Cell contact-mediated signaling of monocytes by stimulated T cells: a major pathway for cytokine induction

T lymphocytes are currently thought to play a pivotal part in the pathogenesis of chronic inflammatory diseases. However, the mechanism(s) by which they exert their pathogenic effect remain(s) elusive. Contact-mediated signaling of monocytes by stimulated T cells is a potent pro-inflammatory mechanism that triggers massive up-regulation of the pro-inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-alpha) that play an important part in chronic destructive diseases such as rheumatoid arthritis and multiple sclerosis. To date cell-cell contact is the only endogenous mechanism to be described that displays such an activity in monocyte-macrophages which are classically stimulated in vitro by bacterial products such as LPS or non-specific stimuli such as phorbol esters or poorly activated by soluble cytokines such as IFN-gamma. Since direct cellular contact occurs at the inflammatory site, we hypothesized that this mechanism is relevant to the pathogenesis of chronic inflammatory disorders. This review aims at summarizing the state of the art and importance of contact-mediated monocyte activation by stimulated T lymphocytes.     

Suppressing IL-32 in monocytes impairs the induction of the proinflammatory cytokines TNFα and IL-1β

Targeting major proinflammatory cytokines such as IL-1β and TNFα is of great interest in patients with chronic inflammatory diseases, including rheumatoid arthritis, colitis, and psoriasis. The cytokine Interleukin (IL)-32 induces proinflammatory cytokines such as TNFα, IL-1β, IL-6, and chemokines. We previously used an IL-32 ligand-affinity column to purify proteinase 3, which is abundantly expressed in neutrophil and monocytic leukocytes but not in other cell types, and found that IL-32 is mainly produced by monocytic leukocytes. This evidence suggested that silencing endogenous IL-32 by short hairpin RNA (shRNA) in monocytic cells might reveal the precise function of endogenous IL-32. Indeed, lipopolysaccharide (LPS)- or phorbol myristate acetate (PMA)-induced proinflammatory cytokine production was significantly inhibited in shRNA/IL-32 stable clones as compared to control clones. Furthermore, macrophages in PMA-differentiated shRNA/IL-32 stable clones displayed remarkably impaired LPS- and IL-1β-induced proinflammatory cytokine production. These data suggest that IL-32 is not only involved in host defense against pathogens, but also might play a role in chronic inflammatory diseases. IL-32 production leads to major proinflammatory cytokine production during the initial immune response.     

Stimulation of human synovial fibroblast DNA synthesis by platelet-derived growth factor and fibroblast growth factor. Differences to the activation by IL-1

The pronounced synovial hyperplasia often found in the joints of patients with rheumatoid arthritis could be explained partially by the action of monocyte-macrophage polypeptides (monokines). This report demonstrates that two cytokines which may be derived from monocyte-macrophage populations, namely platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), stimulate the DNA synthesis and proliferation of human synovial fibroblast-like cells cultured in low (i.e., 1%) fetal bovine serum. Epidermal growth factor, insulin-like growth factor-I, insulin-like growth factor-II (multiplication stimulating activity) and substance P were inactive. Unlike IL-1, PDGF and FGF do not also stimulate PGE2, plasminogen activator, and hyaluronic acid levels. Thus PDGF and FGF, arising from stimulated monocyte-macrophages, may play a role in the stimulation of mesenchymal cell proliferation that often accompanies chronic inflammatory arthritic disease. The synovial cells respond to a variety of cytokines in different ways suggesting multiple-signaling pathways.     

Gastrin-releasing peptide,substance P and cytokines in rheumatoid arthritis

Many studies have shown that modulation of cytokine function is effective in ameliorating symptoms of rheumatoid arthritis. Neuropeptides have recently been shown to have powerful effects on the production and release of cytokines and have also been shown to exert potent proinflammatory and anti-inflammatory effects in animal models of inflammatory diseases. An analysis of cytokine and neuropeptide content of synovial fluid from patients with rheumatoid arthritis has revealed a significant correlation between two neuropeptides, bombesin/gastrin-releasing peptide and substance P, and the proinflammatory cytokine interleukin-6 as well as the erythrocyte sedimentation rate. These findings provide further evidence for a role of neuropeptides and cytokines in the pathophysiology of rheumatoid arthritis, as well as suggesting additional approaches for the development of novel therapeutic interventions.     

TL1A Increases Expression of CD25, LFA-1, CD134 and CD154, and Induces IL-22 and GM-CSF Production from Effector CD4 T-Cells

Elevated levels of the cytokine TL1A is associated with several autoimmune diseases e.g. rheumatoid arthritis and inflammatory bowel disease. However, the exact role of TL1A remains elusive. In this study, we investigated the function of TL1A in a pro-inflammatory setting. We show that TL1A together with IL-12, IL-15 and IL-18 increases expression of the co-stimulatory molecules CD154 (CD40 ligand) and CD134 (OX40) on previously activated CD4⁺ T cells. This indicates that TL1A functions as a co-stimulatory molecule, decreasing the activation threshold of T-cells. We have previously shown that TL1A co-stimulation strongly induces IL-6 in human healthy leukocytes. Interestingly, the cytokine-activated effector T-cells did not produce IL-6 in response to TL1A, indicating distinct effects of TL1A on different cell populations. We further show that this co-stimulation increases the expression of CD25 (IL-2Rα) and CD11a (α-chain of LFA-1) on CD4 T-cells, likely governing increased IL-2/IL-15 sensitivity and cell-cell contact. Along with this, TL1A co-stimulation caused a specific induction of IL-22 and GM-CSF from the activated T-cells. These results substantially contribute to the explanation of TL1A''s role in inflammation. Our results suggest that TL1A should be considered as a target for immunotherapeutic treatment of rheumatoid arthritis and inflammatory bowel disease.     

Calcineurin is expressed and plays a critical role in inflammatory arthritis

Calcineurin is a calcium-activated phosphatase to mediate lymphocyte activation and neuron signaling, but its role in inflammatory arthritis remains largely unknown. In this study, we demonstrate that calcineurin was highly expressed in the lining layer, infiltrating leukocytes, and endothelial cells of rheumatoid synovium. The basal expression levels of calcineurin were higher in the cultured synoviocytes of rheumatoid arthritis patients than those of osteoarthritis patients. The calcineurin activity in the synoviocytes was increased by the stimulation with proinflammatory cytokines such as IL-1beta and TNF-alpha. Moreover, rheumatoid arthritis synoviocytes had an enlarged intracellular Ca(2+) store and showed a higher degree of [Ca(2+)](i) release for calcineurin activity than osteoarthritis synoviocytes when stimulated with either TNF-alpha or phorbol myristate acetate. IL-10, an anti-inflammatory cytokine, failed to increase the Ca(2+) and calcineurin activity. The targeted inhibition of calcineurin by the overexpression of calcineurin-binding protein 1, a natural calcineurin antagonist, inhibited the production of IL-6 and matrix metalloproteinase-2 by rheumatoid synoviocytes in a similar manner to the calcineurin inhibitor, cyclosporin A. Moreover, the abundant calcineurin expression was found in the invading pannus in the joints of mice with collagen-induced arthritis. In these mice, calcineurin activity in the cultured synovial and lymph node cells correlated well with the severity of arthritis, but which was suppressed by cyclosporin A treatment. Taken together, our data suggest that the abnormal activation of Ca(2+) and calcineurin in the synoviocytes may contribute to the pathogenesis of chronic arthritis and thus provide a potential target for controlling inflammatory arthritis.     

Adenoviral transfer of cyclin-dependent kinase inhibitor genes suppresses collagen-induced arthritis in mice

In rheumatoid synovial tissues, synovial fibroblasts are activated by proinflammatory cytokines and proliferate to develop hyperplastic pannus tissues, which irreversibly damage the affected joints. We recently reported that the cyclin-dependent kinase inhibitors p16(INK4a) and p21(Cip1) are not expressed in vivo in rheumatoid synovial fibroblasts, but are readily inducible in vitro. This observation was followed by the successful treatment of rat adjuvant arthritis by local p16(INK4a) gene transfer, showing that the inhibition of the cell cycle of the synovial cells ameliorates the arthritis. In this study, we show that another animal model of rheumatoid arthritis, murine collagen-induced arthritis, can be effectively treated by local gene transfer of p21(Cip1) as well as that of p16(INK4a). The anti-arthritic effects were observed even when the treatment was conducted after the arthritis had developed. Furthermore, the effects included suppression of the expression of proinflammatory cytokines such as IL-1ss, IL-6, and TNF-alpha. Our results demonstrate that the ectopic expression of cyclin-dependent kinase inhibitors not only prevents synovial overgrowth but also ameliorates the proinflammatory milieu in the affected joints. The induction of p21(Cip1) in rheumatoid synovial tissues by pharmacological agents may also be an effective strategy to treat rheumatoid arthritis.     

IL-15 and the initiation of cell contact-dependent synovial fibroblast-T lymphocyte cross-talk in rheumatoid arthritis: effect of methotrexate

To characterize the molecules responsible for synovial fibroblast-T lymphocyte (TL) cross-talk in rheumatoid arthritis (RA), synovial fibroblasts from patients with established RA (RASFibs) were cocultured with TLs from peripheral blood of early RA patients (RAPBTL). TLs from peripheral blood of healthy controls and from synovial fluid of RA served as controls. Adhesion molecules and cytokines were determined by flow cytometry, ELISA, and real-time PCR. RAPBTL (n = 20) induced an up-regulation of ICAM-1, intracellular IL-8, IL-6, IL-15, and surface IL-15 in cocultured RASFibs. In turn, RAPBTL showed an up-regulation of TNF-alpha, IFN-gamma, IL-17, CD25, and CD69 expression. Responses seen with TLs from peripheral blood of healthy controls (n = 20) were significantly lower, whereas responses with TLs from synovial fluid of RA (n = 20) were maximal. Blocking Abs to IL-15 and CD54, but not an isotype-control Ab, down-regulated the increased TL cytokine and activation marker expression. Abs to CD69, CD11a, IL-17, TNF-alpha, and IFN-gamma significantly decreased the up-regulation of RASFib cytokine and CD54 expression. Cocultures using 0.4- micro m inserts did not result in up-regulation of surface molecules or cytokines. Methotrexate significantly inhibited RASFib/TL cross-talk signals and decreased adhesion of TL to RASFibs. In summary, RASFib production of IL-15 induces the proinflammatory cytokines TNF-alpha, IFN-gamma, and IL-17 in cocultured TLs through a cell contact-dependent mechanism. In turn, these cytokines stimulate the expression of IL-15, IL-8, and IL-6 in RASFibs, thereby creating a feedback loop that favors persistent synovial inflammation. Methotrexate seems to disrupt this loop by decreasing cell adhesion.     

Effect of calcitonin gene-related peptide,neuropeptide Y,substance P,and vasoactive intestinal peptide on interleukin-1beta,interleukin-6 and tumor necrosis factor-alpha production by peripheral whole blood cells from rheumatoid arthritis and osteoarthritis patients

In the present study, we have investigated the in vitro effect of calcitonin-related peptide (CGRP), neuropeptide Y (NPY), substance P (SP) and vasoactive intestinal peptide (VIP) at concentrations of 10(-8), 10(-9) and 10(-10) M on the production of different proinflammatory cytokines or chemokines such as IL-1beta, IL-6 and TNFalpha by peripheral whole blood cells from patients with rheumatoid arthritis, as well as from osteoarthritis patients studied as a control group without immunoinflammatory background. We have found that CGRP, NPY, SP and VIP stimulated significantly the production of those cytokines and chemokines in rheumatoid arthritis patients. In general, the stimulation was higher at the 10(-9) M concentration, with SP and VIP, and in rheumatoid arthritis patients compared to osteoarthritis ones. Neuropeptides did not significantly modify the LPS-induced cytokine production by whole blood cells. The results indicate that physiological concentrations of the neuropeptides studied can modulate the inflammatory and immunological response, stimulating significantly the production of inflammatory cytokines by human whole blood cells in rheumatoid arthritis patients, as well as, in a minor way, in osteoarthritis patients.     

Regulation of the interleukin-1 receptor antagonist in THP-1 cells by ligands of the peroxisome proliferator-activated receptor gamma

Monocytes/macrophages (Mphi) play a pivotal role in the persistence of chronic inflammation and local tissue destruction in diseases such as rheumatoid arthritis and atherosclerosis. The production by Mphi of cytokines, chemokines, metalloproteinases and their inhibitors is an essential component in this process, which is tightly regulated by multiple factors. The peroxisome proliferator-activated receptors (PPARs) were shown to be involved in modulating inflammation. PPARgamma is activated by a wide variety of ligands such as fatty acids, the anti-diabetic thiazolidinediones (TZDs), and also by certain prostaglandins of which 15-deoxy-Delta(12,14)-PGJ2 (PGJ2). High concentrations of PPARgamma ligands were shown to have anti-inflammatory activities by inhibiting the secretion of interleukin-1 (IL-1), interleukin-6 (IL-6) and tumour necrosis factor alpha (TNFalpha) by stimulated monocytes.The aim of this study was to determine whether PGJ2 and TZDs would also exert an immunomodulatory action through the up-regulation of anti-inflammatory cytokines such as the IL-1 receptor antagonist (IL-1Ra). THP-1 monocytic cells were stimulated with PMA, thereby enhancing the secretion of IL-1, IL-6, TNFalpha, IL-1Ra and metalloproteinases. Addition of PGJ2 had an inhibitory effect on IL-1, IL-6 and TNFalpha secretion, while increasing IL-1Ra production. In contrast, the bona fide PPARgamma ligands (TZDs; rosiglitazone, pioglitazone and troglitazone) barely inhibited proinflammatory cytokines, but strongly enhanced the production of IL-1Ra from PMA-stimulated THP-1 cells. Unstimulated cells did not respond to TZDs in terms of IL-1Ra production, suggesting that in order to be effective, PPAR ligands depend on PMA signalling. Basal levels of PPARgamma are barely detectable in unstimulated THP-1 cells, while stimulation with PMA up-regulates its expression, suggesting that higher levels of PPARgamma expression are necessary for receptor ligand effects to occur. In conclusion, we demonstrate for the first time that TZDs may exert an anti-inflammatory activity by inducing the production of the IL-1Ra.     

Leflunomide inhibits PDK1/Akt pathway and induces apoptosis of human mast cells

Mast cells release many inflammatory mediators that play an important role not only in allergic diseases but also in chronic inflammatory diseases, autoimmune diseases, and others. A lot of mast cells exist in synovium of rheumatoid arthritis, and it is known that synovitis does not occur in mast cell-deficient mice. Thus, it is thought that mast cells play a very important role in rheumatoid arthritis pathogenesis. Leflunomide is a drug used clinically in the treatment of rheumatoid arthritis. We used clinical doses of 2-cyano-3-hydroxy-N-(4-trifluoromethylphenyl)-butenamide (A77 1726), which is an active metabolite of leflunomide, and decreased the number of viable human primary mast cells in a concentration-dependent manner. This decrease was not reversed by uridine. Inhibition of pyrimidine synthesis by dihydro-orotic acid dehydrogenase inhibition, which is the primary mechanism of action of A77 1726, was not involved. A77 1726 dramatically induced apoptosis of human mast cells and inhibited the phosphorylation of Akt, an important survival signal of mast cells, in a concentration-dependent manner. Caspases 3 and 9, downstream molecules of Akt survival pathway, were also fragmented by A77 1726. In addition, it became evident for the first time that the mechanism involved in this result was the concentration-dependent inhibition of PDK1 phosphorylation, which controls the activation of Akt. These results indicate a new way of controlling mast cells and may therefore be the basis for innovative approaches to the treatment of various diseases related to mast cells.     

Endothelial cell phenotypes in the rheumatoid synovium: activated,angiogenic, apoptotic and leaky

Endothelial cells are active participants in chronic inflammatory diseases. These cells undergo phenotypic changes that can be characterised as activated, angiogenic, apoptotic and leaky. In the present review, these phenotypes are described in the context of human rheumatoid arthritis as the disease example. Endothelial cells become activated in rheumatoid arthritis pathophysiology, expressing adhesion molecules and presenting chemokines, leading to leukocyte migration from the blood into the tissue. Endothelial cell permeability increases, leading to oedema formation and swelling of the joints. These cells proliferate as part of the angiogenic response and there is also a net increase in the turnover of endothelial cells since the number of apoptotic endothelial cells increases. The endothelium expresses various cytokines, cytokine receptors and proteases that are involved in angiogenesis, proliferation and tissue degradation. Associated with these mechanisms is a change in the spectrum of genes expressed, some of which are relatively endothelial specific and others are widely expressed by other cells in the synovium. Better knowledge of molecular and functional changes occurring in endothelial cells during chronic inflammation may lead to the development of endothelium-targeted therapies for rheumatoid arthritis and other chronic inflammatory diseases.     

Soluble TNF-like cytokine (TL1A) production by immune complexes stimulated monocytes in rheumatoid arthritis

TNF-like cytokine (TL1A) is a newly identified member of the TNF superfamily of ligands that is important for T cell costimulation and Th1 polarization. However, despite increasing information about its functions, very little is known about expression of TL1A in normal or pathological states. In this study, we report that mononuclear phagocytes appear to be a major source of TL1A in rheumatoid arthritis (RA), as revealed by their strong TL1A expression in either synovial fluids or synovial tissue of rheumatoid factor (RF)-seropositive RA patients, but not RF-/RA patients. Accordingly, in vitro experiments revealed that human monocytes express and release significant amounts of soluble TL1A when stimulated with insoluble immune complexes (IC), polyethylene glycol precipitates from the serum of RF+/RA patients, or with insoluble ICs purified from RA synovial fluids. Monocyte-derived soluble TL1A was biologically active as determined by its capacity to induce apoptosis of the human erythroleukemic cell line TF-1, as well as to cooperate with IL-12 and IL-18 in inducing the production of IFN-gamma by CD4(+) T cells. Because RA is a chronic inflammatory disease with autoimmune etiology, in which ICs, autoantibodies (including RF), and various cytokines contribute to its pathology, our data suggest that TL1A could be involved in its pathogenesis and contribute to the severity of RA disease that is typical of RF+/RA patients.     

IL-3 attenuates collagen-induced arthritis by modulating the development of Foxp3+ regulatory T cells

IL-3, a cytokine secreted by Th cells, functions as a link between the immune and the hematopoietic system. We previously demonstrated the potent inhibitory role of IL-3 on osteoclastogenesis, pathological bone resorption, and inflammatory arthritis. In this study, we investigated the novel role of IL-3 in development of regulatory T (Treg) cells. We found that IL-3 in a dose-dependent manner increases the percentage of Foxp3(+) Treg cells indirectly through secretion of IL-2 by non-Treg cells. These IL-3-expanded Treg cells are competent in suppressing effector T cell proliferation. Interestingly, IL-3 treatment significantly reduces the severity of arthritis and restores the loss of Foxp3(+) Treg cells in thymus, lymph nodes, and spleen in collagen-induced arthritis mice. Most significantly, we show that IL-3 decreases the production of proinflammatory cytokines IL-6, IL-17A, TNF-α, and IL-1 and increases the production of anti-inflammatory cytokines IFN-γ and IL-10 in collagen-induced arthritis mice. Thus, to our knowledge, we provide the first evidence that IL-3 play an important role in modulation of Treg cell development in both in vitro and in vivo conditions, and we suggest its therapeutic potential in the treatment of rheumatoid arthritis and other autoimmune diseases.     

Selective regulation of cytokine induction by adenoviral gene transfer of IkappaBalpha into human macrophages: lipopolysaccharide-induced, but not zymosan-induced, proinflammatory cytokines are inhibited, but IL-10 is nuclear factor-kappaB independent

Macrophages are the major cytokine producers in chronic inflammatory diseases, but the biochemical pathways regulating cytokine production are poorly understood. This is because genetic tools to dissect signaling pathways cannot be used in macrophages because of difficulties in transfection. We have developed an adenoviral technique to achieve high efficiency gene delivery into macrophages and recently showed that spontaneous TNF-alpha production in rheumatoid arthritis joint cells, chiefly from macrophages, is 75% blocked by adenoviral transfer of IkappaBalpha. In this report we use the same adenovirus to investigate whether the production of a number of proinflammatory cytokines (e.g., TNF-alpha, IL-1beta, IL-6, and IL-8) from human macrophages depends on NF-kappaB. While the cytokine response to certain inducers, such as LPS, PMA, and UV light, is blocked by overexpression of IkappaBalpha, the response to zymosan is not. In contrast, anti-inflammatory mediators (IL-10 and IL-1 receptor antagonist) induced by LPS are only marginally inhibited by IkappaBalpha excess. These studies demonstrate several new points about macrophage cytokine production. First, there is heterogeneity of mechanisms regulating both the proinflammatory and anti-inflammatory cytokines within populations of a single cell type. In addition, the results confirm the utility of the adenoviral technique for functional analysis of cytokine induction. The results also confirm that there are autocrine and paracrine interactions regulating cytokine synthesis within a single cell type. The selectivity of NF-kappaB blockade for proinflammatory but not anti-inflammatory mediators indicates that in macrophages, NF-kappaB may be a good target for the treatment of chronic inflammatory diseases.     

Effect of TNFα on osteoblastogenesis from mesenchymal stem cells

Background

Bone destruction and osteoporosis are accelerated in chronic inflammatory diseases, such as rheumatoid arthritis (RA) and periodontitis, in which many studies have shown the proinflammatory cytokines, especially TNFα, play an important role; TNFα causes osteoclast-induced bone destruction as well as the inhibition of osteoblastogenesis.

Scope of review

Here we review our current understanding of the mechanism of the effect of TNFα on osteoblastogenesis from mesenchymal stem cells (MSCs). We also highlight the function of MSC in the pathogenesis of autoimmune diseases.

Major conclusions

Many studies have revealed that TNFα inhibits osteoblastogenesis through several mechanisms. On the other hand, it has been also reported that TNFα promotes osteoblastogenesis. These discrepancies may depend on the cellular types, the model animals, and the timing and duration of TNFα administration.

General significance

A full understanding of the role and function of TNFα on osteoblastogenesis from MSC may lead to targeted new therapies for chronic inflammation diseases, such as RA and periodontitis.     

Tumor necrosis factor is critical to control tuberculosis infection

Tumor necrosis factor (TNF) is critical and non-redundant to control Mycobacterium tuberculosis infection and cannot be replaced by other proinflammatory cytokines. Overproduction of TNF may cause immunopathology, while TNF neutralization reactivates latent and chronic, controlled infection, which is relevant for the use of neutralizing TNF therapies in patients with rheumatoid arthritis.     

Interleukin-6 biology is coordinated by membrane bound and soluble receptors

Cytokine receptors exist in membrane bound and soluble form. Both forms bind their ligands with comparable affinity. While most soluble receptors are antagonists in that they compete for the ligands with their membrane counterparts, some soluble receptors are agonists. In this case, the complex of ligand and soluble receptor binds on target cells to a second receptor subunit and initiates signal transduction. Soluble receptors of the IL-6 family of cytokines are agonists. In vivo, the IL-6/soluble IL-6R complex stimulates several types of target cells not stimulated by IL-6 alone, since they do not express the membrane bound IL-6R. This process has been named transsignaling. We have shown that in several chronic inflammatory diseases like chronic inflammatory bowl disease, peritonitis and rheumatoid arthritis, transsignaling via the soluble IL-6R complexed to IL-6 is a crucial point in the transition from the acute to the chronic state of the disease. The mechanism by which the IL-6/ soluble IL-6R complex regulates the inflammatory state is discussed.     

Cytokine regulation in RA synovial tissue: role of T cell/macrophage contact-dependent interactions

Several groups have documented the expression of cytokines in rheumatoid arthritis synovial tissue over the past 15 years or so. These studies have indicated that most cytokines examined are expressed at the mRNA levels at least, and many other cytokines are found in abundance as proteins. Our attention has recently focused on the mechanisms that induce and regulate tumour necrosis factor and IL-10. Other workers and ourselves have found that cell-cell contact is an important signal for the induction of cytokines, and our work has demonstrated that tumour necrosis factor and IL-10 production in rheumatoid arthritis synovial joint cells cultures is dependent on T cell/macrophage interaction. In this chapter, we review recent advances in this area and also highlight areas where new therapeutic intervention opportunities arise.