INTRODUCTION

Systemic lupus erythematosus is characterized by a loss of tolerance to nuclear antigens and is accompanied by profound dysregulation of the immune system. Cytokines have been extensively studied in both human and murine lupus. However, in many cases the results for a given cytokine have appeared conflicting due to variation in the type of specimen tested (cells versus serum), the method of detection (mRNA expression versus cytokine protein secretion in vitro), and variability among commercial kits. The reader is referred to several comprehensive reviews on cytokines in lupus.^1–3 This chapter focuses on recent developments, and where possible on areas of emerging consensus.

TH1/TH2 PARADIGM

As initially described, T-cell clones have been divided into two types: Th1 clones [which produce interferon gamma (IFN-g), interleukin (IL)-2, and lymphotoxin] and Th2 clones, which produce IL-4 and IL-5. It was postulated that Th1 cytokines mediate primarily cell-mediated immunity and Th2 cytokines mediate primarily antibody responses.⁴ With the discovery of additional cytokines and the pleiotropic nature of many cytokines, the original concept has become problematic. For example, IFN-g (a prototypic Th1 cytokine) also promotes isotype switching and antibody formation.⁵ Recent evidence indicates that the primary role of IL-2 is in the proliferation and differentiation of Th precursor cells into effector cells rather than as an effector cytokine.⁶ In addition, tumor necrosis factor alpha (TNF-a, a pro-inflammatory cytokine) has a critical role in the induction of cytotoxic T lymphocytes (CTL) in vivo.⁷ As discussed in material following, the complexity and heterogeneity of immune dysregulation in lupus combined with the pleiotropic properties of cytokines [depending on the stage of disease (induction phase versus effector phase)] prevents the classification of lupus as simply a Th1 or Th2 disease.

IL-2

The role of IL-2 in both murine and human lupus has been extensively investigated.^2,3,8 IL-2 is made primarily by T-cells (especially CD4+ T-cells) and promotes T-cell growth. Defective in vitro production of IL-2 following restimulation is a long-standing and robust observation.⁹ Nevertheless, given the central role that T-cells play in SLE pathogenesis (and the central role of IL-2 in T-cell growth and differentiation, activation-induced cell death, the development of regulatory T-cells, and resistance to viral infection) the meaning of defective IL-2 production in SLE is clearly of interest. A number of postulated explanations include intrinsic T-cell defects, T-cell exhaustion, decreased co-stimulation by APC, and excessive suppressor cell function. Several intracellular signaling pathways that contribute to T-cell IL-2 production have been shown to be defective.¹⁰ Recent evidence indicates that defective IL-2 production is a consequence of defective promoter activivity.^8,11

A major question has been whether these defects reflect a primary predisposing defect or instead reflect a secondary defect induced by the altered immunoregulation characteristic of lupus. Supporting a secondary induced defect are the following observations: in vitro IL-2 production is normal in very young MRL/lpr mice, declines rapidly with age-related disease progression, and is related to the presence of CD4+ T cells (which can suppress in vitro IL-2 production by T-cells from MRL/+ mice).¹² Similarly, in the lupus-like disease induced in normal F1 mice following the transfer of homozygous parental strain CD4+ T-cells, recipient F1 mice exhibit defective in vitro IL-2 production in conjunction with the appearance of anti-ssDNA ab as early as two weeks after transfer.¹³ Last, in human SLE intracellular signaling defects resulting in impaired IL-2 production can be induced by IgG from SLE patients.¹⁴ Although these results do not exclude the existence of primary preexisting defects in IL-2 production in some SLE patients, they support the idea that defective in vitro T-cell production of IL-2 can be a consequence of disease.

IFN-G (TYPE II IFN)

IFN-g is a 20- to 25-kd glycoprotein secreted by T-cells and NK cells in response to a variety of stimuli. CD8+ T-cells and Th1 CD4+ cells produce IFN-g during viral infections.¹⁵ The IFN-g receptor consists of the alpha chain (known as IFN-g RI) that binds IFN-g and the associated beta chain (IFN-g RII) required for biologic activity. Binding of IFN-g to its receptor leads to activation of tyrosine kinases JAK1 and JAK2, which then phosphorylate the transcription factor STAT1 that dimerizes and translocates to the nucleus (where it binds and activates the gamma activation site in the promoter of IFN-g inducible genes).

IFN-g has multiple and pleiotropic effects on immune and nonimmune cells, including the ability to inhibit the proliferation of Th2 cells and to induce Ig class switching (promoting IgG2a production).¹⁶ Production of IFN-g during an immune response results in preferential expansion of Th1 cells and promotes macrophage killing in response to intracellular microbes and parasites. IFN-g induces or up-regulates MHC class II expression on immune and nonimmune cells, an effect that may contribute to the pathogenesis of autoimmunity.

IFN-g has a pathogenic, albeit complex, role in SLE. Administration of IFN-g to NZB/NZWF1 mice accelerates the development of glomerulonephritis, whereas treatment of these mice with neutralizing antibody to IFN-g or cDNA encoding INFgR/Fc results in renal remission.^17–19 A complex dichotomous role of IFN-g was seen in MRL-lpr mice in that prophylactic IFN-g administration to preautoimmune mice was beneficial whereas a deleterious effect was seen with IFN-g administration to older mice.²⁰ In the parent-into-F1 model of lupus, IFN-g is critical for up-regulation of Fas and Fas ligand (which prevents lupus development by promoting elimination of autoreactive B-cells by CTL). In the absence of IFN-g, however, mice do not develop lupus in the short term due to B-cell elimination by residual perforin-dependent CTL.²¹

Studies in humans have also been conflicting. Elevated levels of serum IFN-g are seen in lupus patients but may²² or may not be related to disease activity.²³ Increased IFN-g mRNA in PBMC from lupus patients²⁴ and increased intracellular levels in CD4 T-cells from patients with lupus nephritis have also been reported. A mechanism for the increased production of IFN-g in SLE is suggested by studies showing that native nucleosomes and some of their peptides induce a strong IFN-g response in lupus patient T-cells.²⁵

In contrast to the previous, T-cells from lupus patients exhibit a diminished ability to produce IFN-g following in vitro stimulation by mitogens.^26,27 In one study, the in vitro production of IFN-g at basal and stimulated levels did not differ from normal controls overall.²⁸ However, within lupus patients a correlation was seen with disease activity, suggesting that an increase in IFN-g production may occur during exacerbation. In contrast, peripheral blood from lupus patients with active disease contained fewer IFN-g-secreting cells compared to controls,²⁹ and patients with lupus nephritis had lower IFN-g serum levels.³⁰ Polymorphism within the genes for the two IFN-g receptor chains has been associated with the risk of developing SLE.^31,32 A significant difference in the induction of HLA-DR by IFN-g stimulation and a shift to Th2 cytokines was noted in individuals bearing the variant receptor IFN-gR1 Met14/Val14. The greatest risk of development of SLE was detected in the individuals who had the combination of IFN-g R1 Met14/Val14 genotype and IFN-gR2 Gln64/Gln64 genotype.

Aside from differences due to methodology, the pleiotropic effects of IFN-g may explain the conflicting results in human and animal models and this suggests that both decreased and increased IFN-g production contributes to disease pathogenesis at different stages. Higher levels of IFN-g secreted at disease onset may increase MHC class II expression, contributing to the breaking of tolerance to self-antigens and production of pathogenic autoantibodies. Later in the disease, lower production of IFN-g could permit the production of higher levels of Th2 cytokines that in turn permit greater B-cell activity. IFN-g may also amplify effector mechanisms responsible for end-organ damage.

IFN-ALPHA

One of the first cytokine abnormalities to be documented in SLE was an increase in serum IFN-a levels in most SLE patients^33–35 (reported more than 25 years ago). Increased IFN-a levels correlated with disease activity, disease severity, immune activation (as measured by anti-dsDNA titers and complement levels), and clinical features such as fever, rash, and lymphopenia.^33–35 With the advent of IFN-a as a treatment of malignancies and hepatitis C infection 20 years ago, a possible causative link between lupus and IFN-a has been suggested by reports of exacerbation of preexisting autoimmune diseases and/or the occurrence of overt organ-specific and systemic autoimmune diseases with IFN-a therapy.³⁶

Recent studies using microarray technology have demonstrated an IFN-a signature in PBMC from adult and pediatric lupus patients.³⁷ Moreover, the level of IFN-a-induced gene expression correlated with disease activity (suggesting that activation of the IFN-a pathway defines a subgroup of SLE patients with severe disease).³⁸ In addition to the IFN-a gene signature found in SLE patients, gene expression data in lupus-prone mice further substantiated an important role for IFN-a in the pathogenesis of lupus. Results from animal models also suggest a key role for IFN-a in lupus pathogenesis. Homozygous IFNa/b receptor-deleted NZB mice demonstrated an ameliorated disease pattern,³⁹ whereas administration of IFN-a induced an early lethal disease course in pre-autoimmune NZBWF1 mice but not in normal BALB/c mice.⁴⁰

The major source of IFN-a and mechanisms involved in lupus pathogenesis are an area of active investigation. Immune complexes containing anti-dsDNA/dsDNA or apoptotic bodies induce IFN-a production by plasmacytoid dendritic cells (pDC).^35,41 Interestingly, several studies have documented a decreased frequency of pDCs in the blood of SLE patients,⁴² perhaps reflecting an accelerated migration of pDC into tissues such as the skin.⁴³

Regarding pathogenic mechanisms, IFN-a in SLE serum can induce the differentiation of monocytes into dendritic cells with autoantigen-presenting capacity.⁴⁴ In the presence of IFN-a, self-antigens derived from apoptotic cells and expressed on the surface of activated antigen-presenting cells may trigger sustained activation of self-reactive T-cells (which could provide help for autoantibody production by autoreactive B-cells). IFN-a can also enhance B-cell responses and promote immunoglobulin class switching through stimulation of dendritic cells.⁴⁵ Of note, IFN-a also promotes Th1 development and IFN-g production, which in turn promotes Fas ligand and TRAIL expression on NK and activated T-cells (augmenting their capacity to mediate target cell apoptosis).^46,47 How the Th1 actions of IFN-a promote lupus, if at all, is unclear.

IL-12

IL-12 is a heterodimeric cytokine of 70 kDa comprising two covalently linked subunits p35 and p40. The p40 chain is overproduced relative to the p35 chain and forms homodimers that bind to the IL-12 receptor, competing with the bioactive p70 heterodimer for receptor occupancy and thereby serving as a receptor antagonist. IL-12 is predominantly produced by dendritic cells, monocytes, and macrophages, and to a lesser extent by B-cells.⁴⁸ IL-12 is widely accepted as an important regulator of Th1 responses. It also promotes the expansion and survival of activated T-cells and NK cells and modulates the cytotoxic activity of CTLs and NK cells. During the adaptive immune response, IL-12 primes antigen-specific T-cells for high IFN-g production (driving their differentiation toward the Th-1 pathway). IL-12 can also act as an adjuvant for humoral immunity by enhancing production of IgG2a and IgG2b antibodies, and it may enhance antibody production by B-cells.

Several groups have examined IL-12 in the pathogenesis of lupus. IL-12 has been reported as up-regulated in the kidneys of NZB/W F1 mice and MRL/lpr mice⁴⁹ independently of T-cells or T-cell-produced IFN-g.^50,51 Elevated levels of IL-12 in serum from lupus patients have been reported in one study,⁵² whereas impaired production of IL-12 by SLE monocytes or PBMC was observed in vitro (which correlated with disease activity).^53–55 IL-12 production was positively correlated with IFN-g anti-dsDNA antibody and negatively correlated with IL-10.⁵⁶ In that study, IL-10 inhibition or IFN-g addition enhanced IL-12 production (but only the latter restored it to normal levels).

Addition of exogenous IL-12 to lymphocytes derived from lupus patients reduced spontaneous polyclonal production IgG production by B-cells and the number of anti-dsDNA-secreting cells.⁵⁷ This effect was directly mediated by IL-12 and not by IFN-g upregulation or IL-10 down-regulation.

In vivo administration of IL-12 to lupus-like chronic GVHD mice prevented autoantibody production by promoting CTL activity that eliminated activated autoreactive B-cells.⁵⁸ This effect was not blocked by administration of neutralizing anti–IFN-g mAb, indicating a direct CTL-promoting effect of IL-12. However, deletion of IL-12 gene in lupus-prone mice did not significantly influence autoimmunity.⁵⁹ Taken together, it is difficult to determine the exact role of IL-12 in lupus pathogenesis (i.e., whether the alterations in IL-12 cited previously are secondary to lupus-related altered immunoregulation, whether they reflect endogenous compensatory pathways attempting to normalize immune function, or some combination of both). At present, it does not appear that IL-12 defects or excess are central to disease induction. A role for IL-12 in therapy is speculative at present.

IL-4

IL-4 is a multifunctional cytokine with B-cell stimulatory and Th2-promoting properties. IL-4 can rescue B-cells from apoptosis, enhancing their survival,⁶⁰ and is responsible for immunoglobulin isotype switching to IgG1 and IgE.⁶¹ A T-cell suppressor role for this cytokine has also been suggested.⁶²

Conflicting data exists regarding the role of IL-4 in the development of lupus. Elevated levels of IL-4 have been found in the sera of some lupus patients,⁶³ and purified B-cells from lupus patients have been reported to spontaneously produce a soluble factor with IL-4–like activity.⁶⁴ In addition, ex vivo mitogen-induced production of IL-4 was increased in one recent study⁶⁵ (and elevated levels of IL-4 mRNA were reported in another).⁶⁶ In contrast, other studies have demonstrated normal IL-4 mRNA from PBMC of lupus patients,^67,68 and the number of IL-4-producing CD4+ T-cells was significantly decreased in lupus patients.⁶⁹ Polymorphisms in the IL-4 promoter and IL-4 receptor genes have been associated with the development of SLE.^70,71

The preponderance of data from animal models of lupus suggests a role for increased IL-4 production in the pathogenesis of the disease. However, some conflicting results have also been reported. Spleen cells from BXSB lupus-prone mice produce normal amounts of IL-4 following in vitro mitogen stimulation,⁷² whereas increased IL-4 production was reported for lymph node from cells from either C3H/lpr⁷³ or MRL/lpr mice⁷⁴ compared to control mice. Complicating interpretation is the observation that spleen cells from MRL/lpr mice expressed significantly less IL-4 mRNA and less protein upon mitogen stimulation than controls.⁷²

Interleukin-4–positive cells were reported in kidney biopsies from patients with active lupus nephritis in one study,⁷⁵ whereas the expression of IL-4 and IL-4R mRNA correlated negatively with the degree of glomerular injury in another study.⁷⁶ Evidence for a more novel role for IL-4 in the development of lupus nephritis comes from recent studies in which blockade of IL-4 by antibody treatment or of its signaling by inactivation of the Stat6 gene ameliorates glomerulosclerosis and delays or even prevents the development of end-stage renal disease in NZM.2410 mice, despite the presence of high levels of IgG anti-dsDNA.⁷⁷ Thus, IL-4 may serve multiple roles in the development of lupus: it may enhance autoantibody production via its direct B-cell effects, protect against autoimmunity via its T-cell suppressor effect, or perpetuate tissue damage via direct effects on target organs.

IL-5

A possible role for IL-5 in the pathogenesis of lupus has not been extensively evaluated. Elevated IL-5 mRNA has been reported in cutaneous lupus erythematosus,⁷⁸ suggesting a role for Th2 cells in the development of skin involvement in SLE. In the periphery, however, the number of IL-5–secreting T-cells was not significantly different between SLE patients and controls.⁷⁹

A number of studies have suggested that abnormally high levels of IL-5 may play an important role in the abnormal expansion of activated B-cells in lupus. Activation-induced B-cell apoptosis could be rescued with the addition of cytokines such as interleukin IL-5 (or IL-10) in vitro.⁸⁰ NZBW mice congenic for IL-5 transgene exhibited a reduced incidence of lupus nephritis, a decrease in anti-DNA antibody production, and a progressively increased frequency of peripheral B-1 B-cells with age—suggesting that dysregulated continuous high expression of IL-5 in SLE-prone mice may directly or indirectly promote expansion of autoreactive B-1 B-cells and the subsequent suppression of autoimmune disease.⁸¹ The potential relevance of these results to human lupus is unclear.

IL6

IL-6 is a cytokine with pleiotropic effects that shares pro-inflammatory effects with IL-1 and TNF-a and exerts immunoregulatory functions on B- and T-cells. It is produced primarily by monocytes, fibroblasts, and endothelial cells, but also by T-cells, B-cells, and mesangial cells.⁸² IL-6 promotes B-cell maturation to plasma cells and secretion of immunoglobulins. Increasing evidence suggests an important role of IL-6 in the B-cell hyperactivity and immunopathology of SLE. Patients with SLE have increased serum IL-6 levels that correlate with disease activity, with anti-DNA levels,^83,84 with anemia,⁸⁵ and with an increased frequency of IL-6–secreting PBMCs.²⁹ IL-6 production has been detected at the site of various organs involved in lupus: in the cerebrospinal fluid in patients with CNS involvement,⁸⁶ in the urine⁸⁷ or renal glomeruli of patients with lupus nephritis,⁸⁸ and in affected skin.⁸⁹ Serum levels of IL-6–have been correlated with disease activity,⁸³ and urinary levels of IL-6 have been correlated with renal pathology score⁹⁰ (although one study found that urinary but not serum levels correlated with disease activity).⁸⁷ In addition, a rise in plasma IL-6 before lupus exacerbation has been reported by Spronk and colleagues to occur in a subgroup of patients with serositis but not with other manifestations.⁸⁴ Last, with successful treatment of CNS lupus cerebrospinal fluid IL-6 levels decreased significantly.⁹¹

An intrinsic abnormality in lupus B-cell responsiveness to IL-6 has been reported. Cells from normal individuals do not spontaneously express IL-6 receptors. However, the majority of B-cells from lupus patients spontaneously express IL-6 receptors.⁹² Low-density B-cells from healthy subjects did not respond to IL-6, whereas those from patients with active SLE differentiated into immunoglobulin-secreting cells without an additional co-stimulatory signal.⁹³

An association between lupus development and IL-6 has been reported in several murine models. Older autoimmune MRL/lpr mice have increased serum IL-6 and IL-6R in membrane-associated and soluble form.^94–96 In old NZB/W mice, IL-6 blockade reduced (and exogenous IL-6 increased) the ex vivo production of IgG anti-dsDNA antibody.⁹⁷ IL-6 is an early key cytokine in B6.Sle1 lupus, as demonstrated by increased production of IL-6 by splenic B lymphocytes and monocytes and in vitro suppression of ANA production by anti-IL-6 Ab.⁹⁸ In NZB/W mice, administration of recombinant human IL-6 (rhIL-6) accelerated the progression of glomerulonephritis⁹⁹ without changes in anti-dsDNA antibody levels. Chronic IL-6 blockade using anti-IL-6 or anti-IL-6R monoclonal antibody in pre-autoimmune NZB/W mice prevented increases in anti-dsDNA antibody levels, prevented progression of proteinuria, and resulted in increased survival.^100,101 Based on these data, it is possible that IL-6 blockade in human SLE could lead to a decrease in anti-dsDNA antibody levels and interfere with the inflammatory autoimmune process both systemically and locally. A phase I clinical study is currently under way.

IL-10

IL-10 is a potent stimulator of B lymphocyte survival, proliferation, and differentiation and promotes the production of anti-DNA autoantibodies. IL-10 also inhibits activation, cytokine production, and antigen presentation by macrophages and has important interactions in inducing and sustaining immune and inflammatory responses.¹⁰² The inhibitory effect of IL-10 on IL-1 and TNF-a production is crucial to its anti-inflammatory activities. Several lines of evidence suggest that IL-10 may play a role in the pathogenesis of lupus. For example, low-level increases in splenic IL-10 mRNA expression are observed in the P◊F1 model of lupus.¹⁰³ Elevated serum levels of IL-10 and increased spontaneous production of IL-10 in several newly diagnosed untreated patients with SLE (but not in normal controls) have also been reported.¹⁰⁴ Elevated serum levels of IL-10 have been reported in roughly a third of patients with SLE studied, and levels correlated with disease activity as measured by SLEDAI.¹⁰⁵ Disease severity measured by a visual analog scale was reported to correlate with an elevated ratio of IL-10: IFN-g-secreting cells as measured by ELISPOT.²⁹ Similarly, a correlation between changes in disease activity measured by SLEDAI and IL-10 levels over a period of 4 weeks of treatment has also been reported.¹⁰⁶

Furthermore, neutralizing anti-IL-10 antibody delays the onset of proteinuria, glomerulonephritis, and anti-dsDNA antibody production; decreases mortality in NZB/W mice;¹⁰⁷ and inhibits autoantibody production in SCID mice injected with PBMC from SLE patients.¹⁰⁸ The protective effect of anti-IL-10 is due to up-regulation of TNF-a production, because treatment with neutralizing anti-TNF-a mAb resulted in rapid development of autoimmunity. Moreover, AS101 (an immunomodulator that reduces serum levels of IL-10) was beneficial in SCID mice transplanted with mononuclear cells from SLE patients and in NZB/W mice.¹⁰⁹ In contrast, IL-10 had a protective effect in MRL/lpr mice, as evidenced by the fact that IL-10–deficient mice on this background developed more severe lupus and the administration of rIL-10 reduced anti-dsDNA production in MRL/+ mice.¹¹⁰

Interestingly, Llorente and colleagues found high levels of in vitro spontaneous production of IL-10 measured at both protein level and mRNA in 83% of SLE patients and in 75% of healthy members of multiplex families (suggesting that high levels of IL-10 may predispose to disease and precede onset).¹¹¹

Lastly, IL-10 plays an important role in the pathogenesis of immunoregulatory abnormalities in SLE. IL-10 blockade can reverse the impaired allogeneic response in SLE patients.¹¹² Of note, a defect in the responsiveness to IL-10–induced suppression has been reported for IL-6 monocyte production.¹¹³ Such a defect may explain the concomitant elevation of IL-10 and IL-6 in SLE and contribute to perpetuation of polyclonal B-cell activation that characterizes SLE. The effects of IL-10 on B-cell function have lent support for the therapeutic use of IL-10 antagonists in SLE. In a pilot study, treatment of 6 patients with SLE with rh-anti-IL10 antibody achieved a long-lasting reduction of most clinical parameters in 5/6 patients.¹¹⁴ Further studies will be required to evaluate the full potential of anti-IL-10 therapy.