Introduction
RA is an autoimmune disease that is characterized by persistent local and systemic inflammation [
1,
2,
3]. Aggressive forms of RA are thought to be driven by T-lymphocyte activity, leading to the rapid erosion of bone and cartilage. Activated Tm are found in the perivascular regions of the inflamed synovium, and T cells accumulate in the synovial fluid [
4,
5,
6]. It appears that memory T cells retain the potential to traffic through the synovium and lymphatics and recirculate in the blood, as evidenced by the increased numbers of activated memory T cells found in the blood of some RA patients [
6,
7,
8]. It is not known, however, whether Tm migrate into the synovium and undergo activation and further differentiation, or whether activation occurs in regional lymph nodes before migration into the inflammatory site.
In vitro studies [
9,
10] have demonstrated that synovial CD4
+ T cells proliferate poorly and produce decreased levels of IL-2 in response to mitogen or antigen, as compared with peripheral blood T cells from healthy individuals or RA patients. Although such findings suggest that synovial T cells might be partly anergic, more recent studies have indicated that the T cells found in the RA synovium were postactivated cells. Synovial T cells were activated as assessed by expression of CD40 ligand/CD154, and were also efficient helpers for B-cell immunoglobulin production [
9,
10,
11,
12,
13]. Previous studies in animal models [
14,
15] suggested that a balance exists between immunoregulatory IFN-γ-producing Th1 cells and IL-4-producing Th2 cells. Dominance of either subset can result in a chronic disease state. Moreover, induction of Th2 cells in a Th1-mediated disease model of collagen-induced arthritis led to amelioration of autoimmune disease [
16]. Thus, it is possible that, in RA, there is biased differentiation of IFN-γ-secreting proinflammatory Th1 CD4
+ T cells, and insufficient differentiation of immunoregulatory IL-4-producing Th2 cells.
Human memory T cells acquire the ability to secrete IL-4 late during
in vivo differentiation. Our previous studies and work by others have demonstrated that all of the
in vivo differentiated IL-4-producing T cells reside within the mature memory (CD45RO
+, CD27
–) subset of CD4
+ T cells [
17,
18,
19]. Thus, early memory (CD45RO
+, CD27
+) CD4
+ T cells cannot secrete IL-4 after a brief
in vitro stimulation. Previous studies [
17,
18] suggested that early memory T cells represent an uncommitted precursor population, from which both IL-4-producing and IFN-γ-producing effector cells can be generated. The RA synovium appears to contain an increased number of phenotypically mature memory T cells as compared with the blood. However, the majority of memory T cells found in the synovium bear the phenotype of early Tm [
7].
Therefore, the present study was undertaken to examine the cytokine effector status of synovial memory T cells and the functional status of immature memory cell precursors of cytokine-producing effector cells. For these experiments, Tm were isolated from the blood of normal subjects, and from RAPB, RASF and RAST. Intracellular cytokine expression was assessed by flow cytometry immediately after isolation and a brief in vitro stimulation, or after in vitro priming designed to generate IL-4-producing effector T cells.
The results suggest that a deficiency in the generation of adequate numbers of regulatory IL-4-producing effector cells in the synovium might be a contributing factor to the perpetuation of chronic inflammation.
Discussion
RAST and RASF were deficient in IL-4-secreting cells as compared with RAPB after a brief in vitro stimulation. Thus, mature IL-4-secreting effector cells were found to be decreased in the RA synovium but not in the blood.
There are at least three major mechanisms by which IL-4 production might be inhibited in the RA synovium. One mechanism could be through the selective recruitment of IFN-γ-producing effector cells. For example, it has been suggested that the T cells found in the RA synovium have been selectively recruited on the basis of expression of chemokine receptors [
20]. Thus, T cells found in the rheumatoid synovium express CCR5, whereas T cells found at sites of parasitic infection express CCR4 and CCR3 [
20,
21]. However, the role of chemokine receptor expression in the recruitment of T cells to these sites is unknown. This possibility cannot be the full explanation of the current observations in light of the finding that synovial tissue T cells had the capacity to differentiate into IL-4-secreting effector cells in
in vitro cultures. Thus, some synovial cells remained responsive to IL-4- and anti-CD28-mediated signaling in the priming cultures. A second mechanism for suppression of IL-4 production in the rheumatoid synovium could be through regulatory signals received in the local microenvironment. This remains a likely possibility because studies in murine models have shown that only the most highly differentiated T cells obtain a polarized cytokine secretion profile that cannot be altered by external stimuli [
22]. A third possibility is that precursor cells of IL-4 producers rapidly migrate out of the synovium, perhaps being unresponsive to retention signals.
Although the exact mechanism that is operative in the RA synovium remains to be elucidated, our ongoing studies are focused on determining the phenotype and response defect of these highly polarized Th cells.
Tm isolated from rheumatoid blood or synovium contained increased numbers of cells with the capacity to produce IFN-γ after a brief
in vitro stimulation, compared with the same subset isolated from the periphery of normal donors. A strong mitogenic stimulation was employed in order to obtain the maximum cytokine-secreting potential of the effector population. We have previously demonstrated that staining for intracellular cytokines correlated with cytokine secretion [
17]. However, intracellular cytokine analysis proved to be a more sensitive method for detecting cytokines, such as IL-2 and IL-4, and also made it possible to determine the percentage of cells producing each. It should be noted that longer incubations did not result in a substantial increase in the number of cytokine-secreting cells detected in this assay. These studies corroborate previous studies that demonstrated that synovial T cells are enriched in IFN-γ-producing cells on initial isolation [
11]. The results suggest that these memory cells appear to have undergone biased differentiation into Th1 cells, perhaps as a result of signals received in the synovial microenvironment. The small increase in the number of IFN-γ-producing cells found in rheumatoid blood may well reflect the recirculation of memory T cells previously activated in the blood [
7,
23].
Tm were used for these studies because it has been shown [
7,
9] that the majority of CD4
+ cells in the RA synovium are of the memory subset. Therefore, a potential concern was that the
in vitro priming protocol might select for cells that are already committed to IFN-γ or IL-4 production, rather than generate new effector cells. In humans, however, highly polarized effector T cells arise late in the differentiation pathway [
17,
18,
19]. Whereas early memory T cells, identified by the CD45RO
+, CD27
+ phenotype, have the potential to secrete IL-2 and IFN-γ, these cells cannot produce IL-4 and few produce IFN-γ in the absence of IL-2 [
17,
19]. Only mature memory cells, identified by the presence of CD45RO and the loss of CD27 cell-surface expression, have the capacity to secrete IFN-γ or IL-4 alone. We have previously shown that the
in vitro priming protocol used in the present study induces the differentiation of early uncommitted CD4
+, CD27
+ memory T cells into IL-4-producing effector cells [
17]. Thus, it was hypothesized that the uncommitted early CD4
+, CD27
+ memory cells that were isolated from the RA synovium or blood maintained the capacity to differentiate into IL-4-producing effector cells. The majority of T cells that are found in the RA synovium belong to the early memory subset [
7]. Therefore, the defective generation of IL-4 producers from RA synovial fluid under conditions that induced IL-4-producing effector cells from blood memory T cells suggested that the rheumatoid microenviroment played an important role in selecting or modifying the precursor effector memory population.
CD28 is expressed by most human T cells and is thought to be critical for T-cell differentiation. CD28 is also important during T-cell receptor-mediated activation, IL-2 production, and the prevention of anergy [
24]. Recent studies [
25,
26,
27] have indicated that there were increased numbers of CD4
+, CD28
– T cells in the periphery and synovium of RA patients. Therefore, the lack of expression of CD28 on RA CD4
+ T cells represented a potential explanation for the abnormal memory T-cell differentiation observed in RA blood and tissue and the deficiency in IL-4-producing effector cells from the synovial fluid after
in vitro priming. However, CD4
+, CD28
– cells are a minor subset of T cells, representing less than 3% of peripheral CD4
+ T cells from the most chronically active RA patients versus less than 1% from healthy control individuals [
26], and are therefore unlikely to account for the broad defect in memory T-cell differentiation found here. We have observed that CD4
+, CD28
– cells in the periphery represent a unique subset that is restricted to the CD4
+, CD27
– population (Davis L, unpublished observation). As noted above, although there were increased numbers of well-differentiated CD4
+, CD27
– cells in the RA synovium, these cells represented the minority of Tm [
7]. Therefore, the inability to modulate effector cell responses in RASF samples could not be explained by the absence of CD28. Moreover, RASF T cells were capable of cell growth in response to anti-CD28 mAbs and cytokines. This finding is in agreement with previous studies [
28] demonstrating that the CD28 signaling pathway was intact in RA synovial CD4
+ T cells.
The present studies also demonstrated that synovial fluid Tm lacked the capacity to generate IL-4-secreting effector cells in numbers similar to those found in the periphery. Our previous studies [
17] demonstrated that IL-4 was required during the
in vitro priming cultures to generate IL-4-producing effector cells from non-IL-4-producing early memory precursor cells. Therefore, one concern in the current studies was that RA T cells might require exogenous IL-4 to generate IL-4-producing effector cells.
An additional concern was that IFN-γ-producing effector cells might inhibit potential IL-4 precursors from differentiating or expanding in these cultures, although previous studies [
29,
30,
31] demonstrated that IFN-γ indirectly affected the generation of IL-4-secreting cells through the activities of antigen-presenting cells. It should be noted that antigen-presenting cells were depleted from the memory T-cell population before priming in the present studies, and therefore this possibility was only a minor concern.
Both the addition of IL-4 to the priming cultures and the presence of anti-IFN-γ antibody ensured that potential IL-4-secreting precursor populations had optimal conditions for differentiation. Blocking IFN-γ activity during
in vitro priming with a neutralizing anti-IFN-γ antibody had little effect on the number of IFN-γ-secreting effector cells detected on subsequent restimulation. Moreover, the availability of IFN-γ had little impact on the generation of either subset. Thus, the inability to generate IL-4-producing effector cells from RASF precursor cells was not simply explained by the presence of IFN-γ-producing effector cells or the lack of a subset of fully differentiated IL-4-producing T cells in the initial memory cell population. Recent studies [
32] have suggested that there is an intrinsic defect in the development of IL-4-producing effector cells in RA patients. Therefore, it is possible that the apparent disordered differentiation of effector cells becomes more marked at inflammatory sites such as in the rheumatoid synovium [
23,
32].
It is interesting to note that both IL-2 production and IL-4 production appear to be downregulated in RASF T cells. Previous studies [
9] have shown that mature memory (CD45RO
+, CD27
–) CD4
+ T cells isolated from the blood have the same capacity as early memory (CD45RO
+, CD27
+) CD4
+ T cells to produce IL-2. In the RA synovium, IL-2 appears to be downregulated [
9]. It should be noted that IL-2 production was not decreased in T cells obtained from osteoarthritic joints [
9]. In addition, Tm obtained from other tissues, such as inflamed tonsil, expressed levels of IL-2 that were similar to those in blood (Davis L, unpublished observation). Therefore, migration into tissue
per se does not induce downregulation of IL-2 production. Recent studies [
33] have attributed the lack of IL-2 production by synovial CD4
+ T cells to a defect in the T-cell receptor-mediated signal transduction cascade. However, in the present studies we made use of stimuli that bypassed the early steps in the T-cell receptor-mediated signaling cascade and found that there was defective IL-2 production in cells stimulated immediately on isolation from the RA synovium, whereas these same cells were completely competent for IFN-γ production. Thus, it is interesting to speculate that IL-2 and IL-4 are downregulated in the rheumatoid synovium by some active repressor mechanism induced by the synovial microenvironment. Although recent studies have indicated that redox balance alterations were critical in determining whether cells can produce IL-2 [
33,
34], it is not known whether this influences IL-4 production, or has it been determined whether readjustment of redox balance in RA synovial T cells could correct the deficiency in IL-4.
Few studies have used similar techniques to assess cytokine production in blood, synovial tissue, and synovial fluid obtained from RA patients. Immunohistology is routinely carried out on synovial tissue sections, whereas enzyme-linked immunosorbent assays are employed to determine cytokine profiles of serum and synovial fluid [
9,
11,
12,
13,
31]. Therefore, it has remained difficult to determine the potential impact of these compartments on T-cell function in rheumatoid inflammation. In the current studies, RA T cells from all three compartments produced increased amounts of IFN-γ on immediate stimulation. However, the cytokine profiles diverged when assessed after
in vitro priming. Whereas, synovial tissue Tm were deficient in IL-4 producers compared with matching blood, these cells retained the ability to generate IL-4-producing effector cells. In this regard, recent studies [
31] have suggested that synovial tissue cells were selectively responsive to IL-12 produced in the synovium, because IL-12 induced increased production of IFN-γ, but not IL-2 and IL-4. Those studies support the hypothesis that the synovial microenvironment may play a role in skewing T cells toward a Th1 phenotype. The finding that IL-4-producing effector cells could be generated from synovial tissue T cells once removed from that environment suggests that all of the cells were not yet terminally polarized to Th1-like effector cells. Importantly, synovial fluid T cells appeared to have been more affected by the synovial microenvironment than synovial tissue T cells because, regardless of the
in vitro priming conditions, these cells yielded increased percentages of IFN-γ producers and were deficient in IL-4 producers.
The present data suggest that synovial fluid T cells are those that have passed through the synovium, whereas synovial tissue T cells are a mixed population of recently migrated cells and those that have been retained in the synovium. Whether the inability of synovial fluid memory T cells to generate IL-4 producers is the result of activation, differentiation, or prolonged exposure to the synovial microenvironment, the data clearly indicate that a majority of synovial fluid memory T cells appear to be polarized IFN-γ effector cells.
In summary, the present studies show that RA blood and synovial T cells contain increased numbers of polarized IFN-γ effector cells. RA synovial T cells also demonstrated a deficiency in the ability to generate IL-4-producing effector cells. The diminished ability to generate IL-4 effector cells from synovial T cells in vitro suggests that the rheumatoid microenvironment alters T-cell effector function and thereby perpetuates the chronic inflammatory disease state.