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Early explorations of the immune system
In the seventies, at the early time of cytometry few immune cells were characterized and analyzable. The first population described in PMR patients were immunoblasts. At that time, immunoblasts were described as increased in patients compared to controls, as in RA patients, and the increase of immunoblasts was associated with an increased erythrocyte sedimentation rate and a higher diseases activity (47). Immunological mechanisms were also suspected to be responsible of polymyalgia rheumatica and GCA when a Mayo Clinic team described the increase of immune complex in serum of patients with active diseases and the increasing level in patients in remission before relapse (48,49).
In the eighties, lymphocytes subsets were analyzed in PMR, T cells (OKT3+) and CD8+ (OKT8+) T cells were reported as lower than in control, whereas CD4+ (OKT4+) T cells were not altered (50–53). This decrease in CD8+ T cell was then described associated to an increased activation of these cells and under treatment these changes were restored, but only after 24 months of corticosteroids therapy (54). The decrease in CD8+ T cells was also reported in later studies associated to a decrease in CD3+CD16+ or CD56+ cells (55). After 6 months of corticosteroids treatment, the percentage and absolute number of CD8+ T cell increased but the percentage and absolute number of CD3+CD16/56+ remained the same. The authors suggested that despite clinical control of the disease, immunological abnormalities were not totally corrected by corticosteroid treatment. Some authors even suggested the usefulness of CD8+ count for the diagnosis of PMR (56–59). The persistence of a decrease of CD8+ T cell despite corticosteroids was also suggested to be correlated to a more severe and a need for longer corticosteroid treatment (60). But this decrease in CD8+ T cell in patients with early PMR and no treatment was contested and several studies did not observe such decrease (61–64). Impact of the methodology used was questioned to explain such variations in the observations. Moreover, soluble CD8 was also described as increased in PMR patients (65). To reinforce the possible implication of CD8+ T cell in PMR pathophysiology, it has been suggested that agerelated modification in the CD8+ population of T cell might influence the development of PMR (66). T cell receptor repertoire was characterized in PMR patients and most changes seemed related to the age of patients, for healthy controls with paired age presented the same modification (67). Only few beta-chain variable families were decreased in PMR patients. In comparison to adult onset RA, similar alteration in T cell subpopulations were found in PMR and elderly onset RA (68). After many controversies the use of CD8+ T cells for diagnosis or prognosis is no longer a matter of interest. But the fast development of flow cytometry now enables for precise characterization of lymphocytes subsets.
Recent data about immune system’s cells
In the past ten years, with the improvement of flow cytometry techniques, several studies also evaluated the distribution and the characterization of lymphocyte subset in PMR (table I.II). In the end of 2008, and in line with the previous studies about CD8+ T cells, Shimojima et al. suggested that CD8+CD25+ activated T cells were decreased in PMR patients compared to early RA and, at the opposite, CD4+IFNγ+IL-4-, CD8+IFNγ+IL-4- and CD4+TNFα+ were increased in PMR (69). Memory/effector CD3+CD4+CD28- T cells have been reported increased in the peripheral blood of PMR patients compared to healthy controls (70). Naïve CD8+ T cells were decreased in PMR patients and effector/memory CD8+ T cells increased, compared to healthy controls. Together, these studies suggest that effector/memory T cells are increased in patients’ peripheral blood but activated T cells might be studied more in details to conclude to an increase or a decrease. This can light up the results of past studies with inconsistent results. In 2012, for the first time, a decrease in the CD4+CD25highFoxp3+ regulatory T cell subset was described in patients with PMR and GCA, associated to an increase in pro-inflammatory T cell subsets such as Th1 and Th17, suggesting a potential role in the pathophysiology of PMR and GCA (71) and, once again, guiding toward analysis of the inflammatory potential of T cells more than conventional phenotyping. Considering the potential implication of Th17 T cells, ustekinumab has been tested in GCA patients in a prospective open-label trial and enabled a decrease of GC, but ustekinumab has not been tested in PMR patients yet (72,73). In 2013, senescent T cells were reported increased in PMR and GCA patients, with an increase in NKG2D – a marker of immunosenescence – expressing T cells (74). As ligands to NKG2D are expressed in temporal arteries, this may activate T cells in temporal arteries and participate to inflammation in GCA. On the other hand, the role of these cells in PMR is still unknown but the link between ageing and immunological modifications is of great interest.
Cytokines, the messengers of inflammation
In 1993, at the Mayo Clinic, Cornelia Weyand et al. reported an increase in IL-6 level in the serum of patients with PMR and GCA but no increase in TNFα level (91). A decrease after corticosteroids was reported but the mechanism underpinning IL-6 production was supposed to be still active, because the interruption of the treatment resulted in an early increase in IL-6 serum level and in the worsening of disease’s symptoms. The duration of corticosteroid treatment can be discussed. In this study the corticosteroids were quickly stopped and a longer duration of the treatment may have prevented such relapse. For the authors, the production of IL-6 was mainly due to a population of CD14+ cells among peripheral blood mononuclear cells, otherwise monocytes. Other cytokines were studied in the serum of patients with PMR but, apart IL-6, no other cytokine was found increased – such as sIL-2R (92,93), IL-1β (93,94), TNFα (94) – or with an interest for diagnosis or follow-up. IL-6 was also studied in patients with relapsing PMR and was increased in patients with relapse while CRP or ESR might not be modified (95). IL-6 was also reported to be higher in patients with PMR than in patients with elderly onset RA (96). Measurement of the serum IL-6 receptor has been suggested useful for the diagnosis of relapse of PMR and this finding was another element to suggest the use of IL- 6 receptor blockade therapy (97). Since tocilizumab has been used in PMR patients and is still investigated as a steroid sparing agent (8), the evolution of IL-6 under tocilizumab therapy might be of great interest. As already reported, we described the evolution of IL-6 under tocilizumab therapy. Despite an increase of IL-6 in early untreated PMR patients, the level of IL-6 did not change with tocilizumab treatment. But it appears that patients can be divided into two groups. In the first group patients had high level of IL-6 before tocilizumab therapy and the level of IL-6 significantly decreased after treatment. In the other group, IL-6 was not very high compared to controls and the level of IL-6 did not change (or slightly increased) after tocilizumab therapy (77). Despite these discrepancies in IL-6 level in the serum all patients had a PMR diagnosis and fulfilled the Chuang’s criteria. Moreover, during follow-up, the diagnosis was not questioned. Interleukin serum level vary across the day, with the circadian production of cortisol, and might explain the very characteristic inflammatory symptoms of PMR patient. But, only serum circadian variation has been studied (98). The origin of this increase in IL-6 production is not clearly elucidated. No evidence has been found that monocytes from peripheral blood produce more IL-6 in PMR patients and this increase of production might be located in the inflamed tissue (99).
Muscles and synovial fluid, from Plato to PMR pathophysiology
Synovial thickening and inflammation has been described by arthroscopy (100) and this finding is of major importance because, as in RA with synovial thickening, the modifications observed in peripheral blood might be very different from the modifications in the involved tissues and the conclusions made from peripheral blood alone might not be fully relevant to understand PMR pathophysiology. Indeed, as the shadows of Plato’s cave allegory, in peripheral blood we might be looking to very indirect consequences of inflammatory mechanisms hidden in the muscles and synovial tissue. But, unlike the prisoners of Plato’s cave, we might be able to break our chains and to partially understand what take place in these important tissues. Few studies focused on cellular infiltration and cytokine expression in tissue and synovial fluid. Increase in interleukin (IL) 6 in the synovial fluid of a patient with polymyalgia rheumatica was early described (101). The vascular infiltration of patients with PMR and GCA was also studied and revealed infiltration of T cells producing inflammatory cytokines (IL-1, IL-6 and TNFα) in both groups of patients (102). The production of IFNγ differed in patients with GCA and PMR and might be implicated in progression from PMR to GCA, suggesting that PMR is a predisposing condition to vasculitis. In 2010, Kreiner et al. reported an increase in the interstitial level of proinflammatory cytokines, including IL-6, but also IL-1α and β, IL-8, TNFα, compared to healthy controls whereas cytokines’ serum level in PMR patients was not always different from healthy controls (103). These interesting results emphasize the idea of mechanism in periarticular structure poorly reflected by the analysis done in the peripheral blood. Specific pain mechanisms might be playing an important role specifically in the muscles of patients with PMR (104).
Table of contents :
PAGES LIMINAIRES:
RÉSUMÉ
ABSTRACT
MOTS CLES :
KEYWORDS:
ADRESSE DU LABORATOIRE :
REMERCIEMENTS :
SUMMARY OF TABLES:
SUMMARY OF FIGURES:
ABBREVIATIONS:
PART I: INTRODUCTION
THE PATHOPHYSIOLOGY OF POLYMYALGIA RHEUMATICA
INTRODUCTION: POLYMYALGIA RHEUMATICA
WHAT DOES THE IMMUNOGENETIC TELL US ABOUT PMR?
EARLY EXPLORATIONS OF THE IMMUNE SYSTEM
RECENT DATA ABOUT IMMUNE SYSTEM’S CELLS
AUTOANTIBODIES, THE “CHICKEN AND EGG” QUESTION
CYTOKINES, THE MESSENGERS OF INFLAMMATION
MUSCLES AND SYNOVIAL FLUID, FROM PLATO TO PMR PATHOPHYSIOLOGY
THE ROLE OF AN AGEING IMMUNE SYSTEM:
THE INNATE IMMUNE SYSTEM, A MISSING PIECE OF THE PUZZLE?
TOCILIZUMAB, THE FIRST ANTI-INTERLEUKIN-6 RECEPTOR ANTIBODY, FROM 1973 TO NOWADAYS
FROM INTERLEUKIN-6 TO AN ANTIBODY TARGETING THE INTERLEUKIN-6 RECEPTOR
CHARACTERISTICS OF TOCILIZUMAB
INDICATION FOR THE TOCILIZUMAB AND ONGOING DEVELOPMENTS
BONE TURNOVER AND INFLUENCE OF INFLAMMATION, POLYMYALGIA RHEUMATICA, TOCILIZUMAB AND
GLUCOCORTICOIDS ON ITS HOMEOSTASIS
FUNDAMENTALS OF BONE TURNOVER
INFLAMMATION, INTERLEUKIN-6 AND BONE TURNOVER IN THE RHEUMATIC DISEASE
IMPACT OF DRUGS ON BONE TURNOVER
OBJECTIVES OF THE THESIS
PART II: POLYMYALGIA RHEUMATICA: MORE AN INFLAMMATORY THAN AN AUTOIMMUNE DISEASE
A CASE-CONTROL LONGITUDINAL STUDY ON IMMUNOLOGICAL PARAMETERS
ABSTRACT:
INTRODUCTION:
PATIENTS AND METHODS:
PATIENTS:
HEALTHY CONTROLS:
DATA COLLECTION:
INTERLEUKIN-6:
AUTOANTIBODIES ASSAY:
STATISTICAL ANALYSIS:
RESULTS:
FULL BLOOD COUNT IS MAINLY AFFECTED BY INFLAMMATION AND QUICKLY IMPROVED BY TOCILIZUMAB IN POLYMYALGIA
RHEUMATICA PATIENTS.
THE ASSOCIATION BETWEEN INTERLEUKIN-6 AND INFLAMMATORY DISTURBANCES IS NO LONGER OBSERVED AFTER
TOCILIZUMAB THERAPY.
LEVELS OF Γ-GLOBULINS ARE INCREASED IN EARLY PMR, AND A QUICK DECREASE IS OBSERVED AFTER TOCILIZUMAB
THERAPY.
DESPITE Γ-GLOBULIN MODIFICATIONS, NO MARK OF AUTOIMMUNITY IS OBSERVED IN EARLY POLYMYALGIA RHEUMATICA.
DISCUSSION:
REFERENCES:
SUPPLEMENTARY MATERIAL
NEW LEADS AND HYPOTHESIS:
PART III: CORRECTION OF ABNORMAL B CELL SUBSET DISTRIBUTION BY INTERLEUKINE-6 RECEPTOR
BLOCKADE IN POLYMYALGIA RHEUMATICA
A PROSPECTIVE CASE-CONTROL STUDY
ABSTRACT
INTRODUCTION
PATIENTS AND METHODS
PATIENTS, CONTROLS, AND INTERVENTION
LYMPHOCYTE SUBSET ANALYSIS AND CYTOKINE ASSAYS
STATISTICAL ANALYSIS
RESULTS
PATIENTS WITH ACTIVE PMR HAVE SELECTIVE B CELL LYMPHOPENIA AND ALTERATIONS IN B CELL SUBSETS
TOCILIZUMAB THERAPY CORRECTS B CELL SUBSET DISTRIBUTION
INCREASED SERUM IL-6 CORRELATES WITH DISEASE ACTIVITY IN PMR PATIENTS
DISCUSSION
REFERENCES
SUPPLEMENTARY MATERIAL:
FLOW CYTOMETRY
CYTOKINE ARRAY
ELISA
NEW LEADS AND HYPOTHESIS:
PART IV: THE SEMAPHORE STUDY
INTRODUCTION:
PATIENTS AND METHODS:
PATIENTS
FLOW CYTOMETRY:
STATISTICAL ANALYSIS:
RESULTS
PATIENTS’ CHARACTERISTICS:
SURVIVAL ANALYSIS:
INNATE IMMUNE SYSTEM IS IMPAIRED IN CORTICO-DEPENDENT POLYMYALGIA RHEUMATICA PATIENTS:
OVERALL B CELL COMPARTMENT IS NOT AFFECTED, BUT SENESCENT B CELLS ARE INCREASED AND TRANSITIONAL B CELLS
ARE DECREASED IN PATIENTS WITH CORTICO-DEPENDENT PMR.
T CELLS ARE INCREASED BUT WITHOUT A SIGNIFICANT INCREASE OF A SPECIFIC T CELL SUBSET
EXPRESSION OF CD126 DIFFERS IN T CELL SUBSETS
DISCUSSION:
NEW LEADS AND HYPOTHESIS
PART V: TOCILIZUMAB CONTROLS BONE TURNOVER IN EARLY POLYMYALGIA RHEUMATICA .
ABSTRACT:
INTRODUCTION:
METHOD
SAMPLE COLLECTION
AUTOMATED ASSAY AND IL-6 MEASUREMENT
SCANOGRAPHIC BONE ATTENUATION COEFFICIENT ASSESSMENT
STATISTICAL ANALYSIS
RESULTS:
BONE TURNOVER MARKERS AT BASELINE IN PMR
TOCILIZUMAB AND BONE TURNOVER MARKERS
IL-6 RESPONDERS
DISCUSSION
CONCLUSION
REFERENCES
SUPPLEMENTARY MATERIAL
NEW LEADS AND HYPOTHESIS
PART VI: THE PHARMACOKINETIC OF TOCILIZUMAB IN POLYMYALGIA RHEUMATICA QAUNTIFICATION OF THE TOCILIZUMAB IN THE SERUM OF PATIENTS:
MODELLING OF THE PHARMACOKINETIC OF TOCILIZUMAB IN POLYMYALGIA RHEUMATICA:
ANALYSIS OF THE PHARMACODYNAMIC RELATIONSHIP BETWEEN TOCILIZUMAB, CRP,
NEUTROPHILS AND PMR-AS:
PART VII: GENERAL DISCUSSION
PART VIII: CONCLUSION AND PERSPECTIVES
CONCLUSION
PERSPECTIVES
ANNEXES:
ANNEX I: THE PATHOPHYSIOLOGY OF POLYMYALGIA RHEUMATICA, SMALL PIECES OF A BIG PUZZLE
ANNEX II:
ANNEX III: RESUME SUBSTANTIEL EN FRANÇAIS
INTRODUCTION
LA PPR, UNE MALADIE PLUS INFLAMMATOIRE QU’AUTO-IMMUNE : LE TOCILIZUMAB CORRIGE LA DIMINUTION DES LYMPHOCYTES B DANS LA PSEUDO-POLYARTHRITE RHIZOMELIQUE :
CARACTERISATION DES PERTURBATIONS LYMPHOCYTAIRES CHEZ LES PATIENTS AYANT UNE PPR DEPENDANTE DES
CORTICOÏDES ET CARACTERISATION DE L’EXPRESSION DU CD126 :
LE TOCILIZUMAB CORRIGE LES ANOMALIES DE REMODELAGE OSSEUX OBSERVEES DANS LA PPR :
MODELISATION PHARMACOCINETIQUE ET PHARMACODYNAMIQUE DE TOCILIZUMAB DANS LA PPR DEBUTANTE : .
CONCLUSION :
REFERENCES:
