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*
Department of Internal Medicine III and Institute for Clinical Immunology, University of Erlangen-Nuremberg, and
Rheumatologische Gemeinschaftspraxis, Erlangen, Germany; and
Harold C. Simmons Arthritis Research Center, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75235
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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but not IL-2 were evident after priming
with anti-CD3 and anti-CD28. The data suggest that
CD4+ memory T cells from patients with early untreated RA
manifest an intrinsic abnormality in their ability to differentiate
into specific cytokine-producing effector cells that might contribute
to the characteristic Th1-dominated chronic (auto)immune inflammation
in RA. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-2, and TNF-ß, promote macrophage activation, induce
delayed-type hypersensitivity, and are involved in cell-mediated
immunity. Th2 cells have been associated with down-modulation of
macrophage effector functions, they produce the anti-inflammatory
cytokine IL-4, IL-5, and IL-13, and mediate allergic immune responses
(5, 6, 7, 8). A third subset, designated T regulatory cells, produces mainly
IL-10 and might play a role in maintaining peripheral tolerance
(9).
Imbalances in the ratio of activated Th1 vs Th2 cells have been
associated with the development of a variety of pathologic inflammatory
responses (10, 11, 12). For example, Th1-mediated immunity is associated
with the pathogenesis of several organ-specific autoimmune diseases in
animals (13, 14, 15) and has recently been implicated in mediating human
autoimmune diseases such as RA (16, 17). In this regard, elevated
levels of IL-2 and IFN- mRNA have been detected in the peripheral
circulation of patients with RA (17). Moreover, T cells from human
rheumatoid synovial tissue produce mainly IFN- and IL-2 (11, 16, 18, 19, 20, 21). By contrast, T cells producing cytokines characteristic of
activated Th2 cells, in particular IL-4, were rarely found in the
peripheral circulation or in inflamed synovial tissue from patients
with RA (17, 21, 22).
Given the significance that differentiated effector cells play in the outcome of an immune response, it is important to understand the mechanisms that regulate the development of T effector cells. Cytokines appear to be the major factor in determining Th cell polarization from naive T cells. In particular, Th2 cells arise after priming in the presence of IL-4 (23, 24, 25, 26, 27), whereas priming in the absence of IL-4 initiates Th1 cell development (25, 26, 28) that is greatly enhanced by IL-12 (29, 30). Other factors with a regulatory capacity for Th cell differentiation from naive T cells have been identified, such as the intensity of TCR ligation during priming (31, 32) or the nature and intensity of costimulatory signals (33, 34, 35, 36). Recently, it has been shown that in humans, resting memory T cells can also be primed to differentiate into IL-4-producing Th2 cells. Generation of Th2 memory effector cells from early CD27+ memory T cells was dependent on stimulation by CD28, was enhanced by exogenous IL-4, and was inhibited by TCR ligation (37).
As chronic autoimmune responses are perpetuated by repeatedly activated memory T cells, disordered regulation of memory T cell differentiation might promote the pathogenesis of autoimmune diseases. In this regard, large numbers of mature memory T cells have been detected in the peripheral circulation and the synovial tissue of RA patients (38). Although phenotypically mature (CD4+CD27-CD45RBdim) memory T cells from patients with RA fail to produce IL-4, in contrast to those from healthy individuals (39). Thus, mature memory T cells from patients with RA appear to differ functionally from mature memory T cells in healthy individuals. However, the mechanisms responsible for these functional differences have yet to be elucidated.
To test the hypothesis that altered memory effector cell
differentiation contributes to the typical Th1-dominated immune
response in RA, we investigated memory T cell differentiation in RA
patients in vitro. A culture system was employed to assess Th1 and Th2
cell differentiation in vitro using highly purified populations of
CD4+ memory T cells that has previously been used
to identify the signals controlling this process effectively (37). As
it has been demonstrated that chronic activation might cause fixation
of the Th phenotype (40), RA patients were chosen with early disease (6
wk to 12 mo). To eliminate the influence of drugs on the generation of
polarized effector cells, patients were excluded who had previously
been treated with disease-modifying anti-rheumatic drugs (DMARDs),
including methotrexate, or corticosteroids. Whereas Th2 cell
differentiation could be induced in all healthy individuals, RA
patients were identified with an impaired ability to generate Th2
effectors. In some of the patients exogenous IL-4 could overcome the
apparent T cell differentiation defect. However, in eight of 24
patients, no Th2 effectors could be induced. On the contrary, all RA
patients manifested increased in vitro differentiation into specialized
IFN--positive Th1 cells. The data imply an abnormal memory Th
effector cell differentiation in RA that might contribute to the
characteristic Th1-dominated rheumatoid inflammation and, thus, to the
pathogenesis of RA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following mAbs were used for purification of cells, for cell
culture and staining: anti-CD3 (OKT3), anti-CD8 (OKT8),
anti-HLA-DR (L243), and P1.17 (control IgG2a mAb, American Type
Culture Collection, Manassas, VA); anti-CD16, anti-CD19,
FITC-conjugated anti-CD25, FITC-labeled anti-CD30, and
FITC-labeled anti-HLA-DR (Dako Diagnostika, Hamburg, Germany);
anti-CD45RA (111-1C5, a gift from Dr. Ramon Vilella, Barcelona,
Spain); FITC-conjugated anti-CD3, PE-labeled anti-CD4, and
PE-labeled anti-CD45RO (UCHL-1, Sigma-Aldrich, Deisenhofen,
Germany); and anti-CD28 (28.2), FITC-labeled anti-CD69,
PE-labeled anti-IL-4 (MP4–25D2), PE-labeled anti-IL-2
(MQ1–17H12), and FITC-labeled anti-IFN- (4S.B3, PharMingen, San
Diego, CA). Abs used for immunoprecipitation and immunoblotting were
anti-STAT6 (S-20 and M-20) and anti-phosphotyrosine mAb (PY20;
all from Santa Cruz Biotechnology, Santa Cruz, CA).
Study population
The study population consisted of 24 patients with an
established diagnosis of RA as defined by the 1987 revised criteria of
the American College of Rheumatology for the classification of RA (41).
All patients were required to have symptoms of the disease for <12 mo
and had not previously been treated with corticosteroids, DMARDs, or
methotrexate. The mean age of the study population was 52.9 yr (range,
30–71 yr) with a mean disease duration of 5.9 ± 3.8 mo (range, 6
wk to 12 mo). Sixteen of the twenty-four patients had elevated
erythrocyte sedimentation rates (ESR; 
20 mm/h; Westergren), 14
patients had increased levels of C-reactive protein (CRP; 5 mg/l),
and 17 of the patients were seropositive for IgM rheumatoid factor. The
clinical and demographic data of the patients are summarized in
Table I
. Ten healthy age- and sex-matched volunteers
were used as controls. The study protocol was approved by the review
board of the University of Erlangen-Nuremberg, and written informed
consent was obtained from all individuals before entering the
study.
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PBMC were obtained from heparinized venous blood by
centrifugation (30 min, 400 x g) over a Ficoll-Hypaque
gradient (Sigma-Aldrich). Cells were washed with saline and
subsequently incubated with neuraminidase-treated sheep RBC (42). The
rosette-positive cells were further purified by negative selection
panning as previously described (37). In brief, cells were incubated
with saturating amounts of mAb against CD8, CD19, CD16, HLA-DR, and
CD45RA for 15 min on ice in RPMI 1640 medium (Life Technologies,
Eggenstein, Germany) containing 2% pooled normal human serum (NHS),
washed and allowed to bind to plastic petri dishes (Greiner,
Frickenhausen, Germany) coated with goat anti-mouse Igs (Cappel,
West Chester, PA) for 30 min at room temperature. Recovered cells were
washed with PBS and resuspended in RPMI containing 10% NHS. The
homogeneity and purity of the recovered cells were assessed by flow
cytometry. Typically, 95% of the cells were positive for CD3 and
CD4, 90% stained brightly with an mAb to CD45RO, and >98% of the
cells were viable after the purification procedure. The cells were
negative for the activation markers CD25, CD30, CD69, and HLA-DR.
Generation of memory effector T cells
All cell cultures were conducted in RPMI 1640 medium supplemented with penicillin G (100 IU/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), and 10% NHS at 37°C in a humidified atmosphere containing 5% CO2. For the generation of memory effector T cells, a previously described multistep ex vivo cell culture system was used (37) that permitted the differentiation of effector T cells by short term mitogenic stimulation. In brief, mature effector cells were generated by priming purified CD4+ resting memory T cells in flat-bottom cell culture plates (Costar, Cambridge, MA) at a concentration of 0.5 x 106 cells/ml with 1 µg/ml anti-CD28 mAb and 10 U/ml recombinant human IL-2 in the presence or the absence of recombinant human IL-4 (31.25 ng/ml). When anti-CD3 stimulation was used during priming, the plates were coated with OKT3 (1 µg/ml in 50 mM Tris-HCl, pH 9.5) for 24 h at room temperature. Unbound mAb was removed by washing the plates with PBS immediately before the cells were added. After 5 days of priming, the cells were harvested, counted, washed, and rested for 60 h at 37°C at a concentration of 1 x 106 cells/ml in medium supplemented with 10 U/ml rIL-2. The phenotype of the effector populations as defined by the ability of the cells to produce cytokines was determined by intracellular flow cytometry after maximal mitogenic stimulation and compared with that of the starting population. By assessing Th cell differentiation in serial controls, the assay was found to be highly reproducible in individual healthy donors and stable over time.
Flow cytometry
Cells (1 x 105/sample) were stained with saturating amounts of directly labeled mAb and analyzed by FACS (EPICS, Beckman Coulter, Fullerton, CA). To assess the inherent capacity of the cells for cytokine production and to bypass proximal signaling events after TCR/CD3 activation that have been suggested to be impaired in synovial T cells from RA patients (43), cells were stimulated with ionomycin (1 mM; Calbiochem, San Diego, CA) and PMA (20 ng/ml; Sigma-Aldrich) for 5 h in the presence of 2 µM monensin (Sigma-Aldrich) to prevent secretion of cytokines. Cells were harvested, washed, fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS for 15 min at 37°C, and stored at -70°C in 10% DMSO/PBS until analysis. Cells were permeabilized with 0.1% (w/v) saponin (Sigma-Aldrich) in 2% FCS/PBS. Nonspecific binding sites were blocked with 4% mouse and rat serum. Cytoplasmic cytokines were detected by staining with directly labeled mAb against human cytokines for 25 min on ice. Cells were washed with 0.1% saponin/2% FCS/PBS, resuspended in 2% FCS/PBS, and analyzed by flow cytometry. Unstimulated cells that were identically stained and stimulated cells that were permeabilized or not but stained with irrelevant mAb were used as controls for determining background fluorescence.
Immunoprecipitation and Western blotting
Purified CD4+ memory T cells (2–5 x 107; 100 x 106/ml) were deprived of serum for 4 h at 37°C and were stimulated with rIL-4 (100 ng/ml) for 10 min. The cells were lysed in lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 1% SDS, 10 mM NaF, 5 mM EDTA, 1 mM Na3VO4, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 100 µg/ml PMSF in PBS). The lysates were precleared with normal mouse serum and incubated with primary Ab for 1 h at 4°C. Immune complexes were precipitated with recombinant protein A agarose (Pharmacia, Uppsala, Sweden) for 1 h at 4°C, washed three times in lysis buffer, and eluted with SDS-PAGE loading buffer. Proteins were separated in a 7% polyacrylamide gel and transferred onto nitrocellulose. After blocking of nonspecific binding sites (5% milk powder and 0.05% Tween-20 in PBS), the blots were probed with specific Abs for STAT6 or phosphotyrosine (PY20) and developed with enhanced chemiluminescence (Santa Cruz Biotechnology).
Statistical analysis
Differences in data distribution were analyzed by two-tailed Student’s t tests for comparison of frequencies of cells producing particular cytokines between healthy individuals and RA patients, by paired two-tailed t tests for comparison of Th subsets in effector populations with the starting populations within the same group of patients or healthy controls, and by Fisher’s exact test for comparison of the frequencies of HLA-DRB1*01- or *04-positive individuals within different patient groups. p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the inherent capacity of resting peripheral blood
CD4+ memory T cells from RA patients and healthy
controls to produce cytokines, freshly purified cells were stimulated
in vitro and analyzed by flow cytometry. As it has been suggested that
TCR-mediated signaling in RA synovial T cells might be impaired with
respect to phosphorylation of the TCR 
-chain (43), the cytokine
secretion pattern of cells was analyzed throughout the study after
stimulation with PMA and ionomycin, which bypasses the most proximal
signaling events after TCR/CD3 engagement. No difference in the
cytokine secretion pattern was found between cells from RA patients and
cells isolated from healthy controls (Table II).
Moreover, the frequencies of Th1 cells (IFN--positive,
IL-4-negative) or Th2 cells (IL-4-positive, IFN--negative) in
resting memory T cells were similar in patients and controls (Table II). Consequently, resting CD4+ memory T cells
from patients and healthy individuals expressed similar Th1/Th2 cell
ratios after in vitro stimulation (10.5 ± 5.4 vs 9.1 ± 3.6
in RA patients and controls, respectively). Furthermore, no differences
were detected between RA patients and controls with respect to cells
producing IL-4 and IFN-, IL-2 and IFN-, IFN- but no IL-2, or
IL-2 but no IFN- (data not shown).
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To analyze Th2 memory cell differentiation in RA patients we first
established the optimal conditions for Th2 generation in a group of
healthy individuals who were age- and sex-matched with RA patients.
Purified CD4+ memory T cells from the healthy
individuals were primed under various conditions that have previously
been demonstrated to regulate Th2 effector generation from resting
memory T cells, namely stimulation with CD28 and IL-4 in the absence or
the presence of TCR ligation (37). The cytokine secretion pattern of
freshly isolated and ex vivo differentiated memory effector cells from
one healthy individual is shown in Fig. 1. Th2 cell frequencies could not be
increased by priming with anti-CD3 and anti-CD28 (3.1 ±
1.2% in primed cells compared with 3.2 ± 1.6% in freshly
isolated cells in the entire group of healthy individuals; Table III) and were only slightly increased when exogenous
IL-4 was added during priming (4.2 ± 1.8%; data not shown). By
contrast, priming with anti-CD28 in the absence of TCR ligation
induced Th2 cell differentiation and significantly increased the Th2
cell population to 5.7 ± 1.3% (p <
0.04, compared with fresh cells). Priming with anti-CD28 in the
presence of exogenous IL-4 further increased the Th2 cell frequencies
to 9.7 ± 2.7% (p < 0.006 vs fresh
cells; Table III). It should be stressed that in all healthy
individuals, increased numbers of Th2 effector cells could be generated
from resting peripheral blood CD4+ memory T cells
by priming with anti-CD28 in the absence of TCR engagement, with an
increase in Th2 cells of at least 50% routinely noted. Furthermore,
exogenous IL-4 enhanced the efficiency of anti-CD28-induced Th2
cell differentiation in all healthy individuals and resulted in an
additional increase in the number of Th2 cells of >50%.
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Analysis of Th2 effector cell differentiation in RA patients
revealed striking differences compared with that in healthy
individuals. Statistical examination of the Th2 cell differentiation
data from RA patients in response to priming under Th2-inducing
conditions suggested that the data were not normally distributed and
therefore were unlikely to be derived from a single entity but, rather,
represented different cohorts. Patients could be grouped according to
the ability of their memory T cells to differentiate into Th2 effectors
under optimal Th2-inducing conditions. In nine RA patients Th2
effectors were generated by priming with anti-CD28 in the absence
of TCR stimulation in a way comparable to that in healthy controls,
e.g., an increase of Th2 cell frequencies of at least 50% was
initiated by stimulation with anti-CD28 in these patients (Fig. 2A). When IL-4 was added
during priming, a further increase in the Th2 cell frequencies within
the primed population of 50% was achieved (p
< 0.02; Table III
). In contrast to healthy individuals, however, in
this group of RA patients priming with anti-CD28 in the presence of
immobilized anti-CD3 also significantly increased the
IL-4-producing cells (p < 0.05; Table III).
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). There was a significant difference in
the frequencies of Th2 cells after priming with anti-CD28 between
this group of patients and healthy individuals (3.2 ± 1.2 vs
5.7 ± 1.3; p < 0.001). Priming in the presence
of exogenous IL-4 induced Th2 cell differentiation in 7 of these 15
patients (Fig. 2B). In these seven patients a significant
increase in Th2 cells was achieved after culture with anti-CD28 and
rIL-4 compared with that in the starting population
(p < 0.005;Table III). In contrast, however,
in eight RA patients, exogenous IL-4 was without effect with respect to
the generation of Th2 effectors (Fig. 2C). In this group of
patients, the Th2 cell frequencies were 3.0 ± 1.1% in freshly
isolated cells, 3.2 ± 1.5% in CD28-primed effectors, and
3.6 ± 1.4% in effectors that were primed with anti-CD28 and
IL-4 (Fig. 2C). In contrast to the nine patients described
above but similar to healthy individuals, Th2 cell differentiation was
not observed after engagement of the TCR in these 15 patients
(Table III). Altered Th1 cell differentiation in RA patients
TCR stimulation of T cells has been shown to be critical for the
induction of Th1 cell differentiation (31, 37). Thus, Th1 cell
differentiation of RA memory T cells was analyzed after priming with
anti-CD3 and anti-CD28 and was compared with that in healthy
controls. A significant increase in the numbers of IFN--producing
cells occurred after priming with anti-CD3 and anti-CD28 in
controls and patients, with no apparent difference between the groups
(data not shown). To provide a more detailed analysis of the cytokine
secretion profile of individual Th1 cells, freshly isolated
CD4+ memory T cells and effector cells were
double stained with mAb to IL-2 and IFN- and analyzed for the
distribution of IL-2 and IFN- double-producing Th1 cells and more
specialized IFN- single producers. In freshly isolated memory T
cells, no differences were detected between healthy controls and RA
patients or the patient groups in which Th2 cells could or could not be
induced (data not shown). However, after priming, a markedly reduced
number of cells that produced IL-2 only and a significant shift in the
balance of IFN-/IL-2 double producers to IFN- single-producing
Th1 cells were noted in RA patients compared with controls (Fig. 3). Whereas in controls, 18.6 ±
4.6% of the primed effector cells were IL-2 single producers, only
14.2 ± 7.2% of the cells from RA patients exclusively contained
IL-2. Moreover, significantly fewer effector cells were IFN-/IL-2
double producers in RA patients (23.6 ± 8.2%) compared with
those in healthy individuals (33.1 ± 7.0%; p <
0.003). Concomitantly, specialized IFN- single producers were found
more frequently among cells from RA patients than in healthy
individuals (29.0 ± 11.5% vs 22.3 ± 7.5%;
p < 0.05). When patients with impaired Th2 cell
differentiation were analyzed, an even more pronounced shift toward
specialized Th1 cells became obvious (Fig. 4); 18.1 ± 4.7% of the effector
cells from this group of patients were positive for both IFN- and
IL-2, and 32.1 ± 12.4% were specialized IFN- single-producing
Th1 cells.
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Exogenous IL-4 had no effect with respect to Th2 cell
differentiation in eight RA patients. To examine whether an altered
expression or activation of STAT6, which is the critical signaling
molecule downstream of the IL-4R (44, 45, 46), might underlie the impaired
cellular response to IL-4 in these patients, CD4+
T cells from them and from controls were purified and stimulated with
IL-4. STAT6 was immunoprecipitated and probed with an Ab to STAT6 or a
mAb to phosphotyrosine (PY20). No differences in the expression of
STAT6 and its phosphorylation after activation were found between RA
patients and healthy controls (Fig. 5).
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The ex vivo T cell differentiation data were compared with the clinical parameters, such as disease duration, ESR, CRP, and rheumatoid factor, and the demographic data of the patients, such as age, sex, and genetic background. No significant correlation to any of these parameters was found. It should be noted, however, that the frequency of patients with an HLA-DR allele that is associated with aggressive forms of RA (HLA-DRB1*01 and HLA-DRB1*04) was 0.67 (10 of 15) in the patients with impaired Th2 differentiation compared with 0.22 (two of nine) in the group of patients in whom Th2 effectors could be induced (p = 0.09, by Fisher’s exact test).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Th2 cell differentiation could be induced by anti-CD28 in the absence of TCR ligation in all of 10 healthy individuals from the current study in a rather homogeneous fashion. A similar pattern was observed in a group of healthy volunteers that were not age matched with the study population (data not shown), indicating that the induction of Th2 cell differentiation is a common result of priming of memory Th cells from healthy individuals with anti-CD28. The intracellular signals induced by the interactions between CD28 and its natural ligands CD80 and CD86 have been shown to not only complement TCR-mediated signals to allow full activation of T cells but also to play a central role in Th2 cell differentiation. Whereas immune responses driven by Th1 cells could be induced in CD28-deficient mice (49), strong (co)stimulatory signals through CD28 were required to induce Th2 differentiation (34, 35, 50). The proximal and distal events of CD28 signaling are just beginning to be understood. Multiple signaling cascades that may be independent of or dependent on protein tyrosine kinase activation have been demonstrated to be activated by CD28, including activation of phosphoinositide 3-kinase, extracellular signal-regulating kinases 1 and 2, stress-activated protein kinase/c-jun amino terminal protein kinase, and mitogen activation protein kinase 38 (p38/HOG1) (reviewed in Refs. 51 and 52), and it has been shown that CD28-activated signaling cascades regulate several transcription factors involved in cytokine transcription (51, 52). Recently, some evidence has been provided demonstrating that signaling through CD28 in the absence of TCR ligation is sufficient to activate resting T cells, as indicated by cell proliferation, increase of intracellular calcium, and induction of cytokine gene transcription (37, 53). The precise mechanisms, however, that contribute to anti-CD28-induced Th2 cell differentiation remain to be elucidated.
Analysis of freshly isolated resting memory T cells with flow cytometry after staining with mAbs to CD28 revealed no differences in the density of CD28 on the surface of cells from healthy individuals and patients or between individual patient groups (data not shown), confirming previous reports in which no abnormalities in surface CD28 were detected in RA patients (54). Moreover, CD28 signaling in CD4+ memory T cells in RA has been suggested to be functionally intact, as determined by proliferative responses to anti-CD3/anti-CD28-induced activation (55, 56). Consistent with these data, no differences between RA patients and healthy controls in the proliferation of memory cells and the viability of recovered effector cells were observed in the current study after stimulation with anti-CD28 alone or with anti-CD3 and anti-CD28 in combination (data not shown). However, despite similar proliferative responses and comparable densities of surface CD28, memory T cells from the majority of RA patients failed to differentiate into IL-4-producing Th2 effector cells after CD28 stimulation. Thus, CD28-mediated signals in those patients were apparently intact with respect to proliferation of memory T cells, but were impaired with regard to the induction of Th2 differentiation.
An indication of the nature of the defect in CD28 signaling, however,
might derive from the observation that in seven of the 15 patients with
impaired Th2 cell differentiation, priming in the presence of exogenous
IL-4 could overcome the failure to induce Th2 cell differentiation. It
has been demonstrated in healthy individuals that Th2 effector
generation from resting memory T cells is IL-4 dependent (37). Thus, it
can be surmised that in those patients, IL-4R-mediated signals
triggered by exogenous IL-4 are required to supplement CD28 signals for
the initiation of Th2 cell differentiation. Strikingly, however, in the
remaining eight of the 15 patients, even large concentrations of
exogenous IL-4 did not yield Th2 effectors. It has been shown that in
RA patients with long standing disease, T cells derived from the
synovial tissue predominantly produced IL-2, IFN-, and IL-10 (11, 16, 18, 19, 20, 21, 22). Moreover, it was found that those cells were also
resistant to modulation of their phenotype after stimulation under
Th2-inducing conditions (57). However, those cells might have been
primed and activated in vivo and therefore might have lost the
flexibility of functional modulation, as has been demonstrated for
repeatedly activated T cell lines (40). In the current study resting
memory T cells from the peripheral blood of patients with early RA were
analyzed. Some of the patients had symptoms of the disease for as
little as 6 wk, which makes it unlikely that the impairment of Th2
differentiation is secondary to long standing inflammation. Thus, the
finding that the cells from these patients were unable to generate Th2
effector cells even in the presence of exogenous IL-4 strongly suggests
that a disordered potential for memory cell differentiation
contributes not only to the perpetuation but, more importantly, to the
initiation of the unresolved Th1-mediated inflammation in some patients
with RA.
The data imply that in some RA patients memory T cells not only failed to initiate Th2 effector cell differentiation after stimulation with anti-CD28, but also expressed a reduced cellular response to IL-4. Several molecules have been identified that are involved in transmission of signals from the IL-4R to the nucleus. In particular, binding of IL-4 to its receptor initiates phosphorylation of JAK3 and JAK1 that, in turn, phosphorylate STAT6, which exists in a latent form in the cytoplasm (46, 58, 59). Whereas JAK1 and JAK3 are activated by several cytokines, STAT6 confers specificity to IL-4R-mediated signals and is the only presently known STAT protein that is activated in response to IL-4 (58, 59). It has been conclusively shown that STAT6 is required for IL-4 responses and for the development of Th2 cells (44, 45, 46). Moreover, developing Th1 cells express a reduced phosphorylation of JAK3 and subsequently of STAT6, and it has been speculated that this might be a mechanism of phenotype determination (60). Therefore, an alteration in the expression and/or activation of STAT6 in resting memory T cells from RA patients with impaired Th2 differentiation might contribute to reduced IL-4 responsiveness. However, no differences were detected in STAT6 expression and phosphorylation after IL-4-induced activation between patients with impaired Th2 differentiation and controls. Thus, the precise molecular mechanisms of reduced functional cellular responsiveness to IL-4 in these RA patients still must be identified.
In contrast, Th2 cell differentiation could be induced by priming with
anti-CD28 in other patients from the current study and could be
further enhanced by the addition of exogenous IL-4. We have previously
shown that the increase in Th2 cell frequencies in CD28-primed
populations is not simply caused by preferential survival of a subset
of T cells but clearly results from expansion of early uncommitted
CD27+ T cells (37). In these patients, therefore,
CD28-mediated signaling appeared to be intact in resting, uncommitted
memory T cells with regard to proliferation and the initiation of
differentiation. Furthermore, cellular responsiveness to IL-4 in these
patients was comparable with that in normal controls. It is interesting
to note that in these patients even higher numbers of Th2 cells could
be induced by priming with anti-CD28 or anti-CD28 and rIL-4
compared with healthy controls, and that, in contrast to controls, Th2
cells could also be generated when the TCR was stimulated during
priming (Table III). These data imply that the cells from these
patients were more sensitive to Th2-inducing priming conditions. It
will be interesting to determine the consequences of increased Th2
generation. It was previously reasoned that RA is a heterogeneous
entity with distinct histologically defined phenotypes and that several
pathologic mechanisms may cause an RA-like syndrome (61, 62). In those
studies variants of RA have been postulated based upon different
cytokine secretion patterns in tissue sections of synovial biopsies
that could be correlated with the clinical progress of the disease in
the patients. Thus, it might be speculated that the RA patients with
increased Th2 differentiation might suffer from a more self-limited
disease(s), related clinically to RA but not pathogenetically.
Follow-up evaluation of these patients should provide information to
test this hypothesis. In this respect it is interesting to note that
only two of those nine patients were positive for an HLA-DR allele that
is associated with aggressive forms of the disease (HLA-DRB1*01 or
HLA-DRB1*04) compared with 10 of the 15 patients with impaired Th2
differentiation.
Although the ability of memory T cells from RA patients to generate Th2 effectors allowed a clear separation of the patients, no correlations with clinical and demographic data were detected. However, it should be emphasized that the patients from the current study were required to have early disease with symptoms for 6 wk to 12 mo and no previous treatment with corticosteroids and DMARDs. Thus, the patients were analyzed upon their first visit to a rheumatologist when disease might not have proceeded far enough to manifest clear differences between individual patients. Nevertheless, it is clear from the data that patients with similar clinical symptoms might have different cellular pathogenetic mechanisms.
The cytokine secretion profiles of resting memory T cells from RA
patients and healthy individuals stimulated immediately after
purification were comparable with respect to the frequencies of cells
producing IL-2, IL-4, or IFN-. As Th2 differentiation has been shown
to be dependent on IL-4 and could not be induced in the absence of
cells that were able to produce IL-4 (37), the lack of memory cells
capable of producing IL-4 would have provided a simple explanation for
the observed differences in Th2 cell generation. However, as the
frequencies of cells producing IL-4 after stimulation with PMA and
ionomycin were indistinguishable between RA patients and healthy
individuals and among the different patient groups, impaired Th2 cell
differentiation could not simply be attributed to a lack of cells
capable of secreting IL-4 but, rather, is more likely to be related to
an impairment of the cells’ capacity to respond appropriately to CD28
and/or IL-4 signaling. The finding that resting memory T cells from
controls and patients were similar with respect to their cytokine
secretion patterns also indicates that in RA patients Th cell
differentiation was apparently normal to some extent before the disease
had become manifest. Presumably, the resting memory T cells in the
patients were generated in response to some antecedent antigenic
exposure. Clinically, the immune responses of RA patients appear to be
normal before the onset of the disease. It might be speculated,
therefore, that Th cell differentiation is not significantly altered in
RA patients before the initiation of the disease, which could indicate
that the resting memory T cell population from patients with early RA
were generated in response to peptides that were presented by
non-RA-associated MHC class II alleles. Alternatively, the data might
indicate that the T cell differentiation defect in RA is acquired
rather than genetic, which might have substantial implications for the
pathogenesis of the disease.
Supportive of recent suggestions of a Th1-dominated immune response in
the pathogenesis of RA (17, 20, 47, 48), an alteration in the
differentiation of resting memory T cells into Th1 effectors was
obvious in all patients. Whereas the frequencies of cells producing
IFN- were similar in patients and controls after priming with
anti-CD3 and anti-CD28, a marked decrease in IL-2-producing
cells and a significant increase in specialized Th1 cells that produced
IFN- but not IL-2 were evident in RA patients. An even more
pronounced differentiation into specialized Th1 cells producing only
IFN- was noted in patients in whom Th2 cells could not be induced.
These data indicate that an alteration of Th1 cell differentiation is a
common finding in RA, but is enhanced in patients in whom a concomitant
Th2 differentiation defect could be detected. In combination, these
functional abnormalities might result in a marked shift of the Th1/Th2
balance in activated effector cells of these individuals.
In summary, we have provided evidence that memory T cells from patients
with early RA express an altered ability to differentiate into Th
effectors. RA patients could be grouped according to the ability of
their memory T cells to differentiate into Th2 effectors. Whereas in
some patients Th2 differentiation could be induced, the majority of
patients were identified to have a decreased ability to generate Th2
effectors in response to CD28-mediated signals, and some of them also
failed to respond to IL-4. These data suggest a reduced functional
responsiveness to Th2-inducing signals, in particular CD28 engagement
and IL-4, in patients with RA. Moreover, increased generation of Th1
cells specialized in secreting IFN-, but not IL-2, was obvious in
all patients. Altered Th1 cell differentiation and impaired generation
of Th2 cells might contribute to the characteristic Th1-dominated
inflammation in RA. These results imply that abnormalities in the
potential of memory T cells to differentiate into polarized effector
cells may occur early in the course of RA and may play a role in the
subsequent evolution of chronic inflammation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hendrik Schulze-Koops, Department of Internal Medicine III and Institute for Clinical Immunology, University of Erlangen-Nuremberg, Glueckstrasse 4a, D-91054 Erlangen, Germany. E-mail address:
3 Abbreviations used in this paper: RA, rheumatoid arthritis; DMARD, disease-modifying anti-rheumatic drug; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; NHS, normal human serum; CD, cluster of differentiation; JAK, Janus kinase; pTyr, phosphotyrosine.
Received for publication February 12, 1999. Accepted for publication April 8, 1999.
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References |
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