Cytokines in chronic rheumatic diseases: is everything lack of homeostatic balance?

To explore the complex regulation of cytokine activities it may be of help to bear in mind the example of rheumatoid arthritis (RA). A major step forward in RA treatment was achieved when it became possible to control disease manifestations such as joint destruction by blocking TNF. This could indicate that a single cytokine, in this case TNF, drives unopposed a series of events that lead to inflammation and destruction. The situation is less simple inside the joint, however, where proinflammatory cytokines co-exist alongside their endogenous inhibitors. This is a consequence of ongoing processes in which proinflammatory stimuli induce their anti-inflammatory counterparts and the imbalance between the two results in disease.

The present review describes the key levels of imbalance that have been associated with chronic inflammation and tissue destruction. This has to be integrated in general processes of disease initiation through the innate and adaptive immune responses ending in tissue and organ damage (Figure 1).

Chemerin is a plasma protein known for its proinflammatory properties exerted upon binding to the G-protein coupled receptor ChemR23/CMKLR1 - expressed on macrophages and plasmacytoid dendritic cells - where it induces cell migration. Chemerin is secreted as an inactive precursor and is processed by proteases before becoming an active mediator. As for conventional chemokines, the biologically active chemerin binds to ChemR23 with its COOH-terminal portion.

Of interest, different proteases generate different chemerin peptides, which possess opposite functions. Serine proteases mainly produced by activated neutrophils - early mediators in inflammation - therefore generate chemerin 9 (9 AA peptide), which is an agonist in the nanomolar range. Cysteine proteases - mainly produced by macrophages - which arrive later at the inflammatory site, however, generate chemerin 15 (15 AA peptide). This peptide in the picomolar range acts as an antagonist, expressing potent anti-inflammatory activities and contributing to reduce inflammation [34].

A further layer of complexity has been added recently with the description of an additional chemerin receptor named CCRL2, selectively expressed on mouse mast cells. Upon binding to this receptor, chemerin induces neither cell migration nor calcium flux. CCRL2 is therefore supposed to scavenge chemerin. The experimental test of this hypothesis led to the opposite result, however, indicating enhanced inflammation in a rodent model of IgE-mediated passive cutaneous anaphylaxis. A possible explanation could be that mast cells bind the N-terminal portion of chemerin with CCRL2 and present the COOH-terminal portion to cells expressing ChemR23, which are thus potently activated [35].

In the late 1980s Mosmann and colleagues described the Th1/Th2 balance when studying a large series of mouse CD4⁺ T-cell clones [36]. They observed that some clones would produce IFNγ but not IL-4, while others would do the opposite. Therefore, based on the dicotomic production of two key cytokines, it was possible to classify T-cell clones into two groups, which were named Th1 and Th2. The same concepts were verified by studying human T-cell clones [37]. Naïve T cells could be induced to become Th1 or Th2 simply by modifying the cytokine present in the milieu during priming, although the dose of antigen, the amount of co-stimulation, and the age of antigen-presenting cells could also affect polarization.

Of major importance, Th1 cytokines were shown to inhibit Th2 cytokine production and function, and vice versa. This observation included cytokines important for priming: IL-12 and IFNγ for Th1 cells, and IL-4 for Th2 cells. Starting investigations with mouse models of human diseases, it was found that models of multiple sclerosis - such as the antigen-induced experimental acute encephalomyelitis (EAE) - or of RA - such as type II collagen arthritis - were associated with the overexpression of IFNγ but not of IL-4. In sharp contrast, models of allergic diseases such as asthma were associated with IL-4 without IFNγ expression. In these models, forced expression of counteracting T-helper cytokines could in many instances abrogate disease expression [38, 39].

Addition of the Th17 pattern

In 2005 the above classification was amended when it was shown in the mouse that IL-17 was produced by a particular T-helper cell, named Th17 [40, 41] (Figure 3). As early as 1999, however, it was shown that some T-cell clones obtained from the synovium of RA patients were producing IL-17 and differed from the classical Th1/Th2 clones [42]. Indeed, they did not produce IL-4 and produced little, if any, IFNγ.

The Th1/Th2 paradigm was then revisited; key observations were made based on the murine EAE model [43]. This model was previously associated with Th1 responses. Th1 cells are induced by IL-12 produced by monocytes and dendritic cells. IL-12 is a heterodimer composed of p35 and p40 subunits. Protection from EAE was afforded when IL-12 was blocked with anti-IL-12p40. IL-23 is also a heterodimer, however, composed of the IL-12/IL-23 common p40 subunit and the specific p19 subunit. When inhibitors specific to IL-23 or p19-deficient mice were used, it was recognized that IL-23 and not IL-12 was responsible for EAE induction by assisting the expansion of Th17 cells. Many chronic inflammatory diseases previously thought to be associated with Th1 have therefore been reclassified as Th17 diseases [44]. The opposing roles of Th2 and Th17 responses are now clear, since IL-4 strongly inhibits IL-17 differentiation. For Th1 and Th17 cells, a more balanced view is now accepted [45]. In both human and murine conditions, a large proportion of T cells can express simultaneously IFNγ and IL-17. This is clearly seen with T-cell clones from peripheral blood. The simultaneous production of the two cytokines appears uncommon, however, in inflammatory tissues where T cells producing cytokines take on a plasma cell-like appearance, possibly indicating full differentiation with a fixed phenotype [46].

In addition to the production of IL-17 (now referred to as IL-17A), Th17 cells can produce other cytokines - including IL-17F (a close member of the IL-17 family), IL-21, and IL-22. IL-21 acts as an endogenous amplifier of the Th17 lineage [41]. IL-22 appears more specifically associated with skin defense [47]. IL-17A and IL-17F share a large number of functions, with a strong correlation between the genes induced in RA synoviocytes by the two cytokines, IL-17F being less potent [48]. In addition, synergistic activities are seen when combining TNF with IL-17A or IL-17F. IL-17A and IL-17F may, however, have different roles in mouse models of inflammation and host defense [49].

IL-17E (also termed IL-25) is a very different member of the IL-17 family. IL-17E is more a Th2 cytokine, involved in allergic reactions and inhibiting the Th17 pathway [50]. Consequently, there is another balance between the effects of IL-17A and IL-17F and those of IL-17E/IL-25.

Some recently identified cytokines such as IL-27 and IL-35 appear to be more involved in dampening the immune response. IL-27 belongs to the IL-12 cytokine family that also comprises IL-23 and IL-35, all involved in the regulation of T-helper cell differentiation. IL-27 is unique in that it induces Th1 differentiation while simultaneously suppressing immune responses. The immunosuppressive effects of IL-27 depend on inhibition of the development of Th17 cells and induction of IL-10 production [53]. IL-27 exerts potent anti-inflammatory effects in several infectious and experimental autoimmune models. In particular, suppressive effects on helper T cells - which are implicated in the pathogenesis of multiple sclerosis - suggest that IL-27 may be therapeutically relevant in multiple sclerosis. While exciting discoveries have been made, however, these are still at an early stage and further studies are required to understand the pathophysiological roles of IL-27 and its therapeutic potential in humans [54].

The inhibitory cytokine IL-35 contributes to regulatory T-cell function, being specifically produced by Tregs and required for maximal suppressive activity [55]. Ectopic expression of IL-35 confers regulatory activity on naive T cells, whereas recombinant IL-35 suppresses T-cell proliferation. The role of Tregs in RA has been established in both patients and animal models. The Tregs increase in patients who are responding to anti-TNFα therapy. Of the current hypotheses, Treg expansion or transfer may hold promise for the treatment of RA [56].

A further layer of control at the level of expression of cytokines, cytokine inhibitors and acute-phase proteins is provided by hormones. Estrogens as well as androgens inhibit the production of IL-1β and TNFα by monocytes-macrophages. Androgens antagonize stimulatory effects of estrogens. Some studies suggest that estradiol is more inhibitory to Thl cytokines (for example, IFNγ, IL-2) while testosterone is inhibitory to Th2 cytokines (for example, IL-4). On the other hand, cytokines control the hypothalamic-hypophyseal-adrenal gland axis as well as the sex hormones [57]. Vitamins may also affect cytokine production by influencing the polarization of effector CD4⁺ T cells. For instance, retinoic acid enhances Treg expansion while simultaneously inhibiting Th17 cells [58]. Conversely, vitamin D favors Th2 polarization and diverts Tregs from their regulatory function [59, 60]. Finally, prostaglandin E₂ - a metabolite of arachidonic acid - may also affect cytokine production by favoring the expansion of Th17 cells [61].

Chronic inflammatory diseases such as RA are so severe because the disease process affects matrix metabolism. Although RA is seen as a destructive disease, it is not well appreciated that the main problem is in fact the inhibition of repair activity. Any type of chronic joint inflammation, whether infectious, inflammatory, or autoimmune, will result in joint destruction within months or, at best, within a few years, but it will take decades to observe some kind of joint repair - even in conditions like osteoarthritis where repair activity is maintained. In a model of cell interaction between synoviocytes and T-cell clones, it was found that Th1 and Th17 clones induced defects in collagen synthesis in vitro, indicating an inhibition of their repair activity (Figure 1). In sharp contrast, Th2 cells induce collagen synthesis, indicating their beneficial role in repair activity [62]. Very similar conclusions were obtained when monocytes were incubated with Th1 or Th2 clones. The interaction with a Th1 clone led to the production of IL-1, a key marker of destructive inflammation, whereas the use of a Th2 clone led to production of IL-1Ra along with its anti-inflammatory and anti-destructive properties [63].

Wingless integration site (Wnt) proteins make up a family of secreted growth factors, identified in virtually every organism; they regulate key aspects of cellular functions such as growth, differentiation, and death. Several members of the Wnt pathway play an important part in bone remodeling. Dickkopf-1, a soluble inhibitor of the Wnt pathway, controls bone remodeling. Increased Dickkopf-1 levels are linked to bone resorption, and decreased levels are linked to new bone formation. Low-density lipoprotein receptor-related protein 5, the main receptor that mediates Wnt signaling, plays a critical role in bone mass regulation. Gain-of-function mutations of lipoprotein receptor-related protein 5 cause high bone mass phenotypes, whereas loss-of-function mutations are linked to severe osteoporosis [64].

Adipokines are beginning to emerge as mediators of inflammation. Knowledge of their precise activities remains in its infancy, however, and is still controversial [65]. Many of the adipokines appear to have proinflammatory properties. In general, adiponectin is considered anti-inflammatory, and leptin, vistatin and resistin are considered proinflammatory. The formation of adipose tissue could be due to abnormal metabolic processes and, at the local level, due to chronic inflammatory processes such as those occurring in the synovium in RA or osteoarthritis, or in the peritoneal cavity in various inflammatory processes of the digestive system.

Adipocytes are said to produce many hormones and pro-inflammatory mediators. White adipose tissue in humans, however, is assumed to be the main source of IL-1Ra, and also contains IL-10. Furthermore, IFNβ was found to be the principal cytokine inducing IL-1Ra in various white adipose tissues, such as that present in the synovium. It is possible that, in addition to other functions, adipose tissue may be part of a mechanism limiting local inflammation and that fibroblasts in the vicinity may further induce IL-1Ra in adipocytes via the production of IFNβ [66].

Cytokines may have opposing effects on the same cell depending on the circumstances in which they hit their target. The timing and the previous activation status are major determinants of responses that cytokines elicit (Figure 4). Differential outcomes could be sensitization or amplification of proinflammatory signals (that is, priming), reprogramming of signaling resulting in proinflammatory activity of pleiotropic or anti-inflammatory cytokines, and attenuation of anti-inflammatory signals and homeostatic mechanisms. Signal transducer and activator of transcription (STAT) 1 has been shown in vitro and in vivo to be involved in some of these effects. For instance, transient exposure to subactivating concentrations of IFNα or IL-6 primes primary human monocytes for subsequent exposure to IFNγ, resulting in enhanced interferon regulatory factor 1 and indoleamine-2,3-dioxygenase gene expression in a STAT-1-dependent manner [67, 68]. This may explain robust IFN signatures in RA synovium, notwithstanding very low amounts of IFNγ. Enhanced expression of STAT-1-dependent genes upon IFNγ priming of monocytes is a finely tuned process involving Fcγ receptor/DNAX activation protein 12, as demonstrated in Fcγ receptor/DNAX activation protein 12^-/- mice in which the priming effect is lost.

IL-10 contributes to homeostatic responses in proinflammatory conditions. For instance, in human monocytes, Toll-like receptor 2 ligation results in NFκB-dependent TNF production and simultaneously in activator protein-1-dependent IL-10 production [69]. Upon binding to its receptor, IL-10 decreases TNF production in a STAT-3-dependent manner, thus exerting a negative feedback. Pre-exposure of monocytes to IFNα, however, results in IL-10 gaining pro-inflammatory functions. Of interest, this process is STAT 1 dependent. It has therefore been shown in human monocytes primed with IFNα that IL-10 not only fails to reduce the subsequent production of TNF in response to lipopolysaccharide, which may simply indicate a loss of function of the anti-inflammatory activity of IL-10, but in addition primes monocytes to transcribe genes in response to IL-10 usually induced by IFN. It appears that, due to the effect of type I interferons, the balance of IL-10 signaling shifts from STAT 3 (anti-inflammatory) to STAT 1 (proinflammatory) signals. Furthermore, IL-10 induces chemokine production in IFNα-primed macrophages, resulting in recruitment of activated T cells; aberrant IL-10 signaling may therefore contribute to inflammation in conditions with high interferon levels (systemic lupus erythematosus) [70].

The suppressors of cytokine signaling (SOCS) family of intracellular proteins - which encompasses eight members, sharing a central Src homology domain 2 and a C-terminus SOCS box - act as negative regulators of intracellular signaling of the Jak-STAT pathway used by several cytokines. They act by inhibiting the kinase activity, by competing with substrates needed for signal transduction, and by targeting associated proteins to proteasome degradation. Beside negative regulation, SOCS proteins can also affect the quality of signaling. For instance, in the absence of SOCS 3, IL-6 induces a wider transcriptional response, which includes interferon-like gene expression owing to increased STAT 1 phosphorylation. SOCS proteins therefore impact on a number of important mechanisms regulating inflammation and the immune response [71].

Cytokine activities affect most, if not all, biological processes involved in homeostasis as well as in host defense and auto-aggression. A continuous, finely tuned, crosstalk between cytokines, receptors, agonist and antagonist ligands, as well as with mediators belonging to other families of molecules, regulates cytokine biological activities. Furthermore, the context in which cytokines are available, including the temporal sequence of events preceding the availability of a given cytokine, very much impact on their capacity to favor or inhibit inflammation and other biological processes. During the past three decades we have learned that an imbalance in cytokine activities is associated with autoimmune and autoinflammatory disorders. More important, our knowledge of the many levels of cytokine balance has led to the generation of important tools to control inflammatory and destructive diseases. The future will no doubt witness additional major achievements in this area of medicine.

The Scientific Basis of Rheumatology: A Decade of Progress

This article is part of a special collection of reviews, The Scientific Basis of Rheumatology: A Decade of Progress, published to mark Arthritis Research & Therapy's 10th anniversary.

Other articles in this series can be found at: http://arthritis-research.com/sbr