Goronzy, Jörg J.

Abstract Aging and chronic inflammatory syndromes, such as rheumatoid arthritis, are associated with high frequencies of CD4+CD28null T cells, which are rarely seen in healthy individuals younger than 40 years. Inasmuch as rheumatoid arthritis and aging are also associated with elevated levels of TNF-α, we examined whether this proinflammatory cytokine influences CD28 expression. Incubation of T cell lines and clones as well as Jurkat cells with TNF-α induced a reduction in the levels of cell surface expression of CD28. This effect of TNF-α was reversible; however, continuous culture of CD4+CD28+ T cell clones in TNF-α resulted in the appearance of a CD28null subset. In reporter gene bioassays, TNF-α was found to inhibit the activity of the CD28 minimal promoter. Inactivation of the promoter was accompanied by a marked reduction in DNA-protein complex formation by two DNA sequence motifs corresponding to the transcriptional initiator of the CD28 gene. Indeed, in vitro transcription assays showed that nuclear extracts from TNF-α-treated cells failed to activate transcription of DNA templates under the control of a consensus TATA box and the CD28 initiator sequences. In contrast, similar extracts from unstimulated T cells supported transcription. These results demonstrate that TNF-α directly influences CD28 gene transcription. We propose that the emergence of CD4+CD28null T cells in vivo is facilitated by increased production of TNF-α.

Abstract —Giant cell arteritis (GCA) is an inflammatory vasculopathy in which T cells and macrophages infiltrate the wall of medium and large arteries. Clinical consequences such as blindness and stroke are related to arterial occlusion. Formation of aortic aneurysms may result from necrosis of smooth muscle cells and fragmentation of elastic membranes. The molecular mechanisms of arterial wall injury in GCA are not understood. To identify mechanisms of arterial damage, gene expression in inflamed and unaffected temporal artery specimens was compared by differential display polymerase chain reaction. Genes differentially expressed in arterial lesions included 3 products encoded by the mitochondrial genome. Immunohistochemistry with antibodies specific for a 65-kDa mitochondrial antigen revealed that increased expression of mitochondrial products was characteristic of multinucleated giant cells and of CD68+ macrophages that cluster in the media and at the media-intima junction. 4-Hydroxy-2-nonenal adducts, products of lipid peroxidation, were detected on smooth muscle cells and on tissue infiltrating cells, in close proximity to multinucleated giant cells and CD68+ macrophages. Also, giant cells and macrophages with overexpression of mitochondrial products were able to synthesize metalloproteinase-2. Our data suggest that in the vascular lesions characteristic for GCA, a subset of macrophages has the potential to support several pathways of arterial injury, including the release of reactive oxygen species and the production of metalloproteinase-2. This macrophage subset is topographically defined and is also identified by overexpression of mitochondrial genes. Because these macrophages have a high potential to promote several mechanisms of arterial wall damage, they should be therapeutically targeted to prevent blood vessel destruction.

Background —Unstable angina (UA) is associated with systemic inflammation and with expansion of interferon-γ–producing T lymphocytes. The cause of T-cell activation and the precise role of activated T cells in plaque instability are not understood. Methods and Results —Peripheral blood T cells from 34 patients with stable angina and 34 patients with UA were compared for the distribution of functional T-cell subsets by flow cytometric analysis. Clonality within the T-cell compartment was identified by T-cell receptor spectrotyping and subsequent sequencing. Tissue-infiltrating T cells were examined in extracts from coronary arteries containing stable or unstable plaque. The subset of CD4 + CD28 null T cells was expanded in patients with UA and infrequent in patients with stable angina (median frequencies: 10.8% versus 1.5%, P <0.001). CD4 + CD28 null T cells included a large monoclonal population, with 59 clonotypes isolated from 20 UA patients. T-cell clonotypes from different UA patients used antigen receptors with similar sequences. T-cell receptor sequences derived from monoclonal T-cell populations were detected in the culprit but not in the nonculprit lesion of a patient with fatal myocardial infarction. Conclusions —UA is associated with the emergence of monoclonal T-cell populations, analogous to monoclonal gammopathy of unknown significance. Shared T-cell receptor sequences in clonotypes of different patients implicate chronic stimulation by a common antigen, for example, persistent infection. The unstable plaque but not the stable plaque is invaded by clonally expanded T cells, suggesting a direct involvement of these lymphocytes in plaque disruption.

Background —Monocytes are constitutively activated in unstable angina (UA), resulting in the production of IL-6 and the upregulation of acute phase proteins. Underlying mechanisms are not understood. To explore whether the production of the potent monocyte activator IFN-γ is altered in UA, we compared cytokine production by T lymphocytes in patients with UA (Braunwald’s class IIIB) and with stable angina (SA). Methods and Results —Peripheral blood lymphocytes were collected at the time of hospitalization and after 2 and 12 weeks. Cytokine-producing CD4 + and CD8 + T cells were quantified by 3-color flow cytometry after stimulation with phorbol myristate acetate and ionomycin. UA was associated with an increased number of CD4 + and CD8 + T cells producing IFN-γ, whereas patients with SA had higher frequencies of IL-2 + and IL-4 + CD4 + T cells. Expansion of the IFN-γ + T-cell population in UA persisted for at least 3 months. Increased production of IFN-γ in UA could be attributed to the expansion of an unusual subset of T cells, CD4 + CD28 null T cells. Conclusions —Patients with UA are characterized by a perturbation of the functional T-cell repertoire with a bias toward IFN-γ production, suggesting that monocyte activation and acute phase responses are consequences of T-cell activation. IFN-γ is produced by CD4 + CD28 null T cells, which are expanded in UA and distinctly low in SA and controls. The emergence of CD4 + CD28 null T cells may result from persistent antigenic stimulation.

Background —Activation of circulating monocytes in patients with acute coronary syndromes may reflect exposure to bacterial products or stimulation by cytokines such as IFN-γ. IFN-γ induces phosphorylation and nuclear translocation of transcription factor STAT-1, which initiates a specific program of gene induction. To explore whether monocyte activation is IFN-γ driven, patients with unstable (UA) or stable angina (SA) were compared for nuclear translocation of STAT-1 complexes and upregulation of IFN-γ–inducible genes CD64 and IP-10. Methods and Results —Peripheral blood mononuclear cells were stained for expression of CD64 on CD14 + monocytes and analyzed by PCR for transcription of IP-10. Expression of CD64 was significantly increased in patients with UA. Monocytes from UA patients remained responsive to IFN-γ in vitro, with accelerated transcriptional competency of CD64. IP-10–specific sequences were spontaneously detectable in 82% of the UA patients and 15% of SA patients ( P <0.001). Most importantly, STAT-1 complexes were found in nuclear extracts prepared from freshly isolated monocytes of patients with UA, which provides compelling evidence for IFN-γ signaling in vivo. Conclusions —Monocytes from UA patients exhibit a molecular fingerprint of recent IFN-γ triggering, such as nuclear translocation of STAT-1 complexes and upregulation of IFN-γ–inducible genes CD64 and IP-10, which suggests that monocytes are activated, at least in part, by IFN-γ. IFN-γ may derive from stimulated T lymphocytes, which implicates specific immune responses in the pathogenesis of acute coronary syndromes.

Background— Giant cell arteritis is a granulomatous vasculitis of the aorta and its branches that causes blindness, stroke, and aortic aneurysm. CD4 T cells are key pathogenic regulators, instructed by vessel wall dendritic cells to differentiate into vasculitic T cells. The unique pathways driving this dendritic cell–T-cell interaction are incompletely understood, but may provide novel therapeutic targets for a disease in which the only established therapy is long-term treatment with high doses of corticosteroids. Methods and Results— Immunohistochemical and gene expression analyses of giant cell arteritis-affected temporal arteries revealed abundant expression of the NOTCH receptor and its ligands, Jagged1 and Delta1. Cleavage of the NOTCH intracellular domain in wall-infiltrating T cells indicated ongoing NOTCH pathway activation in large-vessel vasculitis. NOTCH activation did not occur in small-vessel vasculitis affecting branches of the vasa vasorum tree. We devised 2 strategies to block NOTCH pathway activation: γ-secretase inhibitor treatment, preventing nuclear translocation of the NOTCH intracellular domain, and competing for receptor-ligand interactions through excess soluble ligand, Jagged1-Fc. In a humanized mouse model, NOTCH pathway disruption had strong immunosuppressive effects, inhibiting T-cell activation in the early and established phases of vascular inflammation. NOTCH inhibition was particularly effective in downregulating Th17 responses, but also markedly suppressed Th1 responses. Conclusions— Blocking NOTCH signaling depleted T cells from the vascular infiltrates, implicating NOTCH- NOTCH ligand interactions in regulating T-cell retention and survival in vessel wall inflammation. Modulating the NOTCH signaling cascade emerges as a promising new strategy for immunosuppressive therapy of large-vessel vasculitis.

Background— In giant cell arteritis (GCA), vasculitic damage of the aorta and its branches is combined with a syndrome of intense systemic inflammation. Therapeutically, glucocorticoids remain the gold standard because they promptly and effectively suppress acute manifestations; however, they fail to eradicate vessel wall infiltrates. The effects of glucocorticoids on the systemic and vascular components of GCA are not understood. Methods and Results— The immunoprofile of untreated and glucocorticoid-treated GCA was examined in peripheral blood and temporal artery biopsies with protein quantification assays, flow cytometry, quantitative real-time polymerase chain reaction, and immunohistochemistry. Plasma interferon-γ and interleukin (IL)-17 and frequencies of interferon-γ-producing and IL-17-producing T cells were markedly elevated before therapy. Glucocorticoid treatment suppressed the Th17 but not the Th1 arm in the blood and the vascular lesions. Analysis of monocytes/macrophages in the circulation and in temporal arteries revealed glucocorticoid-mediated suppression of Th17-promoting cytokines (IL-1β, IL-6, and IL-23) but sparing of Th1-promoting cytokines (IL-12). In human artery-severe combined immunodeficiency mouse chimeras, in which patient-derived T cells cause inflammation of engrafted human temporal arteries, glucocorticoids were similarly selective in inhibiting Th17 cells and leaving Th1 cells unaffected. Conclusions— Two pathogenic pathways mediated by Th17 and Th1 cells contribute to the systemic and vascular manifestations of GCA. IL-17-producing Th17 cells are sensitive to glucocorticoid-mediated suppression, but interferon-γ-producing Th1 responses persist in treated patients. Targeting steroid-resistant Th1 responses will be necessary to resolve chronic smoldering vasculitis. Monitoring Th17 and Th1 frequencies can aid in assessing disease activity in GCA.