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1.  Interaction with activated monocytes enhances cytokine expression and suppressive activity of human CD4+CD45RO+CD25+CD127low regulatory T cells 
Arthritis and rheumatism  2013;65(3):627-638.
Objective
Despite the high frequency of CD4+ T cells with a regulatory phenotype (CD25+CD127lowFoxP3+) in the joints of patients with rheumatoid arthritis (RA), inflammation persists. One possible explanation is that human Tregs are converted into pro-inflammatory IL-17-producing cells by inflammatory mediators and thereby lose their suppressive function. We investigated whether activated monocytes, which are potent producers of inflammatory cytokines and abundantly present in the rheumatic joint, induce pro-inflammatory cytokine expression in human Tregs and impair their regulatory function.
Methods
The presence and phenotype of CD4+CD45RO+CD25+CD127low T cells (memory Tregs) and CD14+ monocytes in the peripheral blood (PB) and synovial fluid (SF) from patients with RA was investigated by flow cytometry. FACS-sorted memory Tregs from healthy controls were co-cultured with autologous activated monocytes and stimulated with anti-CD3 monoclonal antibody. Intracellular cytokine expression, phenotype and function of cells were determined by flow cytometry, ELISA and proliferation assays.
Results
Patients with RA showed higher frequencies of CD4+CD45RO+CD25+CD127low Tregs and activated CD14+ monocytes in SF relative to PB. In vitro-activated monocytes induced an increase in the percentage of IL-17+, IFNγ+ and TNF-α+, but also IL-10+ Tregs. The observed increase in IL-17+ and IFNγ+ Tregs was driven by monocyte-derived IL-1β, IL-6 and TNF-α and was mediated by both CD14+CD16− and CD14+CD16+ monocyte subsets. Despite enhanced cytokine expression, cells maintained their CD25+FoxP3+CD39+ Treg phenotype and showed enhanced capacity to suppress proliferation and IL-17 production by effector T cells.
Conclusion
Tregs exposed to a pro-inflammatory environment show increased cytokine expression as well as enhanced suppressive activity.
doi:10.1002/art.37832
PMCID: PMC3947722  PMID: 23280063
2.  Elevated Levels of CD4+CD25+FoxP3+ T Cells in Systemic Sclerosis Patients Contribute to the Secretion of IL-17 and Immunosuppression Dysfunction 
PLoS ONE  2013;8(6):e64531.
Objective
Immune imbalance between regulatory T (Treg) and Th17 cells is a characteristic of systemic sclerosis (SSc). The functional heterogeneity among Treg can be elucidated by separating Treg into different subsets based on the expression of FoxP3 and CD45RA. The aim of this study was to investigate the role of Treg subsets in the immune imbalance in naïve SSc.
Methods
Peripheral blood mononuclear cells (PBMCs) of 31 SSc patients and 33 healthy controls were analyzed for the expression of CD4, CD25, CD45RA, CTLA-4, FoxP3, and IL-17 using flow cytometry. Treg immunesuppression capacity was measured in co-culture experiments. The expression of FoxP3, CTLA-4, IL-17A, and RORC mRNA was measured by real-time PCR.
Results
The frequency of CD4+CD25+FoxP3+ Treg cells was significantly elevated in patients with SSc (3.62±1.14 vs 1.97±0.75, p<0.001) with diminished immunosuppression capacity. In SSc, the proportion of FoxP3highCD45RA− activated Treg cells (aTreg) was decreased, the proportion of FoxP3lowCD45RA− T cells was increased, and the proportion of FoxP3lowCD45RA+ resting Treg cells (rTreg) was decreased. The immune suppression capacity of aTreg and rTreg was diminished, while FoxP3lowCD45RA− T cells exhibited a lack of suppression capacity. The immune dysfunction of aTreg was accompanied by the abnormal expression of CTLA-4. Th17 cell numbers were elevated in SSc, FoxP3lowCD45RA− T cells produced IL-17, confirming their Th17 potential, which was consistent with the elevated levels of FoxP3+IL-17+ cells in SSc.
Conclusion
A decrease in aTreg levels, along with functional deficiency, and an increase in the proportion of FoxP3lowCD45RA− T cells, was the reason for the increase in dysfunctional Treg in SSc patients, potentially causing the immune imbalance between Treg and Th17 cells.
doi:10.1371/journal.pone.0064531
PMCID: PMC3679128  PMID: 23776439
3.  Interleukin-17–producing T cells are enriched in the joints of children with arthritis, but have a reciprocal relationship to regulatory T cell numbers 
Arthritis and Rheumatism  2008;58(3):875-887.
Objective
To identify interleukin-17 (IL-17)–producing T cells from patients with juvenile idiopathic arthritis (JIA), and investigate their cytokine production, migratory capacity, and relationship to Treg cells at sites of inflammation, as well as to test the hypothesis that IL-17+ T cell numbers correlate with clinical phenotype in childhood arthritis.
Methods
Flow cytometry was used to analyze the phenotype, cytokine production, and chemokine receptor expression of IL-17–producing T cells in peripheral blood and synovial fluid mononuclear cells from 36 children with JIA, in parallel with analysis of forkhead box P3 (FoxP3)–positive Treg cells. Migration of IL-17+ T cells toward CCL20 was assessed by a Transwell assay. Synovial tissue was analyzed by immunohistochemistry for IL-17 and IL-22.
Results
IL-17+ T cells were enriched in the joints of children with JIA as compared with the blood of JIA patients (P = 0.0001) and controls (P = 0.018) and were demonstrated in synovial tissue. IL-17+ T cell numbers were higher in patients with extended oligoarthritis, the more severe subtype of JIA, as compared with patients with persistent oligoarthritis, the milder subtype (P = 0.046). Within the joint, there was an inverse relationship between IL-17+ T cells and FoxP3+ Treg cells (r = 0.61, P = 0.016). IL-17+,CD4+ T cells were uniformly CCR6+ and migrated toward CCL20, but synovial IL-17+ T cells had variable CCR4 expression. A proportion of IL-17+ synovial T cells produced IL-22 and interferon-γ.
Conclusion
This study is the first to define the frequency and characteristics of “Th17” cells in JIA. We suggest that these highly proinflammatory cells contribute to joint pathology, as indicated by relationships with clinical phenotypes, and that the balance between IL-17+ T cells and Treg cells may be critical to outcome.
doi:10.1002/art.23291
PMCID: PMC2675006  PMID: 18311821
4.  Active synovial matrix metalloproteinase-2 is associated with radiographic erosions in patients with early synovitis 
Arthritis Research  2000;2(2):145-153.
Serum and synovial tissue expression of the matrix metalloproteinase (MMP)-2 and -9 and their molecular regulators, MMP-14 and TIMP-2 was examined in 28 patients with inflammatory early synovitis and 4 healthy volunteers and correlated with the presence of erosions in the patients. Immunohistological staining of MMP-2, MMP-14 and TIMP-2 localized to corresponding areas in the synovial lining layer and was almost absent in normal synovium. Patients with radiographic erosions had significantly higher levels of active MMP-2 than patients with no erosions, suggesting that activated MMP-2 levels in synovial tissue may be a marker for a more aggressive synovial lesion.
Introduction:
In cancer the gelatinases [matrix metalloproteinase (MMP)-2 and MMP-9] have been shown to be associated with tissue invasion and metastatic disease. In patients with inflammatory arthritis the gelatinases are expressed in the synovial membrane, and have been implicated in synovial tissue invasion into adjacent cartilage and bone. It is hypothesized that an imbalance between the activators and inhibitors of the gelatinases results in higher levels of activity, enhanced local proteolysis, and bone erosion.
Objectives:
To determine whether the expression and activity levels of MMP-2 and MMP-9, and their regulators MMP-14 and tissue inhibitor of metalloproteinase (TIMP), are associated with early erosion formation in patients with synovitis of recent onset.
Patients and method:
A subset of 66 patients was selected from a larger early synovitis cohort on the basis of tissue availability for the study of synovial tissue and serum gelatinase expression. Patients with peripheral joint synovitis of less than 1 years' duration were evaluated clinically and serologically on four visits over a period of 12 months. At the initial visit, patients underwent a synovial tissue biopsy of one swollen joint, and patients had radiographic evaluation of hands and feet initially and at 1year. Serum MMP-1, MMP-2, MMP-9, MMP-14, and TIMP-1 and TIMP-2 levels were determined, and synovial tissue was examined by immunohistology for the expression of MMP-2 and MMP-9, and their molecular regulators. Gelatinolytic activity for MMP-2 and MMP-9 was quantified using a sensitive, tissue-based gel zymography technique. Four healthy individuals underwent closed synovial biopsy and their synovial tissues were similarly analyzed.
Results:
Of the 66 patients studied, 45 fulfilled American College of Rheumatology criteria for rheumatoid arthritis (RA), with 32 (71%) being rheumatoid factor positive. Of the 21 non-RA patients, seven had a spondylarthropathy and 14 had undifferentiated arthritis. Radiographically, 12 of the RA patients had erosions at multiple sites by 1 year, whereas none of the non-RA patients had developed erosive disease of this extent. In the tissue, latent MMP-2 was widely expressed in the synovial lining layer and in areas of stromal proliferation in the sublining layer and stroma, whereas MMP-9 was expressed more sparsely and focally. MMP-14, TIMP-2, and MMP-2 were all detected in similar areas of the lining layer on consecutive histologic sections. Tissue expression of MMP-14, the activator for pro-MMP-2, was significantly higher in RA than in non-RA patients (8.4 ± 5 versus 3.7 ± 4 cells/high-power field; P = 0.009). In contrast, the expression of TIMP-2, an inhibitor of MMP-2, was lower in the RA than in the non-RA samples (25 ± 12 versus 39 ± 9 cells/high-power field; P = 0.01). Synovial tissue expressions of MMP-2, MMP-14, and TIMP-2 were virtually undetectable in normal synovial tissue samples. The synovial tissue samples of patients with erosive disease had significantly higher levels of active MMP-2 than did those of patients without erosions (Fig. 1). Tissue expression of MMP-2 and MMP-9, however, did not correlate with the serum levels of these enzymes.
With the exception of serum MMP-2, which was not elevated over normal, serum levels of all of the other MMPs and TIMPs were elevated to varying degrees, and were not predictive of erosive disease. Interestingly, MMP-1 and C-reactive protein, both of which were associated with the presence of erosions, were positively correlated with each other (r = 0.42; P < 0.001).
Discussion:
MMP-2 and MMP-9 are thought to play an important role in the evolution of joint erosions in patients with an inflammatory arthritis. Most studies have concentrated on the contribution of MMP-9 to the synovitis, because synovial fluid and serum MMP-9 levels are markedly increased in inflammatory arthropathies. Previously reported serum levels of MMP-9 have varied widely. In the present sample of patients with synovitis of recent onset, serum MMP-9 levels were elevated in only 21%. Moreover, these elevations were not specific for RA, the tissue expression of MMP-9 was focal, and the levels of MMP-9 activity were not well correlated with early erosions. Although serum MMP-2 levels were not of prognostic value, high synovial tissue levels of MMP-2 activity were significantly correlated with the presence of early erosions. This may reflect augmented activation of MMP-2 by the relatively high levels of MMP-14 and low levels of TIMP-2 seen in these tissues. We were able to localize the components of this trimolecular complex to the synovial lining layer in consecutive tissue sections, a finding that is consistent with their colocalization.
In conclusion, we have provided evidence that active MMP-2 complexes are detectable in the inflamed RA synovium and may be involved in the development of early bony erosions. These results suggest that strategies to inhibit the activation of MMP-2 may have the potential for retarding or preventing early erosions in patients with inflammatory arthritis.
PMCID: PMC17808  PMID: 11062605
early synovitis; erosion; metalloproteinase; matrix metalloproteinase-2; rheumatoid arthritis
5.  T Regulatory Cell Numbers and Function in Patients with Antibiotic-Refractory or Antibiotic-Responsive Lyme Arthritis 
Arthritis and rheumatism  2010;62(7):2127-2137.
Objective
In a murine model of antibiotic-refractory Lyme arthritis, the numbers of T regulatory cells (Treg) are dramatically reduced. Our goal was to examine Treg numbers and function in human patients with antibiotic-refractory Lyme arthritis.
Methods
CD4+ T cell subsets were enumerated in peripheral blood (PB) and synovial fluid (SF) in 12 patients with antibiotic-refractory arthritis and 6 with antibiotic-responsive arthritis. Treg function was examined using Borrelia-specific and non-specific Treg proliferation assays.
Results
In both patient groups, IFN-γ+ TH1 cells in SF were abundant and enriched (~50% of CD4+ T cells). In patients with antibiotic-refractory arthritis, the median percentages of FoxP3+ Treg were significantly higher in SF than PB (12% versus 6%) (P<0.01) or in SF in patients with antibiotic-responsive arthritis (12% versus 5%) (P=0.04). Moreover, in the refractory group, a higher percentage of Treg in SF correlated with a shorter duration to resolution of arthritis (r = −0.74, P = 0.006). In contrast, patients with fewer Treg had suboptimal responses to DMARDs and longer duration of arthritis after antibiotics, and they often required synovectomies for arthritis resolution. In each group, Treg in SF dampened B. burgdorferi-specific proliferative responses, and in 2 patients with refractory arthritis, Treg were functional in non-specific suppression assays.
Conclusions
Treg were functional in patients with antibiotic-refractory arthritis, and in some patients, large numbers of these cells in SF appeared to participate in arthritis resolution. However, as in the murine model, patients with refractory arthritis and low numbers of Treg seemed unable to resolve synovial inflammation.
doi:10.1002/art.27468
PMCID: PMC2913315  PMID: 20506317
6.  CD4+CD25+/highCD127low/- regulatory T cells are enriched in rheumatoid arthritis and osteoarthritis joints—analysis of frequency and phenotype in synovial membrane, synovial fluid and peripheral blood 
Introduction
CD4+CD25+/highCD127low/- regulatory T cells (Tregs) play a crucial role in maintaining peripheral tolerance. Data about the frequency of Tregs in rheumatoid arthritis (RA) are contradictory and based on the analysis of peripheral blood (PB) and synovial fluid (SF). Because Tregs exert their anti-inflammatory activity in a contact-dependent manner, the analysis of synovial membrane (SM) is crucial. Published reports regarding this matter are lacking, so we investigated the distribution and phenotype of Tregs in concurrent samples of SM, SF and PB of RA patients in comparison to those of osteoarthritis (OA) patients.
Methods
Treg frequency in a total of 40 patients (18 RA and 22 OA) matched for age and sex was assessed by flow cytometry. Functional status was assessed by analysis of cell surface markers representative of activation, memory and regulation.
Results
CD4+ T cells infiltrate the SM to higher frequencies in RA joints than in OA joints (P = 0.0336). In both groups, Tregs accumulate more within the SF and SM than concurrently in PB (P < 0.0001). Relative Treg frequencies were comparable in all compartments of RA and OA, but Treg concentration was significantly higher in the SM of RA patients (P = 0.025). Both PB and SM Tregs displayed a memory phenotype (CD45RO+RA-), but significantly differed in activation status (CD69 and CD62L) and markers associated with Treg function (CD152, CD154, CD274, CD279 and GITR) with only minor differences between RA and OA.
Conclusions
Treg enrichment into the joint compartment is not specific to inflammatory arthritis, as we found that it was similarly enriched in OA. RA pathophysiology might not be due to a Treg deficiency, because Treg concentration in SM was significantly higher in RA. Synovial Tregs represent a distinct phenotype and are activated effector memory cells (CD62L-CD69+), whereas peripheral Tregs are resting central memory cells (CD62L+CD69-).
doi:10.1186/ar4545
PMCID: PMC4060198  PMID: 24742142
7.  Mast cell activation and its relation to proinflammatory cytokine production in the rheumatoid lesion 
Arthritis Research  1999;2(1):65-74.
Mast cell (MC) activation in the rheumatoid lesion provides numerous mediators that contribute to inflammatory and degradative processes, especially at sites of cartilage erosion. MC activation in rheumatoid synovial tissue has often been associated with tumour necrosis factor (TNF)-α and interleukin (IL)-1β production by adjacent cell types. By contrast, our in situ and in vitro studies have shown that the production of IL-15 was independent of MC activation, and was not related to TNF-α and IL-1β expression. Primary cultures of dissociated rheumatoid synovial cells produced all three proinflammatory cytokines, with production of IL-1β exceeding that of TNF-α, which in turn exceeded that of IL-15. In vitro cultures of synovial macrophages, synovial fibroblasts and articular chondrocytes all produced detectable amounts of free IL-15, macrophages being the most effective.
Introduction:
Increased numbers of mast cells (MCs) are found in the synovial tissues and fluids of patients with rheumatoid arthritis (RA), and at sites of cartilage erosion. MC activation has been reported for a significant proportion of rheumatoid specimens. Because the MC contains potent mediators, including histamine, heparin, proteinases, leukotrienes and multifunctional cytokines, its potential contributions to the processes of inflammation and matrix degradation have recently become evident.
Proinflammatory cytokines are important mediators of inflammation, immunity, proteolysis, cell recruitment and proliferation. Tumour necrosis factor (TNF) reportedly plays a pivotal role in the pathogenesis of RA, especially its ability to regulate interleukin (IL)-1β expression, this being important for the induction of prostanoid and matrix metalloproteinase production by synovial fibroblasts and chondrocytes. IL-15 has been assigned numerous biological effects and has been implicated as an important factor in TNF-α expression by monocyte/macrophages. Some in vitro studies have placed IL-15 upstream from TNF-α in the cytokine cascade, suggesting an interdependence between TNF, IL-1 and IL-15 for the promotion of proinflammatory cytokine expression in the rheumatoid joint.
Aims:
To examine the in situ relationships of TNF-α, IL-1β and IL-15 in relation to MC activation in rheumatoid tissues by use of immunolocalization techniques; and to compare quantitatively the proinflammatory cytokine production by specific cell cultures and rheumatoid synovial explants with and without exposure to a MC secretagogue.
Materials and methods:
Samples of rheumatoid synovial tissue and cartilage–pannus junction were obtained from patients (n = 15) with classic late-stage RA. Tissue sections were immunostained for MC (tryptase) and the proinflammatory cytokines IL-1, TNF-α and IL-15. Rheumatoid synovial tissue explants were cultured in Dulbecco's modified Eagles medium (DMEM) containing either the MC secretagogue rabbit antihuman immunoglobulin (Ig)E, or control rabbit IgG. Primary rheumatoid synovial cell cultures, human articular chondrocytes, synovial fibroblasts and synovial macrophages were prepared as described in the full article. Conditioned culture media from these cultures were collected and assayed for IL-1β, TNF-α and IL-15 using enzyme-linked immunosorbent assay methodology.
Results:
Immunohistological studies of rheumatoid synovial tissues have demonstrated local concentrations of MCs in most specimens of the rheumatoid lesion. Sites of MC activation were associated with localized oedema, and TNF-α, IL-1α and IL-1β production by a proportion of mononuclear inflammatory cells. By contrast, no evidence was found for IL-15 production in tissue sites containing either intact or activated MCs, and IL-15 expression, when observed, bore no relation to tissue sites where TNF-α and IL-1β were evident. The immunodetection of IL-15 was restricted to microfocal sites and was not typical of most junctional specimens, but was associated with a proportion of articular chondrocytes in a minority of junctional specimens.
MC activation within synovial explant cultures was induced by the addition of polyclonal antibody to human IgE. MC activation significantly reduced the levels of TNF-α and IL1β released into the medium, this representing approximately 33% of control values. By contrast, MC activation had little effect on the levels of IL-15 released into the culture medium, the average value being very low in relation to the release of TNF-α and IL-1β . Thus, induced MC activation brings about changes in the amounts of released tryptase, TNF-α and IL-1β , but not of IL-15.
Four preparations of primary rheumatoid synovial cell cultures produced more IL-1β than TNF-α, with only modest values for IL-15 production, indicating that all three cytokines are produced and released as free ligands by these cultures. Of specific cell types that produced IL-15 in vitro, macrophages produced more than fibroblasts, which in turn produced more than chondrocytes. This demonstrates that all three cell types have the potential to produce IL-15 in situ.
Discussion:
The biological consequences of MC activation in vivo are extremely complex, and in all probability relate to the release of various combinations of soluble and granular factors, as well as to the expression of appropriate receptors by neighbouring cells. The subsequent synthesis and release of cytokines such as TNF-α and IL-1 may well follow at specific stages after activation, or may be an induced cytokine response by adjacent macrophagic or fibroblastic cells. However, because no IL-15 was detectable either in or around activated or intact MCs, and the induced MC activation explant study showed no change in IL-15 production, it seems unlikely that the expression of this cytokine is regulated by MCs. The immunohistochemistry (IHC) demonstration of IL-15 at sites of cartilage erosion, and especially by some chondrocytes of articular cartilage, showed no spatial relationship with either T cells or neutrophils, and suggests other functional properties in these locations. The lack of evidence for an in situ association of IL-15 with TNF and IL-1 does not support a role for IL-15 in a proinflammatory cytokine 'cascade', as proposed by other in vitro experiments. We believe that sufficient evidence is available, however, to suggest that MC activation makes a significant contribution to the pathophysiological processes of the rheumatoid lesion.
PMCID: PMC17805  PMID: 11219391
interleukin-15; interleukin-1β; mast cells; rheumatoid arthritis; tumour necrosis factor-α
8.  Plasmodium falciparum–Mediated Induction of Human CD25hiFoxp3hi CD4 T Cells Is Independent of Direct TCR Stimulation and Requires IL-2, IL-10 and TGFβ 
PLoS Pathogens  2009;5(8):e1000543.
CD4+CD25+Foxp3+ regulatory T cells (Tregs) regulate disease-associated immunity and excessive inflammatory responses, and numbers of CD4+CD25+Foxp3+ Tregs are increased during malaria infection. The mechanisms governing their generation, however, remain to be elucidated. In this study we investigated the role of commonly accepted factors for Foxp3 induction, TCR stimulation and cytokines such as IL-2, TGFβ and IL-10, in the generation of human CD4+CD25+Foxp3+ T cells by the malaria parasite Plasmodium falciparum. Using a co-culture system of malaria-infected red blood cells (iRBCs) and peripheral blood mononuclear cells from healthy individuals, we found that two populations of Foxp3hi and Foxp3int CD4+CD25hi T cells with a typical Treg phenotype (CTLA-4+, CD127low, CD39+, ICOS+, TNFRII+) were induced. Pro-inflammatory cytokine production was confined to the Foxp3int subset (IFNγ, IL-4 and IL-17) and inversely correlated with high relative levels of Foxp3hi cells, consistent with Foxp3hi CD4 T cell–mediated inhibition of parasite-induced effector cytokine T cell responses. Both Foxp3hi and Foxp3int cells were derived primarily from proliferating CD4+CD25− T cells with a further significant contribution from CD25+Foxp3+ natural Treg cells to the generation of the Foxp3hi subset. Generation of Foxp3hi, but not Foxp3int, cells specifically required TGFβ1 and IL-10. Add-back experiments showed that monocytes expressing increased levels of co-stimulatory molecules were sufficient for iRBC-mediated induction of Foxp3 in CD4 T cells. Foxp3 induction was driven by IL-2 from CD4 T cells stimulated in an MHC class II–dependent manner. However, transwell separation experiments showed that direct contact of monocytes with the cells that acquire Foxp3 expression was not required. This novel TCR-independent and therefore antigen-non specific mechanism for by-stander CD4+CD25hiFoxp3+ cell induction is likely to reflect a process also occurring in vivo as a consequence of immune activation during malaria infection, and potentially a range of other infectious diseases.
Author Summary
Infection with the malaria parasite Plasmodium falciparum affects 300–600 million people each year. Regulatory T cells (Tregs) expressing the transcription factor Foxp3, which drives genes involved in immunosuppression, are specialized immune cells that can inhibit both protective and harmful inflammatory responses during malaria. While Treg numbers are increased during malaria infection, little is known about how they are induced by the parasite. We addressed this question using an in vitro culture system to model the interaction of the malaria parasite with human immune cells. We found that the parasite induced soluble immune mediators, including the T cell growth-factor IL-2 and the regulatory proteins IL-10 and TGFβ, which drive the induction and expansion of Tregs. These Tregs expressed high levels of Foxp3 and suppressed the production of inflammation and protective immunity-driving mediators by concurrently induced effector T cells. Importantly, we demonstrate that induction of Tregs by the malaria parasite did not necessarily require direct contact with antigen-presenting cells. Our findings suggest that the parasite induces Tregs in an antigen non-specific manner, which may explain why malarial immunosuppression is not confined to malaria-specific immune responses, and provide new insights into the mechanisms governing Treg induction during malaria infection, and potentially other infectious diseases.
doi:10.1371/journal.ppat.1000543
PMCID: PMC2718810  PMID: 19680449
9.  TNFR2 is critical for the stabilization of the CD4+FoxP3+ regulatory T cell phenotype in the inflammatory environment 
Several lines of evidence indicate the instability of CD4+FoxP3+ regulatory T cells (Tregs). We have therefore investigated means of promoting the stability of Tregs. In this study, we found that the proportion of Tregs in mouse strains deficient in TNFR2 or its ligands was reduced in the thymus and peripheral lymphoid tissues, suggesting a potential role of TNFR2 in promoting the sustained expression of FoxP3. We observed that upon in vitro activation with plate-bound anti-CD3 Ab and soluble anti-CD28 Ab, FoxP3 expression by highly purified mouse Tregs was markedly down-regulated. Importantly, TNF partially abrogated this effect of TCR stimulation and stabilized FoxP3 expression. This effect of TNF was blocked by anti-TNFR2 Ab, but not by anti-TNFR1 Ab. Furthermore, TNF was not able to maintain FoxP3 expression by TNFR2-deficient Tregs. In mouse colitis model induced by transfer of naïve CD4 cells into Rag1−/− mice, the disease could be inhibited by co-transfer of WT Tregs, but not by co-transfer of TNFR2-deficient Tregs. Furthermore, in the lamina propria of the colitis model, the majority of WT Tregs maintained FoxP3 expression. In contrast, increased number of TNFR2-deficient Tregs lost FoxP3 expression. Thus, our data clearly show that TNFR2 is critical for the phenotypic and functional stability of Treg in the inflammatory environment. This effect of TNF should be taken into account when designing future therapy of autoimmunity and GVHD by using TNF inhibitors.
doi:10.4049/jimmunol.1202659
PMCID: PMC3552130  PMID: 23277487
T cell; cytokine receptor; tolerance/suppression/anergy
10.  CD8+ Treg Cells Associated with Decreasing Disease Activity after Intravenous Methylprednisolone Pulse Therapy in Lupus Nephritis with Heavy Proteinuria 
PLoS ONE  2014;9(1):e81344.
We focus on the role of CD8+ Treg cell in Intravenous methyl-prednisolone (IVMP) pulse therapy in forty patients with active Class III/IV childhood lupus nephritis (LN) with heavy proteinuria. IVMP therapy for five days. From peripheral blood mononuclear cells (PBMCs) and renal tissues, we saw IVMP therapy definitely restoring both CD4+CD25+FoxP3+ and CD8+CD25+Foxp3+ Treg cell number plus greater expression with intracellular IL-10 and granzyme B in CD8+FoxP3+ Treg from PBMCs. IVMP-treated CD8+CD25+ Treg cells directly suppressed CD4+ T proliferation and induced CD4+CD45RO+ apoptosis. Histologically, CD4+FoxP3+ as well as CD8+FoxP3+ Treg cells appeared in renal tissue of LN patients before IVMP by double immunohistochemical stain. CD8+FoxP3+ Treg cells increased in 10 follow-up renal biopsy specimens after IVMP. Reverse correlation of serum anti-C1q antibody and FoxP3+ Treg cells in PBMNCs (r = −0.714, P<0.01). After IVMP, serum anti-C1q antibody decrease accompanied increase of CD4+FoxP3+ Treg cells. CD8+Treg cells reduced interferon-r response in PBMCs to major peptide autoepitopes from nucleosomes after IVMP therapy; siRNA of FoxP3 suppressed granzyme B expression while decreasing CD8+CD25+Treg-induced CD4+CD45RO+ apoptosis. Renal activity of LN by SLEDAI-2k in childhood LN was significantly higher than two weeks after IVMP (P<0.01). CD8+FoxP3+ Treg cells return in post-IVMP therapy and exert crucial immune modulatory effect to control autoimmune response in LN.
Trial Registration
DMR97-IRB-259
doi:10.1371/journal.pone.0081344
PMCID: PMC3903465  PMID: 24475019
11.  Delayed functional maturation of natural regulatory T cells in the medulla of postnatal thymus: role of TSLP 
BMC Immunology  2006;7:6.
Background
Generation of functional CD4+CD8-CD25+ regulatory T cells (Treg) in the murine thymus depends on FoxP3. Removal of the thymus from neonatal mice has been shown to result in a multiple organ autoimmune disease phenotype that can be prevented by introducing the FoxP3+ Treg population to the animal. It has therefore, been proposed that functional FoxP3+ Treg cells are not made in the neonatal thymus; however, it remains unclear when and where functional FoxP3+CD4+CD8-CD25+ thymocytes are generated in postnatal thymus.
Results
We report that neither FoxP3 mRNA nor protein is expressed in CD4+CD8-CD25+, or CD4+CD8-CD25- thymocytes until 3–4 days post birth, despite the presence of mature CD4+CD8-CD25+/- thymocytes in the thymus by 1–2 days after birth. FoxP3-CD4+CD8-CD25+ thymocytes from day 2 newborn mice show no Treg activity. Interestingly, we are able to detect low numbers of FoxP3+ thymocytes dispersed throughout the medullary region of the thymus as early as 3–4 days post birth. Expression of FoxP3 is induced in embryonic day 17 fetal thymus organ culture (FTOC) after 4–6 days of in vitro culture. Treatment of FTOCs with thymic stromal derived lymphopoietin (TSLP) enhanced expression of FoxP3, and blocking the TSLP receptor reduces FoxP3 expression in FTOC. Furthermore, TSLP stimulates FoxP3 expression in purified CD4+CD8- thymocytes, but not in CD4+CD8+, CD4-CD8+ and CD4-CD8- thymocytes.
Conclusion
Expression of FoxP3 or Treg maturation is ontogenically distinct and kinetically delayed from the generation of CD4+CD8-CD25+ or CD4+CD8-CD25- thymocytes in the postnatal thymus. TSLP produced from medullary thymic epithelia cells (mTEC) contributes to the expression of FoxP3 and the maturation of natural regulatory T cells. Overall, these results suggest that the development of Treg cells requires paracrine signaling during late stages of thymocyte maturation that is distinct from signaling during positive or negative selection.
doi:10.1186/1471-2172-7-6
PMCID: PMC1450317  PMID: 16579866
12.  T-Cell Regulation in Lepromatous Leprosy 
Regulatory T (Treg) cells are known for their role in maintaining self-tolerance and balancing immune reactions in autoimmune diseases and chronic infections. However, regulatory mechanisms can also lead to prolonged survival of pathogens in chronic infections like leprosy and tuberculosis (TB). Despite high humoral responses against Mycobacterium leprae (M. leprae), lepromatous leprosy (LL) patients have the characteristic inability to generate T helper 1 (Th1) responses against the bacterium. In this study, we investigated the unresponsiveness to M. leprae in peripheral blood mononuclear cells (PBMC) of LL patients by analysis of IFN-γ responses to M. leprae before and after depletion of CD25+ cells, by cell subsets analysis of PBMC and by immunohistochemistry of patients' skin lesions. Depletion of CD25+ cells from total PBMC identified two groups of LL patients: 7/18 (38.8%) gained in vitro responsiveness towards M. leprae after depletion of CD25+ cells, which was reversed to M. leprae-specific T-cell unresponsiveness by addition of autologous CD25+ cells. In contrast, 11/18 (61.1%) remained anergic in the absence of CD25+ T-cells. For both groups mitogen-induced IFN-γ was, however, not affected by depletion of CD25+ cells. In M. leprae responding healthy controls, treated lepromatous leprosy (LL) and borderline tuberculoid leprosy (BT) patients, depletion of CD25+ cells only slightly increased the IFN-γ response. Furthermore, cell subset analysis showed significantly higher (p = 0.02) numbers of FoxP3+ CD8+CD25+ T-cells in LL compared to BT patients, whereas confocal microscopy of skin biopsies revealed increased numbers of CD68+CD163+ as well as FoxP3+ cells in lesions of LL compared to tuberculoid and borderline tuberculoid leprosy (TT/BT) lesions. Thus, these data show that CD25+ Treg cells play a role in M. leprae-Th1 unresponsiveness in LL.
Author Summary
Leprosy is a curable infectious disease caused by Mycobacterium leprae (M. leprae) that affects the skin and peripheral nerves. It is manifested in different forms ranging from self-healing, tuberculoid leprosy (TT) with low bacillary load and high cellular immunity against M. leprae, to lepromatous leprosy (LL) with high bacillary load and high antibody titers to M. leprae antigens. However, LL patients have poor cell mediated response against M. leprae leading to delayed clearance of the bacilli. A possible explanation for this bacterial persistence could lie in the presence of more regulatory cells at infection sites and in peripheral blood. This study shows the recovery of the cell mediated response by depletion of CD25+ cells in a subset of LL patients, while another patient subset was not affected similarly. Moreover, an increased frequency of FoxP3+ T cells together with anti-inflammatory macrophages was observed in LL patients' skin biopsies. Thus, these data show that CD25+ Treg cells play a role in M. leprae-unresponsiveness in leprosy patients.
doi:10.1371/journal.pntd.0002773
PMCID: PMC3983090  PMID: 24722473
13.  Ectopic Lymphoid Structures Support Ongoing Production of Class-Switched Autoantibodies in Rheumatoid Synovium 
PLoS Medicine  2009;6(1):e1.
Background
Follicular structures resembling germinal centres (GCs) that are characterized by follicular dendritic cell (FDC) networks have long been recognized in chronically inflamed tissues in autoimmune diseases, including the synovium of rheumatoid arthritis (RA). However, it is debated whether these ectopic structures promote autoimmunity and chronic inflammation driving the production of pathogenic autoantibodies. Anti-citrullinated protein/peptide antibodies (ACPA) are highly specific markers of RA, predict a poor prognosis, and have been suggested to be pathogenic. Therefore, the main study objectives were to determine whether ectopic lymphoid structures in RA synovium: (i) express activation-induced cytidine deaminase (AID), the enzyme required for somatic hypermutation and class-switch recombination (CSR) of Ig genes; (ii) support ongoing CSR and ACPA production; and (iii) remain functional in a RA/severe combined immunodeficiency (SCID) chimera model devoid of new immune cell influx into the synovium.
Methods and Findings
Using immunohistochemistry (IHC) and quantitative Taqman real-time PCR (QT-PCR) in synovial tissue from 55 patients with RA, we demonstrated that FDC+ structures invariably expressed AID with a distribution resembling secondary lymphoid organs. Further, AID+/CD21+ follicular structures were surrounded by ACPA+/CD138+ plasma cells, as demonstrated by immune reactivity to citrullinated fibrinogen. Moreover, we identified a novel subset of synovial AID+/CD20+ B cells outside GCs resembling interfollicular large B cells. In order to gain direct functional evidence that AID+ structures support CSR and in situ manufacturing of class-switched ACPA, 34 SCID mice were transplanted with RA synovium and humanely killed at 4 wk for harvesting of transplants and sera. Persistent expression of AID and Iγ-Cμ circular transcripts (identifying ongoing IgM-IgG class-switching) was observed in synovial grafts expressing FDCs/CD21L. Furthermore, synovial mRNA levels of AID were closely associated with circulating human IgG ACPA in mouse sera. Finally, the survival and proliferation of functional B cell niches was associated with persistent overexpression of genes regulating ectopic lymphoneogenesis.
Conclusions
Our demonstration that FDC+ follicular units invariably express AID and are surrounded by ACPA-producing plasma cells provides strong evidence that ectopic lymphoid structures in the RA synovium are functional and support autoantibody production. This concept is further confirmed by evidence of sustained AID expression, B cell proliferation, ongoing CSR, and production of human IgG ACPA from GC+ synovial tissue transplanted into SCID mice, independently of new B cell influx from the systemic circulation. These data identify AID as a potential therapeutic target in RA and suggest that survival of functional synovial B cell niches may profoundly influence chronic inflammation, autoimmunity, and response to B cell–depleting therapies.
Costantino Pitzalis and colleagues show that lymphoid structures in synovial tissue of patients with rheumatoid arthritis support production of anti-citrullinated peptide antibodies, which continues following transplantation into SCID mice.
Editors' Summary
Background.
More than 1 million people in the United States have rheumatoid arthritis, an “autoimmune” condition that affects the joints. Normally, the immune system provides protection against infection by responding to foreign antigens (molecules that are unique to invading organisms) while ignoring self-antigens present in the body's own tissues. In autoimmune diseases, this ability to discriminate between self and non-self fails for unknown reasons and the immune system begins to attack human tissues. In rheumatoid arthritis, the lining of the joints (the synovium) is attacked, it becomes inflamed and thickened, and chemicals are released that damage all the tissues in the joint. Eventually, the joint may become so scarred that movement is no longer possible. Rheumatoid arthritis usually starts in the small joints in the hands and feet, but larger joints and other tissues (including the heart and blood vessels) can be affected. Its symptoms, which tend to fluctuate, include early morning joint pain, swelling, and stiffness, and feeling generally unwell. Although the disease is not always easy to diagnose, the immune systems of many people with rheumatoid arthritis make “anti-citrullinated protein/peptide antibodies” (ACPA). These “autoantibodies” (which some experts believe can contribute to the joint damage in rheumatoid arthritis) recognize self-proteins that contain the unusual amino acid citrulline, and their detection on blood tests can help make the diagnosis. Although there is no cure for rheumatoid arthritis, the recently developed biologic drugs, often used together with the more traditional disease-modifying therapies, are able to halt its progression by specifically blocking the chemicals that cause joint damage. Painkillers and nonsteroidal anti-inflammatory drugs can reduce its symptoms, and badly damaged joints can sometimes be surgically replaced.
Why Was This Study Done?
Before scientists can develop a cure for rheumatoid arthritis, they need to know how and why autoantibodies are made that attack the joints in this common and disabling disease. B cells, the immune system cells that make antibodies, mature in structures known as “germinal centers” in the spleen and lymph nodes. In the germinal centers, immature B cells are exposed to antigens and undergo two genetic processes called “somatic hypermutation” and “class-switch recombination” that ensure that each B cell makes an antibody that sticks as tightly as possible to just one antigen. The B cells then multiply and enter the bloodstream where they help to deal with infections. Interestingly, the inflamed synovium of many patients with rheumatoid arthritis contains structures that resemble germinal centers. Could these ectopic (misplaced) lymphoid structures, which are characterized by networks of immune system cells called follicular dendritic cells (FDCs), promote autoimmunity and long-term inflammation by driving the production of autoantibodies within the joint itself? In this study, the researchers investigate this possibility.
What Did the Researchers Do and Find?
The researchers collected synovial tissue from 55 patients with rheumatoid arthritis and used two approaches, called immunohistochemistry and real-time PCR, to investigate whether FDC-containing structures in synovium expressed an enzyme called activation-induced cytidine deaminase (AID), which is needed for both somatic hypermutation and class-switch recombination. All the FDC-containing structures that the researchers found in their samples expressed AID. Furthermore, these AID-containing structures were surrounded by mature B cells making ACPAs. To test whether these B cells were derived from AID-expressing cells resident in the synovium rather than ACPA-expressing immune system cells coming into the synovium from elsewhere in the body, the researchers transplanted synovium from patients with rheumatoid arthritis under the skin of a special sort of mouse that largely lacks its own immune system. Four weeks later, the researchers found that the transplanted human lymphoid tissue was still making AID, that the level of AID expression correlated with the amount of human ACPA in the blood of the mice, and that the B cells in the transplant were proliferating.
What Do These Findings Mean?
These findings show that the ectopic lymphoid structures present in the synovium of some patients with rheumatoid arthritis are functional and are able to make ACPA. Because ACPA may be responsible for joint damage, the survival of these structures could, therefore, be involved in the development and progression of rheumatoid arthritis. More experiments are needed to confirm this idea, but these findings may explain why drugs that effectively clear B cells from the bloodstream do not always produce a marked clinical improvement in rheumatoid arthritis. Finally, they suggest that AID might provide a new target for the development of drugs to treat rheumatoid arthritis.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0060001.
This study is further discussed in a PLoS Medicine Perspective by Rene Toes and Tom Huizinga
The MedlinePlus Encyclopedia has a page on rheumatoid arthritis (in English and Spanish). MedlinePlus provides links to other information on rheumatoid arthritis (in English and Spanish)
The UK National Health Service Choices information service has detailed information on rheumatoid arthritis
The US National Institute of Arthritis and Musculoskeletal and Skin Diseases provides Fast Facts, an easy to read publication for the public, and a more detailed Handbook on rheumatoid arthritis
The US Centers for Disease Control and Prevention has an overview on rheumatoid arthritis that includes statistics about this disease and its impact on daily life
doi:10.1371/journal.pmed.0060001
PMCID: PMC2621263  PMID: 19143467
14.  Potential role of regulatory T cells in reversing obesity-linked insulin resistance and diabetic nephropathy 
Diabetes  2011;60(11):2954-2962.
Introduction
FoxP3 expressing regulatory T cells (Tregs) are critical for maintenance of tolerance in rodents and men.
Methods
To characterize the role of Tregs in insulin resistance we first evaluated human visceral adipose tissue for Treg infiltration and secondly used the db/db mouse model.
Results
Obese patients with insulin resistance displayed significantly decreased natural Tregs, but an increase in adaptive Tregs in their visceral adipose tissue as compared to lean controls. To further evaluate the pathogenic role of Tregs in insulin resistance, we used the db/db mouse model. Treg-depletion using an anti-CD25 monoclonal antibody enhanced insulin resistance as shown by increased fasting blood glucose levels as well as an impaired insulin sensitivity. Moreover, Treg-depleted db/db mice developed increased signs of diabetic nephropathy, such as albuminuria and glomerular hyperfiltration. This was paralleled by a pro-inflammatory milieu in both, murine visceral adipose tissue and the kidney. Vice versa, adoptive transfer of CD4+FoxP+ Tregs significantly improved insulin sensitivity and diabetic nephropathy. Accordingly, there was increased mRNA expression of FoxP3 as well as less abundant pro-inflammatory CD8+CD69+ T cells in visceral adipose tissue and kidneys of Treg-treated animals.
Discussion/Conclusion
Our data suggest a potential therapeutic value of Tregs to improve insulin resistance and end organ damage in type 2 diabetes by limiting the pro-inflammatory milieu.
doi:10.2337/db11-0358
PMCID: PMC3198056  PMID: 21911743
15.  Acute-phase serum amyloid A production by rheumatoid arthritis synovial tissue 
Arthritis Research  2000;2(2):142-144.
Acute-phase serum amyloid A (A-SAA) is a major component of the acute-phase response. A sustained acute-phase response in rheumatoid arthritis (RA) is associated with increased joint damage. A-SAA mRNA expression was confirmed in all samples obtained from patients with RA, but not in normal synovium. A-SAA mRNA expression was also demonstrated in cultured RA synoviocytes. A-SAA protein was identified in the supernatants of primary synoviocyte cultures, and its expression colocalized with sites of macrophage accumulation and with some vascular endothelial cells. It is concluded that A-SAA is produced by inflamed RA synovial tissue. The known association between the acute-phase response and progressive joint damage may be the direct result of synovial A-SAA-induced effects on cartilage degradation.
Introduction:
Serum amyloid A (SAA) is the circulating precursor of amyloid A protein, the fibrillar component of amyloid deposits. In humans, four SAA genes have been described. Two genes (SAA1 and SAA2) encode A-SAA and are coordinately induced in response to inflammation. SAA1 and SAA2 are 95% homologous in both coding and noncoding regions. SAA3 is a pseudogene. SAA4 encodes constitutive SAA and is minimally inducible. A-SAA increases dramatically during acute inflammation and may reach levels that are 1000-fold greater than normal. A-SAA is mainly synthesized in the liver, but extrahepatic production has been demonstrated in many species, including humans. A-SAA mRNA is expressed in RA synoviocytes and in monocyte/macrophage cell lines such as THP-1 cells, in endothelial cells and in smooth muscle cells of atherosclerotic lesions. A-SAA has also been localized to a wide range of histologically normal tissues, including breast, stomach, intestine, pancreas, kidney, lung, tonsil, thyroid, pituitary, placenta, skin and brain.
Aims:
To identify the cell types that produce A-SAA mRNA and protein, and their location in RA synovium.
Materials and methods:
Rheumatoid synovial tissue was obtained from eight patients undergoing arthroscopic biopsy and at joint replacement surgery. Total RNA was analyzed by reverse transcription (RT) polymerase chain reaction (PCR) for A-SAA mRNA. PCR products generated were confirmed by Southern blot analysis using human A-SAA cDNA. Localization of A-SAA production was examined by immunohistochemistry using a rabbit antihuman A-SAA polyclonal antibody. PrimaryRA synoviocytes were cultured to examine endogenous A-SAA mRNA expression and protein production.
Results:
A-SAA mRNA expression was detected using RT-PCR in all eight synovial tissue samples studied. Figure 1 demonstrates RT-PCR products generated using synovial tissue from three representative RA patients. Analysis of RA synovial tissue revealed differences in A-SAA mRNA levels between individual RA patients.
In order to identify the cells that expressed A-SAA mRNA in RA synovial tissue, we analyzed primary human synoviocytes (n = 2). RT-PCR analysis revealed A-SAA mRNA expression in primary RA synoviocytes (n = 2; Fig. 2). The endogenous A-SAA mRNA levels detected in individual primary RA synoviocytes varied between patients. These findings are consistent with A-SAA expression in RA synovial tissue (Fig. 1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were relatively similar in the RA synoviocytes examined (Fig. 2). A-SAA protein in the supernatants of primary synoviocyte cultures from four RA patients was measured using ELISA. Mean values of a control and four RA samples were 77.85, 162.5, 249.8, 321.5 and 339.04 μg/l A-SAA, respectively, confirming the production of A-SAA protein by the primary RA synoviocytes. Immunohistochemical analysis was performed to localize sites of A-SAA production in RA synovial tissue. Positive staining was present in both the lining and sublining layers of all eight RA tissues examined (Fig. 3a). Staining was intense and most prominent in the cells closest to the surface of the synovial lining layer. Positively stained cells were evident in the perivascular areas of the sublining layer. In serial sections stained with anti-CD68 monoclonal antibody, positive staining of macrophages appeared to colocalize with A-SAA-positive cells (Fig. 3b). Immunohistochemical studies of cultured primary RA synoviocytes confirmed specific cytoplasmic A-SAA expression in these cells. The specificity of the staining was confirmed by the absence of staining found on serial sections and synoviocyte cells treated with IgG (Fig. 3c).
Discussion:
This study demonstrates that A-SAA mRNA is expressed in several cell populations infiltrating RA synovial tissue. A-SAA mRNA expression was observed in all eight unseparated RA tissue samples studied. A-SAA mRNA expression and protein production was demonstrated in primary cultures of purified RA synoviocytes. Using immunohistochemical techniques, A-SAA protein appeared to colocalize with both lining layer and sublining layer synoviocytes, macrophages and some endothelial cells. The detection of A-SAA protein in culture media supernatants harvested from unstimulated synoviocytes confirms endogenous A-SAA production, and is consistent with A-SAA mRNA expression and translation by the same cells. Moreover, the demonstration of A-SAA protein in RA synovial tissue, RA cultured synoviocytes, macrophages and endothelial cells is consistent with previous studies that demonstrated A-SAA production by a variety of human cell populations.
The RA synovial lining layer is composed of activated macrophages and fibroblast-like synoviocytes. The macrophage is the predominant cell type and it has been shown to accumulate preferentially in the surface of the lining layer and in the perivascular areas of the sublining layer. Nevertheless, our observations strongly suggest that A-SAA is produced not only by synoviocytes, but also by synovial tissue macrophage populations. Local A-SAA protein production by vascular endothelial cells was detected in some, but not all, of the tissues examined. The reason for the variability in vascular A-SAA staining is unknown, but may be due to differences in endothelial cell activation, events related to angiogenesis or the intensity of local inflammation.
The value of measuring serum A-SAA levels as a reliable surrogate marker of inflammation has been demonstrated for several diseases including RA, juvenile chronic arthritis, psoriatic arthropathy, ankylosing spondylitis, Behçet's disease, reactive arthritis and Crohn's disease. It has been suggested that serum A-SAA levels may represent the most sensitive measurement of the acute-phase reaction. In RA, A-SAA levels provide the strongest correlations with clinical measurements of disease activity, and changes in serum levels best reflect the clinical course.
A number of biologic activities have been described for A-SAA, including several that are relevant to the understanding of inflammatory and tissue-degrading mechanisms in human arthritis. A-SAA induces migration, adhesion and tissue infiltration of circulating monocytes and polymorphonuclear leukocytes. In addition, human A-SAA can induce interleukin-1β, interleukin-1 receptor antagonist and soluble type II tumour necrosis factor receptor production by a monocyte cell line. Moreover, A-SAA can stimulate the production of cartilage-degrading proteases by both human and rabbit synoviocytes. The effects of A-SAA on protease production are interesting, because in RA a sustained acute-phase reaction has been strongly associated with progressive joint damage. The known association between the acute-phase response and progressive joint damage may be the direct result of synovial A-SAA-induced effects on cartilage degradation.
Conclusion:
In contrast to noninflamed synovium, A-SAA mRNA expression was identified in all RA tissues examined. A-SAA appeared to be produced by synovial tissue synoviocytes, macrophages and endothelial cells. The observation of A-SAA mRNA expression in cultured RA synoviocytes and human RA synovial tissue confirms and extends recently published findings that demonstrated A-SAA mRNA expression in stimulated RA synoviocytes, but not in unstimulated RA synoviocytes.
PMCID: PMC17807  PMID: 11062604
acute-phase response; rheumatoid arthritis; serum amyloid A; synovial tissue
16.  Specificity of T cells in synovial fluid: high frequencies of CD8+ T cells that are specific for certain viral epitopes 
Arthritis Research  2000;2(2):154-164.
CD8+ T cells dominate the lymphocyte population in synovial fluid in chronic inflammatory arthritis. It is known that these CD8+ T cells are often clonally or oligoclonally expanded, but their specificity and their relevance to the pathogenesis of joint disease has remained unclear. We found that as many as 15.5% of synovial CD8+ T cells may be specific for a single epitope from an Epstein-Barr virus lytic cycle protein. The virus-specific T cells within the joint showed increased expression of markers of activation and differentiation compared with those in the periphery, and retained their functional capacity to secrete proinflammatory cytokines on stimulation. These activated, virus-specific CD8+ T cells could therefore interact with synoviocytes, either by cell-cell contact or by a cytokine network, and play a 'bystander' role in the maintenance of inflammation in patients with arthritis.
Introduction:
Epstein-Barr virus (EBV) is transmitted orally, replicates in the oropharynx and establishes life-long latency in human B lymphocytes. T-cell responses to latent and lytic/replicative cycle proteins are readily detectable in peripheral blood from healthy EBV-seropositive individuals. EBV has also been detected within synovial tissue, and T-cell responses to EBV lytic proteins have been reported in synovial fluid from a patient with rheumatoid arthritis (RA). This raises the question regarding whether T cells specific for certain viruses might be present at high frequencies within synovial fluid and whether such T cells might be activated or able to secrete cytokines. If so, they might play a 'bystander' role in the pathogenesis of inflammatory joint disease.
Objectives:
To quantify and characterize T cells that are specific for epitopes from EBV, cytomegalovirus (CMV) and influenza in peripheral blood and synovial fluid from patients with arthritis.
Methods:
Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were obtained from patients with inflammatory arthritis (including those with RA, osteoarthritis, psoriatic arthritis and reactive arthritis). Samples from human leucocyte antigen (HLA)-A2-positive donors were stained with fluorescent-labelled tetramers of HLA-A2 complexed with the GLCTLVAML peptide epitope from the EBV lytic cycle protein BMLF1, the GILGFVFTL peptide epitope from the influenza A matrix protein, or the NLVPMVATV epitope from the CMV pp65 protein. Samples from HLA-B8-positive donors were stained with fluorescent-labelled tetramers of HLA-B8 complexed with the RAKFKQLL peptide epitope from the EBV lytic protein BZLF1 or the FLRGRAYGL peptide epitope from the EBV latent protein EBNA3A. All samples were costained with an antibody specific for CD8. CD4+ T cells were not analyzed. Selected samples were costained with antibodies specific for cell-surface glycoproteins, in order to determine the phenotype of the T cells within the joint and the periphery. Functional assays to detect release of IFN-γ or tumour necrosis factor (TNF)-α were also performed on some samples.
Results:
The first group of 15 patients included 10 patients with RA, one patient with reactive arthritis, one patient with psoriatic arthritis and three patients with osteoarthritis. Of these, 11 were HLA-A2 positive and five were HLA-B8 positive. We used HLA-peptide tetrameric complexes to analyze the frequency of EBV-specific T cells in PBMCs and SFMCs (Figs 1 and 2). Clear enrichment of CD8+ T cells specific for epitopes from the EBV lytic cycle proteins was seen within synovial fluid from almost all donors studied, including patients with psoriatic arthritis and osteoarthritis and those with RA. In donor RhA6, 9.5% of CD8+ SFMCs were specific for the HLA-A2 restricted GLCTLVAML epitope, compared with 0.5% of CD8+ PBMCs. Likewise in a donor with osteoarthritis (NR4), 15.5% of CD8+ SFMCs were specific for the HLA-B8-restricted RAKFKQLL epitope, compared with 0.4% of CD8+ PBMCs. In contrast, we did not find enrichment of T cells specific for the HLA-B8-restricted FLRGRAYGL epitope (from the latent protein EBNA3A) within SFMCs compared with PBMCs in any donors. In selected individuals we performed ELISpot assays to detect IFN-γ secreted by SFMCs and PBMCs after a short incubation in vitro with peptide epitopes from EBV lytic proteins. These assays confirmed enrichment of T cells specific for epitopes from EBV lytic proteins within synovial fluid and showed that subpopulations of these cells were able to secrete proinflammatory cytokines after short-term stimulation.
We used a HLA-A2/GILGFVFTL tetramer to stain PBMCs and SFMCs from six HLA-A2-positive patients. The proportion of T cells specific for this influenza epitope was low (<0.2%) in all donors studied, and we did not find any enrichment within SFMCs.
We had access to SFMCs only from a second group of four HLA-A2-positive patients with RA. A tetramer of HLA-A2 complexed to the NLVPMVATV epitope from the CMV pp65 protein reacted with subpopulations of CD8+ SFMCs in all four donors, with frequencies of 0.2, 0.5, 2.3 and 13.9%. SFMCs from all four donors secreted TNF after short-term incubation with COS cells transfected with HLA-A2 and pp65 complementary DNA. We analyzed the phenotype of virus-specific cells within PBMCs and SFMCs in three donors. The SFMC virus-specific T cells were more highly activated than those in PBMCs, as evidenced by expression of high levels of CD69 and HLA-DR. A greater proportion of SFMCs were CD38+, CD62L low, CD45RO bright, CD45RA dim, CD57+ and CD28- when compared with PBMCs.
Discussion:
This work shows that T cells specific for certain epitopes from viral proteins are present at very high frequencies (up to 15.5% of CD8+ T cells) within SFMCs taken from patients with inflammatory joint disease. This enrichment does not reflect a generalized enrichment for the 'memory pool' of T cells; we did not find enrichment of T cells specific for the GILGFVFTL epitope from influenza A or for the FLRGRAYGL epitope from the EBV latent protein EBNA3A, whereas we found clear enrichment of T cells specific for the GLCTLVAML epitope from the EBV lytic protein BMLF1 and for the RAKFKQLL epitope from the EBV lytic protein BZLF1.
The enrichment might reflect preferential recruitment of subpopulations of virus-specific T cells, perhaps based on expression of selectins, chemokine receptors or integrins. Alternatively, T cells specific for certain viral epitopes may be stimulated to proliferate within the joint, by viral antigens themselves or by cross-reactive self-antigens. Finally, it is theoretically possible that subpopulations of T cells within the joint are preferentially protected from apoptotic cell death. Whatever the explanation, the virus-specific T cells are present at high frequency, are activated and are able to secrete proinflammatory cytokines. They could potentially interact with synoviocytes and contribute to the maintenance of inflammation within joints in many different forms of inflammatory arthritis.
PMCID: PMC17809  PMID: 11062606
CD8+ T cell; Epstein-Barr virus lytic cycle; human leucocyte antigen peptide tetrameric complex; rheumatoid arthritis; viral immunity
17.  Cytokine, activation marker, and chemokine receptor expression by individual CD4+ memory T cells in rheumatoid arthritis synovium 
Arthritis Research  2000;2(5):415-423.
IL-10, IL-13, IFN-γ, tumor necrosis factor (TNF)-α, LT-α, CD154, and TNF-related activation-induced cytokine (TRANCE) were expressed by 2-20% of rheumatoid arthritis (RA) synovial tissue CD4+ memory T cells, whereas CD4+ cells that produced IL-2, IL-4, or IL-6 were not detected. Expression of none of these molecules by individual CD4+ 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.
Introduction:
In RA large numbers of CD4+ memory T cells infiltrate the inflamed synovium [1,2,3]. The accumulated CD4+ memory T cells in the RA synovium appear to be activated, because they express cytokines and activation markers [4,5,6,7,8]. Expressed cytokines and activation markers should play important roles in the pathogenesis of RA. However, the frequency of cytokine expression by RA synovial CD4+ T cells has not been analyzed accurately. Recently, the roles of chemokine and chemokine receptor interactions in T-cell migration have been intensively examined. Interactions of chemokine and chemokine receptors might therefore be important in the accumulation of the CD4+ T cells in the RA synovium. Accordingly, correlation of cytokine and chemokine receptor expression might be important in delineating the function and potential means of accumulation of individual CD4+ memory T cells in the RA synovium.
In the present study we analyzed cytokine (IL-2, IL-4, IL-6, IL-10, IL-13, IFN-γ , TNF-α , and LT-α ), activation marker (CD154 [CD40 ligand] and TRANCE - also called receptor activator of nuclear factor κ B ligand [RANKL] or osteoclast differentiation factor [ODF]), and chemokine receptor expression by individual CD4+ memory T cells isolated from rheumatoid synovium and blood. To achieve this we employed a single-cell reverse transcription (RT) polymerase chain reaction (PCR) technique. This technique made it possible to correlate mRNAs expressed by individual CD4+ 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.
Cytokine (IL-2, IL-4, IL-6, IL-10, IL-13, IFN-γ, TNF-α, and LT-α ) and activation marker (CD154 and TRANCE) expression by individual CD4+CD45RO+ T cells from RA synovium or blood were analyzed using a single-cell RT-PCR. In brief, single CD4+CD45RO+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:
Cytokine and activation marker expression by individual CD4+CD45RO+T cells from RA synovial tissues was analyzed using a single-cell RT-PCR method. Expression of mRNAs was analyzed in 152 individual synovial tissue CD4+CD45RO+ T cells sorted from three RA patients in which T-cell receptor (TCR) Cβ mRNA was detected. Frequencies of CD4+ memory T cells expressing cytokine and activation marker mRNA in RA synovium are shown in Table 1. IL-2, IL-4, and IL-6 were not expressed by the synovial tissue CD4+CD45RO+ T cells, whereas 2-20% of cells expressed the other cytokine mRNAs.
Few correlations between cytokine and activation marker mRNAs were observed. Notably, no cells contained both IFN-γ and LT-α mRNAs, cytokines that are thought to define the T-helper (Th)1 phenotype [9]. However, the frequency of TRANCE-positive cells in IL-10-positive cells was significantly higher than that in IL-10-negative cells (Table 2). Moreover, the frequency of TRANCE-positive cells in TNF-α-positive cells was also significantly higher than that in TNF-α-negative cells.
Varying percentages of CD4+ 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.
Expression of cytokine and activation marker mRNAs was also analyzed in 48 individual peripheral blood CD4+CD45RO+ T cells from two RA patients. IL-2, IL-4, IL-6, and LT-α were not expressed by the peripheral CD4+CD45RO+ T cells, whereas 4-17% of cells expressed the other markers. The most striking difference between synovial tissue and peripheral blood CD4+ memory T cells was the presence of LT-α expression in the former, but not in the latter. IFN-γ and TNF-α were not expressed by normal peripheral blood CD4+ memory T cells, although they were expressed by RA peripheral blood CD4+ memory T cells.
Discussion:
The present study employed a single-cell PCR technology to analyze cytokine expression by unstimulated RA synovial tissue CD4+ memory T cells immediately after isolation, without in vitro manipulation. The results confirm the Th1 nature of rheumatoid inflammation. It is noteworthy that no individual synovial CD4+ memory T cells expressed both IFN-γ and LT-α mRNAs, even though these are the prototypic Th1 cytokines [9]. 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.
The present data showed that CCR5 expression correlated with IFN-γ but not with LT-α expression by synovial CD4+ memory T cells. It has been reported that CCR5 expression is upregulated in RA synovial fluid and synovial tissue T cells [10,11,12] and that CCR5 Δ 32 deletion may have an influence on clinical manifestations of RA [13], suggesting that CCR5 might play an important role in RA. Recently, it has been claimed that CCR5 was preferentially expressed by Th1 cell lines [14,15]. However, in the present study CCR5 was not expressed by all IFN-γ-expressing cells. Moreover, CCR5 expression did not correlate with expression of LT-α by RA synovial CD4+ memory T cells. Therefore, it is unclear whether CCR5 is a marker of Th1 cells in RA synovium.
IL-10 expression correlated with CCR7 expression by RA synovial CD4+ memory T cells. Recently, it was reported [16] that in the blood CCR7+CD4+ 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.
LT-α expression correlated with CCR6 expression by synovial CD4+ memory T cells. It has been reported that CCR6 is expressed by resting peripheral memory T cells [17], whereas LT-α expression is associated with the presence of lymphocytic aggregates in synovial tissue [7]. The correlation between the expression of these two markers therefore suggests the possibility that CCR6 may play a role in the development of aggregates of CD4+ T cells that are characteristically found in rheumatoid synovium.
TRANCE is known to be expressed by activated T cells, and can stimulate dendritic cells and osteoclasts [18]. Of note, TRANCE-mediated activation of osteoclasts has recently been shown [19] to play an important role in the damage to bone that is found in experimental models of inflammatory arthritis. It is therefore of interest that TRANCE was expressed by 3-16% of the RA synovial CD4+ memory T cells. Of note, 67% of TNF-α-positive cells expressed TRANCE. In concert, TNF-α and TRANCE expressed by this subset of CD4+ memory T cells might make them particularly important in mediating the bony erosions that are characteristic of RA.
Interestingly, there was a correlation between expression of IFN-γ and IL-10 in RA peripheral blood CD4+ memory T cells. In RA peripheral blood, CD154 expression correlated with that of CXCR3 by CD4+ memory T cells. It has been claimed [15] that CXCR3 is preferentially expressed by in vitro generated Th1 cells. However, in the present study CXCR3 did not correlate with IFN-γ expression. Although IFN-γ and TNF-α mRNAs were expressed in vivo by peripheral blood CD4+ T cells from RA patients, LT-α mRNA was not detected, whereas IFN-γ , TNF-α , and LT-α were not detected in samples from healthy donors. These findings indicate that RA peripheral blood CD4+ memory T cells are stimulated in vivo, although they do not express LT-α mRNA. The present studies indicate that the frequencies of CD4+ memory T cells that expressed IFN-γ in the blood and in the synovium are comparable. These results imply that activated CD4+ memory T cells migrate between blood and synovium, although the direction of the trafficking is unknown. The presence of LT-α mRNA in synovium, but not in blood, indicates that CD4+ memory cells are further activated in the synovium, and that these activated CD4+ memory T cells are retained in the synovium until LT-α mRNA decreases.
In conclusion, CD4+ 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.
PMCID: PMC17818  PMID: 11056676
chemokine receptor; cytokine; rheumatoid arthritis; T lymphocyte
18.  Do Natural T Regulatory Cells become Activated to Antigen Specific T Regulatory Cells in Transplantation and in Autoimmunity? 
Antigen specific T regulatory cells (Treg) are often CD4+CD25+FoxP3+ T cells, with a phenotype similar to natural Treg (nTreg). It is assumed that nTreg cannot develop into an antigen specific Treg as repeated culture with IL-2 and a specific antigen does not increase the capacity or potency of nTreg to promote immune tolerance or suppress in vitro. This has led to an assumption that antigen specific Treg mainly develop from CD4+CD25−FoxP3− T cells, by activation with antigen and TGF-β in the absence of inflammatory cytokines such as IL-6 and IL-1β. Our studies on antigen specific CD4+CD25+ T cells from animals with tolerance to an allograft, identified that the antigen specific and Treg are dividing, and need continuous stimulation with specific antigen T cell derived cytokines. We identified that a variety of cytokines, especially IL-5 and IFN-γ but not IL-2 or IL-4 promoted survival of antigen specific CD4+CD25+FoxP3+ Treg. To examine if nTreg could be activated to antigen specific Treg, we activated nTreg in culture with either IL-2 or IL-4. Within 3 days, antigen specific Treg are activated and there is induction of new cytokine receptors on these cells. Specifically nTreg activated by IL-2 and antigen express the interferon-γ receptor (IFNGR) and IL-12p70 (IL-12Rβ2) receptor but not the IL-5 receptor (IL-5Rα). These cells were responsive to IFN-γ or IL-12p70. nTreg activated by IL-4 and alloantigen express IL-5Rα not IFNGR or IL-12p70Rβ2 and become responsive to IL-5. These early activated antigen specific Treg, were respectively named Ts1 and Ts2 cells, as they depend on Th1 or Th2 responses. Further culture of Ts1 cells with IL-12p70 induced Th1-like Treg, expressing IFN-γ, and T-bet as well as FoxP3. Our studies suggest that activation of nTreg with Th1 or Th2 responses induced separate lineages of antigen specific Treg, that are dependent on late Th1 and Th2 cytokines, not the early cytokines IL-2 and IL-4.
doi:10.3389/fimmu.2013.00208
PMCID: PMC3731939  PMID: 23935597
antigen specific Treg; nTreg; Th1-like Treg; Th2-like Treg; immune tolerance
19.  HTLV-1 bZIP Factor Induces Inflammation through Labile Foxp3 Expression 
PLoS Pathogens  2013;9(9):e1003630.
Human T-cell leukemia virus type 1 (HTLV-1) causes both a neoplastic disease and inflammatory diseases, including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 basic leucine zipper factor (HBZ) gene is encoded in the minus strand of the proviral DNA and is constitutively expressed in infected cells and ATL cells. HBZ increases the number of regulatory T (Treg) cells by inducing the Foxp3 gene transcription. Recent studies have revealed that some CD4+Foxp3+ T cells are not terminally differentiated but have a plasticity to convert to other T-cell subsets. Induced Treg (iTreg) cells tend to lose Foxp3 expression, and may acquire an effector phenotype accompanied by the production of inflammatory cytokines, such as interferon-γ (IFN-γ). In this study, we analyzed a pathogenic mechanism of chronic inflammation related with HTLV-1 infection via focusing on HBZ and Foxp3. Infiltration of lymphocytes was observed in the skin, lung and intestine of HBZ-Tg mice. As mechanisms, adhesion and migration of HBZ-expressing CD4+ T cells were enhanced in these mice. Foxp3−CD4+ T cells produced higher amounts of IFN-γ compared to those from non-Tg mice. Expression of Helios was reduced in Treg cells from HBZ-Tg mice and HAM/TSP patients, indicating that iTreg cells are predominant. Consistent with this finding, the conserved non-coding sequence 2 region of the Foxp3 gene was hypermethylated in Treg cells of HBZ-Tg mice, which is a characteristic of iTreg cells. Furthermore, Treg cells in the spleen of HBZ-transgenic mice tended to lose Foxp3 expression and produced an excessive amount of IFN-γ, while Foxp3 expression was stable in natural Treg cells of the thymus. HBZ enhances the generation of iTreg cells, which likely convert to Foxp3−T cells producing IFN-γ. The HBZ-mediated proinflammatory phenotype of CD4+ T cells is implicated in the pathogenesis of HTLV-1-associated inflammation.
Author Summary
Viral infection frequently induces tissue inflammation in the host. HTLV-1 infection is associated with chronic inflammation in the CNS, skin, and lung, but the inflammatory mechanism is not fully understood yet. Since HTLV-1 directly infects CD4+ T cells, central player of the host immune regulation, HTLV-1 should modulate the host immune response not only via viral antigen stimulation but also via CD4+ T-cell-mediated immune deregulation. It has been reported that Foxp3+CD4+ T cells are increased in HTLV-1 infection. It remains a central question in HTLV-1 pathogenesis why HTLV-1 induces inflammation despite of increase of FoxP3+ cells, which generally possess immune suppressive function. We have elucidated here that most of the increased Foxp3+ cells in HBZ-Tg mice or HAM/TSP patients is not thymus-derived naturally occurring Treg cells but induced Treg cells. Since the iTreg cells are prone to lose FoxP3 expression and then become cytokine-producing cells, the increase of iTreg cells could serve as a source of proinflammatory CD4+ T cells. Thus HTLV-1 causes abnormal CD4+ T-cell differentiation by expressing HBZ, which should play a crucial role in chronic inflammation related with HTLV-1. This study has provided new insights into the mechanism of chronic inflammation accompanied with viral infection.
doi:10.1371/journal.ppat.1003630
PMCID: PMC3777874  PMID: 24068936
20.  The effects of 1α,25-dihydroxyvitamin D3 on matrix metalloproteinase and prostaglandin E2 production by cells of the rheumatoid lesion 
Arthritis Research  1999;1(1):63-70.
The biologically active metabolite of vitamin D3, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], acts through vitamin D receptors, which were found in rheumatoid tissues in the present study. IL-1β-activated rheumatoid synovial fibroblasts and human articular chondrocytes were shown to respond differently to exposure to 1α,25(OH)2D3, which has different effects on the regulatory pathways of specific matrix metalloproteinases and prostaglandin E2.
Introduction:
1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], the biologically active metabolite of vitamin D3, acts through an intracellular vitamin D receptor (VDR) and has several immunostimulatory effects. Animal studies have shown that production of some matrix metalloproteinases (MMPs) may be upregulated in rat chondrocytes by administration of 1α,25(OH)2D3; and cell cultures have suggested that 1α,25(OH)2D3 may affect chondrocytic function. Discoordinate regulation by vitamin D of MMP-1 and MMP-9 in human mononuclear phagocytes has also been reported. These data suggest that vitamin D may regulate MMP expression in tissues where VDRs are expressed. Production of 1α,25(OH)2D3 within synovial fluids of arthritic joints has been shown and VDRs have been found in rheumatoid synovial tissues and at sites of cartilage erosion. The physiological function of 1α,25(OH)2D3 at these sites remains obscure. MMPs play a major role in cartilage breakdown in the rheumatoid joint and are produced locally by several cell types under strict control by regulatory factors. As 1α,25(OH)2D3 modulates the production of specific MMPs and is produced within the rheumatoid joint, the present study investigates its effects on MMP and prostaglandin E2 (PGE2) production in two cell types known to express chondrolytic enzymes.
Aims:
To investigate VDR expression in rheumatoid tissues and to examine the effects of 1α,25-dihydroxyvitamin D3 on cultured rheumatoid synovial fibroblasts (RSFs) and human articular chondrocytes (HACs) with respect to MMP and PGE2 production.
Methods:
Rheumatoid synovial tissues were obtained from arthroplasty procedures on patients with late-stage rheumatoid arthritis; normal articular cartilage was obtained from lower limb amputations. Samples were embedded in paraffin, and examined for presence of VDRs by immunolocalisation using a biotinylated antibody and alkaline-phosphatase-conjugated avidin-biotin complex system. Cultured synovial fibroblasts and chondrocytes were treated with either 1α,25(OH)2D3, or interleukin (IL)-1β or both. Conditioned medium was assayed for MMP and PGE2 by enzyme-linked immunosorbent assay (ELISA), and the results were normalised relative to control values.
Results:
The rheumatoid synovial tissue specimens (n = 18) immunostained for VDRs showed positive staining but at variable distributions and in no observable pattern. VDR-positive cells were also observed in association with some cartilage-pannus junctions (the rheumatoid lesion). MMP production by RSFs in monolayer culture was not affected by treatment with 1α,25(OH)2D3 alone, but when added simultaneously with IL-1β the stimulation by IL-1β was reduced from expected levels by up to 50%. In contrast, 1α,25(OH)2D3 had a slight stimulatory effect on basal production of MMPs 1 and 3 by monolayer cultures of HACs, but stimulation of MMP-1 by IL-1β was not affected by the simultaneous addition of 1α,25(OH)2D3 whilst MMP-3 production was enhanced (Table 1). The production of PGE2 by RSFs was unaffected by 1α,25(OH)2D3 addition, but when added concomitantly with IL-1β the expected IL-1 β-stimulated increase was reduced to almost basal levels. In contrast, IL-1β stimulation of PGE2 in HACs was not affected by the simultaneous addition of 1α,25(OH)2D3 (Table 2). Pretreatment of RSFs with 1α,25(OH)2D3 for 1 h made no significant difference to IL-1β-induced stimulation of PGE2, but incubation for 16 h suppressed the expected increase in PGE2 to control values. This effect was also noted when 1α,25(OH)2D3 was removed after the 16h and the IL-1 added alone. Thus it appears that 1α,25(OH)2D3 does not interfere with the IL-1β receptor, but reduces the capacity of RSFs to elaborate PGE2 after IL-1β induction.
Discussion:
Cells within the rheumatoid lesion which expressed VDR were fibroblasts, macrophages, lymphocytes and endothelial cells. These cells are thought to be involved in the degradative processes associated with rheumatoid arthritis (RA), thus providing evidence of a functional role of 1α,25(OH)2D3 in RA. MMPs may play important roles in the chondrolytic processes of the rheumatoid lesion and are known to be produced by both fibroblasts and chondrocytes. The 1α,25(OH)2D3 had little effect on basal MMP production by RSFs, although more pronounced differences were noted when IL-1β-stimulated cells were treated with 1α,25(OH)2D3, with the RSF and HAC showing quite disparate responses. These opposite effects may be relevant to the processes of joint destruction, especially cartilage loss, as the ability of 1α,25(OH)2D3 to potentiate MMP-1 and MMP-3 expression by 'activated' chondrocytes might facilitate intrinsic cartilage chondrolysis in vivo. By contrast, the MMP-suppressive effects observed for 1α,25(OH)2D3 treatment of 'activated' synovial fibroblasts might reduce extrinsic chondrolysis and also matrix degradation within the synovial tissue. Prostaglandins have a role in the immune response and inflammatory processes associated with RA. The 1α,25(OH)2D3 had little effect on basal PGE2 production by RSF, but the enhanced PGE2 production observed following IL-1β stimulation of these cells was markedly suppressed by the concomitant addition of 1α,25(OH)2D3. As with MMP production, there are disparate effects of 1α,25(OH)2D3 on IL-1β stimulated PGE2 production by the two cell types; 1α,25(OH)2D3 added concomitantly with IL-1β had no effect on PGE2 production by HACs. In summary, the presence of VDRs in the rheumatoid lesion demonstrates that 1α,25(OH)2D3 may have a functional role in the joint disease process. 1α,25(OH)2D3 does not appear to directly affect MMP or PGE2 production but does modulate cytokine-induced production.
Comparative effects of 1 α,25-dihydroxyvitamin D3 (1 α,25D3) on interleukin (IL)-1-stimulated matrix metalloproteinase (MMP)-1 and MMP-3 production by rheumatoid synovial fibroblasts and human articular chondrocytes in vivo
Data given are normalized relative to control values and are expressed ± SEM for three cultures of each cell type.
Comparative effects of 1α,25-dihydroxyvitamin D3 (1α,25D3) on Interleukin (IL)-1-stimulated prostaglandin E2 production by rheumatoid synovial fibroblasts and human articular chondrocyte in vivo
Data given are normalized relative to control values and are expressed ± SEM for three cultures of each cell type.
PMCID: PMC17774  PMID: 11056661
1α,25-dihydroxyvitamin D3; matrix metalloproteinase; prostaglandin E2; rheumatoid arthritis
21.  CD4+CD25+ Regulatory Cells Contribute to the Regulation of Colonic Th2 Granulomatous Pathology Caused by Schistosome Infection 
Eggs of the helminth Schistosoma mansoni accumulate in the colon following infection and generate Th2-biassed inflammatory granulomas which become down- modulated in size as the infection proceeds to chronicity. However, although CD4+CD25+FoxP3+regulatory T cells (Tregs) are known to suppress Th1-mediated colitis, it is not clear whether they control Th2 –associated pathologies of the large intestine which characterise several helminth infections. Here we used a novel 3D-multiphoton confocal microscopy approach to visualise and quantify changes in the size and composition of colonic granulomas at the acute and chronic phases of S. mansoni infection. We observed decreased granuloma size, as well as reductions in the abundance of DsRed+ T cells and collagen deposition at 14 weeks (chronic) compared to 8 weeks (acute) post-infection. Th2 cytokine production (i.e. IL-4, IL-5) in the colonic tissue and draining mesenteric lymph node (mLN) decreased during the chronic phase of infection, whilst levels of TGF-β1 increased, co-incident with reduced mLN proliferative responses, granuloma size and fibrosis. The proportion of CD4+CD25+FoxP3+Tregs: CD4+ cells in the mLN increased during chronic disease, while within colonic granulomas there was an approximate 4-fold increase. The proportion of CD4+CD25+FoxP3+Tregs in the mLN that were CD103+ and CCR5+ also increased indicating an enhanced potential to home to intestinal sites. CD4+CD25+ cells suppressed antigen-specific Th2 mLN cell proliferation in vitro, while their removal during chronic disease resulted in significantly larger granulomas, partial reversal of Th2 hypo-responsiveness and an increase in the number of eosinophils in colonic granulomas. Finally, transfer of schistosome infection-expanded CD4+CD25+Tregs down-modulated the development of colonic granulomas, including collagen deposition. Therefore, CD4+CD25+FoxP3+Tregs appear to control Th2 colonic granulomas during chronic infection, and are likely to play a role in containing pathology during intestinal schistosomiasis.
Author Summary
Schistosomiasis is an important parasitic helminth disease afflicting more than 200 million people worldwide. Infections are typically chronic and in the case of Schistosoma mansoni and S. japonicum the majority give rise to an intestinal form of disease caused by the deposition of parasite eggs in the colon and terminal ileum. The eggs cause Th2-associated inflammatory immune granulomas to form, which as the disease develops, are down-regulated by cells of the immune system. However, the mechanisms which underpin the down-regulation of granulomas in the large intestine are not known. In order to investigate the phenomenon of Th2-associated colonic inflammation, we utilized a murine model of infection with S. mansoni and compared immune responses at the acute and chronic phases of infection. We show that a type of regulatory T helper lymphocyte (CD4+CD25+FoxP3+Treg) contributes to regulation of colonic inflammation. These cells modulate anti-egg Th2 responses within the mesenteric lymph nodes and granulomatous pro-fibrotic Th2 responses within the colon. Our study highlights the importance of CD4+CD25+FoxP3+Tregs as a source of regulatory pressure on granuloma formation in the colon and by implication humans with chronic intestinal schistosomiasis.
doi:10.1371/journal.pntd.0001269
PMCID: PMC3153428  PMID: 21858239
22.  Increased CD45RA+FoxP3low Regulatory T Cells with Impaired Suppressive Function in Patients with Systemic Lupus Erythematosus 
PLoS ONE  2012;7(4):e34662.
Background
The role of naturally occurring regulatory T cells (Treg) in the control of the development of systemic lupus erythematosus (SLE) has not been well defined. Therefore, we dissect the phenotypically heterogeneous CD4+FoxP3+ T cells into subpopulations during the dynamic SLE development.
Methodlogy/Principal Findings
To evaluate the proliferative and suppressive capacities of different CD4+ T cell subgroups between active SLE patients and healthy donors, we employed CD45RA and CD25 as surface markers and carboxyfluorescein diacetatesuccinimidyl ester (CFSE) dilution assay. In addition, multiplex cytokines expression in active SLE patients was assessed using Luminex assay. Here, we showed a significant increase in the frequency of CD45RA+FoxP3low naive Treg cells (nTreg cells) and CD45RA−FoxP3low (non-Treg) cells in patients with active SLE. In active SLE patients, the increased proportions of CD45RA+FoxP3low nTreg cells were positively correlated with the disease based on SLE disease activity index (SLEDAI) and the status of serum anti-dsDNA antibodies. We found that the surface marker combination of CD25+CD45RA+ can be used to defined CD45RA+FoxP3low nTreg cells for functional assays, wherein nTreg cells from active SLE patients demonstrated defective suppression function. A significant correlation was observed between inflammatory cytokines, such as IL-6, IL-12 and TNFα, and the frequency of nTreg cells. Furthermore, the CD45RA+FoxP3low nTreg cell subset increased when cultured with SLE serum compared to healthy donor serum, suggesting that the elevated inflammatory cytokines of SLE serum may promote nTreg cell proliferation/expansion.
Conclusions/Significance
Our results indicate that impaired numbers of functional CD45RA+FoxP3low naive Treg cell and CD45RA−FoxP3low non-suppressive T cell subsets in inflammatory conditions may contribute to SLE development. Therefore, analysis of subsets of FoxP3+ T cells, using a combination of FoxP3, CD25 and CD45RA, rather than whole FoxP3+ T cells, will help us to better understand the pathogenesis of SLE and may lead to the development of new therapeutic strategies.
doi:10.1371/journal.pone.0034662
PMCID: PMC3323568  PMID: 22506043
23.  Expansion of regulatory GITR+CD25low/-CD4+ T cells in systemic lupus erythematosus patients 
Introduction
CD4+CD25low/-GITR+ T lymphocytes expressing forkhead box protein P3 (FoxP3) and showing regulatory activity have been recently described in healthy donors. The objective of the study was to evaluate the proportion of CD4+CD25low/-GITR+ T lymphocytes within CD4+ T cells and compare their phenotypic and functional profile with that of CD4+CD25highGITR− T lymphocytes in systemic lupus erythematosus (SLE) patients.
Methods
The percentage of CD4+CD25low/-GITR+ cells circulating in the peripheral blood (PB) of 32 patients with SLE and 25 healthy controls was evaluated with flow cytometry. CD4+CD25low/-GITR+ cells were isolated with magnetic separation, and their phenotype was compared with that of CD4+CD25highGITR− cells. Regulatory activity of both cell subsets was tested in autologous and heterologous co-cultures after purification through a negative sorting strategy.
Results
Results indicated that CD4+CD25low/-GITR+ cells are expanded in the PB of 50% of SLE patients. Expansion was observed only in patients with inactive disease. Phenotypic analysis demonstrated that CD4+CD25low/-GITR+ cells display regulatory T-cell (Treg) markers, including FoxP3, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), transforming growth factor-beta (TGF-β), and interleukin (IL)-10. In contrast, CD4+CD25highGITR− cells appear to be activated and express low levels of Treg markers. Functional experiments demonstrated that CD4+CD25low/-GITR+ cells exert a higher inhibitory activity against both autologous and heterologous cells as compared with CD4+CD25highGITR− cells. Suppression is independent of cell contact and is mediated by IL-10 and TGF-β.
Conclusions
Phenotypic and functional data demonstrate that in SLE patients, CD4+CD25low/-GITR+ cells are fully active Treg cells, possibly representing peripheral Treg (pTreg) that are expanded in patients with inactive disease. These data may suggest a key role of this T-cell subset in the modulation of the abnormal immune response in SLE. Strategies aimed at expanding this Treg subset for therapeutic purpose deserve to be investigated.
doi:10.1186/s13075-014-0444-x
PMCID: PMC4209023  PMID: 25256257
24.  Induced T Regulatory Cells Suppress Osteoclastogenesis and Bone Erosion in Collagen-induced Arthritis Better than Natural T Tegulatory Cells 
Annals of the rheumatic diseases  2012;71(9):1567-1572.
Objective
Osteoclasts are responsible for bone destruction in rheumatoid arthritis (RA) and natural CD4+Foxp3+regulatory T cells (nTregs) can inhibit osteoclastogenesis. This study aims to determine whether TGF-β-induced CD4+Foxp3+regulatory T cells (iTregs) also suppress osteolastogenesis and bone erosion in collagen induced arthritis (CIA).
Methods
Osteoclasts were induced from bone-marrow CD11b+ cells with RANKL and macrophage colony-stimulating factor (M-CSF), and assessed with tartrate-resistant acid phosphatase (TRAP) staining. CD4+ iTregs were generated with TGF-β and added to cultures with different ratios with CD11b+ cells. Transwell and antibody blockade experiments were performed to define the mechanism of action. NF-kB activation was determined by western blot. 3×106 CD4+ iTregs, nTregs or control cells were adoptively transferred to DBA1/J mice on day 14 after immunization with CII/CFA. CIA onset and severity were monitored and bone erosion was examined by CT scan.
Results
Both CD4+ Tregs almost completely suppressed osteoclastogenesis but only iTregs sustained the effect in the presence of IL-6 in vitro. CD4+ iTregs but not nTregs and control cells injected after immunization and before of onset of CIA significantly suppressed disease development. Of note, CT scan showed that the joints in CD4+ iTregs but not nTregs or control cells infused CIA had less bone erosion. Treatment with CD4+ iTregs but not other cells dramatically decreased the levels of NF-kB p65/p50 in osteoclasts in vitro and P65/50 and RANKL expression by synovial tissues in vivo.
Conclusion
Manipulation of CD4+ iTregs may have therapeutic effects on rheumatoid arthritis and other bone erosion related diseases.
doi:10.1136/annrheumdis-2011-201052
PMCID: PMC4038329  PMID: 22764040
25.  Human Regulatory T Cell Suppressive Function Is Independent of Apoptosis Induction in Activated Effector T Cells 
PLoS ONE  2009;4(9):e7183.
Background
CD4+CD25+FOXP3+ Regulatory T cells (Treg) play a central role in the immune balance to prevent autoimmune disease. One outstanding question is how Tregs suppress effector immune responses in human. Experiments in mice demonstrated that Treg restrict effector T cell (Teff) responses by deprivation of the growth factor IL-2 through Treg consumption, resulting in apoptosis of Teff.
Principal Findings
In this study we investigated the relevance of Teff apoptosis induction to human Treg function. To this end, we studied naturally occurring Treg (nTreg) from peripheral blood of healthy donors, and, to investigate Treg function in inflammation in vivo, Treg from synovial fluid of Juvenile Idiopathic Arthritis (JIA) patients (SF-Treg). Both nTreg and SF-Treg suppress Teff proliferation and cytokine production efficiently as predicted. However, in contrast with murine Treg, neither nTreg nor SF-Treg induce apoptosis in Teff. Furthermore, exogenously supplied IL-2 and IL-7 reverse suppression, but do not influence apoptosis of Teff.
Significance
Our functional data here support that Treg are excellent clinical targets to counteract autoimmune diseases. For optimal functional outcome in human clinical trials, future work should focus on the ability of Treg to suppress proliferation and cytokine production of Teff, rather than induction of Teff apoptosis.
doi:10.1371/journal.pone.0007183
PMCID: PMC2746309  PMID: 19779623

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