Rheumatoid synovial CD4+ T cells exhibit a reduced capacity to differentiate into IL-4-producing T-helper-2 effector cells

Abstract

model was employed to generate polarized T-helper (Th) effectors. Normal and RAPB Tm differentiated into both IFN-γ- and IL-4-producing effectors. RA synovial fluid (RASF) Tm demonstrated defective responsiveness, exhibiting diminished differentiation of IL-4 effectors, whereas RA synovial tissue (RAST) Tm exhibited defective generation of IFN-γ and IL-4 producers.

Introduction

]. 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.

T cells, and insufficient differentiation of immunoregulatory IL-4-producing Th2 cells.

].

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.

Antibodies and reagents

].

Patients

All patients had an established diagnosis of RA, as defined by the 1987 revised criteria of the American College of Rheumatology for the classification of RA. All patients had long-standing, active RA. All samples were obtained after informed consent, as approved by the UT Southwestern Institutional Review Board. The data shown were obtained from 14 RA patients (age range 29–78 years). Matching blood and synovial fluid were obtained from six patients. Peripheral blood alone was obtained from one patient and synovial fluid samples were obtained from three patients. Matching blood and synovial tissue samples were obtained from three patients at the time of surgery. An additional synovial tissue sample was obtained from one patient.

Cell preparation

cells.

Cell culture

].

Flow cytometry

cells for each sample.

Statistical analysis

< 0.05 was considered statistically significant.

T cells

T cells isolated from the synovial fluid demonstrated a significant increase in the percentage of cells that produced IFN-γ alone or in combination with IL-2, whereas a decrease was observed in the percentage of cells that secreted IL-2 alone. Few cells from peripheral blood or synovial fluid produced IL-4.

; RA patient 7). As was observed in the synovial fluid, memory T cells isolated from the synovial tissue contained a decreased percentage of IL-2-producing cells and an increased percentage of IFN-γ-producing cells. Thus, T cells isolated from the rheumatoid synovial tissue and fluid demonstrated a polarized Th1 cytokine profile.

; normal1). As observed for blood from RA patients, the majority of cells produced IL-2 alone, whereas fewer cells produced the combination of IL-2 and IFN-γ. Relatively infrequent cells expressed a highly polarized phenotype by producing either IFN-γ alone or IL-4 alone.

= 11) produced IL-2 alone (75 ± 4%; mean ± SEM). Fewer cells produced the combination of IL-2 and IFN-γ (14 ± 3%). Relatively infrequent cells expressed a highly polarized phenotype by producing either IFN-γ (6 ± 1%) or IL-4 alone (4 ± 1%). Less than 1% of the cells produced the combination of IFN-γ and IL-4.

< 0.05) increase in the percentage of cells that secreted IFN-γ alone compared with normal cells (11 ± 2% versus 6 ± 1%), whereas there was no difference between normal blood and RAPB in the number of cells that secreted IL-4 alone (4 ± 1%) or in the frequency of those rare cells that produced the combination of IL-4 and IFN-γ (1 ± 0%).

< 0.05) T cells, whereas no significant difference was observed in the number of cells that produced the combination of IL-4 and IFN-γ (2 ± 0%).

< 0.005). There was no significant difference in the number of synovial tissue cells that produced IL-4 alone (2 ± 1%) or IL-4 and IFN-γ (2 ± 0%), as compared with RASF, normal blood or RAPB.

Thus, T cells isolated from RAST and RASF demonstrated a decrease in IL-2-producing cells, with a concomitant increase in IFN-γ-producing cells when compared with normal blood and RAPB Tm.

].

primed RA T cells was less marked than observed with normal T cells. An increased percentage of IFN-γ producers was observed after priming, and the peak response was achieved in the presence of low concentrations of anti-CD3 (45% versus 11%). Again, the induction of IFN-γ production in RAPB memory T cells was less than noted in normal blood. IL-4-producing cells were generated from RA memory T cells by stimulation with anti-CD28, and these were inhibited by the presence of anti-CD3 mAb. The percentage of IL-4 producers (12%) generated from RA memory T cells was less than was generated from normal T cells (17%).

differentiation, although in lesser numbers than from RAPB and normal blood. Priming in the presence of anti-CD3 reduced the percentages of IL-2-producing and IL-4-producing cells, while increasing the percentage of IFN-γ-secreting cells.

Effect of anti-IFN-γ antibody on the generation of IL-4-secreting effector cells from RA synovial fluid T cells

]. RAPB Tm generated a marked increase in the percentage of IL-4-producing cells when primed with the combination of IL-4 and anti-IFN-γ antibody. In contrast, T cells from the synovial fluid of the same patient generated virtually no IL-4-producing cells and few IL-2-producing cells, despite supplemental IL-4 and anti-IFN-γ antibody.

, synovial T-cell growth was not inhibited by the addition of anti-IFN-γ antibody or IL-4. These data suggest that RAPB Tm have the capacity to generate IL-4-producing or IFN-γ-producing effector cells, whereas memory T cells from RASF are inhibited in their capacity to become IL-4-producing effector cells. Further studies indicated that anti-CD28 mAb and cytokines enhanced synovial fluid Tm growth approximately fourfold above the initial input cell number. Synovial fluid T cells cultured in medium alone underwent a fourfold reduction in cell number, whereas cells cultured with cytokines alone increased in number by an average of twofold (data not shown). These data further support the conclusion that synovial fluid T cells were responsive to signals delivered by the combination of anti-CD28 and cytokines.

Discussion

stimulation. Thus, mature IL-4-secreting effector cells were found to be decreased in the RA synovium but not in the blood.

]. 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.

].

]. 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.

T cells.

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.

] 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.

].

], 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.

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.

suggests that the rheumatoid microenvironment alters T-cell effector function and thereby perpetuates the chronic inflammatory disease state.

Acknowledgements

The authors wish to thank the doctors and staff who participated in the acquistion of the patient samples. We would like to express special gratitude to Dr Richard E Jones (Dallas, Texas) for some of the samples. We would like to thank Angie Mobley of the UT Southwestern Dallas cell analysis facility for technical support for the flow cytometer. This work was supported by the Arthritis Foundation and National Institutes of Health grant AR45293.

Figures and Tables

.

= 4). The total number of cytokine-producing cells detected in each sample is shown in the upper right corner of each graph. The frequency of each subset of cytokine-producing cells is shown (mean ± standard error of the mean). The frequency was derived by using the total number of cytokine-producing cells as the denominator (× 100). Statistically significant differences are described in Results.

-test).

0.05).

0.05) was observed when the cells were cultured in the presence of IL-4 alone or in combination with anti-IFN-γ antibody.

Deficient induction of IL-4 producers from synovial precursors

0.05) was observed in the percentage of synovial IL-4-producing effector cells in the absence or presence of anti-IFN-γ antibody and/or IL-4.

Keywords

• T-helper cells
• cytokines
• rheumatoid arthritis