Inflammatory and helper T lymphocyte responses in human abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is a chronic degenerative disease that usually affects men over 65 years with an estimated prevalence of 5%. Aneurysm rupture represents a catastrophic event which carries a mortality rate of almost 90%. Current therapeutic options for AAAs measuring 5.5 cm in diameter or larger are based on prophylactic surgery, including conventional open reconstruction and endovascular stent-graft insertion. For patients with small asymptomatic AAAs (4.0 up to 5.5 cm in diameter), evidence from two recent large randomized controlled trials indicates no long-term survival benefit from immediate elective surgical repair as compared to imaging surveillance until aneurysm expands to 5.5 cm. This highlights the need for development of novel medical management strategies, including selective pharmacologic approaches, directed at preventing aneurysm expansion. In this regard, it is expected that a detailed knowledge of the pathobiology of human AAA lesion and a better understanding of pathophysiological mechanisms underlying initiation and progression of aneurysmal degeneration, particularly the specific involvement of T lymphocytes, will have special relevance to this challenging issue.

Inflammatory and helper T-cell responses in abdominal aortic aneurysm : controversial issues

Innate and inflammatory responses to endovascular versus open AAA repair. The occurrence of early acute systemic inflammatory responses after conventional open AAA repair is widely recognized and is thought to lead to the development of organ dysfunction and multiple organ failure, responsible for a large proportion of morbidity and mortality associated with aortic surgery. New therapeutic strategies designed to avoid ischemia-reperfusion injury related to aortic cross-clamping and to minimize the degree of tissue damage have thus been developed recently. Specifically, the advent of endovascular techniques has radically extended management options for patients with AAA. Although the method is believed to offer a clear short-term benefit over open repair, notably as regards restricted perioperative haemodynamic parameter fluctuations, reduced blood loss, briefer duration of surgery, shorter hospital stay, and lower 30-day mortality and complication rates, conflicting data are available regarding the exact nature and extent of the inflammatory events arising after such endoluminal procedures ; while several authors have indeed reported that endovascular AAA repair can determine a less intense and extensive inflammatory response, others have unexpectedly observed that the method may elicit a strong inflammatory response, the so-called « postimplantation syndrome ».

Adaptive cellular immune responses in human aneurysmal aortic lesion.

The inflammatory nature of AAA disease has long been suggested by the presence of a great number of CD4+ T lymphocytes in the outer media and adventitia of human AAA lesion. Interestingly, such infiltrating T-cell populations may have significant implications in the process of aneurysm dilation, since cytokines produced by T cells, notably IFN-gamma, have previously been shown to modulate production of matrix-degrading enzymes by resident macrophages and to induce apoptosis of medial SMCs. Through these key pathological mechanisms, T cells could potentially contribute to orchestrate aortic wall connective tissue disordered remodeling and degradation, and promote extensive disruption of elastic media, ultimately leading to aneurysmal degeneration. Nevertheless, despite their relative abundance in human AAA wall tissues, there is limited and controversial information as regards the functional profile of lesional lymphocytes, the exact nature of aortic wall adaptive cellular responses, and the etiologic role of T cells and their cytokines in initiation and progression of the aneurysmal process. Indeed, both Th1-type and Th2-type responses have been identified in human studies and experimental animal models of AAA.

Aims of the work

The main objectives of our work were to explore the innate and adaptive cellular immune responses in human AAA. In the first part of our work, we aimed to examine prospectively innate and inflammatory responses arising in a non-randomised cohort of patients undergoing endovascular versus open AAA repair. In the second part of our work, we focused our efforts on characterizing the nature of adaptive cellular immune responses and the phenotypic and functional repertoire of T cells in human AAA wall tissues obtained from a consecutive series of patients undergoing open AAA repair. Specifically, we sought to determine whether type 1 or type 2 responses occur predominantly in advanced AAA lesion.

Main experimental findings

Limited inflammatory response after endovascular AAA repair. Serial peripheral venous blood samples were collected preoperatively, immediately after declamping or insertion of endograft, and after 1, 3, 6, 12, 24, 48, and 72 hours. We first examined the acute phase reaction and liberation of complement cascade products using turbidimetric method and nephelometry. We found that endovascular repair produced lower postoperative CRP, leucocytosis, neutrophilia, and C3d/C3 ratio as compared to open surgery. We next analyzed surface expression of activation markers on peripheral CD3+ T cells using flow cytometry. We observed a strong upregulation of CD38 after open but not endovascular repair. Analysis of CD69 and CD25 molecules revealed no perioperative fluctuations in any group. We then investigated release of various circulating soluble cell adhesion molecules, proinflammatory cytokines, and chemokines using enzyme-linked immunosorbent assays. We demonstrated that both procedures are characterized by similar increases in ICAM-1 and IL-6 levels. Finally, tendency towards high levels of TNF-alpha and IL-8 was detected in endovascular repair, but data failed to reach statistical significance.

Predominance of type 1 CD4+ T cells in human aneurysmal aortic lesion. We have developed a tissue enzymatic digestion and cell extraction procedure to isolate intact mononuclear cells from aortic wall segments. This original cell isolation protocol enabled us to examine ex vivo the presence, phenotype, and cytokine secretion profile of infiltrating T lymphocytes freshly isolated from human AAA tissues for comparison with their circulating counterparts using flow cytometry. We found that both populations of infiltrating CD4+ and CD8+ T cells display a unique activated memory phenotype, as assessed by an increased expression of CD69 and HLA-DR activation antigens, downregulation of CD62L molecule, and predominant expression of the CD45RO isoform characteristics of memory cells. In addition, we identified the presence in human aneurysmal aortic wall lesion of CD4+ T cells producing high levels of IFN-gamma but not IL-4, reflecting their type 1 nature. In an additional series of experiments, cytokine gene expression was determined in whole aneurysmal and non-diseased aortic samples using LightCycler-based quantitative real-time reverse transcription-polymerase chain reaction. The molecular basis of type 1 or type 2 dominant responses was further specified by analyzing mRNA levels of transcription factors specifically involved in Th1 or Th2 differentiation such as T-bet and GATA-3. We demonstrated that aneurysmal aortic specimens exhibit high transcript levels of IFN-gamma but not IL-4, and consistently overexpressed the IFN-g-promoting cytokine IL-12 and the type 1-restricted transcription factor T-bet, further establishing the prominent type 1 nature of aortic wall responses. Moreover, such selective tissue expression of IL-12 and T-bet in the vessel microenvironment points to a potential role for these signals in directing aortic wall responses towards a type 1 phenotype.

Conclusions

Our findings indicate that endovascular AAA repair is associated with a lesser degree of acute phase reaction, peripheral T-cell activation, and release of complement proteins as compared to conventional open surgery, suggesting that the innate and inflammatory responses to AAA repair are significantly attenuated by the endovascular approach as compared to the traditional open reconstruction. These results support the view that the endoluminal procedure represents an attractive alternative to open surgery for the treatment of large aneurysms. On the other hand, we have demonstrated that Th1 cell infiltrates predominate in human end-stage AAA lesion. These observations are relevant for helping clarify the pathobiology of human AAA tissues and defining prospects for the prevention of aneurysm expansion. Indeed, identification of such infiltrating populations of IFN-gamma-producing CD4+ T cells not only provide new insights into the pathogenesis of the disorder, but could also serve as a basis for the development of novel medical management strategies directed at preventing aneurysm formation and progression, including therapeutic approaches based on the modulation of aortic wall responses and designed to selectively target T-cell activation and cytokine production. In this respect, the present work provides experimental evidence in support of the emerging concept that, although multifactorial, aneurysm disease may be regarded as a Th1-driven immunopathological condition, and suggests that strategies targeting IFN-gamma could be a particularly exciting and fruitful avenue for further investigation. Ongoing clinical and basic research in these areas can be expected to yield design of promising pharmacologic approaches to control AAA expansion. From a clinical perspective, such efforts have the potential to dramatically influence both the outcome and management of this common and life threatening condition.