Lack of correlation between chemokine receptor and Th1/Th2 cytokine ...

International Immunology, Vol. 12, No. 12, pp. 1659–1667

© 2000 The Japanese Society for Immunology

Lack of correlation between chemokine receptor and Th1/Th2 cytokine expression by individual memory T cells Toshihiro Nanki1 and Peter E. Lipsky1 Department of Internal Medicine and Harold C. Simmons Arthritis Research Center, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75235, USA 1Present

address: National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD 20892, USA Keywords: chemokine receptor, cytokine, human, rheumatoid arthritis, Th1, Th2

Abstract Chemokine and chemokine receptor interactions may have important roles in leukocyte migration to specific immune reaction sites. Recently, it has been reported that CXC chemokine receptor (CXCR) 3 and CC chemokine receptor (CCR) 5 were preferentially expressed on Th1 cells, and CCR3 and CCR4 were preferentially expressed on Th2 cells. To investigate chemokine receptor expression by Th subsets in vivo, we analyzed cytokine (IL-2, IL-4 and IFN-γ) and chemokine receptor (CXCR3, CXCR4, CCR3, CCR4 and CCR5) mRNA expression by individual peripheral CD4⍣ memory T cells after short-term stimulation, employing a single-cell RT-PCR method. This ex vivo analysis shows that the frequencies of cells expressing chemokine receptor mRNA were not significantly different between Th1 and Th2 cells in normal peripheral blood. To assess a potential role of in vivo stimulation, we also analyzed unstimulated rheumatoid arthritis synovial CD4⍣ memory T cells. CXCR3, CXCR4, CCR3 and CCR5 expression was detected by individual synovial T cells, but the frequencies of chemokine receptor mRNA were not clearly different between Th1 and non-Th1 cells defined by expression of IFN-γ or lymphotoxin-α mRNA in all RA patients. These data suggest that chemokine receptor expression does not identify individual memory T cells producing Th-defining cytokines and therefore chemokine receptor expression cannot be a marker for Th1 or Th2 cells in vivo. Introduction Migration is necessary for specific subsets of lymphocytes to accumulate at sites of immune reactions. Lymphocyte migration is regulated by various adhesion molecules, as well as the action of chemokines impacting on specific chemokine receptors (1–5). Although the role of adhesion molecules has been studied in detail, the role of chemokines and chemokine receptors is less clear. Chemokine receptors have seven transmembrane domains coupled to a G-protein. Nine CC chemokine receptors (CCR1–9) (6–17) and five CXC chemokine receptors (CXCR1–5) (18–24) have been identified to date, and their diverse actions are the subject of intense investigation. It has been reported that CD4⫹ T cells can be divided into two subsets, Th1 and Th2, by the ability of each subset to

produce specific cytokines (25). In humans, Th1 cells produce IL-2, IFN-γ and lymphotoxin (LT), whereas Th2 cells produce IL-4 and IL-5. Th1 cells mainly participate in cell-mediated immunity and are involved in chronic inflammatory diseases, such as rheumatoid arthritis (RA), whereas Th2 cells regulate immune responses, facilitate IgE production, and are involved in allergic disease (26,27). Consequently, Th1 and Th2 cells may be recruited to different sites of inflammation (5,28,29), perhaps because of their differential expression of adhesion molecules (30,31). In addition to differences in adhesion molecule expression, it has been suggested that Th1 and Th2 cells may respond to specific chemokines because of their unique expression of chemokine receptors. In this regard, it has been reported

Correspondence to: P. E. Lipsky, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Building 10, Room 9N228, 10 Center Drive MSC 1820, Bethesda, MD 20892-1820, USA Transmitting editor: M. Feldmann

Received 11 May 2000, accepted 14 August 2000

1660 Chemokine receptor and cytokine expression that CXCR3 and CCR5 are predominantly expressed by Th1 cells (32–34), whereas CCR3 and CCR4 are predominantly expressed by Th2 cells (32,34–36). However, expression of these chemokine receptors has largely been analyzed using in vitro generated Th1 and Th2 cell lines or clones, that have been stimulated with mitogens and growth-promoting and Th-polarizing cytokines. Therefore, it was possible that these culture conditions rather than the Th cell phenotype influenced chemokine receptor expression. Indeed, it has been reported that stimulation with IL-2 induced CXCR3 and CCR5 expression (37–39). Moreover, the patterns of chemokine receptor expression by polarized Th cells have not been uniform in various reports (32–34). Thus, the correlation between chemokine receptor expression and Th cell phenotype is still unclear, especially in vivo. This issue is of importance because it has been suggested that the phenotype of Th cells at inflammatory sites can be inferred from the pattern of chemokine receptor expression (36–38). Therefore, to investigate the correlation of chemokine receptor and cytokine expression without the influence of prolonged in vitro stimulation and growth, we analyzed individual peripheral CD4⫹ memory T cells for chemokine receptor and cytokine mRNA employing a single-cell RT-PCR method. The results show that the frequencies of CXCR3, CXCR4, CCR3, CCR4 and CCR5 expression were not significantly different between Th0, Th1 and Th2 cells. The data suggest that chemokine receptor expression does not denote specific Th cell differentiation in vivo and therefore that chemokine receptor expression cannot be used as a marker for Th1 or Th2 cell generated in vivo.

Methods Samples Peripheral blood mononuclear cells were separated by FicollHypaque (Pharmacia Biotech, Piscataway, NJ) gradient centrifugation from seven healthy donors. CD4⫹CD45RO⫹ memory T cells were isolated by negative selection (StemSep; StemCell Technologies, Vancouver, Canada). Purity of the separated cells was ⬎95%. CD27–CD4⫹CD45RO⫹ well-differentiated memory cells were separated by flow cytometry using a FACStar Plus (Becton Dickinson, San Jose, CA) after staining negatively selected CD4⫹ T cells (StemSep) with FITC-conjugated anti-CD27 mAb (L128; Becton Dickinson), Quantum Red-conjugated anti-CD4 mAb (Q4120; Sigma, St Louis, MO), and phycoerythrin (PE)-conjugated anti-CD45RO mAb (UCHL-1; Sigma). The isolated T cell subsets were stimulated with 25 ng/ml of phorbol myristate acetate (PMA; Sigma) and 1 µg/ml of ionomycin (Sigma) for 4 h. Synovial tissue was obtained at surgery from three RA patients. RA was diagnosed according to the American College of Rheumatology criteria (40). The synovial tissue was minced and incubated with 0.3 mg/ml collagenase (Sigma) for 1 h at 37°C in RPMI 1640 medium (Life Technologies, Gaithersburg, MD). Partially digested pieces of the tissue were pressed through a metal screen to obtain singlecell suspensions. Mononuclear cells were then isolated by Ficoll-Hypaque gradient centrifugation.

Single-cell sorting and RT-PCR The method for construction of cDNA libraries from single cells was similar to previously reported techniques (41,42). CD4⫹CD45RO⫹ or CD27–CD4⫹CD45RO⫹ single cells were sorted into wells of 96-well PCR plates (Robbins Scientific, Sunnyvale, CA) using the FACStar Plus flow cytometer. The synovial T cells were stained with FITC-conjugated anti-CD4 mAb (Q4120; Sigma) and PE-conjugated antiCD45RO mAb (UCHL-1), and individual CD4⫹CD45RO⫹ T cells were sorted into a 96-well PCR plate using the FACStar Plus. Each well contained 4 µl of lysis buffer [50 mM Tris–HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 10 µM dNTP (Sigma), 5 U/ml PRIME RNase Inhibitor (5⬘ → 3⬘, Boulder, CO), 300 U/ml RNAguard (Pharmacia Biotech), 200 ng/ml oligo(dT)24 (Integrated DNA Technologies, Coralville, IA) and 0.5% NP-40]. The samples were heated to 65°C for 1 min, cooled to 20°C for 3 min and maintained on ice. Two units of AMV reverse transcriptase (Promega, Madison, WI) and 50 U of MMLV reverse transcriptase (Life Technologies) were added, and the samples were incubated at 37°C for 15 min before heat inactivation at 65°C for 10 min. For polyadenylate tailing at the 3⬘ end of the cDNA, 5 µl of tailing buffer (200 mM potassium cacodylate, pH 7.2, 4 mM CoCl2 and 0.4 mM DTT), 2 mM dATP (Boehringer Mannheim, Indianapolis, IN) and 10 U terminal transferase (Boehringer Mannheim) were added, and incubated at 37°C for 20 min followed by heat inactivation at 65°C for 10 min. To amplify the cDNA non-specifically, PCR was performed with 100 µl of 10 mM Tris–HCl, pH 9.0, 50 mM KCl, 2.5 mM MgCl2, 0.01% Triton-X, 1 mM dNTP, 10 U Taq DNA polymerase (Promega) and 2 µM X-(dT)24 primer (ATG TCG TCC AGG CCG CTC TGG ACA AAA TAT GAA TTC-dT24) (Integrated DNA Technologies). 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, 5 U Taq DNA polymerase was added, followed by an additional 25 cycles of PCR. For gene-specific amplification, 1 µl of non-specifically amplified cDNA was amplified by PCR in 25 µl of 10 mM Tris– HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 0.01% Triton-X, 200 µM dNTP and 0.625 U Taq DNA polymerase. The cycling program was: 94°C for 1 min, annealing temperature (58°C: IL-2, IL-4, IFN-γ, CCR5 and β-actin; 60°C: LT-α, CXCR3 and CXCR4; 61°C: TCR Cβ; 62°C: CCR3) for 1 min, 72°C for 1 min, for 35 cycles followed by a final extension for 7 min. The primers were designed to be within 600 bp of the 3⬘ end of each mRNA. For nested amplification, 1 µl of amplified PCR reaction mixture was added to a second PCR reaction mixture (50 µl of 10 mM Tris–HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 0.01% Triton-X, 200 µM dNTP and 1.25 U Taq DNA polymerase). The cycling program was: 94°C for 1 min, annealing temperature (60°C: IL-2, IL-4, IFN-γ, LT-α, CXCR3, CXCR4, CCR5 and β-actin; 61°C: TCR Cβ; 62°C: CCR3) for 1 min, 72°C for 1 min for 35 cycles followed by a final extension for 7 min. The PCR products were then separated by electrophoresis through 2.0% agarose. The primers used were IL-2, 5⬘-TAC AAG AAT CCC AAA CTC ACC AGG A, 3⬘-GTC AGT GTT GAG ATG ATG CTT TGA CA, 3⬘ (nested)TGG TTG CTG TCT CAT CAG CAT ATT CA; IL-4, 5⬘-CTT TGC

Chemokine receptor and cytokine expression 1661 TGC CTC CAA GAA CAC AAC T, 3⬘-TCT CAT GAT CGT CTT TAG CCT TTC CA, 3⬘ (nested)-TCC TTC ACA GGA CAG GAA TTC AAG C; IFN-γ, 5⬘-AAG GCT TTA TCT CAG GGG CCA ACT, 5⬘ (nested)-CAA GAT CCC ATG GGT TGT GTG TTT, 3⬘-TGG AAG CAC CAG GCA TGA AAT CTC; LT-α, 5⬘-CCT GAA CCA TCC CTG ATG TCT G, 3⬘-AAA TAG TCC CCT CCC TGC CTC T, 3⬘ (nested)-TCT AGT CAT CCC CCA AGC TCC TC; CXCR3, 5⬘-CAC TGC CCT TCT CAT TTG GAA ACT, 5⬘ (nested)-AGT ACA AGG CAT GGC GTA GAG GG, 3⬘-GCA AAT ATA GAG GTC TTG GGG AC; CXCR4, 5⬘-GGA CCT GTG GCC AAG TTC TTA GTT, 3⬘-ACT GTA GGT GCT GAA ATC AAC CCA, 3⬘ (nested)-CAG CTG GGG ATC ATT TCT AGC TTT; CCR3, 5⬘-CTA AGG TCA TTA CCA CAG GCC AGG, 5⬘ (nested)-GCA GCG TAC TCA TCA TCA ACC C, 3⬘-AGC AGG GAA AGA ACT AGG CAC ATT; CCR5, 5⬘-CTC AGG GAA TGA AGG TGT CAG A, 5⬘ (nested)-AGC CTC TGA ATA TGA ACG GTG AGC, 3⬘-TGC TAC TGT TGC ACT CTC CAC AAC T; β-actin, 5⬘-GTC CTC TCC CAA GTC CAC ACA, 5⬘ (nested)TTG TTA CAG GAA GTC CCT TGC CAT, 3⬘-CTG GTC TCA AGT CAG TGT ACA GGT AA; TCR Cβ, 5⬘-TCA AGT CCA GTT CTA CGG GCT C, 5⬘ (nested)-CTC TCG GAG AAT GAC GAG TGG AC, 3⬘-TCA TAG AGG ATG GTG GCA GAC A. Cytokine (IL-2, IL-4 and IFN-γ) and chemokine receptor (CXCR3, CXCR4, CCR3, and CCR5) expression by peripheral CD4⫹ memory or CD27–CD4⫹ memory T cells was analyzed in β-actin⫹ wells. The cytokine and chemokine receptor expression by synovial CD4⫹ memory T cells were analyzed in TCR Cβ⫹ wells. As negative controls, 1–2 wells without cells and 2–4 wells without reverse transcriptase per donor were analyzed. Genespecific PCR products were not detected from these negative controls, 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 or chemokine receptor from a total of three donors were sequenced. All the sequences of the PCR products were identical to the previously published sequences (data not shown). Analysis of CCR4 expression To analyze expression of CCR4 and cytokines from single cells, PCR amplification from individual cell cDNA was carried out without the initial non-specific amplification, since the nucleotide sequence of the 3⬘ end of CCR4 mRNA has not been reported. Therefore, individual CD4⫹ memory T cells were sorted into wells containing 5 µl of lysis buffer [50 mM Tris–HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.8 mM dNTP, 5 U/ml PRIME RNase Inhibitor, 300 U/ml RNAguard, 10 µg/ml oligo(dT)12–18 (Pharmacia Biotech) and 2% NP-40]. The samples were heated to 65°C for 1 min, cooled to 20°C for 3 min and maintained on ice. Reverse transcriptase (10 U, SuperScript II; Life Technologies) was added and the samples were incubated at 42°C for 50 min before heat inactivation at 65°C for 15 min. RNase H (0.1 U; Pharmacia Biotech) was added and incubated at 37°C for 20 min. The final volume of each sample was 10 µl; 2 µl of the cDNA was used for gene-specific amplification. PCR conditions were the same as for the gene-specific PCR after non-specific amplification (annealing temperature for CCR4

amplification was 58°C in the first PCR and 60°C in the nested PCR). Primers used were CCR4 5⬘-GTG GTT CTG GTC CTG TTC AAA TAC, 3⬘-CGT GGA GTT GAG AGA GTA CTT GGT T, 5⬘ (nested)-TAC TAT GCA GCA GAC CAG TGG GTT T and 3⬘ (nested)-GGT TGC GCT CAG TAT AAC AAG TGC T. Statistical analyses To compare frequencies of chemokine receptor-expressing cells between different T cell subsets, Fisher’s exact probability test was used. To compare frequencies of cytokineproducing cells between CD27–CD4⫹CD45RO⫹ and total CD4⫹CD45RO⫹ T cells, the χ2 test was used. Results Comparison of chemokine receptor and cytokine expression by unstimulated and stimulated peripheral CD4⫹ memory T cells We initially compared chemokine receptor expression between unstimulated and stimulated peripheral CD4⫹ memory T cells employing the single-cell RT-PCR method. For these experiments, CD4⫹ memory T cells were stimulated with PMA and ionomycin for 4 h to up-regulate cytokine gene expression. Chemokine receptor (CXCR3, CXCR4, CCR3 and CCR5) and cytokine (IL-2, IL-4 and IFN-γ) expression was determined (Fig. 1). The frequencies of cells expressing chemokine receptors increased somewhat in stimulated CD4⫹ memory T cells (Table 1). Cytokine expression was only detected after stimulation. In stimulated CD4⫹ memory T cells, there was no obvious relationship between chemokine receptor and cytokine expression. Comparison of chemokine receptor expression in Th subsets Cytokine and chemokine receptor expression by stimulated peripheral CD4⫹CD45RO⫹ cells was analyzed from three additional normal donors. Frequencies of the cytokine and chemokine receptor positive cells in a total of 193 cells are shown in Table 2. The CD4⫹ memory T cells were classified into Th0 (IL-4⫹ and IFN-γ⫹), Th1 (IL-4– and IFN-γ⫹), Th2 (IL-4⫹ and IFN-γ–) and IL-2 only-producing cells by cytokine production pattern (Table 3). Six Th0, 44 Th1, five Th2 and 36 IL-2 only-producing cells were identified. The frequency of CXCR3, CXCR4, CCR3 and CCR5 expression was not significantly different in the Th0, Th1 and Th2 subsets (Table 4). In contrast, the frequency of CCR3 expression by IL-2 only-producing CD4⫹ memory T cells was lower than that in Th0 or Th1 cells (P ⬍ 0.05). The Th1 cells were divided into IL-2⫹ (IL-2⫹ and IFN-γ⫹) and IL-2– cells (IFN-γ only). Twenty-two IL-2⫹ Th1, and 22 IL2– Th1 cells were identified. The frequencies of chemokine receptor expression were not significantly different between these two subsets (data not shown). Cytokine and chemokine receptor expression of peripheral CD27–CD4⫹ memory T cells Th2 cells are uncommon in circulating CD4⫹ memory T cells and therefore definitive conclusions about chemokine receptor expression by this subset could not be drawn. Therefore, to investigate chemokine receptor expression by Th2 cells in

1662 Chemokine receptor and cytokine expression Table 2. Frequencies of cytokine and chemokine receptor expression from stimulated peripheral CD4⫹ memory T cells Donor

IL-2 IL-4 IFN-γ CXCR3 CXCR4 CCR3 CCR5

1 (n ⫽ 34)

2 (n ⫽ 31)

5 0 8 5 7 6 1

10 6 8 1 2 1 1

(15%) (0%) (24%) (15%) (21%) (18%) (3%)

3 (n ⫽ 48)

4 (n ⫽ 80)

Total (n ⫽ 193)

(32%) 18 (38%) (19%) 0 (0%) (26%) 13 (27%) (3%) 2 (4%) (6%) 8 (17%) (3%) 4 (8%) (3%) 7 (15%)

35 (44%) 5 (6%) 21 (26%) 5 (6%) 11 (14%) 8 (10%) 8 (10%)

68 (35%) 11 (6%) 50 (26%) 13 (7%) 28 (15%) 19 (10%) 17 (9%)

Table 3. Frequencies of each Th subset in peripheral CD4⫹ memory T cells

Fig. 1. Cytokine (IL-2, IL-4 and IFN-γ) and chemokine receptor (CXCR3, CXCR4, CCR3 and CCR5) expression by peripheral CD4⫹CD45RO⫹ cells. (A) Unstimulated cell. (B) Cells stimulated by PMA and ionomycin for 4 h. Peripheral CD4⫹CD45RO⫹ single cells were sorted, and cytokine and chemokine receptor expression was analyzed employing RT-PCR. PCR products were separated by electrophoresis through 2.0% agarose. As a negative control, one well which did not contain a cell was analyzed (last lane, C).

Cytokine

Donor

IL-2 IL-4 IFN-γ

1 2 3 4 Total (n ⫽ 34) (n ⫽ 31) (n ⫽ 48) (n ⫽ 80) (n ⫽ 193)

Th0 ⫹ ⫹ ⫹ – ⫹ ⫹ Th1 ⫹ – ⫹ – – ⫹ T h2 ⫹ ⫹ – – ⫹ – IL-2 only ⫹ – – No Cytokines – – –

0 (0%) 0 (0%)

3 (10%) 0 (0%) 0 (0%) 0 (0%)

3 (4%) 0 (0%)

6 (3%) 0 (0%)

2 (6%) 2 (6%) 8 (17%) 10 (13%) 22 (11%) 6 (18%) 3 (10%) 5 (10%) 8 (10%) 22 (11%) 0 (0%) 0 (0%)

3 (10%) 0 (0%) 0 (0%) 0 (0%)

1 (1%) 1 ( 1%)

4 (2%) 1 (1%)

3 (9%)

2 (6%) 10 (21%) 21 (26%) 36 (19%)

23 (68%) 18 (58%) 25 (52%) 36 (45%) 102 (53%)

Table 4. Comparison of chemokine receptor expression between Th subsets Table 1. Comparison of cytokine and chemokine receptor expression between unstimulated and stimulated peripheral CD4⫹ memory T cells


Unstimulated (n ⫽ 33)

Stimulated (n ⫽ 34)a

0b (0%c) 0 (0%) 0 (0%) 2 (6%) 6 (18%) 2 (6%) 3 (9%)

5 0 8 5 7 6 1

(15%) (0%) (24%) (15%) (21%) (18%) (3%)

aCD4⫹CD45RO⫹ memory T cells were stimulated with PMA and ionomycin for 4 h. bNumber of positive wells. cFrequency of cytokine or chemokine receptor positive wells in βactin⫹ wells.

CXCR3 CXCR4 CCR3 CCR5

Th0 (n ⫽ 6)

Th1 (n ⫽ 44)

Th2 (n ⫽ 5)

IL-2 only (n ⫽ 36)

1 2 2 1

6 (14%) 10 (23%) 8 (18%) 7 (16%)

0 0 0 0

3 5 1 2

(17%) (33%) (33%) (17%)

(0%) (0%) (0%) (0%)

(8%) (14%) (3%)a (6%)

aCCR3 expression in IL-2 only-producing cells was significantly less than in the Th0 and Th1 cells (P ⬍ 0.05).

greater detail, we analyzed peripheral CD27–CD4⫹CD45RO⫹ T cells from two additional donors, since this population is known to be enriched in Th2 cells (43,44). CD27– cells comprised only 7 and 15% of CD4⫹CD45RO⫹ T cells from these two donors, consistent with previously published results (45). Frequencies of the cytokine and chemokine receptor positive cells in a total of 237 stimulated CD27–

Chemokine receptor and cytokine expression 1663 Table 5. Frequencies of cytokine and chemokine receptor expression in peripheral CD27–CD4⫹ memory T cells

Table 7. Comparison of chemokine receptor expression between Th subsets in peripheral CD27–CD4⫹ memory T cells

Donor


5 (n ⫽ 109)

6 (n ⫽ 128)

Total (n ⫽ 237)

85 (78%) 31 (28%) 66 (61%) 11 (10%) 13 (12%) 4 (4%) 7 (6%)

87 (68%) 16 (13%) 47 (37%) 23 (18%) 15 (12%) 8 (6%) 11 (9%)

172 (73%)a 47 (20%)a 113 (48%)a 34 (14%)b 28 (12%) 12 (5%)b 18 (8%)

Frequencies of IL-2-, IL-4-, IFN-γ- and CXCR3-producing cells in CD27–CD4⫹ memory T cells were significantly higher than found in total CD4⫹ memory cells, and CCR3 expression in CD27–CD4⫹ memory cells was significantly less than in total CD4⫹ memory cells (aP ⬍ 0.001, bP ⬍ 0.01).


Th0 (n ⫽ 21)

Th1 (n ⫽ 92)

Th2 (n ⫽ 26)

IL-2 only (n ⫽ 57)

2 2 1 3

16 14 4 6

1 2 2 1

8 5 3 5

(10%) (10%) (5%) (14%)

(17%) (15%) (4%)a (7%)

(4%) (8%) (8%) (4%)

(14%) (9%) (5%) (9%)

aFrequency of CCR3 expression by T 1 cells from CD27–CD4⫹ h memory cells was significantly lower than that of Th1 cells from total CD4⫹ memory cells (P ⬍ 0.05).

Table 8. Comparison of CCR4 expression between Th subsets in peripheral CD4⫹ memory T cells Th0 (n ⫽ 1) Th1 (n ⫽ 14) Th2 (n ⫽ 4) IL-2 only (n ⫽ 39)

Table 6. Frequencies of each Th subset in peripheral CD27– CD4⫹ memory T cells Cytokine IL-2

IL-4

Th0 ⫹ ⫹ ⫹ Th1 ⫹ – – Th2 ⫹ ⫹ ⫹ IL-2 only ⫹ – No cytokines –

Donor IFN-γ

5 (n ⫽ 109) 6 (n ⫽ 128) Total (n ⫽ 237)

⫹ ⫹

17 (16%) 0 (0%)

4 (3%) 0 (0%)

21 (9%) 0 (0%)

⫹ ⫹

40 (37%) 9 (8%)

33 (26%) 10 (8%)

73 (31%) 19 (8%)

– –

11 (10%) 3 (3%)

10 (8%) 2 (2%)

21 (9%) 5 (2%)

–

17 (16%)

40 (31%)

57 (24%)

–

12 (11%)

29 (23%)

41 (17%)

CD4⫹CD45RO⫹ T cells are shown in Table 5. The frequency of IL-2-, IL-4- and IFN-γ-producing cells in the CD27–CD4⫹ memory T cells was significantly (P ⬍ 0.001) higher than found in the whole CD4⫹CD45RO⫹ memory T cell population. The ratio of IL-2:IL-4:IFN-γ producing cells in the CD27–CD4⫹ memory T cells was 3.7:1:2.4, whereas the ratio in total CD4⫹ memory T cells was 6.2:1:4.5. CXCR3 expression in the CD27– CD4⫹ memory T cells was also significantly higher than in the whole CD4⫹ memory T cells (P ⬍ 0.01), whereas CCR3 expression in the CD27–CD4⫹ memory T cells was significantly lower than in the whole CD4⫹ memory T cell (P ⬍ 0.01). Within the CD27–CD4⫹CD45RO⫹ memory T cells, 21 Th0, 92 Th1, 26 Th2 and 57 IL-2 only-producing cells were identified (Table 6). The ratio of Th0:Th1:Th2:IL-2 only-producing cells in the CD27–CD4⫹CD45RO⫹ T cells was 0.8:3.5:1:2.2, whereas the ratio in the whole CD4⫹ memory T cells was 1.2:8.8:1:7.2. The frequency of CCR3 expression by Th1 cells from CD27–

CCR4

1 (100%)

9 (64%)

1 (25%)

21 (54%)

Table 9. Correlation between IFN-γ or LT-α and chemokine receptor expression by RA synovial CD4⫹ memory T cellsa


IFN-γ⫹ (n ⫽ 18)

IFN-γ– (n ⫽ 134)

LT-α⫹ (n ⫽ 11)

LT-α– (n ⫽ 141)

4 15 1 8

21 101 6 24

2 10 0 1

23 106 7 31

(22%) (83%) (6%) (44%)b

(16%) (75%) (4%) (18%)

(18%) (91%) (0%) (9%)

(16%) (75%) (5%) (22%)

aRA synovial CD4⫹ memory T cells were analyzed directly after isolation with no in vitro stimulation. bCCR5 expression by IFN-γ⫹ cells was significantly higher than by IFN-γ– cells (P ⬍ 0.05).

CD4⫹ memory T cells was less than that by Th1 cells from the total CD4⫹ memory T cells (P ⬍ 0.05) (Table 7). However, the frequencies of chemokine receptor expression by Th2 cells were not significantly different compared with those expressed by Th0 or Th1 cells. Cytokine and CCR4 expression Frequencies of CCR4⫹ cells by Th0, Th1, Th2 and IL-2 onlyproducing cells are shown in Table 8. CCR4 expression was detected within all the subsets, and the frequencies were not significantly different. Chemokine receptor and IFN-γ or LT-α expression of RA synovial CD4⫹ memory T cells The cytokine and chemokine receptor expression of freshly isolated RA synovial CD4⫹CD45RO⫹ T lymphocytes was also analyzed from three RA patients. IL-2 and IL-4 mRNA were not detected in the synovial T cells. The frequency of CCR5⫹ cells in IFN-γ⫹ cells was significantly higher than that in IFNγ– cells (Table 9), whereas LT-α expression did not correlate with chemokine receptor expression. However, the correlation

1664 Chemokine receptor and cytokine expression Table 10. Correlation of CCR5 and IFN-γ or LT-α expressions in each different patient CCR5 expression RA1 IFN-γ⫹ IFN-γ– LT-α⫹ LT-α–

4/11a

(36%) 6/39b (15%) 1/6 (17%) 9/44 (20%)

RA2 0/3 (0%) 6/36 (17%) 0/1 (0%) 6/38 (16%)

RA3

Total (100%)c

4/4 12/59 (20%) 0/4 (0%) 16/59 (27%)

8/18 (44%)d 24/134 (18%) 1/11 (9%) 31/141 (22%)

of CCR5⫹ cells in IFN-γ⫹ cells/number of IFN-γ⫹ cells. of CCR5⫹ cells in IFN-γ– cells/number of IFN-γ– cells. cP ⬍ 0.005. dP ⬍ 0.05. aNumber bNumber

between CCR5 and IFN-γ expression was found in only two of three patients (RA1 and RA3) analyzed (Table 10). Discussion We employed a single-cell PCR method to assess the correlation between chemokine receptor expression and the cytokine production pattern of individual peripheral memory T cells. Using in vitro generated Th1 or Th2 cell lines or clones, previous studies reported that CXCR3 and CCR5 were preferentially expressed by Th1 cells, and CCR3 and CCR4 were expressed by Th2 cells. The current data show that expression of no specific chemokine receptor correlated with Th1 or Th2 function defined by cytokine mRNA pattern. These data imply that chemokine receptor expression cannot be a marker for in vivo generated Th1 or Th2 cells. To analyze cytokine and chemokine receptor expression, we stimulated the peripheral CD4⫹ memory T lymphocytes for 4 h with PMA and ionomycin. This stimulation increased the frequency of chemokine receptor expression modestly, but was necessary to detect cytokine mRNA. It was, therefore, possible that the stimulation altered the relationship between chemokine receptor and cytokine expression, and the results must be interpreted with this caveat. Previously, the chemokine receptor expression of Th1 or Th2 cells was analyzed mainly by in vitro generated Th1 or Th2 cell lines or clones (32–36). However, in vitro generated Th1 or Th2 cells may be influenced by the culture condition, especially by cytokines. CXCR3 was identified as a ligand for two chemokines, IFN-γ-inducible protein 10 and monokine induced by IFN-γ (20). CXCR3 is expressed mainly by memory T cells in the periphery (34,37). CXCR3 expression could be induced by culture for 3 weeks with IL-2 and down-regulated by stimulation with anti-CD3 (37,38). Furthermore, it was reported that CXCR3 was preferentially expressed by Th1 compared to Th2 cell lines generated from cord blood T cells stimulated with phytohemagglutinin (PHA) with IL-12 or IL-4 respectively, followed by expansion with IL-2 (32). However, contrary results were noted with Th1 and Th2 clones generated from peripheral blood lymphocytes following antigenic stimulation, both of which expressed CXCR3 (33). Finally, CXCR3 was found to be expressed at high levels on Th0 and Th1, and at low levels on Th2 cells generated from cord blood

T cells by stimulation with PHA plus IL-2 and IL-12 or IL-4 respectively, followed by re-stimulation with PHA and irradiated allogeneic peripheral blood mononuclear cells (34). These results suggest that different modes of stimulation employed to generate Th1 or Th2 cells might be a more important influence on chemokine receptor expression than a specific relationship to a functional phenotype and are consistent with the current data that the frequency of CXCR3 expression was not different between Th subsets in fresh peripheral CD4⫹ memory T cells. These results indicate that CXCR3 expression does not correlate with the cytokine production pattern by peripheral CD4⫹ memory T cells. It should be noted, however, that CXCR3 was up-regulated by the brief in vitro stimulation. Moreover, CXCR3 expression did not correlate with IFN-γ or LT-α expression by RA synovial CD4⫹ memory T cells. Thus, CXCR3 could not be a marker for Th1 T cells in vivo. CXCR4 was cloned as an orphan receptor (21–23,46) and the ligand was found to be stromal derived factor-1, originally identified as a growth factor for murine pre-B cells (47,48). Expression of CXCR4 was up-regulated by stimulation with anti-CD3 and CD28 mAb or IL-2 (39,49). Subsequently, it was reported that IL-4 induced CXCR4 expression on CD4⫹ T cells and that only Th2 clones expressed CXCR4 (50). However, Th1 clones could also be induced to express CXCR4 by culture with IL-4. These results suggested that CXCR4 expression is not a marker for Th2 cells and is consistent with the current results. CCR3 has been identified as a ligand for a number of chemokines, including eotaxin, RANTES, monocyte chemotactic protein (MCP)-2, MCP-3 and MCP-4 (8–10,51). By flow cytometric analysis, small numbers of peripheral memory T cells have been reported to express CCR3 (34). Moreover, CCR3 was shown to be expressed preferentially by Th2 cell lines or clones (32,34–36). Thus, cell lines established from CCR3⫹ peripheral T cells produced mainly IL-4, whereas cell lines established from CCR3– T cells mainly secreted IFN-γ (35). However, only a minority of the cells established from CCR3⫹ or CCR3– T cells secreted IL-4 and IFN-γ respectively. Subsequently, it was shown that some Th1 clones expressed CCR3 (36). The current data showed that the frequency of CCR3 expression was not different between Th1 and Th2 cells in the peripheral CD4⫹ memory T cell compartment, although CCR3 was up-regulated by the brief in vitro stimulation. Furthermore, 5% of RA synovial CD4⫹ memory T cells expressed CCR3, although as shown here and previously reported (52,53) RA is a disease characterized by a Th1dominant response in the synovium. Moreover, 6% of IFN-γ⫹ synovial CD4⫹ memory T cells expressed CCR3. The results suggested that CCR3 is not a marker for Th2 cells in vivo. Memory T cells that produced only IL-2 expressed CCR3 less frequently than Th0 and Th1 cells. This subset may have the ability to mature into either Th1 and Th2 cells (54). After maturation into Th1, but before maturation to the stage that CD27 is down-regulated CCR3 expression may be up-regulated. It is noteworthy that maturation to either Th2 or to CD27– is not association with up-regulation of CCR3 expression. These results indicated that CCR3 is not a marker for activated Th2 cells in either the peripheral blood or rheumatoid synovium. In fact, IL-2 and IL-4 enhanced CCR3 expression by

Chemokine receptor and cytokine expression 1665 T cells (55). Thus, culture to establish Th2 clones may affect CCR3 expression. CCR4 was identified as a ligand for a number of chemokines, including RANTES, macrophage inflammatory protein (MIP)-1α, MCP-1, macrophage-derived chemokine, and thymus and activation-regulated chemokine (11,56,57). It was reported that CCR4 was also preferentially expressed on Th2 cells generated in vitro (32,34,58). The current data indicated that the frequency of CCR4 expression by peripheral CD4⫹ memory T cells was not different between Th subsets. Recently it was shown that 22% of RA synovial fluid T cells expressed CCR4 (59) and 25% of juvenile RA synovial fluid CD4⫹ T cells expressed CCR4 (60). Again, in vitro stimulation may have influenced expression of this chemokine receptor as it does not appear that CCR4 expression is a marker associated with Th1 or Th2 cells in periphery. CCR5 is a ligand for a variety of chemokines, including RANTES, MIP-1α and MIP-1β (12), and is expressed by CD45RO⫹ T cells in the periphery (34,39,61). CCR5 expression can be induced by culture with IL-2 (37,39). Moreover, it has been claimed that CCR5 was preferentially expressed by Th1 cell lines (32,33), although subsequently it was reported that CCR5 was expressed by both Th1 and Th2 cells (34). Different modes of stimulating the generation of Th1 or Th2 cells might, therefore, have influenced the CCR5 expression pattern. The current data showed that CCR5 expression did not correlate with Th subsets in peripheral CD4⫹ memory cells, although CCR5 was down-regulated by the brief in vitro stimulation. However, CCR5 expression correlated with IFN-γ expression in some, but not all RA synovial samples and not with LT-α, which is also thought to be a Th1 cytokine, by synovial CD4⫹ memory T cells. Moreover, only 44% of IFN-γ expressing cells expressed CCR5 and 18% of IFN-γ– cells expressed CCR5. Furthermore, CCR5 expression did not correlate with IFN-γ expression by circulating RA blood CD4⫹ memory T cells (data not shown). Therefore, expression of CCR5 by synovial CD4⫹ memory T cells does not appear to be a reliable marker of activated Th1 cells in the synovium. These data indicate that although CCR5 expression by CD4⫹ memory T cells in RA synovial tissue correlated with IFN-γ expression in some patients, it is unlikely that this chemokine receptor could be a Th1 marker. Although CCR5 expression did not correlate with IFN-γ expression by peripheral CD4⫹ memory T cells, there was a correlation in one of three RA synovial CD4⫹ memory T cell samples. RA synovial CD4⫹ T cells might be stimulated by inflammatory cytokines and antigens in the synovium that could affect chemokine receptor and cytokine expression. Alternatively, CCR5- and IFN-γ-expressing cells could preferentially accumulate in the synovium of some RA patients. CD27–CD4⫹ memory T cells are a well-differentiated population that uniquely contains IL-4-producing Th2 cells (43) and comprises ~10–20% of peripheral CD4⫹ memory T cells (45). This population was therefore analyzed to obtain sufficient Th2 cells for an accurate assessment of their chemokine receptor expression. The frequency of IL-2-, IL-4- and IFN-γproducing cells was higher in the CD27–CD4⫹CD45RO⫹ memory T cell subset than that in the total CD4⫹ T memory cell population. Moreover, the ratio of Th0:Th1:Th2:IL-2 only-

producing cells indicated that Th2 cells were highly enriched in this population. It was noteworthy that the frequency of CCR3 expression by Th1 cells within the CD27–CD4⫹ memory cells was lower than that from total CD4⫹ memory T cells. Since CD27–CD4⫹ memory cells represent a final step in the process of CD4⫹ T cell differentiation occurring after multiple rounds of antigenic stimulation (62), recurrent stimulation appears to result in down-regulation of CCR3 by Th1 cells that persist in this subset. Interestingly, CXCR3 expression was up-regulated by CD27–CD4⫹ memory T cells compared to total CD4⫹ memory T cells. This suggests the possibility that well-differentiated T cells might express CXCR3 more than poorly differentiated T cells. We defined Th1 or Th2 cells by mRNA cytokine expression, and correlated chemokine receptor and cytokine mRNA expression. It must be emphasized that these studies did not examine the potential correlation between the expression of chemokine receptor and cytokine proteins by individual cells. Because of the complex regulation of expression of these molecules, differences could have emerged if the expression of chemokine receptor and cytokine protein had been determined. To accomplish this, however, would have required in vitro stimulation as cytokine expression could not be detected by flow cytometry without in vitro stimulation. Indeed, the in vitro stimulation that was employed to up-regulate cytokine expression, clearly modified chemokine receptor expression. The relevance of this to the analysis was emphasized by recent findings that cell-surface CCR5 expression could be down-regulated by in vitro stimulation (39,63). Although we employed in vitro stimulation with peripheral cells, the ex vivo analysis of cells from rheumatoid synovium was carried out without stimulation so that the characteristics of in vivo stimulation could be determined. This could only be carried out at the single-cell level using PCR analysis of mRNA. However, confirmation of these results using analysis of protein expression remains an important goal. The single-cell RT-PCR method is a powerful tool to analyze expression of many genes from one cell and to analyze correlations between gene expression (41,42). Moreover, the method appears to be capable of detecting relative differences in the amount of mRNA for different genes in the same cell, since amplification efficiencies of different cDNA are normalized by limiting the length of the cDNA and tailing the cDNA. Using this technique, the frequencies of cytokine expressing memory T cells were almost identical to that detected by intracellular staining after a similar brief in vitro stimulation (64). It should be noted, however, that cell-surface expression of CXCR3 has been reported to be higher than detected by this mRNA analysis (34,37). Efficiency for detecting CXCR3 mRNA by this method may be lower than cell-surface staining or mRNA expression may be regulated differently from cell-surface protein expression. In contrast, the frequencies of mRNA expression of CXCR4, CCR3 and CCR5 were almost the same as those previously reported for cell-surface expression (34,63). Therefore, the single-cell RTPCR method appears to provide a reasonably accurate assessment of cytokine and chemokine receptor expression by individual memory T cells. In conclusion, cytokine and chemokine receptor expression of peripheral CD4⫹ memory T cells has been analyzed

1666 Chemokine receptor and cytokine expression employing a single-cell RT-PCR method. The results showed that chemokine receptor expression does not correlate with Th defining cytokine production, suggesting that chemokine receptor expression cannot be used as a marker for Th1 or Th2 cells in vivo. Acknowledgements We thank Drs Kenji Hayashida, Laurie Davis, Hermann Girschick, Nancy Farner and Kiyoshi Shimooku for critical discussion, Ms Angie Mobley for assistance with cell sorting, and Ms Christine Pavlovitch, Rehana Hussain and Michelle McGuire for their technical support. This work was supported by NIH grant AR-39169.

Abbreviations CCR CXCR LT MCP MIP PE PHA PMA RA

CC chemokine receptor CXC chemokine receptor lymphotoxin monocyte chemotactic protein macrophage inflammatory protein phycoerythrin phytohemagglutinin phorbol myristate acetate rheumatoid arthritis

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