosyltransferase and 0.5 nM dUTP-biotin (Boehringer Mannheim Biochemicals Corp., Indianapolis, IN) (31, 32). Specimens were washed twice with PBS/1% BSA and incubated with
a 1:50 dilution of avidin-tricolor (Caltag Labs., South San Francisco, CA) at 4°C for 30 min. Cells were washed twice and analyzed by flow cytometry.
Synovial fluid lymphocytes were
examined from seven patients with Lyme arthritis of 6 mo
to 3.2-y duration. These contained a predominance of CD4+
over CD8+ T cells in only four of seven cases (Fig. 1 A,
Table 1), compared to a consistent CD4 predominance in
PBL. Also present in the synovial mononuclear cells was a
remarkable percentage of
T cells (18.9 ± 6.8%) (Fig. 1 A,
Table 1), compared to ~1-5% in PBL (Reference 1 and
see Fig. 3). The synovial
population was largely devoid of
surface CD4, and only a minor proportion (~20% on average) expressed low to intermediate levels of CD8 (Fig. 1 B).
In addition, whereas
T cells from PBL express predominantly the V
2 gene product (33), Lyme arthritis synovial
fluid
cells were primarily of the V
1 subset, with lesser
proportions of V
2 and V
3 cells. This was determined by
both flow cytometry using V
-specific antibodies (Fig. 1 A),
and semi-quantitative PCR using specific V
primers (Fig. 2).
|
Stimulation of Lyme arthritis synovial fluid mononuclear
cells with a sonicate of B. burgdorferi (strain N40) induced
vigorous proliferation (Table 1), yielding a two- to threefold increase in cell number over 6 d. During this period,
the composition of T cell subsets shifted considerably. Although the percentage of CD8+ cells changed only slightly,
there was frequently a striking loss in the proportion of
CD4+ cells by as much as threefold. Thus, despite the increase in total lymphocyte number during the 6-d culture,
there was frequently little change or even a decrease in the
absolute number of CD4+ cells, as illustrated by patient no. 2 in Table 2. This was paralleled by a reciprocal increase in
T cells, in some cases to as much as 50% of the cultured
synovial lymphocytes (Fig. 1, Table 1). These continued to
be mostly V
1 cells as determined by both antibody (Fig. 1 A)
and PCR (Fig. 2) analysis.
The loss of CD4+ synovial cells might have resulted
from unresponsiveness of this subset to B. burgdorferi, and
hence overgrowth by the CD8+ and + subsets. However, this seems unlikely since we have previously observed
that PBL also proliferate strongly to B. burgdorferi with an
expansion of predominantly CD4+ cells (28). Alternatively,
because PBL contain only a small proportion of
cells
(1), the
subset might be responsible for the loss of CD4+
cells in Borrelia-activated synovial cultures. Consistent with this notion was the one case (patient no. 6) where the percentage of
T cells did not increase following stimulation
with B. burgdorferi. In this instance, the proportion of CD4+
cells actually increased from 36.5 to 51.4% (Table 1).
To more directly address the possibility
that synovial cells might be cytolytic toward the CD4+
subset,
T cell clones were derived from synovial fluids
of two Lyme arthritis patients using a sonicate of B. burgdorferi and irradiated autologous PBL. A panel of 18 Borreliaresponsive
clones was established, the majority of which
express V
1 and lack surface CD4 and CD8. DNA sequencing of the
chain from seven clones confirmed that
they all express V
1, but were otherwise each unique and
contained varying degrees of N region diversity (Roessner,
K., manuscript in preparation).
T cells frequently manifest cytolytic activity toward a
broad array of target cells (2, 16). Such a spectrum of cytolysis might occur when a target molecule is widely expressed, as is the case with the apoptosis-inducing molecule, Fas (17). As shown in Fig. 3, Fas expression by fresh
CD4+ PBL was low to negligible, but was present on a
large proportion of CD4+ synovial lymphocytes. By contrast, the CD8+ and
+ subsets of PBL or synovial lymphocytes displayed considerably lower levels of Fas.
Surface expression of FasL protein by B. burgdorferi-reactive and CD4+
T cell clones was examined by flow
cytometry using two methods, a Fas-Fc fusion protein as
well as a purified anti-human FasL rabbit antiserum. Control staining for Fas-Fc was determined using a human IL4
receptor-Fc (IL4R-Fc) fusion protein (as surface-bound IL4 would not be anticipated for a secreted cytokine). Fig.
4 A (column 3) illustrates results of staining using the FasFc fusion protein, on represetative
(114B) and
(2.11)
synovial T cell clones. By this method, surface FasL protein
was expressed on a considerably higher proportion of the
cells than on the B. burgdorferi-reactive
T cell clones
seven days after the last stimulation. Similar findings were
seen with an additional two
and two
synovial T cell
clones. In contrast, the levels of surface Fas antigen on the
clones were somewhat less than on the
clones, (Fig.
4 A, column 4).
The anti-FasL antibody confirmed the disparity in surface
FasL expression between synovial versus
T cell clones.
Fig. 4 B (column 1) shows that 7 d after antigenic stimulation of the Borrelia-reactive
(114B) and
(2.11)
clones, surface FasL was present on the
clone, but was
only marginally detectable on the
clone. This finding
was consistent for three
and three
clones studied.
However, the
clones were capable of induction of FasL
upon stimulation, as shown after 3 h of activation with
PMA and ionomycin. In agreement with a recent report
(27), FasL expression on the T cell line, Molt 4, was enhanced by blocking metalloprotease activity with EDTA
(Fig. 4 B, column 4). This was less consistently observed
for the
T cell clones, and was not observed for the
clones. It was particularly striking that the levels of FasL on
the
clones remained detectable for at least 10 d following stimulation with B. burgdorferi (Fig. 4 B, column 1).
This is in distinct contrast to
T cells which express FasL
only transiently after activation (21; Roessner, K., unpublished observations).
To further explore whether the Lyme
arthritis synovial fluid T cell clones might be cytolytic
toward T lymphocytes expressing high levels of surface Fas,
the Jurkat T cell line was initially used as a representative
Fashigh target. Fig. 5 A shows that the
clones manifested
very efficient cytolytic activity toward Jurkat cells, with
50% maximal lysis achieved at an effector/target ratio between 10:1 and 3:1. This finding was remarkably consistent
for each of five different V
1 clones tested from two patients. In contrast, Borrelia-reactive CD4+
T cell clones
manifested little, if any, cytolysis of Jurkat cells (data not
shown). Cytolysis by the
clones was not inhibited by
antibodies to TCR-
, HLA class I or II, but was blocked
by anti-LFA-1 antibody (Fig. 5 C ), supporting the notion
that cytolysis was dependent on cell contact.
The potential contribution of Fas to cytolysis by cells
was examined using three approaches. Initially, two Faslow
variants of Jurkat cells, H7 and B4, were independently derived by radiation mutagenesis followed by selection with
lytic anti-Fas antibody, M2. H7 expresses 3% of the levels
of Fas found on wild-type Jurkat cells, whereas B4 displays 1%
(Fig. 5 B). Fig. 5 A demonstrates that the efficiency of cytolysis of both Faslow variants was diminished approximately
two- to threefold compared to that observed with wildtype Jurkat cells. However, lysis of the Jurkat Faslow variants
was not completely eliminated, suggesting that part of the
cytolytic activity of the
clones was independent of Fas.
This was supported by anti-Fas antibody blocking studies.
Inhibition of Jurkat cell cytolysis by the clones was
also achieved using a nonlytic anti-Fas antibody, M38 (24).
Fig. 5 D shows that the blocking of cytolysis with M38
was partial, achieving 30-50% inhibition at the highest
concentration of antibody (10 µg/ml), whereas control
mouse Ig did not block cytolysis. In vitro cytolysis consists
of a calcium-independent component mediated by Fas and
a calcium-dependent component delivered by perforin
(18). Blocking perforin action by chelation of calcium with EGTA also resulted in partial inhibition of Jurkat cytolysis, which could then be blocked almost completely by
the further addition of anti-Fas antibody (Fig. 5 D). A third
method of disrupting Fas-FasL interaction used the Fas-Fc
fusion protein. Fig. 5 E shows that Fas-Fc, but not IL4RFc, partially blocked cytolysis of Jurkat cells by the
clones, though to a slightly lesser extent than did nonlytic
anti-Fas antibody.
The above findings show that clones derived from
synovial fluid express prolonged and high levels of FasL and
suggest that
cells preferentially lyse Fashigh cells. To directly assess whether uncloned synovial
cells function in
a similar manner, FasL expression was determined on synovial lymphocytes after Borrelia stimulation. As shown in
Fig. 6 A, 7 d after activation, FasL expression was confined
exclusively to a major proportion of the
cells. FasL was
still expressed by at least 50% of the synovial
cells for as
long as 11 d after Borrelia stimulation.
To further assess the contribution of the cells to the
loss of synovial CD4+ cells, the
subset was depleted by
flow cytometric sorting and compared to a nondepleted
sample of the same specimen after five days of stimulation
with B. burgdorferi. During this period, the
cells in the
nondepleted synovial sample expanded from 4.3% to 11%
(Fig. 6 B). This was accompanied by a decreased proportion of CD4+ cells, from 35.6 to 25.3%. In striking contrast, the
-depleted population contained only 4%
cells after 5 d and manifested a predominance of CD4+
cells (40.8%)(Fig. 6 B). In addition, the CD4+ cells in the
4-day cultures contained a subpopulation of CD4low cells
which comprised a greater proportion of the total CD4+
cells in the
-replete than the
-depleted specimen (Fig.
6 B, arrow inset). These CD4low cells represented apoptotic
cells, as determined by the TUNEL assay combined with
surface staining and analyzed by flow cytometry (Fig. 6 C ).
Smaller proportions of apoptotic cells were also observed in
the CD8+ and
+ subsets. Observations similar to these
have been made with
depletion of two additional Lyme
synovial fluid specimens, as well as by noting a depletion of
CD4+ cells when V
1 cloned T cells were added to cultures of PBL that have been stimulated with B. burgdorferi
(data not shown).
To assess whether the appearance of the apoptotic CD4low
subset in the -replete cultures was in part Fas-mediated,
FasL was blocked using the Fas-Fc fusion protein. Synovial
fluid lymphocytes were stimulated with B. burgdorferi in the
presence of either no additives, Fas-Fc, or control mouse
IgG. As shown in Fig. 7, the appearance of apoptotic
CD4low cells occurred beginning about five days after Borrelia stimulation. The proportion of this subset increased dramatically thereafter in all cultures except that containing
Fas-Fc. The findings support the view that the
subset
induces apoptosis of synovial CD4+ cells at least partly
through Fas/FasL interactions.
The collective observations suggest an immunoregulatory circuit whereby synovial V1 T cells bearing high levels of FasL selectively restrict the expansion of infiltrating
inflammatory Fashigh CD4+ lymphocytes through cytolysis
in a Fas-dependent manner. The findings are in agreement
with recent studies showing that FasL mRNA expression
by T cells is highest in the
subset (21). Not only were
levels of surface FasL high on the V
1 clones, they remained elevated for considerably longer periods than similarly activated
T cells. This may serve to explain the
broad spectrum of cytolytic activity that has frequently
been observed for many
cells (2, 16). The results parallel
other recent descriptions of immunosuppression resulting
from constitutive expression of FasL by Sertoli cells in the
testis (22), and by components of the eye (23).
The current findings may also bear on observations that
collagen-induced arthritis in mice (34) and adjuvant arthritis in rats (35) are both more severe following administration of anti- antibody. Collagen-induced arthritis is also
more aggressive in mice bearing a genetic deletion of the
locus (Lefrancois, L., personal communication). Similar results have been observed in a model of orchitis in which
depletion accelerated the inflammatory response (36).
T
cells have also been observed to modulate the functional
profile of CD4+ cells. In certain instances this has manifested as selectively inhibiting TH2-dependent cytokine responses, such as IgE production in an allergy model (37)
and Coxsackievirus-induced myocarditis (38). The resulting TH1 bias may be due solely to the production of the
TH1-type cytokine, IFN
, by
cells (37), but may also reflect a greater sensitivity of TH2 cells to Fas-mediated apoptosis. In this regard, it is noteworthy that B. burgdorferi-
reactive CD4+ T cells from Lyme arthritis patients express
a TH1 cytokine phenotype (39). Studies are in progress to
determine whether a TH1 enrichment results in the residual
CD4+ synovial T cells following stimulation with B. burgdorferi.
Lyme arthritis synovial T cells also represent a rare instance where
T cell clones obtained from a human infectious disease manifest a proliferative response in the presence of the causative agent. This does not establish that
Lyme arthritis synovial
cells are responding directly to a
Borrelial component. It is entirely possible that B. burgdorferi induces the appearance of surface molecules to which
V
1 cells respond secondarily. Cutaneous lesions in leprosy
also contain
T cells that react to the causative agent,
Mycobacterium leprae (9). The repertoire of
cells that react
to mycobacterial products is restricted in both humans and
mice (11, 40), and in some instances involves recognition of nonpeptide components such as prenyl pyrophosphates
(15, 41). Conceivably,
cells in Lyme arthritis may also
recognize nonprotein components of B. burgdorferi. On balance, the current findings are consistent with the concept
that
cells participate in the defense against infectious
agents while modulating the immune response through
Fas-mediated apoptosis.
Address correspondence to Dr. Ralph C. Budd, Division of Immunology, The University of Vermont College of Medicine, Given Medical Building Room C-303, Burlington, VT 05405-0068.
Received for publication 22 May 1996
This work was supported by National Institutes of Health grant AR43520 and the Arthritis Foundation.We thank Colette Charland (The University of Vermont College of Medicine, Burlington, VT) for assistance with flow cytometry and Roberta Christie (The University of Vermont College of Medicine, Burlington, VT) for preparation of the manuscript.
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