Inhibition of IL-32 activation by α-1 antitrypsin suppresses alloreactivity and increases survival in an allogeneic murine marrow transplantation model

Abstract

Interleukin (IL)-32 was originally identified in natural killer cells and IL-2-activated human T lymphocytes. As T cells are activated in allogeneic transplantation, we determined the role of IL-32 in human mixed lymphocyte cultures (MLCs) and GVHD. In allogeneic MLCs, IL-32 increased two-fold in responding T cells, accompanied by five-fold increases of TNF␣, IL-6, and IL-8. After allogeneic hematopoietic cell transplantation, IL-32 mRNA levels in blood leukocytes were statistically significantly higher in patients with acute GVHD (n ‫؍‬ 10) than in serial samples from patients who did not develop acute GVHD (n ‫؍‬ 5; P ‫؍‬ .02). No significant changes in IL-32 levels were present in patients with treated (n ‫؍‬ 14) or untreated (n ‫؍‬ 8) chronic GVHD, compared with healthy controls (n ‫؍‬ 8; P ‫؍‬ .5, and P ‫؍‬ .74, respectively). As IL-32 is activated by proteinase-3 (PR3), we determined the effect of the serine protease inhibitor ␣-1 antitrypsin (AAT) on IL-32 levels and showed suppression of IL-32 and T-lymphocyte proliferation in MLCs. In an MHC-minor antigen disparate murine transplant model, preconditioning and postconditioning treatment with AAT resulted in attenuation or prevention of GVHD and superior survival compared with albumin-treated controls (80% vs 44%; P ‫؍‬ .04). These findings suggest that AAT modulates immune and inflammatory functions and may represent a novel approach to prevent or treat GVHD. (Blood. 2011;118(18):5031-5039) IntroductionAn extensive literature describes the role of proinflammatory cytokines in the manifestations of conditioning-related toxicity in patients undergoing hematopoietic cell transplantation (HCT) and the immunologic interactions of donor and recipient cells that follow HCT. We were interested in determining specifically the involvement of IL-32 in the "cytokine storm" that has been described in the peri-HCT and post-HCT period. 1 For one, we and others have shown that tumor necrosis factor ␣ (TNF), which is consistently up-regulated in transplant recipients, is a potent inducer of IL-32. 2 Conversely, IL-32 has been shown to induce TNF, 2-5 suggesting the possibility of an amplification loop between these two cytokines. Secondly, IL-32 was originally identified in IL-2-activated T lymphocytes and natural killer (NK) cells, 6 supporting a potential role in T-cell activation and function after allogeneic HCT. Furthermore, IL-32 is present in supernatants of IL-12, IL-18, and IL-12 plus IL-18-stimulated human NK cells and in 7 the supernatant of concanavalin A-stimulated human PBMCs. 7 In addition, in patients with myelodysplastic syndrome who exhibit excessive apoptosis in hematopoietic cells, we reported, in agreement with others, that silencing of endogenous IL-32 significantly reduced apoptosis and the expression of other proinflammatory mediators. 3,8 Based on these data, we postulated a role for IL-32 in alloactivation and in GVHD. As IL-32 is activated via partial cleavage by proteinase-3 (PR3), 9 we hypothesized that the naturally occurring serine protease...

α-Linoleic Acid Enhances the Capacity of α1-Antitrypsin to Inhibit Lipopolysaccharide-Induced IL-1β in Human Blood Neutrophils

“…Considering protein levels, we found no differences in cytosolnuclear profiles of RelA/p65, IκBα, and IκBβ between LPS and LPS/A1AT-or Human-plasma-purified A1AT used as therapy not only inhibits neutrophil elastase and proteinase 3 but also expresses broad antiinflammatory and immunomodulatory effects, including modulation of IL-1β levels. (11,(39)(40)(41) This latter observation prompted us to investigate the effect of currently used preparations of A1AT in treating humans on processing secretion of active IL-1β in LPS-activated human blood neutrophils in vitro. It is important to point out that a major source of IL-1β (39,42,43); moreover, neutrophil-derived IL-1β mediates further neutrophil recruitment and activation.…”

“…In brief, increasing diabetes, gouty arthritis, and ischemia-reperfusion injuries. (11)(12)(13)(14)(15)(16)(17) Despite growing reports on the anti inflammatory properties demonstrated for A1AT, the mechanism of a direct effect of A1AT on cells remains to be defined. Both in vitro and in vivo studies provide evidence that the beneficial effects of A1AT are mostly related to suppression of Toll-like receptor (TLR) agonist-induced innate immune cell activation.…”

Expanding the Clinical Indications for α1-Antitrypsin Therapy

“…When examining the cellular targets of AAT, one finds that these primarily include members of the innate immune system, such as macrophages and neutrophils, as well as B lymphocytes and dendritic cells. In contrast, responses of purified T lymphocytes are consistently unaffected by AAT (7,8,(10)(11)(12)(13), allowing for a variety of responses to IL-2, as well as to concanav alin A and anti-CD3/CD28 stimulation, to persist. This cell-specific discretion, together with the ability to protect tissues from injury, sets AAT in a unique niche among modulators of the immune system, a separate entity to other antiinflammatory agents and classic immunosuppressants.…”

Anti-inflammatory and immunomodulatory properties of α1-antitrypsin without inhibition of elastase

“…For example, the addition of exogenous AAT in vitro inhibits the release of IL-8 by monocytes (9) and the expression of HIV-1 (10). In animal models, the administration of AAT prevents murine islet cell allografts from rejection (11), blocks β-cell apoptosis (12), and suppresses alloreactivity in allogeneic marrow transplantation models (13,14). In other models, AAT therapy reduces TNF-α-or endotoxin-induced lethality, cigarette smoke-induced emphysema and inflammation, and suppresses bacterial proliferation during infections (15)(16)(17).…”