Role of IL-7 and TSLP

in immunopathology of (rheumatoid) arthritis

The research described in this thesis was financially supported by the Dutch Arthritis

Association (Reumafonds). Printing of this thesis was funded by Amgen Inc.

ISBN: 978-90-393-52-199 Printed by Proefschriftmaken.nl All rights reserved. No part of this book may be reproduced of transmitted in any form or by any means, without permission from the author. S.A.Y. Hartgring University Medical Center Utrecht Rheumatology & Clinical Immunology PO Box 85500 (F02.127) 3508 GA Utrecht The Netherlands

Role of IL-7 and TSLP

in immunopathology of (rheumatoid) arthritis

De rol van IL-7 en TSLP in immuunpathologie bij (reumatode) artritis

(met een samenvatting in het Nederlands)

Proefschrift

ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof.dr. J.C. Stoof, ingevolge het besluit van het college voor promoties

in het openbaar te verdedigen op donderdag 3 december 2009 des middags te 12.45 uur

door

Sarita Aimee Yvonne Hartgring

geboren op 28 augustus 1980 te Utrecht

Promotoren: Prof.dr. F.P.J.G. Lafeber

Prof.dr. J.W.J. Bijlsma

Co-promotor: Dr. J.A.G. van Roon

Contents

Chapter 1. Introduction: Role of IL-7 and TSLP in immunopathology of

rheumatoid arthritis Chapter 2. Persistence of IL-7 activity and levels on TNF blockade in patients

with rheumatoid arthritis Chapter 3. Elevated expression of IL-7 receptor in inflamed joints mediates IL-

7 induced immune activation in RA Chapter 4. Numbers of CD25+ Foxp3+ T cells that lack the IL-7R in RA

patients are increased and have impaired suppressive function Chapter 5. Severity of proteoglycan-induced arthritis correlates with IL-7 and is

diminished by IL-7 neutralization Chapter 6. Blockade of the IL-7 receptor prevents collagen-induced arthritis

and is associated with reduction of T-cell activity and proinflammatory mediators

Chapter 7. IL-7 receptor ligands IL-7 and TSLP differentially promote collagen-

induced arthritis Chapter 8. Receptors for IL-7 and TSLP promote proteoglycan-induced arthritis

and immunopathology Chapter 9. Summary and Discussion: Counteracting IL-7- and TSLP-driven

immune activation as novel strategies to prevent joint inflammation in RA Nederlandse samenvatting Dankwoord List of publications Curriculum vitae

7

25

43

63

75

91

109

127

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167

175

181

183

6

1

1

Introduction

Role of IL-7 and TSLP

in immunopathology of (rheumatoid) arthritis

SAY Hartgring

JWJ Bijlsma

FPJG Lafeber

JAG van Roon

Parts of this chapter have been published in

the Annals of the Rheumatic Diseases 2006;65 Suppl 3:iii69-iii74

Chapter 1

9

Rheumatoid arthritis (RA) is characterized by persistent inflammation of the joints, which results in progressing cartilage and bone destruction (1). Several studies revealed an important role for CD4 T cells, B cells, and macrophages in the inflamed joints of RA patients (2-8). Large numbers of these cells have been demonstrated in the synovial fluid and synovial tissue of RA patients. Their numbers and/or activity have been shown to correlate with clinical symptoms (3-5, 7, 9, 10). Several anti T cell (CTLA4Ig, Abatacept), anti B cell (antiCD20, rituximab) and anti-macrophage (anti-TNF, infliximab, adalimumab; anti-IL-6R, tocilizumab) therapies in RA patients have resulted in good clinical responses (8, 11, 12) in substantial proportions of patients. Despite this success a large number of patients does not or only partially respond to therapy. Moreover, effects of therapy are transient requiring repeated drug administration. This implicates that it is still worthwhile to target other mediators and mechanisms that are indicated to play an important role in the immunopathology of RA. Recent findings indicate that interleukin-7 (IL-7) and thymic stromal lymphopoeitin (TSLP), potent immunoregulatory cytokines, could play an important and unique role in immunopathology of RA as well as other (rheumatic) inflammatory diseases.

Role of IL-7 in immunity and inflammation IL-7 is a member of the IL-2 family and signals through the IL-7 receptor- chain (IL-7R, CD127), in conjunction with the common gamma chain (c). This common chain is shared by receptors for IL-2, IL-4, IL-9, IL-15, and IL-21. IL-7/IL-7R expression For IL-7 to signal properly, both IL-7R and c are required. Since c is ubiquitously expressed by lymphocytes, surface expression of IL-7R on a lymphocyte indicates that the cell is able to respond to IL-7 (13). IL-7R is expressed on immature B and T cells and most mature T cells (13, 14), but also on dendritic cells (DCs) and activated monocytes (15). Epithelial cells in lymphopoietic tissues such as bone marrow, thymus, spleen, and gut produce IL-7. Apart from the sites of lymphopoiesis, several cell types throughout the body, such as keratinocytes, hepatocytes, and endothelial cells as well as cells from the immune system including monocytes, and (follicular) DCs have the potential to produce IL-7 (13, 16, 17). Role of IL-7 in lymphoid development The non-redundant role for IL-7 in lymphopoesis is most clearly demonstrated by mice deficient in either IL-7 or IL-7R. In these mice, B- and T-cell development is severely impaired and T cells are completely absent (18). Both IL-7-increased thymic output and peripheral expansion of T cells have been shown to contribute to expansion and maintenance of the T-cell pool (13, 19). With progressing age and strong thymus atrophy (20) the expansion and maintenance of the T-cell pool becomes largely dependent on thymic-independent pathways (13, 21), with a prominent role for IL-7. IL-7 plays an important role in T-cell homeostasis in humans and mice by provision of signals for T-cell

Introduction

10

proliferation, growth, and survival of both developing and mature cells (13, 22, 23). Thus, IL-7 plays a major role in regulating the amount of T cells in the periphery. This can be seen most strikingly in lymphodepleted hosts, where IL-7 has been shown to be critical in T-cell recovery (24). Recently, IL-7 injections in humans clearly indicated the strong capacity of IL-7 to regulate T-cell numbers, primarily through peripheral expansion of the T-cell pool. T-cell receptor spectratyping revealed that there was increased T-cell receptor diversity of the peripheral T-cell pool following IL-7 therapy compared to that observed prior to IL-7 therapy. Interestingly, despite CD4+ expansion, there was no significant increase in regulatory T-cell numbers as monitored by FoxP3 expression. Recently Fox-P3-expressing regulatory T cells were shown to largely lack the IL-7R (25). Thus, IL-7 therapy selectively expands populations with the highest levels of CD127 expression, resulting in measurable increases in T-cell receptor repertoire diversity (26). The effects of IL-7 on B cell development differ significantly between mice and humans. Although in IL-7 knockout mice clear effects on B cell development have been shown and immature human B cells can respond to IL-7 (27), IL-7R deficiency in humans does not affect B cell development (28, 29). IL-7R deficient individuals only have abnormalities in T-cell development as measured by diminished CD3 T-cell numbers and reduced lymphocyte proliferation to mitogen and allogenic cells, whereas B-cell numbers are normal (28, 29). IL-7-induced activation of human T cells and myeloid cells Apart from stimulating mechanisms that regulate T-cell numbers, IL-7 has been shown to stimulate several effector functions of not only T cells, but also other cells of both the acquired and innate immune system. Although IL-7 can stimulate IL-4 production, human in vitro studies have shown that IL-7 primarily induces Th1 cytokine secretion (IFN production) both by human CD4 and CD8 T cells in the absence of IL-4 production (30). In mixed mononuclear cell cultures from peripheral blood of healthy controls IL-7 induces IFN but no IL-4 secretion (van Roon, unpublished data). Besides cytokine secretion, IL-7 also stimulates cytotoxic activity mediated by CD8 T cells (31, 32). In addition to affecting function of CD4 and CD8 T cells, IL-7 also augments function of natural killer (NK) cells (33). In humans, this activity seems to be restricted to the function of mature NK cells, since disruption of the IL-7 pathway in IL-7R deficient individuals does not lead to impaired NK development (28). The effect of IL-7 on mature B cells is largely unknown. Recently it was described that following injections of IL-7 to human individuals no significant expansion of mature B-cell populations was observed (26). Mature B cells do not express the IL-7R, however this receptor might mediate T cell-dependent B cell activation. This is suggested from the fact that despite the lack of direct effect of IL-7R deficiency on B cell numbers, this deficiency does influence B cell activity as Ig levels in IL-7R-deficient humans are reduced (28). It is however unclear whether these effects are mediated by IL-7 or the related IL-7R ligand TSLP (see below).

Chapter 1

11

In addition to the acquired immune system IL-7 has also been described to activate cells from the innate immune system. IL-7 induces secretion of IL-1, IL-1, IL-6, IL-8, MIP-1, and TNF by human monocytes (31, 34, 35). However, it should be noticed that high, possibly supraphysiologic, levels of IL-7 (50-100 ng/ml) are required to induce this cytokine secretion, approximately 100- to 1000-fold higher than observed for activation of T cells (31, 36, 37). This may be related to the much lower to absent IL-7R expression on monocytes as compared to T cells (38). In contrast to T cells and monocytes, IL-7 does not have any direct effect on granulocyte activity, related to the absence of IL-7R expression on these cells (39). Role of IL-7 in experimental inflammatory conditions In mice, in vivo administration of IL-7 has also demonstrated strong effects on cells from the acquired and innate immune system. Administration of IL-7 resulted in increased numbers of T cells, B cells, NK cells, and macrophages (40). T cells from IL-7-treated mice had enhanced proliferative responses to various stimuli in vitro and were able to potentiate T-cell mediated cytotoxicity (CTL) responses in vivo (40). Bone marrow transplant (BMT) studies, in which mice were treated with IL-7 after BMT, resulted in acceleration of lymphocyte regeneration and improvement of T- and B-cell function (41). An in vivo mouse model of IL-7 transfected glioma cells showed reduction of tumorigenicity that could be reversed by injecting an anti-IL-7 antibody at the tumour site. IL-7 can also promote delayed-type hypersensitivity reactions in mice (42). Finally, IL-7 has been suggested to play a role in models of inflammatory autoimmune disease, as it has been shown that the persistence of IL-7-dependent colitogenic memory CD4 T cells is critical to the maintenance of experimental colitis (43). Effects of IL-7 in rodents and primates differ markedly in some aspects, as demonstrated by analysis of IL-7R-deficient humans and mice (13, 28, 29). Therefore, analysis of IL-7-induced effects in (non-human) primates is also important. In baboons IL-7 increased virus-specific IFN producing CD4 T-cell numbers (44). IL-7 treatment of baboons (after total body irradiation (TBI) and subsequent CD34 cell transplantation) and Indian rhesus macaques, increased CD4 and CD8 lymphocytes populations and lymph nodes were enlarged compared to untreated animals (45, 46). IL-7 furthermore increased the ability of CD4 and CD8, central memory and effector memory, T cells to produce the pro-inflammatory cytokines TNF and IFN (45). In contrast with IL-7-stimulated T-cell reconstitution upon TBI followed by CD34 cell transplantation, IL-7 did not increase B-cell, monocyte, and NK-cell counts in baboons (46). The above data indicate that IL-7 is an important immunoregulatory cytokine, which stimulates immunity that could contribute to inflammation and inflammation-induced immunopathology in RA as well as other chronic inflammatory (rheumatic) diseases.

Introduction

12

Role of TSLP in immunity and inflammation The IL-7-related cytokine, thymic stromal lymphopoietin (TSLP) is also a member of the IL-2 family. TSLP also interacts with the IL-7R, but signals by formation of a heterodimer with a c-like chain, called the TSLP receptor (47-49). TSLPR/TSLP expression The TSLPR chain has been shown to be expressed by DCs, monocytes, pre-activated T cells and mast cells (15, 50, 51). Moreover, NK cells are implicated to respond to TSLP as well, indicating them to express the TSLPR (52).TSLP is not produced by many immune cells. Mast cells are in fact the only hematopoietic cells expressing the cytokine (53). Non-heamatopoietic cells expressing TSLP are lung fibroblasts, smooth muscle cells, skin keratinocytes and bronchial epithelial cells (54). Mast cells are shown to upregulate TSLP expression upon stimulation by IgE crosslinking (54). Cytokines such as IL-1, TNF, as well as Toll-like receptor (TLR)-3 signalling (e.g. via virus derived double-stranded RNA) activate bronchial epithelial cells through to produce TSLP (55, 56). Also, allergen-derived proteases were described to induce TSLP expression in airway epithelial cells, thus suggested to be involved in Th2-driven responses in allergic diseases (57). Thus far the only negative regulator of TSLP production by epithelial cells that has been reported is 9-cis-retinoic acid (58). TSLP (over-)expression is seen in typical Th2 mediated disorders. In normal skin, histologic staining does not show any TSLP at all. In patients with atopic dermatitis, the cytokine is not seen in normal skin, but is highly expressed in acute and chronic atopic dermatitis lesions (54). Also, high levels of TSLP in humans are associated with airway inflammatory disease (53). Opposite to allergy, 70% of patients with Crohn disease that is indicated to be Th1/Th17 driven, had no TSLP mRNA expression in the gut epithelial tissue, suggesting that dysregulation of TSLP expression might contribute to pathology or that pathology dysregulates TSLP expression (59). Role of TSLP in lymphoid development As indicated, IL-7 signals through the IL-7 receptor (IL-7R), a heterodimeric protein consisting of the IL-7R-chain and a common cytokine receptor -chain (c) (60).Therefore, IL-7 and IL-7R knockout mice show different phenotypes, as TSLP signaling is abrogated in the receptor knockout as well (18). TSLP-/- (double knockout) mice show no changes in lymphocyte development. A combination of c and TSLP results in more severe impairment of lymphocyte development than in mice only deficient in c (18). IL-7-deficient mice show less severe impairment of lymphocyte development than IL-7R-/- mice, suggesting that TSLP can substitute for IL-7 to some extent (18). In addition, over-expression of TSLP in IL-7-/- mice can rescue B- and T-cell development (61) providing more evidence that TSLP can substitute for IL-7. In humans, no TSLP or IL-7 specific deficiencies have been described. Patients with an IL-7R mutation have been described and are impaired in T-cell development. As indicated above, this impairment of IL-7R leads to T-B+NK+ severe combined immune deficiency (SCID), confirming that potentially IL-7 and/or TSLP play a role in human T-cell

Chapter 1

13

development (29). However, the presence of B cells in SCID patients shows that IL-7 and/or TSLP are not absolutely required for B-cell lymphopoiesis in humans. The role of TSLP in homeostatic peripheral expansion (HPE) is shown in sublethally irradiated mice, but effects are less profound as for IL-7 (18). In addition, TSLPR knockouts show weakened recovery of lymphocyte numbers, also indicating that this cytokine is required for HPE. TLSP-induced activation of human T cells and myeloid cells In human in vitro assays, TSLP fails to directly induce T-cell activation, in particular Th2 proliferation that is suggested to be a prominent feature of TSLP in mice (see below) (62). This inability to respond to TSLP could be contributed to the observation that TSLPR is not expressed on naive T cells, but only on pre-activated cells (63). Stimulating these activated cells with TSLP, increases their expression of the IL-2R, thus increasing the sensitivity for IL-2 and with that augmenting proliferation (51). Despite the fact that TSLP is unable to directly drive naive cells towards a Th2 cytokine production, it can in presence of DCs. CD11c myeloid DCs stimulated with TSLP show upregulated expression of HLA-DR, CD40, CD80, CD86, and CD83 and secrete high levels of the chemokines thymus-and activation regulated chemokine (TARC) and macrophage-derived chemokine (MDC). TSLP-activated DCs stimulate production of in particular Th2 cytokines (IL-4, IL-5, and IL-13) when added to allogenic naive CD4 T cells (54). This induction is shown to depend on the expression of OX40 ligand (62) and is inhibited by IL-12 (64). Although TSLP-primed human DCs have the capacity to induce Th2 responses, studies that have used these cells to stimulate autologous naive CD4 T cells have demonstrated a predominant induction of Th1 activity along with Th2 activity (65). In addition, activation of TSLP-primed DCs by soluble CD40L enhances IL-12 production that shifts the Th1 and Th2 cytokine production towards Th1 cytokine production. This was indicated by reduced IL-4 and IL-13, and increased IFN production by autologous CD4 T cells. TSLP also stimulates the capacity of TLR3-activated DCs to induce Th17 cytokine production (IL-17 and IL-22) by allogenic CD4 T cells (66). Apart from CD11c myeloid DCs, TSLP has been reported to stimulate TARC secretion by monocytes, although at a 10- times lower level than myeloid DCs (15). Role of TSLP in experimental inflammatory conditions Different from human, TSLP In mice directly induces a Th2 phenotype (IL-4 production) in CD4 T cells (67). In vivo, TSLP-TSLPR interactions are critical for immunity to the intestinal pathogen Trichuris. Monoclonal antibody-mediated neutralization of TSLP, or deletion of the TSLPR in normally resistant mice resulted in defective expression of Th2 cytokines and persistent infection. Susceptibility was accompanied by elevated expression of IL-12/IL-23p40, IFN, and IL-17A, and development of severe intestinal inflammation. Critically, neutralization of IFN in Trichuris-infected TSLPR-/- mice restored Th2 cytokine responses and resulted in worm expulsion, demonstrating TSLPR-independent pathways for Th2 cytokine production (68). Additionally, TSLPR-/- mice also displayed elevated production of IL-12/IL-23p40 and IFN, and developed heightened intestinal inflammation upon exposure to dextran sodium sulfate, demonstrating the immunoregulatory role for TSLP in a mouse

Introduction

14

model of inflammatory bowel disease (68). Therefore, TSLP is suggested to be protective for inflammatory bowel disease (59), which is considered Th1 or Th17 driven (59, 69).TSLP has been implicated in atopic conditions. High levels of TSLP are associated with airway inflammatory disease in mice (53, 62). Moreover, TSLPR-/- mice are unable to develop inflammatory allergic responses in the lung (70) and over-expression of TSLP induces both spontaneous airway inflammation and atopic dermatits (71, 72). The above data indicate TSLP to potently activate cells from the myeloid lineage that induce strong T-cell activation. TSLP is primarily suggested to play a role in Th2-driven responses, although it is able to significantly promote Th1 and Th17 activity as well. Taken together, IL-7R ligands IL-7 and TSLP are clearly involved in immunity in several inflammatory diseases. IL-7 clearly activates T cells and causes T-cell dependent activation of cells from the myeloid lineage (DCs, monocytes) and is associated with primarily Th1/Th17-driven immunopathology. TSLP potently activates cells from the myeloid lineage that induce strong T-cell activation and is suggested to primarily play a role in Th2-driven responses (Fig. 1).

Figure 1. IL-7 and TSLP regulate Th1/17 and Th2 driven responses

Epithelial cellsFibroblasts

KeratinocytesMast cells

Epithelial cellsFibroblastsMacrophages/DCsEndothelial cells

IL-7R

Th1/Th17 activationmacrophage activation

osteoclast induction

Th1/Th17-drivenimmunopathology

IL-7

c TSLPR

Th2 cell activationmast cell activation

dendritic cell-mediated allergy

Th2-drivenallergic responses

TSLP

IL-7R

Figure 1. IL-7 and TSLP regulate Th1/17 and Th2 driven responses

Epithelial cellsFibroblasts

KeratinocytesMast cells

Epithelial cellsFibroblastsMacrophages/DCsEndothelial cells

IL-7R

Th1/Th17 activationmacrophage activation

osteoclast induction

Th1/Th17-drivenimmunopathology

IL-7

c TSLPR

Th2 cell activationmast cell activation

dendritic cell-mediated allergy

Th2-drivenallergic responses

TSLP

IL-7R

Chapter 1

15

Role of IL-7 in inflammation in (rheumatoid) arthritis In addition to the potent immunoregulatory role of IL-7/IL-7R pathways in inflammation in several immune diseases, recent studies indicate IL-7 could also contribute in a unique manner to inflammation and tissue destruction in RA. IL-7/ IL-7 receptor expression in RA Serum IL-7 levels in RA patients were higher as compared to healthy controls, and correlated positively with markers of inflammation (C-reactive protein, marker of acute phase responses). Although a number of groups have reported increased levels of serum IL-7 levels in patients with RA and juvenile idiopathic arthritis (36, 73, 74), conflicting data on serum concentrations have been reported (75). Such differences in IL-7 levels may be due to heterogeneity in drug use between the studies as explained below. In support of a role of IL-7 in RA synovitis are the strongly elevated IL-7 levels in RA synovial fluid (SF) (up to 480 pg/ml) compared to IL-7 levels in SF of osteoarthritis patients (a joint disease with mild or no inflammation)(37). In synovial tissue (ST) biopsies of RA patients high IL-7 levels are expressed by macrophages, fibroblasts and endothelial cells throughout the tissue. Numbers of IL-7+ cells strongly correlated with the presence of CD68+ macrophages in lining and sub-lining. Double staining demonstrated that CD68+ macrophages were major producers of IL-7 (37). In addition, DC-like cells in the lymphoid follicles were found to express IL-7. Recently we supported the latter observation by showing that in vitro GM-CSF/IL-4-generated DCs from RA patients were indeed significant producers of IL-7. In addition, IL-7 production by DCs from healthy controls was previously shown by other groups (17, 76). Limited data are available on the IL-7R expression in RA patients. Previously we have demonstrated that the majority of T cells from the synovial fluid expresses the IL-7R (36). In addition we demonstrated that CD25+ T cells both from the synovial fluid and the peripheral blood of RA patients largely lack the IL-7R, whereas CD25- T cells express IL-7R at a high level (77). IL-7 activity on human CD4 T cells and monocytes from RA patients in vitro RA is characterized by a diverse auto-reactive T-cell response against numerous self antigens expressed in the inflamed joints (e.g. collagen type II, heat-shock proteins, and aggrecan) (78). However, although detectable, such T cells are present in a low frequency and are part of an oligo/polyclonal intra-articular T-cell response (79). Although several groups have tried to explain this widespread T-cell response, a causal linkage has not been provided. Irrespectively, in fact, the T-cell contribution in RA may have been underestimated since, merely based on the use of T-cell receptor mediated mitogenic stimuli, hypo-responsiveness of intra-articular T cells from RA patients has been suggested (80). This contrasts the observation that a large activated T-cell pool is present in RA joints. Until now the number of factors that explain these (hyper)activated T cells are limited (81, 82). Our data demonstrated that intra-articular CD4 T cells are hyper-responsive to IL-7 (37). In co-cultures of monocytes/macrophages and CD4 T cells, T-cell activation was shown to require cell contact and was related to the IL-7-induced expression of co-stimulatory molecules on

Introduction

16

SF macrophages, such as CD40, CD86 and in particular CD80 (37). In addition, upregulation of co-stimulatory molecules such as LFA-1 and CD69 was observed and could play an important role in (CD4) T-cell activation. This IL-7-induced contact-dependent activation, which is associated by monocyte activation (measured by up-regulation of CD80 and CD40), is also associated with TNF production (38). IL-7 (at similar concentrations) fails to induce TNF secretion by isolated T cells, or monocytes cultured separately. These data supported the previous data showing that IL-7 induces TNF secretion by mononuclear cells from the synovial fluid of RA patients (36). Previously we demonstrated that IL-7 promotes arthritogenic Th1 cell activity in cultures of mononuclear cells from RA patients. IL-7 primes T cells for IFN and TNF production in contrast to IL-4 production (36). IFN induction by IL-7 is IL-12-dependent since blockade of IL-12 significantly reduced IFN production. Interestingly, IL-7-induced TNF production is not inhibited by IL-12-blockade (36). This may be related to the induction of other regulatory cytokines. Using cytokine arrays we found that IL-7 can stimulate Th1 activity (IFN production) but possibly also Th17 activity since also IL-17 production was increased by IL-7. This was in the absence of induction of Th2 activity (no IL-4, IL-5) of RA PB and SF mononuclear cells. Th1 activity was associated by induction of Th1 cell differentiating factors such as IL-12 and small amounts of IL-18. Interestingly, IL-7 also induced chemokines (MIG and MIP1) that can cause chemotaxis of in particular Th1 cells. Possible role of IL-7 in immunopathology in RA Since IL-7 is an inducer of TNF, which has been shown to be a pivotal inducer of inflammation and joint destruction in a large proportion of RA patients, this implies that IL-7 could also contribute to TNF-dependent inflammation and joint destruction. However, independent of TNF IL-7 could also promote joint destruction by the induction of other mediators such as for example IL-17. In addition by induction of many other proinflammatory cytokines, possibly independent of TNF, IL-7 might promote inflammation and consequently inflammation-induced destruction of joint tissues such as cartilage and bone. Another mechanism contributing to joint destruction that IL-7 might induce in particular is the activation of fibroblasts. Th1 cells, either by cell contact or cytokine secretion (eg. IFN), have been shown to activate fibroblasts (83). Since IL-7 induces Th1 activation, it is anticipated that IL-7 might also induce fibroblast activation and possibly fibroblast-induced destruction of cartilage and bone matrices, however, direct proof needs to be provided. In this respect, the independence of IL-7 from TNF remains to be demonstrated. The notion that TNF, in contrast to IL-7, does not induce but inhibits Th1 development, points towards IL-7-induced TNF-independent effects that could occur (84). Apart from fibroblast activation IL-7 recently has been shown to play a pivotal role in osteoclastogenesis and bone loss. In mice IL-7 induces bone loss through increased osteoclastogenesis, whereas it was found that IL-7R-deficient mice show greatly increased femoral trabecular bone volume compared with wild-type and heterozygous littermates (85). The IL-7-induced bone loss in mice is mediated by induction of RANKL and TNF production from T cells (86). These secreted cytokines were found to induce osteoclasts from monocytes and bone marrow B-cell precursors (86). Similarly, IL-7 induced osteoclast

Chapter 1

17

formation from human monocytes in a T cell-dependent way that was strongly (approximately 50%) dependent on RANKL (87). Together, this suggests that IL-7 could contribute to bone destruction in RA by induction of T cell-dependent osteoclastogenesis. In addition to the production of IL-7 by cells from the synovial tissue, increased IL-7 mRNA expression by articular cartilage chondrocytes from RA patients compared to OA patients has been observed (88). In chondrocytes from cartilage tissue of a RA patient we recently have demonstrated IL-7 protein expression by immunohistochemistry. The role of this IL-7 in RA cartilage remains to be elucidated, however recent findings suggest that IL-7 might directly cause cartilage damage in RA (89). Long and colleagues recently demonstrated that IL-7 protein can be produced by articular chondrocytes. Production is increased upon stimulation with fibronectin fragments and a combination of IL-1 and IL-6. Most interesting, endogenous production of IL-7 by cartilage tissue is higher when obtained from older donors or from patients with osteoarthritis (OA). Through chondrocyte-expressed IL-7R, this IL-7 is demonstrated to induce production of matrix metallo proteinase-13 (MMP-13) associated with enhanced release of proteoglycans from the cartilage matrix of (aged) healthy controls and OA patients. Thus it is also conceivable that IL-7 both in a paracrine an autocrine manner contributes to joint tissue destruction in RA as well as other joint diseases. The above data indicate that IL-7 may induce cell contact dependent, spreading of the auto-immunity and immuno-pathology via an alternative, primarily cytokine-driven route. This unique route could operate, at least in a subpopulation of patients or in certain stages of the disease, largely independent of TNF-induced immunopathology. Moreover, IL-7/IL-R pathways may indirectly via inflammation but also directly be involved in bone and cartilage destruction. These pathways may not only play a role in patients with RA but also in other chronic inflammatory (rheumatic) diseases.

Role of TSLP in inflammation in (rheumatoid) arthritis Although TSLP has also been linked to inflammatory arthritis, the functional properties of TSLP in arthritic conditions are poorly studied. Recently, we and others have demonstrated increased levels of TSLP compared to osteoarthritis patients (van Roon et al unpublished observation (90). TNF and Toll-like receptor ligands stimulate TSLP production in RA- and OA-derived synovial fibroblasts (90, 91). Whereas TSLP only causes minimal activation of mononuclear cells from peripheral blood, we recently demonstrated that TSLP activates CD11c+ DCs from healthy controls and RA patients (van Roon et al., unpub. results). These TSLP-primed DCs potently stimulated T cell proliferation and cytokine production by CD4 T cells. Finally, in a recent paper it has been postulated that TSLP may have an arthritogenic effect in collagen antibody induced arthritis (CAIA). However, because this study is lacking the appropriate controls and experimental setup these data are difficult to interpret (90). Together these data point towards a potential role for TSLP in RA as well, however, the exact role of TSLP in RA patients and experimental arthritis needs further study.

Introduction

18

In summary, based on our published data and preliminary work as well as recent data from other groups the following concept is proposed (Fig. 2) : In auto-immune diseases such as RA an unknown trigger X may cause (self) antigen-specific T-cell activation [1], resulting in cytokine production (e.g. by IFN, IL-17, and GM-CSF) and cell contact that lead to activation of cells such as monocytes, macrophages and dendritic cells [2], which is associated with IL-7 and TNF production (17). Increased IL-7 (not TNF) (37, 84) induces cell contact-dependent cytokine-activated (37, 92) T cells causing a spreading of T-cell activation (proliferation, survival, differentiation) associated with auto-antigenic recognition (possibly intermediate affinity self antigens) (93). Such cytokine-activated by-stander T-cells in turn stimulate monocytic cells and B-cells. As a consequence monocytes can differentiate into macrophages and osteoclasts that mediate inflammation and joint destruction (37, 86, 87). Activated B cells in their turn are potent antigen presenting cells and can develop into plasma cells secreting pathogenic autoantibodies [6] (8). Finally, activated T-cells interact (via cell contact and cytokine production) with and activate synovial fibroblasts which can be associated with additional IL-7 production [7] (83). Both stimulated IL-7 production by fibroblasts and macrophages (which could also be produced by alternative routes, Y and Z) further enhance IL-7-driven immunopathology. TSLP that might be induced upon Toll-like receptor stimulated fibroblasts [7] or by surrounding epithelial cells and mast cells [8] is suggested to induce myeloid DC-driven Th2 activation associated with IL-4 production (54, 62).

Figure 2. Summarizing concept of the mechanism by which IL-7 and TSLP play a role in the immunopathology of autoimmune disease, such as rheumatoid arthritis.

cell-cell contact soluble mediators

IFN, IL-17, GM-CSF

[1] Trigger X

auto-reactive/ TCR-activated T cell

diverse cytokine-activated bystander autoimmunity

[2]

[4]

[3]

[5] IFN IL-17

sRANKL

IL-4 +

+

+ +

Immunopathology macrophages, osteoclasts, fibroblasts

[6]

TSLP IL-7

driven pathways

TNF driven

pathway

pathogenic autoantibodies

Fibroblast

IFN

Trigger Z (e.g. TLR agonists)

[8] TSLP

Trigger Y (e.g. TLR agonists)

epithelial cells mast cells

IL-7R c

IL-7R IL-7R

TSLPR

vascular activation chemotaxis angiogenesis catabolic cytokines / enzymes

expansion

T cell IL-7

[7] IL-7

TSLP

T cell B cell

IL-7

TNF

Monocyte Macrophage

mDC

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Summary and hypothesis IL-7 is a potent immunoregulatory cytokine that is produced by cells of the immune system and tissue cells at the inflammatory site of several rheumatic disorders, correlating with parameters of disease. IL-7 activates T cells and seems to cause primarily T cell-dependent B cell, and macrophage activation. In addition, IL-7 can induce bone loss by stimulating RANKL-dependent osteoclastogenesis. IL-7 is suggested to induce both TNF-dependent and independent inflammatory responses and immunopathology. Considering the lack of response or partial response to anti-TNF therapy and other more recently available biologicals in a considerable number of patients the elucidation of the role IL-7 in immunopathology is of major value. As such, studying the capacity of IL-7 and IL-7R blockade to reduce arthritis and joint pathology in experimental animal models for arthritis is a necessity. Since TSLP is a very potent immunoregulatory cytokine that shares the IL-7R with IL-7, it is also essential to study in more detail the effects of TSLP in arthritic conditions. In addition the capacity of TSLP to promote Th2 responses in mice strongly suggests regulatory capacities in arthritic conditions considering the potential of Th2 cell activity to regulate experimental and (rheumatoid) arthritis. The present thesis starts from the hypothesis that IL-7 promotes and TSLP reduces immunopathology of RA and experimental arthritis by differentially regulating T cell-driven immunity. IL-7 is indicated to induce Th1/Th17-driven immune responses to contribute to immunopathology; TSLP will induce a Th2-driven immune response that regulates immunopathology. To test this hypothesis and to investigate the role of IL-7 and TSLP the following questions were addressed:

To what extent do IL-7 levels and IL-7 activity persist upon TNF blockade? (chapter 2)

Are IL-7R-expressing cells present in the inflamed joints of RA patients and does this receptor mediate immune activation in these patients? (chapter 3)

What are the functional properties of IL-7R+ and IL-7R- T cells in patients with RA? (chapter 4)

Can neutralization of IL-7 prevent proteoglycan-induced arthritis, a chronic relapsing model for RA? (chapter 5)

In what way do the IL-7R ligands IL-7 and TSLP influence experimental collagen-induced arthritis? (chapter 6)

Does blockade of the IL-7R inhibit experimental collagen-induced arthritis? (chapter 7)

How does TSLPR deficiency affect proteoglycan-induced arthritis as compared to blockade of the IL-7R? (chapter 8)

Introduction

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Introduction

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2

Persistence of IL-7 activity and levels

on TNF blockade in patients with rheumatoid arthritis

JAG van Roon 1

SAY Hartgring 1

MJ Wenting 1

KMG Jacobs 1

PP Tak 2

JWJ Bijlsma 1

FPJG Lafeber 1

1 Dept. Rheumatology & Clinical Immunology, University Medical Center Utrecht, the Netherlands

2 Dept. Clinical Immunology & Rheumatology, Academic Medical Cente, Amsterdam, the Netherlands

Annals of the Rheumatic Diseases

2007 May; 66(5): 664-669.

Chapter 2

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Abstract Objectives To identify the mechanism of interleukin (IL)-7-stimulated tumour necrosis factor (TNF) production and to determine the relationship between intra-articular IL-7 and TNF expression levels in patients with rheumatoid arthritis (RA). In addition, the effect of TNF blockade on IL-7 activity and on IL-7 levels was studied. Methods The effect of IL-7 on isolated CD4 T cells and CD14 monocytes/macrophages was studied. IL-7 and TNF levels were measured in the synovial fluid of patients with RA. In RA synovial tissue, IL-7 and TNF expression was assessed in addition to IL1, numbers of inflammatory cells and adhesion molecule expression. The extent to which TNF blockade could prevent IL-7-induced lymphocyte responses was studied in vitro. In addition, regulation of serum IL-7 levels on anti-TNF therapy (adalimumab) was studied. Results IL-7 induced cell contact-dependent TNF production by cocultures of T cells and monocytes, but not by T cells and monocytes cultured separately. IL-7 and TNF levels in RA synovial fluid and synovial tissue significantly correlated. IL-7-stimulated lymphocyte responses were not inhibited by TNF blockade. Circulating IL-7 levels were significantly reduced in patients who successfully responded to anti-TNF treatment. However, IL-7 levels persisted in non-responders. Conclusion The present data suggest that IL-7 is an important inducer of T cell-dependent TNF production in RA joints. This may contribute to the correlation of intra-articular IL-7 and TNF in these joints. Furthermore, the persistence of IL-7-induced inflammatory activity on TNF blockade in vitro and persistence of IL-7 levels and disease activity in anti-TNF non-responders suggest that IL-7 might additionally promote TNFindependent inflammation.

IL-7 levels and activity persist upon TNF blockade

28

Introduction Rheumatoid arthritis (RA) is a chronic disabling type of arthritis that affects ~1% of the adult population. RA is characterised by persistent inflammation of the joints, often resulting in continuously progressing tissue destruction (1). Numerous studies revealed a pivotal role for CD4 T cells and macrophages in RA synovitis (2-6) associated with abundant production of catabolic enzymes and proinflammatory cytokines (27) including tumour necrosis factor (TNF) (8-15). Clinical studies support the importance of TNF in inflammatory and tissue-destructive processes in RA patients (16). Despite the success of anti-TNF treatment, a considerable number of patients do not respond or only improve partially (16-18). The lack of efficacy of anti-TNF treatment in certain patients might be due to persisting TNF-independent proinflammatory activity induced by mediators other than TNF. Additionally, such mediators may contribute to continuous TNF induction, preventing an adequate response to anti-TNF treatment. Recently, several studies indicated that interleukin (IL)-7 might be such a mediator, contributing to chronic inflammation in RA. IL-7 belongs to the IL-2 family of cytokines that includes IL-2, IL-4, IL-9, IL-15, IL-21 and thymic stromal lymphopoietin. IL-7 mediates its effects through the IL-7 receptor (IL-7R), which consists of the common cytokine c chain (c) and the IL-7R chain (19). IL-7 is produced by stromal cells at lymphopoietic sites and plays a role in the regulation of peripheral homeostasis of the CD4 T-cell pool. IL-7 is a growth factor for T cells in early T-cell development, and promotes proliferation, survival and differentiation of mature naive and memory T cells (20). In addition, high concentrations of IL-7 induce cytokine production by monocytes from healthy individuals (21). In patients with arthritis (RA and juvenile idiopathic arthritis (JIA)), increased levels of IL-7 have been shown compared with healthy controls (22-24) and correlated with increased disease activity (22, 24) In addition, strongly increased IL-7 levels were found in the synovial fluid (SF) of RA patients and JIA patients compared with patients with osteoarthritis and oligoarticular patients, respectively (25, 26). Furthermore, abundant expression of IL-7 by macrophages, endothelial cells and fibroblasts was detected in the synovial tissue of patients with RA (25, 27). The purpose of this study was to define the mechanism by which IL-7 induces TNF production by monocytes and CD4 T cells, to investigate the relationship between intra-articular IL-7 and TNF levels. Also, the TNF dependency of IL-7-induced lymphocyte activation was tested in vitro. Finally, the persistence of IL-7 levels on TNF blockade was studied in patients treated with the anti-TNF monoclonal antibody Adalimumab.

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Methods Patients. Table 1 shows the demography of patients with RA. Patients with RA were classified according to the 1987 revised American College of Rheumatology criteria (28). Patients who donated peripheral blood (PB) or synovial fluid (SF) for cell cultures or analysis of IL-7 and TNF by ELISA were randomly selected from our outpatient clinic. Synovial tissue biopsy specimens were taken from a cohort of patients with persistent synovitis of the knee. Anti-TNF-treated patients had previously failed to at least three conventional anti-rheumatic drugs. Written consent was obtained from the patients according to the Helsinki declaration, and the University Medical Center Utrecht medical ethics committee approved the design of the studies. Cytokine assessment by enzyme-linked immunosorbent assay. Prior to cytokine analysis, SF of RA patients(n= 30) was treated with hyaluronidase (20 U/ml; type IV, Sigma, Munich, Germany) for 20 min at 37C to reduce viscosity. IL-7 and TNF in SF were measured with a commercially available ELISA according to the manufacturers instructions (Diaclone, Besancon, France, and Biosource Europe, Nivelles, Belgium, respectively). Specificity was tested as described previously (25). IL-7 levels in serum samples from anti-TNF-treated patients with RA were measured using a different ELISA kit (R&D, Minneapolis, MN) as this measures IL-7 in the serum more sensitively than the above-described ELISA kit (Diaclone). One possible explanation for the observed difference in sensitivity is the use of different IL-7-specific monoclonal antibodies recognising dissimilar epitopes that could be influenced in their own way by IL-7-binding molecules such as those previously described (eg, glycoseaminoglycans such as heparin and chondroitin sulphate) (29). To demonstrate the specificity of anti-TNF treatment on IL-7 levels, circulating levels of IL-15 (R&D) and IL-18 (MBL, Woburn, MA) were also measured with commercially available ELISA kits. Immunohistology of synovial tissue. RA knee synovial tissue biopsy specimens were obtained, stored and prepared for immunohistochemical analysis as described previously (25, 30). Biopsy sections (n= 23) were incubated with polyclonal rabbit anti-human IL-7 antibody (H-151, Santa Cruz Biotechnology, Santa Cruz, CA), followed by a two-step immunophosphatase staining method as described previously. Numbers of IL-7 cells were counted independently by two observers (JvR, MW) who were blinded to the patients identity. Cells were counted in 3-5 tissue sections per patient that included in each section intimal lining layer and synovial sublining. Numbers of cells were calculated per mm2 as an average of the analyzed tissue sections. In an additional set of slides, serial sections were stained with the following mouse monoclonal antibodies (mAbs): anti-CD3 (SK7; Becton-Dickinson, San Jose, California, USA) to detect T lymphocytes, anti-CD68 (EBM11; Dako, Glostrup, Denmark) for macrophages in the intimal lining layer and synovial sublining, and anti-CD55 (clone 67; Serotec, Oxford, UK), which recognises fibroblast-like synoviocytes. Staining was also done with mAbs against the proinflammatory cytokines TNF (52B83; Monosan, Uden, The Netherlands) and IL-1b (2D8; Immunokontact, Frankfurt, Germany). Staining was performed according to a three-step immunoperoxidase method, as described

IL-7 levels and activity persist upon TNF blockade

30

previously (31) For control sections, the primary antibodies were omitted or irrelevant isotype-matched antibodies were used. Sections stained were coded and randomly analyzed by one blinded observer (MW) using digital image analysis, as described previously (32) Measurements for CD markers were expressed in cell counts/mm2 and for cytokines in integrated optical density/mm2. Fluorescence-activated cell sorting analysis. The expression of IL-7R (CD127) on CD4 T cells and CD14 monocytes/ macrophages from PB was analyzed directly after isolation by fluorescence-activated cell sorting (FACS) analysis. Cells were triple stained with CD4-PE-Cy5, CD14-FITC (Dako) and CD127-PE (Immunotech, Marseille, France). FITC/PE-labelled isotype controls (Immunotech) were used for control staining. For intracellular TNF detection of co-cultured CD4 and CD14 cells, FACS analysis was used. During the last 4h of a 3-day culture period in the presence or absence of IL-7 (Peprotech, Rocky Hill, New Jersey, USA), cells were exposed to 10 mg/ml Brefeldin A (ICN Pharmaceuticals, Costa Mesa, CA) to block protein secretion and enhance intracellular cytokine staining. The cells were fixed and permeabilised with a fixation and permeabilisation kit, according to the manufacturers instructions (Caltag Laboratories, Burlingame, CA). During fixation, cells were stained with fluorochrome-labelled CD4 and CD14 surface antibodies. Fluorochrome-labelled anti-TNF-PE and isotype control antibodies (R&D) were added during the permeabilisation step to stain TNF intracellular. Fluorescence was analyzed by FACS analysis. The mean fluorescence intensity of TNF produced by CD4 and CD14 cells in the presence of IL-7 was expressed as the percentage compared with cells in the absence of IL-7. Cell isolation. Heparinised PB or SF was diluted 1:1 with RPMI 1640 medium (Gibco BRL, Life Technologies, Mezelbeke, Belgium) containing penicillin (100 U/ml), streptomycin (100 mg/ml) and glutamine (2 mM). Mononuclear cells (MC) were isolated by density centrifugation using Ficoll-Paque (Pharmacia, Uppsala, Sweden). CD4 and CD14 cells were isolated from peripheral blood mononuclear cells through negative selection by means of microbead-activated cell sorting as described previously (25). Cell cultures. To determine proliferation of synovial fluid mononuclear cells (SFMC), these cells were cultured in 96-well plates (1*106 cells/ml; 20 ml/well) for 3 days. The cells were cultured in RPMI supplemented with penicillin, streptomycin, glutamine and 10% pooled fetal calf serum (Gibco BRL) in the presence or absence of IL-7 (Peprotech), in the presence or absence of anti-TNF (cA2, 10 mg/ml, Centocor, Malvern, PA). Proliferation was measured as described previously (25). To analyze cytokine production, isolated cells (0.5*106 cells/ ml; 1 ml/well) were also cultured in 24-well plates. To study the influence of direct cellcell contact, CD4 and CD14 cells were co-cultured for 3 days in these 24-well plates in the presence or absence of a transwell (6.5 mm, 0.4 mm pore size, Corning, NY) and with or without IL-7. CD14 cells were placed in the lower compartment and CD4 cells in the upper compartment, preventing direct cell-cell contact between the two cell fractions, but allowing effects mediated via soluble factors.

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Statistical analysis. Statistical analysis of paired evaluations was performed using the non-parametric Wilcoxon signed ranks test. Correlation analysis between the numbers of IL-7 cells and inflammatory markers was done by Spearmans correlation analysis for nonparametric data and Pearsons correlation analysis for parametric data. Differences were considered statistically significant at p

IL-7 levels and activity persist upon TNF blockade

32

Results IL-7 stimulates T cell-dependent TNF production by monocytes/ macrophages. Since monocytes/macrophages are major producers of TNF, we investigated how IL-7 influences TNF production by monocytes and compared this with production by CD4 T cells. IL-7 did not stimulate TNF production of CD14 monocytes/ macrophages or CD4 T cells cultured alone (Fig. 1A). However, IL-7 did stimulate TNF production hen monocytes/macrophages were co-cultured with CD4 T cells. To measure the specificity of this TNF induction, IL-1 was also measured. In all cases, IL-1 levels stayed below the detection limit. Interruption of direct cellcell contact (by use of a semipermeable membrane in a transwell culture system) almost completely prevented IL-7-stimulated TNF production (Fig. 1A). TNF production was associated with T cell activation (measured by proliferation and major histocompatibility complex class II expression) and monocyte activation (measured by CD40 and CD80 induction) (data not shown).

CRP: C-reactive protein, ESR: erythrocyte sedimentation rate, RF: rheumatoid factor.For age, disease duration, ESR, and CRP levels, mean (SD) values are given.Numbers of female/male and RF positive/negative patients are also given. ESR and CRP levels were available for 25 patients. ESR and CRP levels were available for 19 patients.

24.9 28.636.8 41.4 27.2 34.5CRP (mg/l)

44.1 29.142.3 33.4 32.1 20.8*ESR (mm/1st h)

16/613/1019/11 RF (+/2)

17/516/723/7Sex ratio (female/male)

14 99 12 12 15 Disease duration

52 (13)60 (11)64 (10) Age, mean (SD)

222330Number

Serum anti-TNFstudy baseline

values

Synovial tissue

Synovial fluid

Table 1. Demography of patients with rheumatoid arthritis (RA)

CRP: C-reactive protein, ESR: erythrocyte sedimentation rate, RF: rheumatoid factor.For age, disease duration, ESR, and CRP levels, mean (SD) values are given.Numbers of female/male and RF positive/negative patients are also given. ESR and CRP levels were available for 25 patients. ESR and CRP levels were available for 19 patients.

24.9 28.636.8 41.4 27.2 34.5CRP (mg/l)

44.1 29.142.3 33.4 32.1 20.8*ESR (mm/1st h)

16/613/1019/11 RF (+/2)

17/516/723/7Sex ratio (female/male)

14 99 12 12 15 Disease duration

52 (13)60 (11)64 (10) Age, mean (SD)

222330Number

Serum anti-TNFstudy baseline

values

Synovial tissue

Synovial fluid

Table 1. Demography of patients with rheumatoid arthritis (RA)

Chapter 2

33

To detect whether CD4 T cells or CD14 monocytes/macrophages produced TNF, intracellular TNF was measured (by FACS analysis, Fig. 1B). In patients with RA who secreted high TNF levels in the IL-7-stimulated co-cultures, we detected a consistent and significant increase in TNF production by CD14 monocytes in all individuals (35.9 (8.2%), expressed as percentage vs unstimulated co-cultures, p

IL-7 levels and activity persist upon TNF blockade

34

TNF

Opt

Den

sity

(*10

3 )

IL-7+ cells/mm2

r = 0.491p = 0.024

0

50

100

150

200

250

0 1000 2000 3000 4000 5000 6000

Synovial tissue

0

0.1

0.2

0.3

0.4

0.5

0.6

IL-7 (pg/ml)

TNF

(ng/

ml)

0 100 200 300 400 500

r=0.824p

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*Statistically significant correlations.

ICAM: intercellular adhesion molecule, TNF: tumour necrosis, VCAM: vascular cell adhesion molecule

0.040*0.430E-selectin

0.3440.207VCAM-1

0.6230.108ICAM-1

0.939-0.017CD22

0.2980.227CD8

0.2190.266CD4

0.5960.117CD3

IL-7 levels and activity persist upon TNF blockade

36

Persistent IL-7 levels on anti-TNF mAb treatment in non-responding patients. Apart from the TNF-independent induction of proinflammatory activity by IL-7 in vitro, it was investigated whether IL-7 persisted in patients with RA who were treated with anti-TNF mAb. This is of particular interest as previously TNF was shown to stimulate IL-7 production (of RA fibroblasts) in vitro (27). According to the European League Against Rheumatism response criteria (33), 7 non-responders and 15 moderate/good responders were identified (disease activity scores (DAS) are shown in Fig. 4A. In responders, IL-7 levels significantly decreased upon anti-TNF treatment (at all time points after the start of treatment). However, in non-responders, IL-7 levels did not change significantly on treatment (Fig. 4B). After 2 weeks of treatment, the change in IL-7 levels significantly correlated with the change in erythrocyte sedimentation rate (ESR) and DAS (r=0.633, p,0.01; r=0.438, p

Chapter 2

37

weeks after start of anti-TNF therapy

0

20

40

60

80

100

120

0 2 6 12

DA

S (%

vs

base

line)

*

******

***

RespondersNon-Responders

A B C

IL-7

R

expr

essi

on (M

FI) CD4+ T cells

CD14+ cells

0

5

10

15

20

25

30

35

cyto

kine

con

c. (%

vs

base

line)

0

50

100

150

200

250

0

50

100

150

200

250 IL-7 IL-18D

*** *

Figure 4. Anti-tumour necrosis factor (TNF) monoclonal antibody treatment (adalimumab, 40 mg subcutaneous, every other week) reduces serum interleukin (IL)-7 levels in clinical responders, whereas in clinical non-responders IL-7 levels persist. IL-7R expression levels on T cells and monocytes/ macrophages in responders were not significantly changed. Based on the European League Against Rheumatism (EULAR) response criteria, patients were judged as non-responders (n= 7) and responders (n= 15). As expected, in responders, a strong suppression of disease activity score (DAS) was observed, which was significantly different from that in non-responders, who showed only modest changes in disease activity. The average absolute DAS score at baseline did not differ significantly between responders and non-responders (mean (SD), 6.7 (1) vs 5.8 (1.3), respectively, A) Serum IL-7 levels in responders at 2, 6 and 12 weeks significantly decreased on anti-TNF treatment compared with those at baseline (100%). Non-responders did not show significant changes in IL-7 levels compared with those at baseline. The difference in percentage change of IL-7 levels between responders and non-responders was significantly different at 2 and 6 weeks after the start of treatment. Baseline IL-7 values between anti-TNF responders and non-responders were not significantly different (14.9 (13.6) and 13.2 (10.7) pg/ml, respectively, B). Serum IL-18 levels in responders at 2, 6 and 12 weeks did not significantly alter on anti-TNFtreatment compared with those at baseline (100%). Baseline IL-18 values between anti-TNFresponders and non-responders were not significantly different (358 (300) and 557 (487) pg/ml, respectively, C). IL-7R expression levels (mean fluorescence intensity (MFI)) on CD4 T cells and CD14 monocytes/macrophages on treatment. No significant change in IL-7R expression on T cells was observed. The modest IL-7R expression on monocytes (compared to isotype control, indicatedby the dashed line) was only slightly increased in non-responders after 2 and 6 weeks of anti-TNFtreatment (both p

IL-7 levels and activity persist upon TNF blockade

38

Discussion T-cell activation has been observed to induce IL-7 secretion by dendritic cells. Blockade of IL-7 in these cultures prevented T-cell activation (37, 38). Recently, we have found that maturation of RA dendritic cells in vitro by activation through Toll-like receptors is associated with significantly increased IL-7 protein levels. Although the exact triggers for the IL-7 production that induce inflammation in RA are unknown, recently the cell types producing IL-7 have been identified. Apart from fibroblasts (25, 27) and endothelial cells, professional antigen-presenting cells such as macrophages and dendritic-like cells also produce IL-7 in RA synovial tissue (25, 38). The present study demonstrates that IL-7 stimulates the production of TNF by monocytes requiring cell contact with CD4 T cells, a mechanism that has been recognised to be crucial in RA (13-15). Furthermore, in RA SF, and tissue IL-7 expression correlates with expression of TNF. Apart from inducing TNF, IL-7 can induce proinflammatory activity that persists on blockade of TNF. The most important finding is that non-responsiveness to anti-TNF treatment is related to persistent IL-7 levels. Previously, a high concentration of IL-7 (100 ng/ml) was shown to induce cytokine secretion (including TNF) by isolated monocytes from healthy controls (21). Lower concentrations ((10 ng/ml) did not induce TNF secretion. The present data are in line with this study, indicating that RA monocytes/ macrophages when cultured separately cannot be stimulated by IL-7 to induce TNF secretion in a concentration up to 10 ng/ml. However, in the presence of CD4 T cells, this lower IL-7 concentration induces high amounts of TNF production. The above-described T cell contact-dependent effects may be related to the expression of IL-7R primarily on CD4 T cells, in contrast with monocytes that lack IL-7R expression. This indicates that IL-7 (produced by cells such as antigen-presenting cells) may primarily act on T cells to induce T cell contact-dependent activation of other cell types such as monocytes. This mechanism of action could also occur in RA joints, since synovial CD4 T cells and macrophages from the SF show similar IL-7R expression patterns as their circulating counterparts (data not shown). The correlation of IL-7 and TNF expression in RA joints may be due to the capacity of IL-7 to induce TNF (this study, 24) or, vice versa, due to the capacity of TNF to induce IL-7 (27) Alternatively, common or distinct triggers may induce both IL-7 and/or TNF, independent of the mutual action of both cytokines. Our data show that in a substantial proportion of patients TNF blockade results in a decrease of serum IL-7. This reduction may be due to the prevention of a direct effect of TNF on several cell types to produce IL-7. Reduction of IL-7 in case of anti-TNF treatment may subsequently contribute to reduction of inflammation and disease activity. In our study, a reduction in IL-7 levels correlated with a reduction in disease parameters (ESR, DAS) after 2 weeks of treatment. Based on these data, it is suggested that downregulation of IL-7 by anti-TNF may contribute to disease inhibition. In addition, considering the potent proinflammatory effects of IL-7, it is indicated that insufficient IL-7 reduction could contribute to persistent disease activity in a substantial number of patients.

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Although anti-TNF treatment downregulates circulating IL-7 levels, it can be questioned whether anti-TNF treatment leads to sufficient suppression of IL-7 expression at the site of inflammation. Previously we have reported increased IL-7 levels in the SF of patients with RA who were using anti-TNF drugs when compared with patients who were not treated with TNF blocking agents (25). Since circulating levels of IL-7 may have different sources other than the joint (eg, lymphopoietic sites), there may be dissociation between intra-articular and circulating IL-7 levels. Persistent local IL-7 could thus mediate persisting and residual inflammation. Measurement of IL-7 in the synovial tissue of anti-TNF-treated patients in a controlled study will be needed to demonstrate whether indeed IL-7 production persists in RA joints on anti-TNF treatment. The persistence of serum IL-7 levels in patients who do not respond clinically to anti-TNF treatment is an interesting observation. In these patients, IL-7 production seems to be induced by a TNF-independent pathway. As shown in the present study, IL-7 may subsequently induce proinflammatory responses that are also TNF independent. IL-7-driven pathways may be present both in responding and non-responding patients, explaining either the partial responses or the lack of response to anti-TNF treatment. Apart from patients with RA, increased IL-7 levels are found in the circulation or at the inflammatory site of several other (auto) immune-mediated diseases, such as psoriasis and JIA (22, 39). Since in these diseases anti-TNF treatment is used as an anti-inflammatory drug and IL-7 may be an important proinflammatory mediator, detailed analysis of the role of IL-7 in the immunopathogenesis of RA and these diseases may lead to novel treatment strategies.

Acknowledgements We thank dr. N. Jahangier and dr. A. van Rijthoven for providing patient material, and dr. D. Fitzpatrick and dr. C. Willis (Amgen Inc.) for critical reading of the manuscript. The Dutch Arthritis Association and Amgen Inc. contributed financially to this work.

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33. van Gestel AM, Prevoo ML, t