Cytokine, activation marker, and chemokine receptor expression by individual CD4+ memory T cells in rheumatoid arthritis synovium

Abstract

cells correlated with the exception of TRANCE and IL-10, and TRANCE and TNF-α . A correlation between expression of IL-10 and CCR7, LT-α and CCR6, IFN-γ and CCR5, and TRANCE and CXCR4 was also detected. memory T cells in the RA synovium. memory T cells in the synovium and blood. Synovial tissues from three RA patients and peripheral blood mononuclear cells from two RA patients and a normal donor were analyzed. T cells was sorted into each well of a 96-well PCR plate using a flow cytometer. cDNA from individual cells was prepared, and then the cDNA was nonspecifically amplified. The product was then amplified by PCR using gene-specific primers to analyze cytokine and activation marker expression. T cells, whereas 2-20% of cells expressed the other cytokine mRNAs. ). Moreover, the frequency of TRANCE-positive cells in TNF-α-positive cells was also significantly higher than that in TNF-α-negative cells. memory T cells expressed CC and CXC chemokine receptors. The frequency of CCR5-positive cells in IFN-γ-positive cells was significantly higher than that in IFN-γ-negative cells, whereas the frequency of CCR6-positive cells in LT-α-positive cells was significantly higher than that in LT-α-negative cells, and the frequency of CCR7-positive cells in IL-10-positive cells was significantly higher than that in IL-10-negative cells. Furthermore, the frequency of CXCR4-positive cells in TRANCE-positive cells was significantly higher than that in TRANCE-negative cells. memory T cells. ]. These results imply that, in the synovium, regulation of IFN-γ and LT-α must vary in individual cells, even though both Th1 cytokines can be produced. memory T cells. Therefore, it is unclear whether CCR5 is a marker of Th1 cells in RA synovium. memory T cells express lymph-node homing receptors and lack immediate effector function, but efficiently stimulate dendritic cells. These cells may play a unique role in the synovium as opposed to in the blood. By producing IL-10, they might have an immunoregulatory function. In addition, IL-10 expression also correlated with expression of TRANCE. Although it is possible that IL-10 produced by these cells inhibited T-cell activation in the synovium, TRANCE expressed by these same cells might function to activate dendritic cells and indirectly stimulate T cells, mediating inflammation in the synovium. These results imply that individual T cells in the synovium might have different, and sometimes opposite functional activities. T cells that are characteristically found in rheumatoid synovium. memory T cells might make them particularly important in mediating the bony erosions that are characteristic of RA. memory T cells are retained in the synovium until LT-α mRNA decreases. memory T cells are biased toward Th1 cells in RA synovium and peripheral blood. In the synovium, IFN-γ and LT-α were produced by individual cells, whereas in the rheumatoid blood no LT-α-producing cells were detected. Furthermore, there were modest correlations between individual cells that expressed particular cytokines, such as IL-10, and certain chemokine receptor mRNAs.

Introduction:

memory T cells in the RA synovium.

memory T cells in the synovium and blood.

Materials and method:

Synovial tissues from three RA patients and peripheral blood mononuclear cells from two RA patients and a normal donor were analyzed.

T cells was sorted into each well of a 96-well PCR plate using a flow cytometer. cDNA from individual cells was prepared, and then the cDNA was nonspecifically amplified. The product was then amplified by PCR using gene-specific primers to analyze cytokine and activation marker expression.

Results:

T cells, whereas 2-20% of cells expressed the other cytokine mRNAs.

). Moreover, the frequency of TRANCE-positive cells in TNF-α-positive cells was also significantly higher than that in TNF-α-negative cells.

memory T cells expressed CC and CXC chemokine receptors. The frequency of CCR5-positive cells in IFN-γ-positive cells was significantly higher than that in IFN-γ-negative cells, whereas the frequency of CCR6-positive cells in LT-α-positive cells was significantly higher than that in LT-α-negative cells, and the frequency of CCR7-positive cells in IL-10-positive cells was significantly higher than that in IL-10-negative cells. Furthermore, the frequency of CXCR4-positive cells in TRANCE-positive cells was significantly higher than that in TRANCE-negative cells.

memory T cells.

Discussion:

]. These results imply that, in the synovium, regulation of IFN-γ and LT-α must vary in individual cells, even though both Th1 cytokines can be produced.

memory T cells. Therefore, it is unclear whether CCR5 is a marker of Th1 cells in RA synovium.

memory T cells express lymph-node homing receptors and lack immediate effector function, but efficiently stimulate dendritic cells. These cells may play a unique role in the synovium as opposed to in the blood. By producing IL-10, they might have an immunoregulatory function. In addition, IL-10 expression also correlated with expression of TRANCE. Although it is possible that IL-10 produced by these cells inhibited T-cell activation in the synovium, TRANCE expressed by these same cells might function to activate dendritic cells and indirectly stimulate T cells, mediating inflammation in the synovium. These results imply that individual T cells in the synovium might have different, and sometimes opposite functional activities.

T cells that are characteristically found in rheumatoid synovium.

memory T cells might make them particularly important in mediating the bony erosions that are characteristic of RA.

memory T cells are retained in the synovium until LT-α mRNA decreases.

memory T cells are biased toward Th1 cells in RA synovium and peripheral blood. In the synovium, IFN-γ and LT-α were produced by individual cells, whereas in the rheumatoid blood no LT-α-producing cells were detected. Furthermore, there were modest correlations between individual cells that expressed particular cytokines, such as IL-10, and certain chemokine receptor mRNAs.

Introduction

memory T cells in the RA synovium.

stimulation, and to correlate cytokine and chemokine receptor expression.

memory T cells from RA blood, with the exception that no cells expressing LT-α were detected. There were modest correlations between individual cells that expressed particular cytokine and chemokine receptor mRNAs.

Specimens

].

Also, peripheral blood mononuclear cells were separated by ficoll-hypaque gradient centrifugation from two RA patients and a normal donor.

Single cell sorting and reverse transcription polymerase chain reaction

flow cytometer.

; Integrated DNA Technologies Incorporated). Twenty-five cycles of amplification were performed with 1 min at 94°C, 2 min at 42°C, and 6 min at 72°C, plus 10 s extension per cycle. Afterward, 5U Taq DNA polymerase was added, followed by an additional 25 cycles of PCR.

.

To confirm that the PCR products were amplified from the corresponding genes, the nucleotide sequences of the PCR products were analyzed. More than five PCR products of each cytokine from a total of two or three donors were sequenced. All the sequences of the PCR products were identical to the previously published sequences (data not shown).

To confirm that each well contained only one cell after sorting, TCR Vβ mRNA was analyzed by single-cell RT-PCR using Vβ family-specific primers. In the wells analyzed, only one TCR Vβ was detected (data not shown).

Statistical analyses

To analyze correlations between cytokines and chemokine receptor expressions, and to compare frequencies of chemokine receptor-expressing cells between different T-cell subsets, Fisher's exact probability test was used.

memory T cells

.

T cells, whereas 2-20% of cells expressed the other mRNAs.

memory T cells

). Moreover, the frequency of TRANCE-positive cells in TNF-α-positive cells was also significantly higher than that in TNF-α-negative cells.

memory T cells

).

memory T cells from RA patients and a normal donor

memory T cells.

<0.005). No other correlations were detected (data not shown).

Discussion

]. These results imply that, in the synovium, regulation of IFN-γ and LT-α must vary in individual cells, even though both Th1 cytokine mRNAs can be expressed.

memory T cells, although it correlated with IFN-γ . Therefore, it is unclear whether CCR5 is a marker of Th1 cells in RA synovium.

memory T cells expressed CCR7 and there was a correlation between IL-10 production and CCR7 expression, these cells may play a unique role in the synovium as opposed to in the blood. By producing IL-10, they may exert an immunoregulatory function. In addition, it is interesting to note that IL-10 expression also correlated with expression of TRANCE. Although it is possible that IL-10 produced by these cells inhibited T-cell activation in the synovium, TRANCE expressed by these same cells might function to activate dendritic cells and indirectly stimulate T cells, mediating inflammation in the synovium. These results imply that individual T cells in the synovium might have different, and sometimes opposite functional activities.

T cells that are characteristically found in rheumatoid synovium.

memory T cells might make them particularly important in mediating the bony erosions that are characteristic of RA.

memory T cells are retained in the synovium until LT-α mRNA decreases.

memory T cells are biased toward Th1 cells in RA synovium and peripheral blood. In the synovium, IFN-γ and LT-α were produced by individual cells, whereas in the rheumatoid blood no LT-α-producing cells were detected. Furthermore, there were modest correlations between individual cells that expressed particular cytokines and certain chemokine receptor mRNAs.

Acknowledgement

We thank Drs Kenji Hayashida, Hermann Girschick, and Sule Yavuz for critical discussion, Ms Angie Mobley for assistance with cell sorting, and Ms Christine Pavlovitch, Rehana Hussain, and Michelle McGuire for their technical support.

Figures and Tables

Tcells were sorted, and cytokine and activation marker expressions were immediately determined in the TCR Cβ (TCRBC) mRNA-positive wells employing single-cell RT-PCR. Amplified PCR products were separated by electrophoresis through 2.0% agarose. As negative controls, one well that did not contain a cell (C1) and one well that was not prepared with reverse transcriptase (C2) were analyzed.

T cells in synovial tissue of RA patients

Values are expressed as number (percentage) of cytokine-positive cells in TCR Cβ-positive cells.

memory T cells

< 0.05.

Primers used for PCR amplification

memory T cells

< 0.05.

memory T cells

< 0.05.

T cells in peripheral blood of RA patients and a normal donor

Values are expressed as number (percentage) of cytokine-positive cells in TCR Cβ-positive cells.

memory T cells of RA peripheral blood

< 0.005.

Keywords

• chemokine receptor
• cytokine
• rheumatoid arthritis
• T lymphocyte