Interaction of bovine uterine luminal protein with interleukin-2 and the interleukin-2 receptor of T lymphocytes

Bovine uterine luminal proteins (ULP) collected on Day 17 of pregnancy were tested for inhibition of binding of interleukin-2 (IL-2) to the IL-2 receptor (IL-2R) of bovine (CLC) and human (HLC) T lymphocytes and for binding to IL-2. Additional experiments assessed IL-2 binding to the p55 alpha chain (Tac protein) of the IL-2R of HLC. High- and low-molecular weight (Mr) ULP components (H-ULP greater than 248,000 Mr and L-ULP 21,000 Mr, respectively) inhibited (p less than 0.05 and 0.01, respectively) the binding of 125I-IL-2 to the IL-2R of CLC, whereas only H-ULP inhibited (p less than 0.05) binding to the IL-2R (presumably, the p75 beta chain) of HLC. H-ULP failed (p greater than 0.05) to bind to the p55 alpha chain of the IL-2R of HLC. For IL-2 binding, L-ULP failed (p greater than 0.05) to bind 125I-IL-2 in short (2 h)-term and long (45 h)-term experiments, whereas binding was evident (p less than 0.05) for H-ULP at 2 h of incubation. For H-ULP, mean (+/- SEM) percentages for bound and unbound 125I-IL-2 were 70.1 +/- 11.4 and 29.9 +/- 11.4, respectively. Further purification of H-ULP yielded a component (1.76 x 10(6) Mr) that bound 11.7% of 125I-IL-2 and inhibited (p less than 0.01) thymidine uptake and binding of 125I-IL-2 to the IL-2R of CLC. H-ULP-mediated suppression of lymphocyte proliferation may result from blocking IL-2R recognition of IL-2 as well as binding to IL-2, whereas suppression by L-ULP may predominantly result from blocking IL-2R.     

Expression of PTHrP and its cognate receptor in the rheumatoid synovial microcirculation

Genome sequence analyses revealed the occurrence of two paralogous ppa genes potentially encoding distinct Family I inorganic pyrophosphatases (sPPases, EC3.6.1.1) in the marine unicellular cyanobacteria Prochlorococcus marinus strains MED4 and MIT9313 and Synechococcus sp. WH8102. Protein sequence alignment and phylogenetic analysis indicated that the ppa gene proper of cyanobacteria (ppa1) encodes a presumably inactive mutant enzyme whereas the second gene (ppa2) might encode an active sPPase closely related to those of some proteobacteria. Heterologous expression of the two cloned P. marinus MED4 ppa genes in Escherichia coli confirmed this proposal, only the inactive ppa1 product being immunodetected by anti-cyanobacterial sPPase antibodies. A possible scenario of ppa gene inactivation and replacement in the context of the postulated rapid diversification of marine unicellular cyanobacteria, the most abundant photosynthetic prokaryotes in the oceans, is discussed.     

Expression of interleukin-2 receptor (IL-2R) in feline peripheral blood lymphocytes

A monoclonal anti-interleukin-2 receptor (IL-2R) antibody has been identified as a putative antibody against the human IL-2R. In the present study, anti-Tac antibody CD-25 was used to determine cell expressing IL-2 receptor in feline peripheral blood lymphocytes by means of direct immunofluorescence tests and complement-mediated lymphocytotoxicity tests. With complement-mediated lymphocytotoxicity, approximately 18% of feline peripheral blood lymphocytes expressed the receptors. By the direct immunofluorescence test, we found approximately 22% of IL-2R positive cells in lymphocytes of feline peripheral-blood.     

Expression of interleukin-2 receptor on blood lymphocytes stimulated with allogeneic lymphocytes or autologous tumor cells

Summary The kinetics of interleukin-2 receptor (IL-2R) expression and the [³H]dT incorporation of blood lymphocytes after the first and the second stimulation with allogeneic leukocytes (primary and secondary MLC) or with the autologous tumor cells (primary and secondary MLTC) were compared. The expression of IL-2R paralleled the induction of DNA synthesis. The proportion of IL-2R⁺ cells of the unprimed donors peaked earlier in the secondary MLC as compared to the primary MLC (on days 3 and 5 respectively). In MLC of alloimmunized healthy individuals and in the MLTC of cancer patients the highest proportions of IL-2R⁺ cells were detected between days 2 and 3 after both the first and second stimulations. Thus the first in vitro stimulation in the MLTC showed similar kinetics to those of the secondary MLC of unprimed individuals and to the primary MLC response of the allo-immunized individuals. The findings in the MLTC substantiate the hypothesis that cancer patients can be sensitized to their own tumors. The kinetics of the appearance of the IL-2R together with the characteristics of the IL-2-propagated cultures provide useful information for the strategy of expansion of auto-tumor reactive lymphocyte populations.     

Characterization of the natural killer-like lymphocytes in rheumatoid synovial fluid

IgG Fc- cytotoxic cells found in the synovial fluid of patients with rheumatoid arthritis have natural killer (NK)-like characteristics but can kill NK-resistant cell lines as well. The phenotype of these cells was defined by complement-mediated lysis with monoclonal antibodies. The synovial fluid killer cell activity was significantly reduced by treatment with complement and OKT11 and 4F2, but the cytotoxic T cells did not express the NK-related antigens OKM1 and Leu-7, nor the cytotoxic T lymphocyte-specific antigen, OKT8. These results demonstrate that the synovial fluid killer cells resemble the activated T cells generated in an autologous mixed leukocyte reaction or in the treatment of peripheral blood mononuclear cells with interleukin 2, and they are distinct from the conventional NK cells found in blood.     

Atypical T cells in rheumatoid synovial membranes

Atypical mononuclear cells (AMC) with Sézary-cell-like morphology were demonstrated to be present in rheumatoid synovial membranes. These cells have exclusively T cell membrane characteristics, like identical cells previously found in rheumatoid synovival fluids. AMC were predominantly present in transitional areas between nodular lymphocytic aggregates and plasma cell rich areas. In ultrathin sections AMC accounted for approximately 30% of all lymphocyte cells; in studies of cells isolated from rheumatoid synovial membranes AMC comprised 30--40% of the E-rosette-forming cells. The hypothesis that these cells represent reactive T cells is discussed.     

Displacement of linker for activation of T cells from the plasma membrane due to redox balance alterations results in hyporesponsiveness of synovial fluid T lymphocytes in rheumatoid arthritis

The T lymphocytes that reside in the synovium of the inflamed joints in patients with rheumatoid arthritis display severe hyporesponsiveness upon antigenic stimulation, which is probably due to their constant subjection to high levels of oxidative stress. Here we report that the synovial fluid T lymphocytes exert severely impaired phosphorylation of the adaptor protein linker for activation of T cells (LAT), a crucial component of the TCR-mediated signaling pathways. In healthy T lymphocytes, LAT is a membrane-bound protein and becomes phosphorylated by zeta-associated protein of 70 kDa (ZAP-70) upon TCR engagement. The molecular basis underlying the deficient phosphorylation of LAT and consequently the hyporesponsiveness of the synovial fluid T lymphocytes lies in the membrane displacement of LAT. We demonstrate that the subcellular localization of LAT is sensitive to changes in the intracellular levels of the antioxidant glutathione. The membrane anchorage of LAT, and consequently the phosphorylation of LAT and the cellular activation of the synovial fluid T lymphocytes upon TCR engagement, is restored in synovial fluid T lymphocytes after supplementation of the intracellular glutathione levels with N-acetyl-l -cysteine. These data suggest a role for the membrane displacement of LAT in the hyporesponsiveness of the synovial fluid T lymphocytes as a consequence of oxidative stress.     

Expression of interleukin-2 receptor by activated peripheral blood lymphocytes upregulated by the plasma level of interleukin-2 in patients with recurrent aphthous ulcers

This study used flow cytometry to determine the peripheral blood lymphocyte subsets and a sandwich enzyme immunoassay to measure the plasma levels of interleukin-2 (IL-2) and soluble IL-2 receptor (sIL-2R) in 34 patients in different stages of recurrent aphthous ulcers (RAU) and in 32 age/sex-matched normal control subjects. In the exacerbation stage of RAU, a significant increase in the percentages of CD3+ (p < 0.001), CD4+ (p < 0.001), CD4+ IL-2R+ (p < 0.001), CD8+ IL-2R+ (p < 0.01) and IL-2R+ cells (p < 0.001), in the CD4+/CD8+ (p < 0.01) and CD4+/CD3+ CD8+ ratios (p < 0.01), and in the plasma level of IL-2 (p < 0.001) was found in the patients as compared with the levels in the normal control subjects. However, in the post-exacerbation stage of RAU, there was a significant decrease in the percentage of CD4+ cells (p < 0.05) and in the CD4+/CD8+ (p < 0.01) and CD4+/CD3+ CD8+ ratios (p < 0.001), as well as a significant increase in CD8+ cells (p < 0.001) in the patients, as compared with the levels in the normal control subjects. Because the CD4+, CD4+ IL-2R+ and CD8+ IL-2R+ cell counts and the plasma level of IL-2 increased simultaneously in the patients in the exacerbation stage of RAU, we suggest that the markedly increased plasma level of IL-2 may have been secreted by the increased number of activated CD4+ cells, and that the expression of IL-2R by activated peripheral blood lymphocytes was upregulated by the plasma level of IL-2 in patients with RAU. In addition, the increase and then decrease of the CD4+/CD8+ ratio in the RAU patients and the increased number of CD4+ IL-2R+ and CD8+ IL-2R+ activated T cells in the RAU patients support the role of cell-mediated cytotoxicity in the immunopathogenesis of RAU.     

Induction of cyclo-oxygenase by interleukin-1 in rheumatoid synovial cells

The ability of interleukin-1 (IL-1) to stimulate prostaglandin E2 (PGE2) production by human rheumatoid adherent synovial cells was found to be time-dependent and sensitive to protein synthesis inhibitors. Cells incubated with exogenous arachidonic acid (10 microM) showed no increase in PGE2 production. However, with IL-1 (2.5 U/ml) and exogenous arachidonic acid there was a marked increase, with levels reaching twice that for cells incubated with IL-1 alone. Aspirin pre-treatment studies and the use of [acetyl-14C]aspirin showed that IL-1 increased PGE2 production through the induction of cyclo-oxygenase.     

Cell-to-cell contacts between lymphoreticular cells in rheumatoid synovial membrane

Nodular type and diffuse perivascular lymphoid cell infiltrates in rheumatoid synovial membranes from 72 patients were examined under the electron microscope. Attempts were made to describe the ultrastructural relations of the cells examined and to classify the different types of cell-to-cell contracts between them. Members of a complex series, composed of gradually differentiating forms of cell contacts beginning from simple membrane adherences and interdigitations and gap junctions; minute filamentous intercellular bridges: subsurface confronting cisternae and desmosome-like structures, are described and suggested as function-dependent morphological representatives of sites of cellular interactions.     

Induction of cytotoxic T lymphocytes specific to malignant glioma using T2 cells pulsed with HLA-A2-restricted interleukin-13 receptor alpha 2 peptide in vitro

Interleukin-13 receptor α2 （IL-13Rα2） is a glioma-restricted cell-surface epitope not otherwise detected within the central nervous system. The present study is a report of a novel approach of targeting malignant glioma with IL-1 3Rα2-specific cytotoxic T lymphocyte （CTL） induced from the peripheral blood mononuclear cells of healthy donors by multiple stimulations with human leukocyte antigen （HLA）-A2-restricted IL-1 3Rα2345-353 peptide-pulsed T2 cells. The induced CTL showed specific lysis against T2 cells pulsed with the peptide and HLA-A2^＋ glioma cells expressing IL- 1 3Rα2345-353, while HLA-A2 glioma cell lines that express IL-13Rα2345-353 could not be recognized by CTL. The peptide-specific activity was inhibited by anti-HLA class I monoclonal antibody. These results suggest that the induced CTL specific for IL-1 3Rα2345-353 peptide could be a potential target of specific immunotherapy for HLA-A2 patients with malignant glioma.     

The combined effects of anti-TNFalpha antibody and IL-1 receptor antagonist in human rheumatoid arthritis synovial membrane

TNFalpha and IL-1 are the pivotal cytokines involved in rheumatoid arthritis (RA). More recently, the biological therapy targeting TNFalpha or IL-1 has been impressively effective for many RA patients, however, it remains insufficient in some patients. In the present study, we examined the combined effects of two agents against TNFalpha and IL-1 in human RA synovial membrane. Synovial explants (an ex vivo model) and synovial fibroblasts (an in vitro model) were prepared from 11 RA patients, and then anti-TNFalpha antibody (Anti-TNFalpha) and IL-1 receptor antagonist (IL-1Ra), either alone or in combination, were added to the synovial explants and fibroblasts. IL-6 and MMP-3 production were measured after incubation. As a result, their production significantly decreased by the combination of agents compared with the control group in both the synovial explants and fibroblasts. The efficacy of this combination was also observed for IL-6 production compared with each agent alone in the synovial explants, and for IL-6 and MMP-3 production compared with each agent alone in the synovial fibroblasts. Therefore, the combination of Anti-TNFalpha and IL-1Ra appears more beneficial in synovial membrane, particularly when compared with a single agent alone.     

Interleukin-12 induces gene expression in interleukin-2 stimulated human T lymphocytes

IL-12 is a critical immunoregulatory cytokine that promotes cell-mediated immune responses by inducing the differentiation of Th1 cells. To better clarify the molecular basis of IL-12 action, we compared the gene expression in human T lymphocytes activated by IL-2 and IL-12. mRNAs from T lymphocytes activated by either IL-2 alone or IL-2 plus IL-12 were transcribed into cDNAs. A differential mRNA display was conducted. As a result, differential display of five cDNA fragments was obtained. Sequence analysis suggests that they had high homology with recorded genes as found by a computer search against GenBank. Two full genes of the five fragments were cloned, which activation-induced C-type lectin and glucose transporter-like protein. Interestingly, these proteins were expressed in the T cells stimulated by IL-2 and IL-12, but not in the T cells stimulated by IL-2 alone. These results suggest that C-type lectin and glucose transporter-like protein may play an important role in the T lymphocyte activation induced by IL-12.     

Dominant and shared T cell receptor beta chain variable regions of T cells inducing synovial hyperplasia in rheumatoid arthritis.

Previously, we demonstrated the presence of at least two distinct subpopulations of patients with rheumatoid arthritis (RA) employing a cell-transfer experiment using severe combined immunodeficient (SCID) mice. One group of patients, whose T cells derived from the rheumatoid joints, induced synovial hyperplasia (SH) in the SCID mice (the positive group). The other group did not display the induction of SH (the negative group). TCR/Vbeta gene usage analysis indicated that some dominant T cell subpopulations were oligoclonally expanding only in the rheumatoid joints, and not in the periphery of the patients of the positive group. Moreover, these T cell subpopulations were not seen in the joints of patients in the negative group or in non-RA patients. In addition, the preferential uses of certain TCR/Vbetas (Vbeta8, Vbeta12, Vbeta13, and Vbeta14) genes were demonstrated in these T cells. In this study, to investigate whether these T cells are driven by a certain antigen(s), the third complementarity determining regions (CDR3s) of TCR/Vbeta, especially Vbeta8 and Vbeta14 PCR products, were cloned and sequenced. As a result, a dominant CDR3 sequence, CASS-PRERAT-YEQ, was found in Vbeta14+ T cells from the rheumatoid joint of a patient (Patient 1) of the positive group with a Vbeta14 skew. The identical CDR3 sequence also predominated in Vbeta14+ T cells from the rheumatoid joint of another patient (Patient 7) of the positive group with a Vbeta14 skew. In addition, in the patients (Patients 4, 7, 8) of the positive group with a Vbeta8 skew, other dominant CDR3 sequences, CASS-ENS-YEQ and CASS-LTEP-DTQ, were found as in the case of Vbeta14. However, no identical CDR3 sequences were detected dominantly in the joints of the patients in the negative group or in non-RA patients. A Vbeta14+ T cell clone (TCL), named G3, with the identical CDR3 sequence, CASS-PRERAT-YEQ, was isolated successfully from Patient 1, and cell transfer of G3 with autologous irradiated peripheral mononuclear cells induced SH in the SCID mice. Taken together, these results suggest that T cells inducing SH, thought to be pathogenic for RA, might be driven by a certain shared antigen(s).     

Chemokine secretion of rheumatoid arthritis synovial fibroblasts stimulated by Toll-like receptor 2 ligands

To analyze the role of Toll-like receptors (TLR) in the pathogenesis of rheumatoid arthritis, we have assessed the effects of stimulation of cultured synovial fibroblasts by the TLR-2 ligand bacterial peptidoglycan. By using high density oligonucleotide microarray analysis we identified 74 genes that were up-regulated >2.5-fold. Fourteen CC and CXC chemokine genes were among the genes with the highest up-regulation. Quantitative real-time PCR analysis confirmed up-regulation of granulocyte chemotactic protein (GCP)-2, RANTES, monocyte chemoattractant protein (MCP)-2, IL-8, growth-related oncogene-2, and to a lesser extent, macrophage-inflammatory protein 1alpha, MCP-1, EXODUS, and CXCL-16. GCP-2, RANTES, and MCP-2 were detected in culture supernatants of synovial fibroblasts stimulated with peptidoglycan. Chemokine secretion induced by stimulation of rheumatoid arthritis synovial fibroblasts via TLR-2 was functionally relevant as demonstrated by chemotaxis assays. GCP-2 and MCP-2 expression, which have not been reported previously in rheumatoid arthritis, was demonstrated in synovial tissue sections of patients diagnosed with rheumatoid arthritis but not in those with osteoarthritis. Correspondingly, synovial fluid levels were significantly higher in patients diagnosed with rheumatoid arthritis as compared with osteoarthritis. Thus, we present evidence for an induction of chemokine secretion by activation of synovial fibroblasts via TLR-2, possibly contributing to the formation of inflammatory infiltrates characteristically found in rheumatoid arthritis joints.     

Cells of the synovium in rheumatoid arthritis. T lymphocytes

Recent findings have substantiated the importance of T lymphocytes to the pathogenesis of rheumatoid arthritis (RA). Here, we review emerging data regarding genetic predisposition, spontaneous animal models of arthritis, and cell-cell interactions that implicate T cells as driving synovial inflammation and joint destruction. Information regarding the proinflammatory role of interleukin-17-producing T cells and the functional state of regulatory T cells both in animal models and in patients with RA is also discussed. In light of the overwhelming evidence that disrupted T-cell homeostasis greatly contributes to joint pathology in RA, the therapeutic potential of targeting activators of pro-inflammatory T cells or their products is compelling.     

Effect of interleukin-2 on the expression of cell cycle genes in human T lymphocytes

We have studied the expression of seven cell cycle-dependent genes in phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells, in macrophage-depleted cultures and in macrophage-depleted cultures plus Interleukin-2 (IL-2). The expression of all seven genes is increased in PHA stimulated peripheral cells. Only two (2F1 and the IL-2 receptor) are increased in PHA-stimulated macrophage depleted cultures. Addition of IL-2 to these cultures increased the RNA levels of four genes (KC-1, c-myc, beta-actin and IL-2R), but has no effect on three others (4F1, 2F1, and JE-3). The results indicate that the expression of these cell cycle genes is regulated by different components of the mitogenic stimulus.     

Cytochemical investigation of cathepsin B in rheumatoid synovial membrane and fluid

Cathepsin B was cytochemically investigated in the cells of synovial membranes and in the cell pellet of synovial fluids obtained from 50 patients with rheumatoid arthritis and eight patients with various nonrheumatoid arthropathies. The activity of Cathepsin B was estimated by using the substrate N-alpha-benzoyl-DL-arginine-naphthylamide HCl and diazoic dye Fast Corinth V in phosphate buffer pH 6.0 in the presence of EDTA and cysteine. A significant activity of cathepsin B was shown in lining mesothelial cells, in macrophages of the submesothelial infiltrations, as well as in fibroblasts prominent in the deep areas of rheumatoid synovial membranes. In the cell pellets of synovial fluids the highest activity of cathepsin B was found in the macrophages and polymorphonuclear leukocytes, accompanied by a variable activity in lymphocytes. The considerable activity of cathepsin B, an enzyme with degradative action upon collagen and proteoglycans, in the main cellular populations of rheumatoid synovial membranes and fluids, suggests its involvement in the genesis and maintenance of rheumatoid lesions.     

Ligation of the T cell receptor complex results in phosphorylation of Smad2 in T lymphocytes

TGF-beta modulates immune responses by regulating T cell function. The Smad family of proteins has been recently shown to transduce signals for the TGF-beta superfamily and Smad2 mediates TGF-beta signaling. Here, we showed that TGF-beta phosphorylated Smad2 and induced interaction between Smad2 and Smad4 in primary T cells and the Jurkat T cell line. Interestingly, ligation of the T cell receptor (TCR)/CD3 complex with anti-CD3 mAb also phosphorylated Smad2, but failed to induce interaction between Smad2 and Smad4 in the Jurkat T cell line. Phosphorylation of Smad2 via the TCR/CD3 complex was not abrogated by treatment with neutralizing antibody against TGF-beta. Furthermore, PD98059, a MEK inhibitor, suppressed Smad2 phosphorylation by stimulation with anti-CD3 mAb in Jurkat T cell line. These findings indicated that not only TGF-beta but also stimulation via the TCR/CD3 complex phosphorylated Smad2 through mitogen-activated protein (MAP) kinase cascades, suggesting that Smad2 may function in both TGF-beta- and TCR/CD3 complex-mediated signaling pathways in T cells.     

Expression of human interleukin-2 receptor cDNA in E. coli

cDNAs for human interleukin-2 receptor were recently cloned and sequenced (Leonard et al., 1984, Nature 311, 626-631; Nikaido et al., 1984, Nature 311, 631-635; Cosman et al., Nature 312, 768-771). In the studies reported here, we describe the expression of a cDNA clone for the human interleukin-2 receptor in E. coli using an "open reading frame" expression vector pMR100. The inserted cDNA was expressed in E. coli transformants as a tripartite fusion polypeptide fused to the lambda cI protein at its amino terminus and to beta-galactosidase at its carboxy terminus. We demonstrate that the bacterially produced IL-2 receptor protein can bind to IL-2.