Molecular Interactions between T Cells and Fibroblast-Like Synoviocytes
2007; Elsevier BV; Volume: 171; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2007.070004
ISSN1525-2191
AutoresChinh Tran, Steven K. Lundy, Peter White, Judith Endres, Christopher D. Motyl, Raj Gupta, Cailin Moira Wilke, Eric A. Shelden, Kevin C. Chung, Andrew G. Urquhart, David A. Fox,
Tópico(s)Immune Cell Function and Interaction
ResumoThe mechanism of fibroblast-like synoviocyte (FLS) transformation into an inflammatory phenotype in rheumatoid arthritis (RA) is not fully understood. FLS interactions with invading leukocytes, particularly T cells, are thought to be a critical component of this pathological process. Resting T cells and T cells activated through the T-cell receptor have previously been shown to induce inflammatory cytokine production by FLS. More recently, a distinct population of T cells has been identified in RA synovium that phenotypically resembles cytokine-activated T (Tck) cells. Using time lapse microscopy, the interactions of resting, superantigen-activated, and cytokine-activated T cells with FLS were visualized. Rapid and robust adhesion of Tck and superantigen-activated T cells to FLS was observed that resulted in flattening of the T cells and a crawling movement on the FLS surface. Tck also readily activated FLS to produce interleukin IL-6 and IL-8 in a cell contact-dependent manner that was enhanced by exogenous IL-17. Although LFA-1 and ICAM-1 co-localized at the Tck-FLS synapse, blocking the LFA-1/ICAM-1 interaction did not substantially inhibit Tck effector function. However, antibody blocking of membrane tumor necrosis factor (TNF)-α on the Tck surface did inhibit FLS cytokine production, thus illustrating a novel mechanism for involvement of TNF-α in cell-cell interactions in RA synovium and for the effectiveness of TNF-α blockade in the treatment of RA. The mechanism of fibroblast-like synoviocyte (FLS) transformation into an inflammatory phenotype in rheumatoid arthritis (RA) is not fully understood. FLS interactions with invading leukocytes, particularly T cells, are thought to be a critical component of this pathological process. Resting T cells and T cells activated through the T-cell receptor have previously been shown to induce inflammatory cytokine production by FLS. More recently, a distinct population of T cells has been identified in RA synovium that phenotypically resembles cytokine-activated T (Tck) cells. Using time lapse microscopy, the interactions of resting, superantigen-activated, and cytokine-activated T cells with FLS were visualized. Rapid and robust adhesion of Tck and superantigen-activated T cells to FLS was observed that resulted in flattening of the T cells and a crawling movement on the FLS surface. Tck also readily activated FLS to produce interleukin IL-6 and IL-8 in a cell contact-dependent manner that was enhanced by exogenous IL-17. Although LFA-1 and ICAM-1 co-localized at the Tck-FLS synapse, blocking the LFA-1/ICAM-1 interaction did not substantially inhibit Tck effector function. However, antibody blocking of membrane tumor necrosis factor (TNF)-α on the Tck surface did inhibit FLS cytokine production, thus illustrating a novel mechanism for involvement of TNF-α in cell-cell interactions in RA synovium and for the effectiveness of TNF-α blockade in the treatment of RA. The normal synovial lining is one or two cell layers thick, contains both synovial macrophages and fibroblast-like synoviocytes (FLS), and is responsible for maintaining synovial fluid homeostasis. In rheumatoid arthritis (RA), the synovial lining transforms into the synovial pannus, a multicellular tissue composed of hyperplastic FLS closely intertwined with invading lymphocytes and antigen-presenting cells. The current consensus is that FLS are not merely bystanders in the inflammatory process but are active participants in joint destruction.1Firestein GS Invasive fibroblast-like synoviocytes in rheumatoid arthritis. Passive responders or transformed aggressors?.Arthritis Rheum. 1996; 39: 1781-1790Crossref PubMed Scopus (564) Google Scholar, 2Kontoyiannis D Kollias G Fibroblast biology. Synovial fibroblasts in rheumatoid arthritis: leading role or chorus line?.Arthritis Res. 2000; 2: 342-343Crossref PubMed Scopus (37) Google Scholar, 3Meinecke I Rutkauskaite E Gay S Pap T The role of synovial fibroblasts in mediating joint destruction in rheumatoid arthritis.Curr Pharm Des. 2005; 11: 563-568Crossref PubMed Scopus (49) Google Scholar, 4Mor A Abramson SB Pillinger MH The fibroblast-like synovial cell in rheumatoid arthritis: a key player in inflammation and joint destruction.Clin Immunol. 2005; 115: 118-128Crossref PubMed Scopus (338) Google Scholar, 5Müller-Ladner U Kriegsmann J Franklin BN Matsumoto S Geiler T Gay RE Gay S Synovial fibroblasts of patients with rheumatoid arthritis attach to and invade normal human cartilage when engrafted into SCID mice.Am J Pathol. 1996; 149: 1607-1615PubMed Google Scholar However, the mechanisms underlying this inflammatory transformation are not yet well understood. The most prominent invading lymphocyte in the RA synovium is the T cell.6Abrahamsen TG Froland SS Natvig JB Pahle J Elution and characterization of lymphocytes from rheumatoid inflammatory tissue.Scand J Immunol. 1975; 4: 823-830Crossref PubMed Google Scholar, 7Bankhurst AD Husby G Williams Jr, RC Predominance of T cells in the lymphocytic infiltrates of synovial tissues in rheumatoid arthritis.Arthritis Rheum. 1976; 19: 555-562Crossref PubMed Scopus (66) Google Scholar, 8Van Boxel JA Paget SA Predominantly T-cell infiltrate in rheumatoid synovial membranes.N Engl J Med. 1975; 293: 517-520Crossref PubMed Scopus (296) Google Scholar T-cell interactions with professional antigen-presenting cells have been well characterized, but in vitro studies have also shown that T cells can adhere to and activate FLS. Resting, unstimulated T cells have been shown to alter gene expression in FLS and cause release of interleukin IL-6, IL-8, and prostaglandin E2,9Yamamura Y Gupta R Morita Y He X Pai R Endres J Freiberg A Chung K Fox DA Effector function of resting T cells: activation of synovial fibroblasts.J Immunol. 2001; 166: 2270-2275PubMed Google Scholar and this is augmented by the T-cell cytokine IL-17,9Yamamura Y Gupta R Morita Y He X Pai R Endres J Freiberg A Chung K Fox DA Effector function of resting T cells: activation of synovial fibroblasts.J Immunol. 2001; 166: 2270-2275PubMed Google Scholar which is present in RA synovium.10Chabaud M Durand JM Buchs N Fossiez F Page G Frappart L Miossec P Human interleukin-17: a T cell-derived proinflammatory cytokine produced by the rheumatoid synovium.Arthritis Rheum. 1999; 42: 963-970Crossref PubMed Scopus (810) Google Scholar This effector function of resting T cells was not confined to a unique T-cell subset, as CD4+, CD8+, CD45RA+, and CD45RO+ T cells all had a similar capacity to activate FLS. T cells recovered from RA synovial fluid likewise elicited strong inflammatory responses when cultured with FLS, in particular up-regulating the immune-modulating cytokine IL-15.11Miranda-Carús ME Balsa A Benito-Miguel M Perez de Ayala C Martin-Mola E IL-15 and the initiation of cell contact-dependent synovial fibroblast-T lymphocyte cross-talk in rheumatoid arthritis: effect of methotrexate.J Immunol. 2004; 173: 1463-1476PubMed Google Scholar As a prototype for autoreactive T cells that are potentially arthritogenic, collagen II-stimulated T cells enhanced FLS production of many inflammatory mediators, and this effector function appeared to be proportional to the length of T-cell activation with collagen.12Kim WU Cho ML Jung YO Min SY Park SW Min DJ Yoon JH Kim HY Type II collagen autoimmunity in rheumatoid arthritis.Am J Med Sci. 2004; 327: 202-211Crossref PubMed Scopus (54) Google Scholar A distinct T-cell activation state has been characterized for its ability to induce tumor necrosis factor TNF-α production from monocytes. These cytokine-activated T cells (Tck) were activated in vitro by a cocktail of IL-6, IL-2, and TNF-α, without T-cell antigen receptor (TCR) ligation. Similarities of function and surface markers have been established between Tck cells and a population of T cells that are abundant in RA synovia.13Brennan FM Hayes AL Ciesielski CJ Green P Foxwell BM Feldmann M Evidence that rheumatoid arthritis synovial T cells are similar to cytokine-activated T cells: involvement of phosphatidylinositol 3-kinase and nuclear factor κB pathways in tumor necrosis factor alpha production in rheumatoid arthritis.Arthritis Rheum. 2002; 46: 31-41Crossref PubMed Scopus (134) Google Scholar However, interactions of Tck cells with FLS have not yet been described. Conversely, FLS have also been shown to positively stimulate T cells. Simple co-culture of T cells with FLS enhances T-cell survival and prevents apoptosis.14Scott S Pandolfi F Kurnick JT Fibroblasts mediate T cell survival: a proposed mechanism for retention of primed T cells.J Exp Med. 1990; 172: 1873-1876Crossref PubMed Scopus (67) Google Scholar, 15Salmon M Scheel-Toellner D Huissoon AP Pilling D Shamsadeen N Hyde H D'Angeac AD Bacon PA Emery P Akbar AN Inhibition of T cell apoptosis in the rheumatoid synovium.J Clin Invest. 1997; 99: 439-446Crossref PubMed Scopus (234) Google Scholar More robust responses have also been reported. FLS can induce naive T-cell proliferation by presentation of superantigen.16Tsai C Diaz Jr, LA Singer NG Li LL Kirsch AH Mitra R Nickoloff BJ Crofford LJ Fox DA Responsiveness of human T lymphocytes to bacterial superantigens presented by cultured rheumatoid arthritis synoviocytes.Arthritis Rheum. 1996; 39: 125-136Crossref PubMed Scopus (58) Google Scholar, 17Brinckerhoff CE Guyre PM Increased proliferation of human synovial fibroblasts treated with recombinant immune interferon.J Immunol. 1985; 134: 3142-3146PubMed Google Scholar When co-cultured with FLS, T cells up-regulate the activation markers CD69 and CD25,11Miranda-Carús ME Balsa A Benito-Miguel M Perez de Ayala C Martin-Mola E IL-15 and the initiation of cell contact-dependent synovial fibroblast-T lymphocyte cross-talk in rheumatoid arthritis: effect of methotrexate.J Immunol. 2004; 173: 1463-1476PubMed Google Scholar, 18Corrigall VM Solau-Gervais E Panayi GS Lack of CD80 expression by fibroblast-like synoviocytes leading to anergy in T lymphocytes.Arthritis Rheum. 2000; 43: 1606-1615Crossref PubMed Scopus (31) Google Scholar as well as many cytokines, such as IL-17, TNF-α, and interferon-γ.11Miranda-Carús ME Balsa A Benito-Miguel M Perez de Ayala C Martin-Mola E IL-15 and the initiation of cell contact-dependent synovial fibroblast-T lymphocyte cross-talk in rheumatoid arthritis: effect of methotrexate.J Immunol. 2004; 173: 1463-1476PubMed Google Scholar, 12Kim WU Cho ML Jung YO Min SY Park SW Min DJ Yoon JH Kim HY Type II collagen autoimmunity in rheumatoid arthritis.Am J Med Sci. 2004; 327: 202-211Crossref PubMed Scopus (54) Google Scholar FLS can also present peptides derived from type II collagen and human cartilage glycoprotein 39 to T-cell hybridomas specific to those peptides.19Tran CN Davis MJ Tesmer LA Endres JL Motyl CD Smuda C Somers EC Chung KC Urquhart AG Lundy SK Kovats S Fox DA Presentation of arthritogenic peptide to antigen-specific T cells by fibroblast-like synoviocytes.Arthritis Rheum. 2007; 56: 1497-1506Crossref PubMed Scopus (72) Google Scholar These antigen-dependent interactions of FLS with T cells are contact-dependent and can be blocked by neutralizing antibodies to class II major histocompatibility complex19Tran CN Davis MJ Tesmer LA Endres JL Motyl CD Smuda C Somers EC Chung KC Urquhart AG Lundy SK Kovats S Fox DA Presentation of arthritogenic peptide to antigen-specific T cells by fibroblast-like synoviocytes.Arthritis Rheum. 2007; 56: 1497-1506Crossref PubMed Scopus (72) Google Scholar and LFA-1 or ICAM-1.16Tsai C Diaz Jr, LA Singer NG Li LL Kirsch AH Mitra R Nickoloff BJ Crofford LJ Fox DA Responsiveness of human T lymphocytes to bacterial superantigens presented by cultured rheumatoid arthritis synoviocytes.Arthritis Rheum. 1996; 39: 125-136Crossref PubMed Scopus (58) Google Scholar The current studies were undertaken to better define the molecular basis of T-cell/FLS functional interactions. We first sought to directly record in vitro interactions of resting, superantigen-activated, and Tck T cells with FLS by time lapse microscopy. Both Tck and superantigen-activated T cells formed tight associations with FLS that followed similar morphology and kinetics. By further characterizing the interaction of FLS with Tck, it was found that Tck were able to stimulate FLS to produce proinflammatory cytokines in a contact-dependent manner similar to previous observations with resting and antigen-stimulated T cells. However, unlike other T-cell/FLS interactions that were blocked with antibodies to LFA-1/ICAM-1, Tck cell interactions with FLS were not substantially inhibited by antibodies to LFA-1 or ICAM-1 but were in part dependent on surface TNF-α expression by Tck. All procedures involving specimens obtained from human patients were performed under protocols approved by the University of Michigan Institutional Review Board. FLS were obtained from human synovial tissue obtained at arthroplasty or synovectomy from RA joints. RA diagnosis was based on the presence of at least four of the seven American College of Rheumatology criteria for RA.20Arnett FC Edworthy SM Bloch DA McShane DJ Fries JF Cooper NS Healey LA Kaplan SR Liang MH Luthra HS The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis.Arthritis Rheum. 1988; 31: 315-324Crossref PubMed Scopus (18643) Google Scholar FLS were isolated by digestion of synovial tissue with 1 mg/ml of type II collagenase (Worthington Biochemical, Lakewood, NJ) for 2 to 24 hours followed by adherence in cell culture flasks and vigorous washes to remove debris and nonadherent cells.21Zimmermann T Kunisch E Pfeiffer R Hirth A Stahl H-D Sack U Laube A Liesaus E Roth A Palombo-Kinne E Emmrich F Kinne RW Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary culture—primary culture cells markedly differ from fourth-passage cells.Arthritis Res. 2001; 3: 72-76Crossref PubMed Scopus (155) Google Scholar Adherent cells were grown to confluency in CMRL medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (Atlanta Biological, Lawrenceville, GA), 2 mmol/L glutamine (Cambrex, East Rutherford, NJ), 50 U/ml penicillin (Cambrex), and 50 μg/ml streptomycin (Cambrex) and then passaged into fresh flasks. RA FLS used before passage 4 were depleted of CD14-positive cells at passage 1 by magnetic bead separation (Miltenyi Biotech, Auburn, CA) to remove contaminating monocyte/macrophage lineage cells. T cells were obtained from peripheral blood by negative selection using RosetteSep, human T-cell enrichment cocktail (Stemcell Technologies, Vancouver, BC, Canada) and separation on a Ficoll gradient following the manufacturer's protocol. In brief, freshly isolated blood from healthy volunteers was incubated with antibodies for 20 minutes at room temperature. Blood was diluted 1:1 with 2% newborn calf serum/phosphate-buffered saline (PBS) before overlay onto room temperature Ficoll and centrifugation at 1200 relative centrifugal force with the brake off. The buffy coat containing only T cells was recovered and used for subsequent experiments. Trest were unstimulated T cells. Tck were cytokine-activated T cells stimulated by a cocktail of 25 ng/ml TNF-α, 100 ng/ml IL-6, and 25 ng/ml IL-2 in RPMI 1640 medium containing 10% fetal calf serum, 2 mmol/L l-glutamine, penicillin, and streptomycin for 8 days13Brennan FM Hayes AL Ciesielski CJ Green P Foxwell BM Feldmann M Evidence that rheumatoid arthritis synovial T cells are similar to cytokine-activated T cells: involvement of phosphatidylinositol 3-kinase and nuclear factor κB pathways in tumor necrosis factor alpha production in rheumatoid arthritis.Arthritis Rheum. 2002; 46: 31-41Crossref PubMed Scopus (134) Google Scholar and washed three times before use. Tsea were T cells co-cultured with FLS in the presence of 40 ng/ml of the superantigen Staphylococcal enterotoxin A.16Tsai C Diaz Jr, LA Singer NG Li LL Kirsch AH Mitra R Nickoloff BJ Crofford LJ Fox DA Responsiveness of human T lymphocytes to bacterial superantigens presented by cultured rheumatoid arthritis synoviocytes.Arthritis Rheum. 1996; 39: 125-136Crossref PubMed Scopus (58) Google Scholar FLS were seeded into 12-well plates at a density of 10,000 cells/well and allowed to adhere for 48 hours before the addition of 250,000 cytokine-activated (Tck) cells. Where indicated, recombinant human IL-17 (R&D Systems, Minneapolis, MN) was added to co-cultures to yield a final concentration of 20 ng/ml. Final culture volumes were 1.5 ml/well. Co-culture times were between 24 and 72 hours depending on the assay. Co-culture supernatants were harvested and assayed by enzyme-linked immunosorbent assay (ELISA). ELISAs for IL-6 and IL-8 were performed following the manufacturer's protocols (BD Biosciences, San Jose, CA). Capture antibodies were coated on enzyme immunoassay plates at a 1:250 dilution in carbonate buffer (pH 9.5) overnight at 4°C. Plates were washed and blocked with PBS:10% fetal calf serum for 1 hour at room temperature before the addition of culture supernatants and recombinant standards followed by reincubation at room temperature. After 2 hours, culture supernatant was washed away and 1:250 dilutions of biotinylated detection antibodies and streptavidin-horseradish peroxidase were added for 1 hour at room temperature. Plates were then washed seven times, 0.1 ml of a 1:1 mixture of TMB substrate was added to each well, and substrate conversion was terminated by addition of 0.1 ml of 2 N H2SO4. Absorbance was read at 450 nm, and sample values were compared with the standard curves. RA FLS (10,000/well) were plated on 24-well ImageLock plates (Essen Instruments, Ann Arbor, MI) and cultured in CMRL media containing 10% fetal bovine serum and antibiotics in an atmosphere of 5% CO2 for 2 days. Culture medium was removed, and FLS were washed twice with serum-free RPMI 1640 medium before the addition of 250,000 prewarmed, 5(6)-carboxyfluorescein diacetate N-succinimidyl ester-labeled, T cells in RPMI 1640 medium containing 10% fetal calf serum, 2 mmol/L l-glutamine, penicillin, and streptomycin. Plates containing T cells and FLS were immediately placed into IncuCyte-FLR imaging system (Essen Instruments) that is equipped with an LED for excitation at a nominal center wavelength of 470 nm and a FWHM bandwidth of 25 nm. This source is filtered with a short-pass filter with a cutoff wavelength of 490 nm, which ensures that there are no tails of the LED illumination that would get through the emission filter. The detection filter used is a bandpass filter with a 75-nm width centered at 545 nm. The chamber is designed to fit into a standard, humidified, CO2 incubator, and a moving objective allows the cell culture to be stationary while images are captured at different positions from well to well. Images (1-μm resolution) were collected at 10-minute intervals starting 30 minutes after addition of the plate to the IncuCyte-FLR chamber. Movies were generated using IncuCyte software (Essen) at three frames/second, which is equivalent to 30 minutes of culture/second. Still images were cropped and expanded to four times the size of the original images and then exported to Microsoft PowerPoint (Microsoft, Redmond, WA). For co-culture studies comparing CD11a and ICAM-1 localization, FLS were transfected with an expression vector for a fusion protein of ICAM-1 and eGFP. This expression vector was termed CD54-eGFP and was created as follows. Primers were constructed against the sequence described by accession BC015969 to polymerase chain reaction (PCR) amplify the open reading frame (without the stop codon) of human ICAM-1. A HindIII restriction site added to the beginning of 5′ primer and an AgeI site added to the tail of the 3′ primer facilitated ligation of the amplified fragment into the pEGFP-N1 expression vector (Clontech, Palo Alto, CA). After ligation, the insert sequence was verified to be free of mutation. This resulted in a CD54-eGFP fusion protein expression vector, with ICAM-1 at the N terminus and eGFP at the C terminus (intracellular) and a short peptide linker (GSTPVAT) in between. Transfection was performed using an adult human dermal fibroblast kit (Amaxa, Gaithersburg, MD), and manufacturer's protocols were observed. FLS (20,000 to 50,000 cells) were grown on glass coverslips in six-well plates for 48 hours. Medium was changed, and 500,000 to 1 million Tck cells were added to the cultures. Co-cultures were allowed to interact for 1 to 24 hours before coverslips were washed with PBS and fixed in 4% paraformaldehyde/PBS for 1 hour at room temperature or overnight at 4°C. After fixation, coverslips were washed in 2% newborn calf serum/PBS to remove residual paraformaldehyde and blocked in 5% nonfat milk/PBS for 30 minutes. Coverslips were again washed with 2% newborn calf serum/PBS. Primary antibody used for staining was mouse anti-human CD11a (clone HB202; a kind gift of Bruce Richardson, University of Michigan, Ann Arbor, MI).16Tsai C Diaz Jr, LA Singer NG Li LL Kirsch AH Mitra R Nickoloff BJ Crofford LJ Fox DA Responsiveness of human T lymphocytes to bacterial superantigens presented by cultured rheumatoid arthritis synoviocytes.Arthritis Rheum. 1996; 39: 125-136Crossref PubMed Scopus (58) Google Scholar Secondary antibody used was goat anti-mouse IgG Alexa 594 (Invitrogen-Molecular Probes, Carlsbad, CA). Confocal imaging was performed using an FV-500 microscope (Olympus, Center Valley, PA) with excitation by argon 488 nm and HeNeG 543 lasers (×60 magnification, 1.4 NA, UPLAN APO). FLS were seeded into 12-well plates at a density of 10,000 per well and allowed to adhere for 48 hours to the bottom chamber. Transwell inserts with a 0.4-μm membrane pore size were placed on top of the wells containing FLS. These transwell inserts had been placed in medium for 24 hours before the addition to plates containing FLS, to saturate the membrane. Tck (n = 500,000) were added to the bottom chamber or into the transwell insert. Total volume in the bottom chamber was 1.5 ml. Total volume in the transwell was 0.5 ml. IL-17 was added to the transwell insert at a 0.04, 0.4, and 4 ng/ml concentration in 0.5 ml but would diffuse through the transwell to give a final concentration of 0.01, 0.1, and 1.0 ng/ml, respectively. FLS were seeded into 12-well plates at a density of 10,000 cells/well and allowed to adhere for 48 hours. Antibodies used for blocking studies were anti-huCD11a (anti-LFA-1, clone HB202; B. Richardson, University of Michigan), anti-huCD49d (anti-VLA-4, clone BU49; Ancell, Bayport, MN), anti-huTNF-α (clones 28401 or 6401; R&D Systems), or control IgG from normal mouse serum (Hybridoma Core, University of Michigan). Tck were incubated at room temperature with 10 μg/ml of blocking antibodies for 2 hours and then washed before addition of 250,000 Tck cells to washed adherent FLS lines. Supernatants were collected after 72 hours of co-culture and tested for IL-6 and IL-8 by ELISA. In some experiments, one of two TNF-α blocking antibodies from different clones (28401 or 6401; R&D Systems) or control mouse IgG were used to coat Tck at 1 μg/ml for 30 minutes at room temperature. Tck cells (250,000 cells/well) were then added to FLS-containing wells with additional antibody added to the co-culture to maintain a final antibody concentration of 1 μg/ml. IL-17 was titrated into these co-cultures in the following concentrations: 0.02, 0.2, and 2.0 ng/ml (we found similar results using 2 and 20 ng/ml IL-17, data not shown). Co-cultures were then maintained for 24 to 72 hours before collection of supernatants. For studies that examined blocking of surface TNF-α, Tck were incubated for 30 minutes with control or TNF-α-specific antibodies. Excess unbound antibody was removed by washes with 2% newborn calf serum in PBS. These preblocked Tck were used to stimulate FLS. Alternatively, anti-TNF-α or control MsIg antibodies were added directly to the co-cultures of Tck and RA FLS at a final concentration of 1 μg/ml. Supernatants were harvested after 36 hours of co-culture and IL-8 was detected by ELISA. Trest and Tck were obtained from the same donor. U937 cells were treated with phorbol 12-myristate 13-acetate (2 μg/ml) for 2 hours before harvest. RNA was extracted from 5 million cells of each cell type using the RNeasy kit (Qiagen, Valencia, CA). Reverse transcription was performed on 2 μg of RNA from each cell type for 50 minutes at 37°C. A second reverse transcription was performed after treating the RNA with 1 U of RNase to exclude the possibility of amplifying contaminating genomic DNA. The resultant cDNA products were diluted 2:3 before use in PCR. One μl of the diluted cDNA was used for each PCR reaction. TNF-α primer sequences were 5′-CAGAGGGAAGAGTTCCCCAG-3′ for the upstream primer and 5′-CCTTGGTCTGGTAGGAGACG-3′ for the downstream primer.22Hauber HP Mikkila A Erich JM Kroger N Meyer A Schoder V Zander AR Pforte A TNFα, interleukin-10 and interleukin-18 expression in cells of the bronchoalveolar lavage in patients with pulmonary complications following bone marrow or peripheral stem cell transplantation: a preliminary study.Bone Marrow Transplant. 2002; 30: 485-490Crossref PubMed Scopus (22) Google Scholar β-Actin served as the control. The β-actin primers were 5′-GTGGGGCGCCCCAGGCACCA-3′ for the upstream primer and 5′-CTCCTTAATGTCACGCACGATTTC-3′ for the downstream primer. PCR amplification was performed in a PX2 thermocycler (Thermo Hybaid, Franklin, MA) as follows: first step, one cycle of 94°C for 3 minutes; second step, 30 cycles (1 minute at 94°C, 1 minute at 60°C, 40 seconds at 72°C); and third step, one cycle extension for 5 minutes at 72°C. PCR products were analyzed by electrophoresis on a 2% agarose gel containing 1× SYBR Safe DNA gel stain (Invitrogen), and band size was compared with a 100-bp molecular weight marker (Promega, Madison, WI). Previous work in our laboratory used two types of T cells, Trest and Tsea,9Yamamura Y Gupta R Morita Y He X Pai R Endres J Freiberg A Chung K Fox DA Effector function of resting T cells: activation of synovial fibroblasts.J Immunol. 2001; 166: 2270-2275PubMed Google Scholar, 16Tsai C Diaz Jr, LA Singer NG Li LL Kirsch AH Mitra R Nickoloff BJ Crofford LJ Fox DA Responsiveness of human T lymphocytes to bacterial superantigens presented by cultured rheumatoid arthritis synoviocytes.Arthritis Rheum. 1996; 39: 125-136Crossref PubMed Scopus (58) Google Scholar which could be viewed as representing polar states of T-cell stimulation from naïve to full activation. An alternative of T-cell activation has been described, termed cytokine-activated T cells (Tck). Tck are generated by stimulation independent of the TCR by a cocktail of cytokines: TNF-α, IL-6, and IL-2. These cytokines, especially TNF-α and IL-6, are found within synovial tissues. Tck have been documented to possess effector function identical to T cells purified from RA synovial fluid, most notably in the induction of TNF production by monocytes. The stimulatory mechanism of these cells is clearly distinct from T cells activated through CD3/CD28 cross-linking.13Brennan FM Hayes AL Ciesielski CJ Green P Foxwell BM Feldmann M Evidence that rheumatoid arthritis synovial T cells are similar to cytokine-activated T cells: involvement of phosphatidylinositol 3-kinase and nuclear factor κB pathways in tumor necrosis factor alpha production in rheumatoid arthritis.Arthritis Rheum. 2002; 46: 31-41Crossref PubMed Scopus (134) Google Scholar We therefore sought to explore Tck interactions with FLS. The method of generation of Tck yielded a nearly homogeneous population of 94% CD3-positive T cells with little or no contamination by B cells, monocytes, or fibroblasts (see Supplemental Table 1 at http://ajp.amjpathol.org). To observe the physical interactions between FLS and differentially activated T cells, co-cultures were filmed using time lapse microscopy (see Supplemental Video S1A–C at http://ajp.amjpathol.org). Three activation states of T cells were used, unstimulated (Trest), Staphylococcal enterotoxin A-stimulated (Tsea), and cytokine-activated T cells (Tck). Representative images from time lapse microscopy of the co-cultures show significant cellular interaction of the 5(6)-carboxyfluorescein diacetate N-succinimidyl ester-labeled Tck and Tsea but not Trest cells with FLS within the first 30 minutes of co-culture (Figure 1, A–C). When the activated T cells adhered to FLS, they acquired an enlarged, darker appearance (indicated by white arrows) than T cells with only minimal interactions. In contrast, Trest cells maintained a smaller, brighter appearance despite being proximal to FLS, and unlike their activated counterparts, did not appear to crawl over the surface of the FLS. Further evidence of a difference in interactions between Trest and activated T cells can be witnessed by following the movement of individual cells in relation to the movement of the FLS (see Supplemental Video S1A–C at http://ajp.amjpathol.org). When FLS moved in the presence of Trest, the T cells tended to break contact with the FLS and move to another position in the well. In contrast, Tck and Tsea tended to stay attached to one FLS throughout the 24-hour assay period despite the movement of the FLS. No significant differences between the interactions of Tsea or Tck with FLS were observed using time lapse microscopy. FLS cultures were stimulated by the addition of Tck and allowed to interact for 36 hours. The cytokine IL-17, produced by RA synovial tissues,10Chabaud M Durand JM Buchs N Fossiez F Page G Frappart L Miossec P Human interleukin-17: a T cell-derived proinflammatory cytokine produced by the rheumatoid synovium.Arthritis Rheum. 1999; 42: 963-970Crossref PubMed Scopus (810) Google Scholar has been shown to cooperate with Trest in the activation of FLS9Yamamura Y Gupta R Morita Y He X Pai R Endres J Freiberg A Chung K Fox DA
Referência(s)