Cytokines I

Sock's Rheumatoid Arthritis Links - RA

Cytokines I

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Cytokines. Most immune cells secrete or stimulate the production of powerful immune factors called cytokines. In small amounts, cytokines are indispensable for maintaining the balance of the body during immune responses, infections, injuries, tissue repair, blood clotting, clearing of debris from inflamed blood vessels, and other aspects of healing. If overproduced, however, they can cause serious damage, including dangerous levels of inflammation and cellular injury.Cytokines are very important in the destructive process of rheumatoid arthritis, particularly those known as interleukins (ILs)--notably IL1 and IL6--and tumor necrosis factor (TNF). TNF is now known to be the major cause of joint damage and various systemic manifestations of RA, including weight loss.

Leukocytes. The leukocytes, the other major white blood cells in the body, are also spurred into action by the over-zealous T cells. Leukocytes stimulate the production of key players in the inflammatory process:

Leukotrienes attract white blood cells to the area.
Prostaglandins open blood vessels and increase blood flow.
Nitric Oxide is a gas that is important in blood vessel flexibility and dilation. In excessive amounts, however, it becomes a damaging substance that may play a major destructive role in RA.

It is unclear how the autoimmune processes associated with RA are initiated. Some postulate that infectious agents may be involved, as many infectious arthritides (ie, rheumatic fever caused by streptococcal infection, Lyme arthritis, and postviral arthritis) have characteristics similar to those of RA.

What has become clearer are the mediators in the pathways leading to RA. Since RA is an autoimmune disorder, it is characterized by the inability of the immune system to discriminate between self and non-self. This results in the destruction of both self and non-self tissues by the immune system, since the immune system views all tissues as potential targets. The immune system has both humoral and cellular branches. The humoral branch is responsible for the formation of antibodies. In patients with RA, antibodies called rheumatoid factors are often present. Although they do not necessarily correlate with disease activity, they are a useful marker for RA, and seropositive patients tend to have a more progressive course of disease.

The cellular branch of the immune system involves polymorphonuclear cells, macrophages, and lymphocytes. The inflammatory response of RA is inherent to the cellular branch. Antigen-presenting cells, which include macrophages, engulf antigens and present them to T lymphocytes. T lymphocytes attach to antigens at the major histocompatibility complex (MHC) of the cell wall, activating the T cell. The activated T cell then stimulates T and B cell production, thereby stimulating the inflammatory process. Activated T cells and macrophages release vasoactive substances such as histamine, prostaglandins, and cytokines. These substances can promote cellular destruction and increase blood flow, resulting in the characteristic sequelae of RA.

New evidence about cytokines has revealed that a disequilibrium of pro-inflammatory versus anti-inflammatory synovial cytokines is a major factor in the disease process of RA. RA appears to be associated with a predominance of pro-inflammatory cytokines in the synovium. Thus, newer pharmacotherapies for RA are focusing on balancing pro- versus anti-inflammatory cytokines. For example, agents that antagonize the pro-inflammatory cytokines TNF-alpha and IL-1 have recently been produced. These agents, as previously mentioned, include etanercept and infliximab, both TNF-alpha antagonists, and anakinra, an IL-1 receptor antagonist (IL-1ra).

While the toxicity of TNF-alpha precluded its use in cancer chemotherapy, it immediately pointed to the potential importance of TNF-alpha blockade in the treatment of other diseases. Tumor necrosis factor-alpha was quickly recast as a primary acute inflammatory mediator, and was soon shown to be a mediator of chronic inflammatory disease as well. Hence, development of highly specific techniques for blocking TNF-alpha production or activity became an important goal. The 2 approaches used for TNF-alpha blockade involved antibodies and soluble receptors.

Soluble TNF receptors. Fusion proteins based on the TNF receptor were first developed in an academic laboratory. Peppel and colleagues used a multistep polymerase chain reaction (PCR) method to produce a cDNA sequence encoding the extracellular domain of the human TNFR2 attached to a sequence encoding the Fc portion and hinge region of a mouse immunoglobulin G1 (IgG1) heavy chain through an oligomer encoding a thrombin-sensitive peptide linker. This construct was placed downstream from a cytomegalovirus promoter sequence, and expressed in Chinese hamster ovary cells. These cells secreted a protein capable of binding TNF-alpha and inactivating it both in vitro and in vivo. Indeed, the molecule was very stable when administered to mice.

Design of TNF-alpha antagonists based on the fusion of TNFR ectodomains to the heavy chain of an antibody molecule has changed the course of rheumatoid arthritis (RA), ankylosing spondylitis, and Crohn's disease, each of which seem to depend on TNF-alpha for pathogenic progression. Anti-TNF-alpha antibodies. Anti-TNF-alpha antibodies, most notably infliximab and adalimumab have been developed for the treatment of RA.

Synthesis of TNF-alpha is induced not only by LPS, but also by many other molecules of microbial origin. The basis of this induction was elucidated with the discovery of the LPS receptor. This receptor was found to be a Toll-like receptor 4 (TLR4), one member of a family of proteins, each responsible for the detection of a distinct set of microbial molecules. These receptors are the "eyes" of the innate immune system, and each of them provokes TNF-alpha synthesis when activated. Blockade of TNF-alpha synthesis may provide an alternative approach to decreasing inflammation in patients with RA and other related diseases.

The clinical results support the conclusion that we are at last approaching a profound understanding of the place that TNF-alpha occupies in inflammation, and may hope to identify the primary lesions that cause such inflammatory diseases as RA. Determination of these lesions will advance the development of rational therapies with the potential to interrupt the pathogenesis of these conditions.

Studies in well-defined animal models of arthritis make it clear that tumor necrosis factor (TNF) is involved in early joint swelling. However, TNF alone is neither arthritogenic nor destructive and exerts its arthritogenic potential through the induction of interleukin-1 (IL-1). Intriguingly, TNF-independent IL-1 production is found in many model situations, including pathways driven by macrophages, T cells and immune complexes.

Its relevance is underlined by the great efficacy of anti-IL-1 therapy and a total lack of chronic, erosive arthritis in IL-1ß-deficient mice. Osteoclast-mediated bone erosion is stimulated by IL-1 as well as by the combination of TNF and T-cell-derived IL-17. Cartilage erosion is dependent on IL-1ß and is greatly enhanced in the presence of immune complexes.

The synovial reaction in RA patients is characterized by the abundance of many cytokines, chemokines and growth factors. It is now generally accepted that TNF and IL-1 are master cytokines in the process of chronic joint inflammation and the concomitant erosive changes in cartilage and bone. Proinflammatory and destructive properties were first demonstrated in culture studies in vitro and the arthritogenic potential of TNF and IL-1 was substantiated by arthritis induction in rodents.

Arthritis could be elicited by local injection of recombinant cytokines in the knee joint this observation was underlined by the occurrence of chronic, erosive arthritis in transgenic mice displaying general TNF overexpression. Interestingly, the dominant expression of TNF-mediated pathology in joint tissues in these transgenic mice is still largely unexplained. More recently, further proof of arthritogenicity was obtained from the induction of arthritis by local overexpression of cytokines in joint tissues by using viral vectors.

Intriguingly, IL-1 is much more potent than TNF in inducing cartilage destruction in vivo. Tiny amounts of IL-1 are sufficient to cause proteoglycan synthesis inhibition in chrondrocytes, whereas a roughly 1001000-fold higher dose of TNF is needed to obtain the same effect. Importantly, synergy between IL-1 and TNF has been demonstrated. Apart from potency differences, it is clear that it is hard to measure significant TNF levels in inflamed synovial tissue or synovial fluid of RA patients and the levels are certainly not higher than those of IL-1.

Most effects might be related to membrane-bound forms of cytokines, which are hard to measure. In contrast, impact on articular cartilage from synovium-derived mediators probably needs trafficking of soluble forms. The situation might be different at sites of pannus overgrowth, where close contact between synovial cells and chondrocytes does occur.

A strong argument for the limited, direct role of TNF in arthritis has emerged from elegant studies in TNF transgenic mice. Joint inflammation was completely arrested when these mice were treated with antibodies against anti-IL-1 receptor. This argues that the pathology runs through the induction of IL-1, which is the real arthritogenic trigger, either alone or in synergy with TNF. TNF levels were still high after treatment with antibodies against IL-1 receptor, which implies that TNF alone is hardly arthritogenic.

Both animal model studies and clinical observations have contributed greatly to the identification of TNF and IL-1 as useful therapeutic targets. Apart from the obvious demonstration that arthritis in TNF transgenic mice could be blocked with anti-TNF antibodies, it was a major breakthrough to note that collagen type II arthritis, the classical RA model in rodents, could be suppressed with anti-TNF antibodies or TNF soluble receptors. This identified a key role of TNF in autoimmune arthritis.

Further studies on this model revealed that TNF blockade was efficient when started before or shortly after the onset of arthritis, whereas anti-IL-1 treatment was at least as efficient and also arrested advanced arthritis and joint destruction. Studies in TNF receptor knockout mice have demonstrated that the incidence and severity of collagen arthritis were less in such mice. However, once the joints were affected, full progression to erosive damage was seen in an apparently TNF-independent fashion.

Similar studies with neutralizing antibodies have been performed in a range of arthritis models. The relative roles of TNF and IL-1 in early joint inflammation were variable in different models, but the crucial role of IL-1 in late arthritis and erosive joint destruction was a consistent finding. This implies that overkill by other mediators might occur in the inflammatory process, and that the stimulus, type of process and probably also the stage of the arthritis are major determinants of the mediator profile. Intriguingly, IL-1 seems to be a suitable downstream target in joint erosion.

In addition to the evidence from studies on animal models, the cytokines TNF and IL-1 were demonstrated in increased quantities in RA synovial tissue, along with the presence of cell-associated receptors for these cytokines. The remarkable anti-inflammatory activity of a first neutralizing monoclonal anti-TNF antibody in RA patients revealed the potential of anti-cytokine therapy and has subsequently stimulated the development of improved anti-TNF reagents such as fully humanized antibody and engineered fusion proteins of TNF soluble receptors and Fc fragments, with reduced immunogenicity and a prolonged half-life.

There is no doubt that TNF blockers provide impressive protection against pain and joint swelling in most RA patients. It is also evident that anti-TNF therapy is not effective in all RA patients, nor does it control arthritis in all affected joints of good responders.

The initial studies targeting IL-1 were performed with soluble IL-1 type I receptor. Clinically relevant effects were not seen, which was disappointing at the time and raised questions about the relevance of IL-1 as a therapeutic target in human RA. However, it is now understood that the choice of type I receptor was unfortunate because this soluble receptor has a high affinity for IL-1 receptor antagonist (IL-1ra), thus scavenging the endogenous IL-1 inhibitor. In that sense, the decoy type II receptor might make a better inhibitor, but it has the disadvantage that it has a lower affinity for IL-1. Studies are awaited with optimal, engineered IL-1 receptor fragments or potent, neutralizing anti-IL-1 antibodies.

Apart from studies with soluble receptors, clinical trials have also employed IL-1ra, the effect on joint inflammation being limited. A significant reduction of joint erosion was evident. IL-1ra has a weak pharmacokinetic profile and it is still unclear whether the limited effect on joint inflammation is akin to the RA process or related to suboptimal blocking of IL-1. Comparisons with animal model studies teach us that continued high dosing is crucial in fully controlling IL-1.

Collagen arthritis could not be controlled with a repeated daily injection of IL-1ra, but great suppressive effects were achieved with IL-1ra supplied with an osmotic minipump. Similarly, local IL-1ra overexpression in the knee joint with viral vectors showed proper efficacy in this model. Until high-quality IL-1 blockers become available for clinical trials, conclusions on the relative roles of TNF and IL-1 in RA patients have to be made with great care.

Remarkably, the recent evaluation of joint erosions after the treatment of RA patients with anti-TNF provided the first evidence for a joint protective effect, as reported at the 1999 ACR meeting. This was shown for a combination of anti-TNF antibodies with methotrexate, and also for a single treatment with antibodies as well as TNF soluble receptor. Unfortunately, the actual data have not yet been published, hampering detailed attention in this review.

The finding might fit with the hypothesis that TNF overproduction in RA synovial tissue is mainly caused by deranged behaviour of synoviocytes, generating too much TNF. If this is so, TNF will drive IL-1 production and TNF blockade will be sufficient to control this TNFIL-1 pathway.

Intriguingly, it is in line with the initial hallmark observation made in RA synovial cell cultures: the addition of neutralizing anti-TNF antibodies strikingly reduced the production of IL-1. Unfortunately, this observation was made with isolated cell cultures, has not been confirmed by others and awaits confirmation for intact synovial tissue.

Anti-TNF antibodies used in clinical studies display cytotoxic effects. This implies that one mechanism of the anti-TNF effect could be linked to binding to TNF-bearing cells and subsequent killing of these cells, potentially including TNF/IL-1-producing cells or neighbouring cells. In addition, the TNF soluble receptor used in some of the anti-TNF trials not only binds to TNF but also scavenges lymphotoxin.

The latter might have an impact on T-cell-driven pathways. Significantly, TNF rather than lymphotoxin seems to be the major cytokine expressed in RA synovial tissue. A final comment to be made here is that analysis of anti-erosive efficacy in clinical trials is based mainly on bone erosions. Focal damage of cartilage is more difficult to score on X-rays. It remains to be seen whether the relative dominance of TNF and IL-1 involvement and amplifying elements by pathways mediated by T cells and immune complexes are similar or different in the destruction of cartilage and bone in RA.

Proinflammatory and destructive properties were first demonstrated in culture studies in vitro and the arthritogenic potential of TNF and IL-1 was substantiated by arthritis induction in rodents.

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. Attention has recently focused on the mechanisms that induce and regulate tumour necrosis factor and IL-10.

Investigators have found that cellcell contact is an important signal for the induction of cytokines, and their 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.

It is now well accepted that the spontaneous production of proinflammatory cytokines (in particular, tumour necrosis factor [TNF] and IL-1) produced locally in the inflamed synovial joint contribute directly/indirectly to the pathogenesis of rheumatoid arthritis (RA).

These observations have arisen from ex vivo studies on human synovial cultures, immunohistochemical and mRNA analysis of synovium, and in vivo studies in animal models of arthritis.

These investigations led to the development of several TNF and IL-1 inhibitors, two of which are currently licensed Remicade (chimeric anti-TNF antibody) and Enbrel (TNF-receptor fusion protein). While such therapies targeting TNF in chronic inflammatory disease are very successful, it is also apparent that long-term blockade of a cytokine such as TNF, which is important in innate and acquired immunity, may lead to an increase in latent and/or opportunistic infections.

This is now apparent, with a small but significant increase in unusual infections, as well as the re-emergence of latent tuberculosis, particularly in Central and Eastern Europe. There is thus a need to understand what mechanisms lead to the production of proinflammatory cytokines in RA synovial tissue, and further to determine how this is linked to homeostatic regulation.

It has been observed that while the production of proinflammatory cytokines and enzymes is increased in RA, this is offset to some degree by the action of the endogenous anti-inflammatory cytokines and cytokine inhibitors. Of particular importance in this respect is IL-10, an important regulator of TNF-a and IL-1ß spontaneously produced by macrophages in the rheumatoid joint.

Thus, if endogenous IL-10 is blocked in RA synovial cell cultures, the spontaneous production of both TNF and IL-1 increases significantly. There is therefore an important need to develop therapies that block proinflammatory pathways but leave unaffected those pathways that regulate immunoregulatory cytokines such as IL-10. Histological studies of synovium in RA have indicated that this tissue is very cellular, and that several different cell types including macrophages and T cells are in close proximity.

This may suggest that contact signals between macrophages and T cells could be of importance in vivo in modulating macrophage function. Studies have found that TNF-a production in RA synovium is T-cell dependent, as removal of CD3-positive T cells from RA synovial mononuclear cells resulted in significant reduced macrophage TNF-a production. Furthermore, this signal was abrogated if physical contact between the two cell types was blocked.

Direct-contact-mediated interactions have been studied by several groups using transformed T-cell lines and monocytic lines, and have been found to play a role in inducing the synthesis of several cytokines including IL-1ß, TNF-a, IL-10 and metalloproteinases. Investigative scientists have studied T cell/monocyte cognate interactions using cells isolated from the peripheral blood of normal donors.

Importantly, they observed that the manner in which T cells were activated influenced the profile of cytokines induced in the monocytes. Thus, if blood T cells were activated with cross-linked anti-CD3, this induced the production of TNF-a and IL-10 in monocytes.

However, if the T cells were stimulated with a cocktail of cytokines (TNF-a, IL-2 and IL-6) for 8 days (bystander activation), TNF-a production followed but IL-10 production did not.

These observations suggested that cytokine-stimulated T cells (Tck) may be the actual T cells in RA synovial tissue that induce macrophage TNF-a production, because they induce an unbalanced, proinflammatory cytokine response from monocytes and they could be part of a vicious cycle.

Indeed, in addition to the mechanism of T-cell activation determining the cytokine profile produced by monocytic cells, the corresponding T-cell phenotype would also appear to be important, as one study suggested a differential regulation of monocyte-derived cytokine production by Th1-like and Th2-like cells.

This study describes CD4+ Th1 clones inducing high levels of IL-1ß production by THP-1 monocytes, whereas Th2 clones induced higher levels of IL-1ra. This implies that Th1 cells are proinflammatory whereas Th2 cells are anti-inflammatory.

The hypothesis that RA T cells mimic the action of Tck cells is attractive since T cells found in RA synovium have unusual characteristics. The T cells are relatively small and noncycling, but have features of activation, with over one-half expressing HLA class II, VLA antigens, CD25 and CD69. T-cell receptor analysis has not revealed a consistent pattern, and responses to putative autoantigens have not been easy to reproduce.

Based on these features and the low capacity of T cells to produce T-cell-derived cytokines, it has been proposed that T cells in the joint may not be involved in the later stages of the disease. Scientists have, however, proposed that they are involved in disease pathology through the activation of macrophages to induce TNF. The environment of the RA synovium is favourable to Tck cells, as it is rich in cytokines.

Unutmaz et al. described bystander-activated T cells generated from normal peripheral blood mononuclear cells (PBMC) with IL-2, IL-6 and TNF-al. They found that IL-15 could, by itself, mimic the IL-6/TNF-a/IL-2 cocktail used to activate Tck. IL-15 is of particular interest as it is found in RA synovium and can activate peripheral blood T cells to induce TNF-a synthesis in U937 cells or in adherent RA synovial cells in a contact-dependent manner.

Other cognate cell-to-cell interactions occur in the synovial joint, which contribute to the disease pathology observed in RA. These interactions include endothelial cell/T cell and fibroblast/T cell interactions. During the early stages of inflammation there is a large cellular infiltration from the peripheral circulation to the synovial joint, where interactions between T cells and vascular endothelium drive further extravasation and infiltration by the expression of cell adhesion molecules, chemokines and cytokines.

In addition, the earliest infiltrating cells, neutrophils, can be activated by contact-mediated interaction with T cells, as determined by the ability of these neutrophils to be primed for respiratory burst by formylmethionine leucine phenylalanine peptide.

As the pathology of RA progresses to chronic inflammation and pannus forms at the cartilagepannus junction, the interactions between T cells and fibroblasts/macrophages predominate. The interaction between stimulated T cells and dermal fibroblasts or synoviocytes has recently been shown to induce MMP-1 (collagenase) and TIMP-1, with an imbalance in favour of the proinflammatory MMP-1, also inhibiting the synthesis of type I and type III collagen by fibroblast cells.

Studies undertaken thus far have documented the potent stimulatory activity of T cells on monocyte cytokine production during the pathology of RA. The abundance of T cells and monocytes in the peripheral circulation, which have the potential to physically interact with each other, however, does not seem to induce cytokine production. This led to the hypothesis and subsequent characterisation of an inhibitory factor, apolipoprotein A-1, preventing monocyte activation in the plasma/serum.

The presence of apolipoprotein A-1 and subsequent inhibition of T-cell-mediated macrophage activation may suggest this molecule to have a useful anti-inflammatory therapeutic effect in chronic inflammatory diseases such as RA.

As contact-dependent signals have been demonstrated to be of importance, much attention has focused on the probable candidate molecules on the surface of cells mediating these functions. Specific surface interaction molecules that have been reported to mediate induction of monocyte cytokine synthesis include CD69, LFA-1, CD44, CD45, CD40, membrane TNF, and signalling lymphocytic activation molecule.

Further studies demonstrated that T cells activated through the T-cell receptor complex induced monocyte IL-10 synthesis, which was partially dependent on endogenous TNF-a and IL-1, and that T-cell membrane TNF-a was an important contact-mediated signal. However, IL-10 synthesis still occurred when TNF-a and IL-1 were neutralised, suggesting that there are other TNF/IL-1-independent signals required for IL-10 synthesis.

While TNF clearly plays a role in IL-10 production, there are other signals independent of TNF and IL-1 that may be involved. Of particular interest are members of the TNF/TNF-receptor family, which include CD40, CD27, CD30, OX-40 and LTß. The ligands of these TNF-receptor molecules have been described as upregulated on T-cell activation.

In addition, CD40L, 4-1BB, CD27L and CD30 have been described to be released as soluble molecules after activation. The interaction between CD40L and CD40 has been suggested to be important for inducing both IL-1 and IL-12 synthesis following T-cell interaction with monocytes and, more recently, to mediate IL-10 production by human microglial cells on interaction with anti-CD3-stimulated T cells.

In addition, it have recently shown that CD40L/CD40 interaction mediates cognate induction of macrophage IL-10. The potential involvement of these ligand/counter ligand pairs on cells interacting in synovial membrane tissue is known.

In addition to the stimulus encountered by the T cells, data has indicated the importance of the differentiation state of the monocyte in determining cytokine profiles in response to activated T cells.

Scientists observed that CD40 signalling augmented lipopolysaccharide (LPS)-induced IL-10 production by monocytes, but also observed that CD40 ligation induced IL-10 production by differentiated monocytes (macrophages) in the absence of LPS. Indeed, the priming mechanism of the macrophage determined the cytokine profile: macrophage-colony-stimulating factor preprogrammed macrophages to produce both IL-10 and TNF-a on stimulation by CD40L or Tck, whereas IFN-? priming resulted predominantly in TNF-a.

IFN-?-primed macrophages, however, can produce an endogenous IL-10 activity that is not secreted into the supernatant on cell contact with either Tck or CD40L transfectants, as neutralisation of endogenous IL-10 resulted in a marked increase in TNF-a production. This observation may agree with the report of membrane-associated IL-10 and may highlight differences in the ability of these two types of macrophage-like cells to process cytokines.

Macrophage-colony-stimulating factor can usually be readily detected in the RA joint, while IFN-? is scarce. This may indicate why both TNF-a and IL-10 are found in synovial membrane cell cultures. This preprogramming of macrophages would appear to be irrespective of the triggering stimulus because macrophages stimulated by either Tck, CD40 ligation or LPS result in similar cytokine profiles. It would thus appear that the route of differentiation of the monocyte is critical in the induction of IL-10 in cognate interactions between activated T cells and macrophages.

TNF-a synthesis in monocytes in response to some stimuli (LPS) but not others (zymosan or CD45 ligation) is NF-?B dependent. It is thus of interest to observe that Tck-induced, but not T-cell-receptor-dependent stimulated T cell (Ttcr)-induced, TNF-a production in monocytes is NF-?B dependent. Furthermore, whereas phosphatidyl-inositol 3-kinase (PI3K) inhibitors blocked Ttcrinduction of TNF-a, they paradoxically augmented TNF-a in monocytes stimulated by Tck cells.

Importantly,it was found that RA T cells behaved like Tck cells, in that the induction of TNF-a in resting peripheral blood monocytes was NF-?B dependent but superinduced if PI3K was blocked. An identical result was observed if NF-?B or PI3K was blocked in the RA synovial cell cultures.

IL-10 production in monocytes/macrophages is equally complex. In response to LPS, IL-10 production is dependent on endogenous IL-1 and TNF-a.

Furthermore, there is selective mitogen-activated protein kinase (MAPK) utilisation where IL-10 production was dependent on p38 MAPK, and TNF-a production was dependent on both p38 MAPK and p42/44 MAPK. The involvement of p38 MAPK activity in IL-10 production subsequently led to the characterisation of the downstream effector, hsp27, as an anti-inflammatory mediator.

Little is known, however, regarding the involvement of the PI3K pathway in macrophage production of IL-10. PI3K and its downstream substrate p70S6K mediate IL-10-induced proliferative responses but not anti-inflammatory effects. A recent study has described Tck-induced macrophage IL-10 production to be dependent on PI3K and p70S6K, whereas TNF-a production is negatively regulated by PI3K and is p70S6K dependent.

This suggests that IL-10 and TNF-a share a common component, p70S6K, but differentially utilise PI3K activity. These results are reproduced in the spontaneous cytokine production by RA synovial mononuclear cells (RA-SMCs) and by cocultures of RA synovial T cells with macrophages (RA-T/macrophage cocultures), further suggesting the relevance of this Tck/macrophage cognate coculture system as a model for cytokine production occurring in the inflamed synovium of the rheumatoid joint.

Although many other studies have implicated other signalling cascades in the induction of IL-10 production, not much work exists on the signalling required in macrophages stimulated by cognate interactions with fixed activated T cells. PKC and cAMP signalling have been implicated in IL-10 and TNF-a production and are currently under investigation in our group. Preliminary results would suggest that both these cascades differentially regulate IL-10 and TNF-a.

Studies undertaken by other groups have reported the involvement of the cAMP/PKA pathway in the induction of IL-10 production by human PBMC. The membrane-permeable dibutyryl cAMP was capable of elevating IL-10 mRNA and of augmenting LPS-induced IL-10 production, but on its own was incapable of producing IL-10 protein.

This work also demonstrated a role for PKC in the induction of IL-10 by LPS using the PKC inhibitors, calphostin C and H-7. This result contradicts some data but may reflect that this study used PBMC, a heterogeneous population, as compared with purified monocyte-derived macrophages.

In addition, the selective inhibition of phosphodiesterase type IV by rolipram was found to augment LPS-induced IL-10 production by murine peritoneal macrophages. The mechanism of this was thought to be as a consequence of LPS inducing the anti-inflammatory mediator, prostaglandin E2, which in turn upregulates intracellular cAMP via stimulation of adenylate cyclase activity.

The cognate activation of macrophages by T cells has focused almost exclusively on the membrane interactions mediating the macrophage effector function, such as nitric oxide release, phagocytosis, B-cell help and, more recently, the cytokine profiles induced. The control of T-cell-induced IL-10 and TNF-a production by monocyte-derived macrophages is complex and is regulated at many levels.

These levels include priming of the monocyte/macrophage, T-cell stimulation, and hence specific ligand/receptor interaction, and the resulting signal cascades and crosstalk between them. The continued study of these contact-mediated interactions, the signal transduction distal to the receptor and how they compare between the induction of IL-10 and TNF-a may discover potential therapeutic targets selectively affecting proinflammatory TNF-a production without affecting anti-inflammatory IL-10 production. Such targets will prove to be of great benefit in the treatment of such chronic inflammatory diseases as RA.

T-cell-mediated autoimmune responses are considered to play a role in the pathogenesis of rheumatoid arthritis (RA). Activation of T lymphocytes requires two signals from antigen-presenting cells. The first signal, the binding of the T-cell receptor to its antigen major histocompatibility complex ligand, provides specificity of antigens.

The second signal is mediated by costimulatory molecules, of which a family of proteins called B7 appears to be the most potent. The B7 costimulatory pathway involves at least two molecules, B7-1 (CD80) and B7-2 (CD86), on antigen-presenting cells, both of which can interact with their counter-receptors, CD28 and CTLA-4, on T cells.

The interaction of the CD28 receptor on the lymphocyte with receptors of the B7 family on the antigen-presenting cell is one of the most important of these costimulatory pathways. This signal induces T-cell activations and clonal expansion and inhibits T-cell apoptosis. Activation of the T-cell receptor without costimulation of the CD28 receptor does not induce activation but instead induces anergy or cell death .

Recent studies have shown that patients with RA carry a subset of CD4+ T cells CD4+CD28- T cells that lacks the receptor CD28. Cells of this CD4+CD28- subset have several features differentiating them from classic T helper cells.

They do not depend on the B7/CD28 pathway for activation, do not express the CD80 receptor, are incapable of activating B cells, have significant cytolytic activity, and express high levels of IFN-? and IL-2. Thus, the presence of significant numbers of CD4+CD28- T cells could shift immune response from B-cell activation and production of immunoglobulins toward activation of type-1 T helper cells and production of IFN-? and involvement of macrophages releasing matrix-degrading proteases.

CD4+CD28- T cells are infrequent in healthy individuals (comprising 0.12.5% of T cells) [5], whereas higher levels have been seen in patients with unstable angina, multiple sclerosis, Wegener's granulomatosis, and rheumatoid arthritis with extra-articular manifestations.

Study highlights: Clonal expansion of CD4+CD28- T cells is a characteristic finding in patients with rheumatoid arthritis (RA). Expanded CD4+ clonotypes are present in the peripheral blood, infiltrate into the joints, and persist for years. CD4+CD28- T cells are oligoclonal lymphocytes that are rare in healthy individuals but are found in high percentages in patients with chronic inflammatory diseases.

The size of the peripheral blood CD4+CD28- T-cell compartment was determined in 42 patients with RA and 24 healthy subjects by two-color FACS analysis. The frequency of CD4+CD28- T cells was significantly higher in RA patients than in healthy subjects.

Additionally, the number of these cells was significantly higher in patients with extra-articular manifestations and advanced joint destruction than in patients with limited joint manifestations. The results suggest that the frequency of CD4+CD28- T cells may be a marker correlating with extra-articular manifestations and joint involvement.

In this study, the CD4+CD28- T-cell frequency in patients with RA and healthy subjects was evaluated. The frequency of CD4+CD28- T lymphocytes in the control group was similar to the frequencies found by other investigators.

Among RA patients, the frequency of these lymphocytes was significantly higher than in the controls. Nevertheless, the CD4+CD28- T-cell compartment differed depending on extra-articular manifestations and joint involvement. The lowest frequency of CD4+CD28- T cells was in RA patients with limited joint manifestations. Significantly higher numbers of CD28- lymphocytes were present in patients with advanced joint involvement and extra-articular manifestations.

The association of CD4+CD28- T cells with disease status has given rise to the hypothesis that these cells directly contribute to disease manifestations. Patients with nodulosis and extra-articular manifestations of RA have grossly expanded populations of CD4+CD28- T cells.

In patients with coronary artery disease, the frequency of these cells correlates with the risk of acute coronary syndromes. Such syndromes develop if the atherosclerotic plaque is inflamed and develops a fissure or ulceration, with subsequent thrombosis. Clonal expansion of CD4+CD28- T cells has been found in the inflamed plaque of such patients.

The lack of CD28 expression on CD4+ T cells is a very unusual feature for the mature CD4 T cell. T-cell function has been intimately linked to the CD28 molecule. Thus clonally expanded T cells in RA patients are characterized not only by abnormal growth behavior but also by unusual functional properties.

The presence of large numbers of these T cells in RA patients is likely to influence immune responsiveness and alter mechanisms of inflammation, which depend on T-cell regulation.

In contrast with classic T cells, CD4+CD28- T cells produce a high amount of IFN in the absence of costimulatory pathway. The expansion of this cell population is genetically determined. CD28 deficiency is due to a transcriptional block resulting from the loss of nuclear transcription factors binding to two distinct regulatory motifs in the promoter region of the CD28 gene. The repression of CD28 transcription may be also the consequence of chronic exposure to TNF-a, which leads to the blocking of CD28 transcription.

Despite the loss of the CD28 molecule, these CD4+ T cells are functionally active and have the ability to release cytokines in the absence of a costimulatory pathway.

CD4+CD28- T cells contribute to the cell infiltrate and exhibit increased survival after apoptotic stimuli. Resistance to apoptosis in CD28- T cells is due to elevated expression of antiapoptotic protein Bcl-2 and Fas-associated with death domain-like IL-1-converting enzyme inhibitory protein (FLIP). The absence of CTLA4 surface expression on CD28- T cells may also play a role in their prolonged proliferative response and resistance to activation-induced cell death.

Moreover, these cells are characterized by intracellular storage of the cytolytic proteins perforin and granzyme B and are functionally specialized for cytotoxic activity. Perforin was found in CD4+CD28- peripheral blood lymphocytes, and CD4+ perforin-positive T cells were present in the synovial tissue, where their frequency correlated with the expansion of the CD4+CD28- T-cell compartment in the periphery.

CD4+CD28- T cells have several characteristics of natural killer (NK) cells, including the cell-surface expression of regulatory killer activating and inhibitory receptors, CD8aa homodimers, and molecule 161, which enhance their ability to infiltrate tissue. The presence of CD8aa homodimers as well as regulatory killer activating and inhibitory receptors on CD28- T cells suggests that the functional properties of these cells are under the control of MHC class I molecules.

The expansion and activation of these cells in RA may therefore reflect a coordinated action of MHC-class-II- and MHC-class-I-mediated stimulation of T-cell receptors and killer inhibitory receptors (KIRs), respectively. The gene for the killer-cell immunoglobulin-like receptor KIR2DS2 was found to be a genetic risk factor of vasculitis manifestations in patients with RA.

The accumulation of NK-receptors expressing CD4 cells in synovial tissue is compatible with a direct contribution of these cells to the tissue lesions.

Study results confirm previous reports that the role of CD4+CD28- T cells in RA pathogenesis may be related to their cytotoxic capability, which may contribute to extra-articular manifestations. The higher frequency of these cells in patients with severe joint involvement and rapid joint progression confirm observations that the frequency of CD4+CD28- T cells may correlate with the risk of occurrence of joint erosions in RA.