MMP (Matrix Metalloprotease)-9-Producing Monocytes Enable T Cells to Invade the Vessel Wall and Cause Vasculitis.

RATIONALE: Giant cell arteritis (GCA)-a primary vasculitis of medium and large arteries-is associated with vessel wall damage, elastic membrane fragmentation, and vascular remodeling. Proteinases are believed to contribute to pathogenesis by degrading extracellular matrix and causing tissue injury. OBJECTIVE: The MMP (matrix metalloproteinase)-9-a type IV collagenase-is produced in the vasculitic lesions of GCA. It is unknown which pathogenic processes are MMP-9 dependent. METHODS AND RESULTS: The tissue transcriptome of GCA-affected temporal arteries contained high amounts of MMP-9 transcripts, and immunostaining for pro-MMP-9 localized the enzyme to wall-infiltrating macrophages. MMP-2 and MMP-9 transcripts were also abundant in monocytes and monocyte-derived macrophages from patients with GCA. Patient-derived monocytes outperformed healthy monocytes in passing through engineered basement membranes. GCA CD (cluster of differentiation) 4+ T cells required MMP-9-producing monocytes to penetrate through matrix built from type IV collagen. In vivo functions of MMP-9 were tested in a human artery-SCID (severe combined immunodeficiency) chimera model by blocking enzyme activity with a highly specific monoclonal antibody or by injecting rMMP-9 (recombinant MMP-9). Inhibiting MMP-9 activity profoundly suppressed vascular injury, decreased the density of inflammatory infiltrates ( P<0.001), reduced intramural neoangiogenesis ( P<0.001), and prevented intimal layer hyperplasia ( P<0.001). rMMP-9 amplified all domains of vasculitic activity, promoted assembly of T-cell infiltrates ( P<0.05), intensified formation of new microvessels ( P<0.001), and worsened intimal thickening ( P<0.001). Systemic delivery of N-acetyl-proline-glycine-proline-a matrikine produced by MMP-9-mediated gelatinolysis-had limited vasculitogenic effects. CONCLUSIONS: In large vessel vasculitis, MMP-9 controls the access of monocytes and T cells to the vascular wall. T cells depend on MMP-9-producing monocytes to pass through collagen IV-containing basement membrane. Invasion of vasculitogenic T cells and monocytes, formation of neoangiogenic networks, and neointimal growth all require the enzymatic activity of MMP-9, identifying this protease as a potential therapeutic target to restore the immunoprivilege of the arterial wall in large vessel vasculitis.

Inhibition of JAK-STAT Signaling Suppresses Pathogenic Immune Responses in Medium and Large Vessel Vasculitis.

BACKGROUND: Giant cell arteritis, a chronic autoimmune disease of the aorta and its large branches, is complicated by aneurysm formation, dissection, and arterial occlusions. Arterial wall dendritic cells attract CD4+ T cells and macrophages to form prototypic granulomatous infiltrates. Vasculitic lesions contain a diverse array of effector T cells that persist despite corticosteroid therapy and sustain chronic, smoldering vasculitis. Transmural inflammation induces microvascular neoangiogenesis and results in lumen-occlusive intimal hyperplasia. We have examined whether persistent vessel wall inflammation is maintained by lesional T cells, including the newly identified tissue-resident memory T cells, and whether such T cells are sensitive to the cytokine-signaling inhibitor tofacitinib, a Janus kinase (JAK) inhibitor targeting JAK3 and JAK1. METHODS: Vascular inflammation was induced in human arteries engrafted into immunodeficient mice that were reconstituted with T cells and monocytes from patients with giant cell arteritis. Mice carrying inflamed human arteries were treated with tofacitinib or vehicle. Vasculitic arteries were examined for gene expression (reverse transcription polymerase chain reaction), protein expression (immunohistochemistry), and infiltrating cell populations (flow cytometry). RESULTS: Tofacitinib effectively suppressed innate and adaptive immunity in the vessel wall. Lesional T cells responded to tofacitinib with reduced proliferation rates (<10%) and minimal production of the effector molecules interferon-γ, interleukin-17, and interleukin-21. Tofacitinib disrupted adventitial microvascular angiogenesis, reduced outgrowth of hyperplastic intima, and minimized CD4+CD103+ tissue-resident memory T cells. CONCLUSIONS: Cytokine signaling dependent on JAK3 and JAK1 is critically important in chronic inflammation of medium and large arteries. The JAK inhibitor tofacitinib effectively suppresses tissue-resident memory T cells and inhibits core vasculitogenic effector pathways.

Clinical and pathological evolution of giant cell arteritis: a prospective study of follow-up temporal artery biopsies in 40 treated patients.

Although clinical signs and symptoms of giant cell arteritis improve promptly after starting glucocorticoid therapy, reports have suggested that the vascular inflammation may persist. To assess the duration and quality of histopathologic changes in treated patients, we prospectively obtained second temporal artery biopsies in patients treated for 3 to 12 months after their first diagnostic biopsy. Forty patients (28 women, 12 men, median age 77 years) agreed to have a second temporal artery biopsy randomly assigned to 3, 6, 9, or 12 months subsequent to the first. Clinical and laboratory evaluation of the patient cohort revealed a typical rapid response and continued suppression of clinical manifestations as a result of glucocorticoid treatment. Histopathologic findings, evaluated in a blinded manner by a cardiovascular pathologist, showed unequivocal findings of vasculitis in 7/10 patients with second temporal artery biopsy at 3 months, 9/12 at 6 months, 4/9 at 9 months, and 4/9 at 12 months. Lymphocytes were present in all positive initial biopsies and remained the dominant cell population in chronically treated patients. Granulomatous inflammation decreased in a time-dependent manner from 78 to 100% at initial biopsy to 50% at 9 months and 25% at 12 months. The increased medial fibrosis noted in the second biopsies (60 vs 33% in primary temporal artery biopsies) suggested that the finding may represent a chronic finding in arteritis. In summary, the response to glucocorticoids in giant cell arteritis was frequently discordant. Clinical manifestations were readily suppressed, but vascular changes were gradual and often incomplete.

DNA methylation analysis of the temporal artery microenvironment in giant cell arteritis.

OBJECTIVE: To investigate the inflammatory response in giant cell arteritis (GCA) by characterising the DNA methylation pattern within the temporal artery microenvironment. METHODS: Twelve patients with non-equivocal histological evidence for GCA and 12 age-matched, sex-matched and ethnicity-matched controls with normal biopsies were studied. DNA was extracted from the affected portions of temporal artery tissue in patients with GCA and from histologically confirmed normal arteries in controls. Genome-wide DNA methylation status was evaluated using the Illumina Infinium HumanMethylation450 BeadChip Array. Differentially methylated loci between affected and unaffected arterial tissues were identified, and subsequent bioinformatic analysis performed. Immunohistochemistry was used to examine tissue expression patterns in temporal artery biopsies. RESULTS: We identified 1555 hypomethylated CG sites (853 genes) in affected temporal artery tissue from patients with GCA compared with normal controls. Gene ontology enrichment analysis of hypomethylated genes revealed significant representation in T cell activation and differentiation pathways, including both TH1 and TH17 signatures. Our DNA methylation data suggest a role for increased activity of the calcineurin/nuclear factor of activated T cells (NFAT) signalling pathway in GCA, confirmed by immunohistochemistry showing increased expression and nuclear localisation of NFAT1. NFAT signalling downstream targets such as interleukin (IL)-21/IL-21R and CD40L were overexpressed in GCA-affected arteries. Further, proinflammatory genes including TNF, LTA, LTB, CCR7, RUNX3, CD6, CD40LG, IL2, IL6, NLRP1, IL1B, IL18, IL21, IL23R and IFNG were hypomethylated in the cellular milieu of GCA arteries. CONCLUSIONS: We characterised the inflammatory response in GCA-affected arteries using 'epigenetic immunophenotyping' and identified molecules and pathways relevant to disease pathogenesis in GCA.

Blocking interferon γ reduces expression of chemokines CXCL9, CXCL10 and CXCL11 and decreases macrophage infiltration in ex vivo cultured arteries from patients with giant cell arteritis.

BACKGROUND: Interferon γ (IFNγ) is considered a seminal cytokine in the pathogenesis of giant cell arteritis (GCA), but its functional role has not been investigated. We explored changes in infiltrating cells and biomarkers elicited by blocking IFNγ with a neutralising monoclonal antibody, A6, in temporal arteries from patients with GCA. METHODS: Temporal arteries from 34 patients with GCA (positive histology) and 21 controls were cultured on 3D matrix (Matrigel) and exposed to A6 or recombinant IFNγ. Changes in gene/protein expression were measured by qRT-PCR/western blot or immunoassay. Changes in infiltrating cells were assessed by immunohistochemistry/immunofluorescence. Chemotaxis/adhesion assays were performed with temporal artery-derived vascular smooth muscle cells (VSMCs) and peripheral blood mononuclear cells (PBMCs). RESULTS: Blocking endogenous IFNγ with A6 abrogated STAT-1 phosphorylation in cultured GCA arteries. Furthermore, selective reduction in CXCL9, CXCL10 and CXCL11 chemokine expression was observed along with reduction in infiltrating CD68 macrophages. Adding IFNγ elicited consistent opposite effects. IFNγ induced CXCL9, CXCL10, CXCL11, CCL2 and intracellular adhesion molecule-1 expression by cultured VSMC, resulting in increased PBMC chemotaxis/adhesion. Spontaneous expression of chemokines was higher in VSMC isolated from GCA-involved arteries than in those obtained from controls. Incubation of IFNγ-treated control arteries with PBMC resulted in adhesion/infiltration by CD68 macrophages, which did not occur in untreated arteries. CONCLUSIONS: Our ex vivo system suggests that IFNγ may play an important role in the recruitment of macrophages in GCA by inducing production of specific chemokines and adhesion molecules. Vascular wall components (ie, VSMC) are mediators of these functions and may facilitate progression of inflammatory infiltrates through the vessel wall.

IL-33 is overexpressed in the inflamed arteries of patients with giant cell arteritis.

OBJECTIVE: To study the expression of interleukin (IL)-33 and to evaluate its relationship with macrophage polarisation in artery biopsy specimens from patients with giant cell arteritis (GCA). METHODS: IL-33, ST2, p-STAT-6 and perivascular IL-1 receptor-associated kinase 1 (p-IRAK1) tissue distribution was evaluated by immunohistochemistry. Inducible nitric oxide synthase and CD163 were also used by immunohistochemistry to evaluate the M1 and M2 polarisation, respectively. Quantitative gene expression analysis of IL-33, T-helper (Th)2-related transcription factor STAT6, Th2 cytokines (IL-4, IL-5, IL-25) and interferon (IFN)-γ was performed in artery biopsy samples obtained from 20 patients with GCA and 15 controls. Five additional patients who had received prednisone when the temporal artery biopsy was performed were also enrolled. RESULTS: IFN-γ and IL-33 were significantly overexpressed in the inflamed arteries of GCA patients. IL-33 overexpression was not accompanied by a concomitant increase of Th2 cytokines. Neovessels scattered through the inflammatory infiltrates were the main sites of IL-33 expression. The expression of IL-33 receptor ST2 and of p-IRAK1 was also increased in GCA patients. Arteries from glucocorticoid-treated patients had a lower expression of IL-33. IL-33 was accompanied by the expression of p-STAT6 and a clear M2 macrophages polarisation. CONCLUSIONS: A role for IL-33 in the inflammation of GCA patients is supported by these findings.

Toll-like receptors 4 and 5 induce distinct types of vasculitis.

Large vessel vasculitides, such as Takayasu arteritis and giant cell arteritis, affect vital arteries and cause clinical complications by either luminal occlusion or vessel wall destruction. Inflammatory infiltrates, often with granulomatous arrangements, are distributed as a panarteritis throughout all of the artery's wall layers or cluster in the adventitia as a perivasculitis. Factors determining the architecture and compartmentalization of vasculitis are unknown. Human macrovessels are populated by indigenous dendritic cells (DCs) positioned in the adventitia. Herein, we report that these vascular DCs sense bacterial pathogens and regulate the patterning of the emerging arteritis. In human temporal artery-SCID chimeras, lipopolysaccharides stimulating Toll-like receptor (TLR)4 and flagellin stimulating TLR5 trigger vascular DCs and induce T-cell recruitment and activation. However, the architecture of the evolving inflammation is ligand-specific; TLR4 ligands cause transmural panarteritis and TLR5 ligands promote adventitial perivasculitis. Underlying mechanisms involve selective recruitment of functional T cell subsets. Specifically, TLR4-mediated DC stimulation markedly enhances production of the chemokine CCL20, biasing recruitment toward CCL20-responsive CCR6(+) T cells. In adoptive transfer experiments, CCR6(+) T cells produce an arteritis pattern with media-invasive T cells damaging vascular smooth muscle cells. Also, CCR6(+) T cells dominate the vasculitic infiltrates in patients with panarteritic giant cell arteritis. Thus, depending on the original danger signal, vascular DCs edit the emerging immune response by differentially recruiting specialized T effector cells and direct the disease process toward distinct types of vasculitis.

Inflammatory Cell Composition and Immune-Related microRNA Signature of Temporal Artery Biopsies From Patients With Giant Cell Arteritis.

Objectives: The aim of this study was to quantitatively assess distinct immune cell subsets comprising inflammatory infiltrate in temporal artery biopsies (TABs) from patients with giant cell arteritis (GCA), and to link the obtained histopathological data with expression profiles of immune-related microRNAs (miRNAs). Methods: The study included 68 formalin-fixed, paraffin-embedded TABs from treatment-naïve patients, including 30 histologically positive GCA and 16 negative GCA TABs, and 22 control non-GCA TABs. Quantitative assessment of histological parameters was performed using histopathological and immunohistochemical techniques. miRNA expression analysis was performed by quantitative real-time PCR. Results: Intense transmural mononuclear inflammatory infiltrates in TAB-positive GCA arteries were predominantly composed of CD3+, CD4+ and CD8+ T lymphocytes, and CD68+ macrophages, accompanied by a strong nuclear overexpression of the nuclear factor of activated T cells, cytoplasmic 1 (NFATC) in the lymphocyte infiltrate fraction. Furthermore, TAB-positive GCA arteries were characterized by significant overexpression of nine pro-inflammatory miRNAs (miR-132-3p/-142-3p/-142-5p/-155-5p/-210-3p/-212-3p/-326/-342-5p/-511-5p) and a significant under-expression of six regulatory immune-related miRNAs (miR-30a-5p/-30b-5p/-30c-5p/-30d-5p/-30e-5p/-124-3p), whose expression levels significantly associated with most evaluated histopathological parameters. Notably, we revealed miR-132-3p/-142-3p/-142-5p/-155-5p/-212-3p/-511-5p as major promoters of arterial inflammation and miR-30a-5p/-30c-5p/-30d-5p as putative regulators of NFATC signaling in TAB-positive GCA arteries. Conclusion: Overall, we demonstrated that an altered arterial tissue-specific pro-inflammatory miRNA signature favors enhanced T cell-driven inflammation and macrophage activity in TAB-positive GCA arteries. Moreover, dysregulation of several immune-related miRNAs seems to contribute crucially to GCA pathogenesis, through impairing their regulatory activity towards T cell-mediated immune responses driven by the calcineurin (CaN)/NFAT signaling pathway, indicating their therapeutic, diagnostic and prognostic potential.

The Immunopathology of Giant Cell Arteritis Across Disease Spectra.

Giant cell arteritis (GCA) is a granulomatous systemic vasculitis of large- and medium-sized arteries that affects the elderly. In recent years, advances in diagnostic imaging have revealed a greater degree of large vessel involvement than previously recognized, distinguishing classical cranial- from large vessel (LV)- GCA. GCA often co-occurs with the poorly understood inflammatory arthritis/bursitis condition polymyalgia rheumatica (PMR) and has overlapping features with other non-infectious granulomatous vasculitides that affect the aorta, namely Takayasu Arteritis (TAK) and the more recently described clinically isolated aortitis (CIA). Here, we review the literature focused on the immunopathology of GCA on the background of the three settings in which comparisons are informative: LV and cranial variants of GCA; PMR and GCA; the three granulomatous vasculitides (GCA, TAK, and CIA). We discuss overlapping and unique features between these conditions across clinical presentation, epidemiology, imaging, and conventional histology. We propose a model of GCA where abnormally activated circulating cells, especially monocytes and CD4+ T cells, enter arteries after an unknown stimulus and cooperate to destroy it and review the evidence for how this mechanistically occurs in active disease and improves with treatment.

Distinct vascular lesions in giant cell arteritis share identical T cell clonotypes.

Giant cell arteritis (GCA) is a spontaneous vasculitic syndrome that specifically targets the walls of medium and large arteries. Vascular lesions are characterized by patchy granulomatous infiltrates composed of T cells, macrophages, histiocytes, and giant cells. To test the hypothesis that a locally residing antigen recruits T cells into the vessel walls, we have analyzed T cell receptor (TCR) molecules of tissue infiltrating T cells. A total of 638 CD4+ T cell clones were isolated from temporal artery specimens of three patients with GCA. Analysis of TCR molecules for the usage of V beta 1-V beta 20 revealed that all TCR V beta elements were represented, demonstrating that interleukin 2 (IL-2)-responsive T cells infiltrating the tissue are highly diverse. To detect expanded T cell specificities, we made use of the patchy character of the inflammatory disease and compared the TCR repertoire of T cells established from independent vasculitic foci of the same artery. Sequence analysis of TCR V beta chains documented that individual TCR specificities were present in multiple copies, indicating clonal expansion. T cells with identical beta chains were isolated from distinct inflammatory foci of the same patient. These specificities represented only a small fraction of tissue-infiltrating T cells and involved the V beta 5.3 gene segment in the two patients sharing the HLA-DRB1*0401 allele. The third complementarity determining region of clonally expanded TCR beta chains was characterized by a cluster of negatively and positively charged residues, suggesting that the juxtaposed antigenic peptide is charged. The sharing of identical T cell specificities by distinct and independent regions of the granulomatous inflammation suggests that these T cells are disease relevant and that their repertoire is strongly restricted. These data suggest that an antigen residing in the arterial wall is recognized by a small fraction of CD4+ T cells in the inflammatory process characteristic for GCA.

ROS-producing immature neutrophils in giant cell arteritis are linked to vascular pathologies.

Giant cell arteritis (GCA) is a common form of primary systemic vasculitis in adults, with no reliable indicators of prognosis or treatment responses. We used single cell technologies to comprehensively map immune cell populations in the blood of patients with GCA and identified the CD66b+CD15+CD10lo/-CD64- band neutrophils and CD66bhiCD15+CD10lo/-CD64+/bright myelocytes/metamyelocytes to be unequivocally associated with both the clinical phenotype and response to treatment. Immature neutrophils were resistant to apoptosis, remained in the vasculature for a prolonged period of time, interacted with platelets, and extravasated into the tissue surrounding the temporal arteries of patients with GCA. We discovered that immature neutrophils generated high levels of extracellular reactive oxygen species, leading to enhanced protein oxidation and permeability of endothelial barrier in an in vitro coculture system. The same populations were also detected in other systemic vasculitides. These findings link functions of immature neutrophils to disease pathogenesis, establishing a clinical cellular signature of GCA and suggesting different therapeutic approaches in systemic vascular inflammation.

Genomics of human intracranial aneurysm wall.

BACKGROUND AND PURPOSE: The pathogenesis of intracranial aneurysms (IAs) remains elusive. Most studies have focused on individual genes, or a few interrelated genes or products, at a time in human IA. However, a broad view of pathologic mechanisms has not been investigated by identifying pathogenic genes and their interaction in networks. Our study aimed to analyze global gene expression patterns in the IA wall. METHODS: To our knowledge, our group was the first to perform Illumina microarray analysis on human IA via comparison of aneurysm wall and superficial temporal artery tissues from 6 consecutive patients. We adopted stringent statistical criteria to the individual genes; genes with a false discovery rate <0.01 and >2-fold change were selected as differentially expressed. To identify the overrepresented biologic pathways with the differentially expressed genes, we performed hypergeometric testing of the genes selected by relaxed criteria of P<0.01 and fold change >1.5. RESULTS: There are 326 distinct differentially expressed genes between IA and superficial temporal artery tissues (>2-fold change) with a false discovery rate <0.01. Analysis of the Kyoto Encyclopedia of Genes and Genomes pathways revealed the most impacted functional pathways: focal adhesion, extracellular matrix receptor interaction, and cell communication. Analysis of the Gene Ontology also supported the involvement of another 2 potentially important pathways: inflammatory response and apoptosis. CONCLUSIONS: The differentially expressed genes in the aneurysm wall may shed light on aneurysm pathobiology and provide novel targets for therapeutic intervention. These data will help generate hypotheses for future studies.

An uneven expression of T cell receptor V genes in the arterial wall and peripheral blood in giant cell arteritis.

The aim of the study was to investigate T cell receptor (TCR) usage at the time of diagnosis of giant cell arteritis (GCA) and to estimate the degree of clonality of T-cells infiltrating the lesion. Seven patients with biopsy-proven giant cell arteritis were included in the study. Immunocytochemistry in biopsies from the temporal arteries and flow cytometric analysis of peripheral blood lymphocytes (PBL) was performed using monoclonal antibodies specific for CD3, CD4 and CD8 and 13 TCR Valpha and Vbeta gene segment products. The CDR3 fragment length polymorphism was assessed by gel electrophoresis of PCR-amplified TCR segments. The T lymphocytes were found to be concentrated to the adventitia rather than the media or intima. Six of the seven patients with GCA had expansions of T lymphocytes, expressing selected TCR V genes in the arterial wall. None of these expansions was found in PBL. The infiltrating T-cells were poly- or oligoclonal. In conclusion, the dominating part of the inflammatory infiltrate in GCA emanates from the adventitial microvessels. There is an uneven expression of TCR V genes by T lymphocytes in the inflammatory infiltrates as compared to peripheral blood T lymphocytes at the time of diagnosis, consistent with an antigen-driven immunological reaction in the arterial wall.

Temporal small arterial inflammation is common in patients with giant cell arteritis.

Giant cell arteritis (GCA) primarily involves medium-to-large arteries. Small-vessel inflammation is a recognized phenomenon occurring in association with GCA. However, its significance is poorly elucidated. Histologic sections and medical records of105 temporal artery specimens were retrospectively reviewed between 2008 and 2017 to examine associated clinical manifestations and laboratory data including antinuclear antibody and p-antineutrophilic cytoplasmic antibody titers. Immunohistochemical staining for CD4 and CD8 was performed in select cases to assess the nature of the inflammatory response. Seventy-eight patients meeting the diagnostic criteria of temporal arteritis were included in the analysis. Twenty-eight specimens demonstrated temporal arteritis with small arterial inflammation (SAI), and 50 specimens showed temporal arteritis without SAI. Eight (28.6%) of 28 patients with SAI presented with jaw claudication, whereas 5 (17.9%) were febrile at presentation. In contrast, in 50 patients without SAI, jaw claudication and fever were seen in 11 and 2 cases, respectively (P = .01 and P = .0047, respectively). No statistically significant difference was noted between other symptoms and laboratory indices between the 2 groups. Elevated p-antineutrophilic cytoplasmic antibody titers in GCA may be associated with concomitant polymyalgia rheumatica or treatment-resistant disease. We also identified a higher count of CD4 and CD8 T cells in SAI cases, although the ratio of CD4/CD8 T lymphocytes was within normal limits. In conclusion, simultaneous involvement of arterioles and medium- to large-sized arteries is common in GCA and may be associated with treatment-refractory disease. Documentation of small arterial involvement in GCA will help the clinicians to manage the disease more effectively.

Analysis of In Vivo Serpin Functions in Models of Inflammatory Vascular Disease.

Serpins have a wide range of functions in regulation of serine proteases in the thrombotic cascade and in immune responses, representing up to 2-10% of circulating proteins in the blood. Selected serpins also have cross-class inhibitory actions for cysteine proteases in inflammasome and apoptosis pathways. The arterial and venous systems transport blood throughout the mammalian body representing a central site for interactions between coagulation proteases and circulating blood cells (immune cells) and target tissues, a very extensive and complex interaction. While analysis of serpin functions in vitro in kinetics or gel shift assays or in tissue culture provides very necessary information on molecular mechanisms, the penultimate assessment of biological or physiological functions and efficacy for serpins as therapeutics requires study in vivo in whole animal models (some also consider cell culture to be an in vivo approach).Mouse models of arterial transplant with immune rejection as well as models of inflammatory vasculitis induced by infection have been used to study the interplay between the coagulation and immune response pathways. We describe here three in vivo vasculitis models that are used to study the roles of serpins in disease and as therapeutics. The models described include (1) mouse aortic allograft transplantation, (2) human temporal artery (TA) xenograft into immunodeficient mouse aorta, and (3) mouse herpes virus (MHV68)-induced inflammatory vasculitis in interferon-gamma receptor (IFNγR) knockout mice.

IL-6 expression is correlated with increased T-cell proliferation and survival in the arterial wall in giant cell arteritis.

Giant cell arteritis (GCA) is the most common vasculitis in adults affecting large and medium-sized arteries. IL-6 and T cell accumulation within the arterial wall contribute to the pathogenesis of GCA, and blockade of IL-6 activity is efficacious in its treatment. We examined the relationship between levels of IL-6 expression and immunological processes that control the expansion of T cells in GCA-positive temporal artery biopsies. CD4 T cells accumulated in clusters within the media and deep intima of all GCA lesions. There was a significant positive correlation between the expression of IL-6 mRNA and increased frequency of proliferating CD4 T cells. The expansion of T cells can be inhibited by T regs but IL-6 expression was not correlated with differences in T reg accumulation. Increased IL-6 levels were also significantly correlated with lower frequencies of CD4 T cells undergoing apoptotic cell death. In conclusion, IL-6 may contribute to the accumulation of CD4 T cells in GCA by supporting their proliferation and survival within the arterial wall through mechanisms that are independent of effects on local T reg expansion.

Expression and Function of IL12/23 Related Cytokine Subunits (p35, p40, and p19) in Giant-Cell Arteritis Lesions: Contribution of p40 to Th1- and Th17-Mediated Inflammatory Pathways.

Background: Giant-cell arteritis (GCA) is considered a T helper (Th)1- and Th17-mediated disease. Interleukin (IL)-12 is a heterodimeric cytokine (p35/p40) involved in Th1 differentiation. When combining with p19 subunit, p40 compose IL-23, a powerful pro-inflammatory cytokine that maintains Th17 response. Objectives: The aims of this study were to investigate p40, p35, and p19 subunit expression in GCA lesions and their combinations to conform different cytokines, to assess the effect of glucocorticoid treatment on subunit expression, and to explore functional roles of p40 by culturing temporal artery sections with a neutralizing anti-human IL-12/IL-23p40 antibody. Methods and results: p40 and p19 mRNA concentrations measured by real-time RT-PCR were significantly higher in temporal arteries from 50 patients compared to 20 controls (4.35 ± 4.06 vs 0.51 ± 0.75; p < 0.0001 and 20.32 ± 21.78 vs 4.17 ± 4.43 relative units; p < 0.0001, respectively). No differences were found in constitutively expressed p35 mRNA. Contrarily, p40 and p19 mRNAs were decreased in temporal arteries from 16 treated GCA patients vs those from 34 treatment-naïve GCA patients. Accordingly, dexamethasone reduced p40 and p19 expression in cultured arteries. Subunit associations to conform IL-12 and IL-23 were confirmed by proximity-ligation assay in GCA lesions. Immunofluorescence revealed widespread p19 and p35 expression by inflammatory cells, independent from p40. Blocking IL-12/IL-23p40 tended to reduce IFNγ and IL-17 mRNA production by cultured GCA arteries and tended to increase Th17 inducers IL-1ß and IL-6. Conclusion: IL-12 and IL-23 heterodimers are increased in GCA lesions and decrease with glucocorticoid treatment. p19 and p35 subunits are much more abundant than p40, indicating an independent role for these subunits or their potential association with alternative subunits. The modest effect of IL-12/IL-23p40 neutralization may indicate compensation by redundant cytokines or cytokines resulting from alternative combinations.

Functional profile of tissue-infiltrating and circulating CD68+ cells in giant cell arteritis. Evidence for two components of the disease.

Macrophages represent a critical component in the inflammatory lesions of giant cell arteritis. By combining immunohistochemistry and in situ hybridization, we have analyzed the functional heterogeneity of tissue-infiltrating macrophages in patients with untreated vasculitis. 20% of macrophages in temporal artery tissue synthesized IL-6-specific mRNA and produced IL-6 and IL-1 beta proteins. IL-6 and IL-1 beta production was not limited to CD68+ cells in the lymphoid aggregates but was a feature of CD68+ cells dispersed throughout the tissue. 50% of tissue-infiltrating CD68+ cells synthesized 72-kD type IV collagenase. Only a small subset of CD68+ cells produced cytokines as well as collagenase, indicating functional specialization or distinct differentiation stages of CD68+ cells in the inflamed tissue. Activation of CD68+ cells was not restricted to tissue-infiltrating cells. Expression of IL-6 and IL-1 beta was found in 60-80% of circulating monocytes of patients with untreated giant cell arteritis, whereas collagenase production was restricted to tissue macrophages. IL-6 and IL-1 beta production by the majority of circulating monocytes was a shared feature of patients with giant cell arteritis and polymyalgia rheumatica but was not found in rheumatoid arthritis. These data suggest that giant cell arteritis has two components of disease, an inflammatory reaction in vessel walls and a systemic activation of monocytes. Systemic monocyte activation can manifest independently without vasculitis as exemplified in patients with polymyalgia rheumatica.