The role of interleukin-6 classical and trans-signaling in allogeneic stem cell transplantation

Abstract

IL-6 family cytokines share structural similarities and utilize glycoprotein 130 (gp130) for signal transduction. IL-6 itself has both pro- and anti-inflammatory effects. IL-6 trans-signaling is mediated by the soluble IL-6 receptor (IL-6R) and is responsible for most of its proinflammatory effects, while the anti-inflammatory classical IL-6 signaling is mediated by membrane-bound IL-6R. Availability of soluble IL-6R regulates the balance between classical and trans-signaling. Dysregulation of this balance has been implicated in immune-mediated diseases, including graft-versus-host disease (GVHD) that occurs after allogeneic stem cell transplantation (ASCT) and can results in disabling and life-threating complications. The pathogenesis of GVHD is very complex, and IL-6 seems to contribute to this process. However, the role of classical and IL-6 trans-signaling in GVHD has not been investigated previously in either clinical studies or animal models. The aim of the thesis was, therefore, to investigate whether various forms of IL-6 signaling and various IL-6 family members influence outcomes after ASCT. In the first study, we investigated effects of serum levels of IL-6 family cytokines on outcomes after ASCT in a population of 100 consecutive allotransplant recipients. C-reactive protein (CRP) levels partly reflect IL-6-trans-signaling. We show that pretransplant CRP and IL-6 levels showed significant correlation for allotransplant recipients, but only CRP levels were significantly associated with treatment-related mortality (TRM) in multivariate analyses. Of the other IL-6 family cytokines, only for high IL-31 could a significant association with clinical outcome (increased TRM) be observed. In the second study we investigated how genetic variations in the IL-6R genes of donors andrecipients influenced pretransplant level of IL-6 family cytokines, pretransplant CRP levels and posttransplant outcome. Ten single nucleotide polymorphisms (SNPs) with and without known association to immune-mediated diseases/biological effects were selected. Homozygosity for themajor alleles of the IL-6R SNPs rs2228145 and rs4845618 was associated with high pre- and posttransplant CRP serum levels and decreased sIL-6R levels but did not influence transplant outcomes. Homozygosity for the minor allele of rs4379670 was associated with decreased pretransplant CRP levels, whereas rs4845618 donor genotype was associated with aGVHD. Finally, the recipient genotype of the IL-6R SNP rs432950 was associated with the probability to wean of immunosuppression. The effects of G-CSF administration on systemic levels of IL-6 family cytokines in healthy stem cells were investigated in the third study. G-CSF administration significantly increased the levels of both IL-6 and CRP, whereas the levels of the other IL-6 family cytokines were not significantly altered. G-CSF was also able to potentate IL-6 release from in vitro cultured monocytes, fibroblasts and mesenchymal stem cells stimulated by various Toll-like receptor agonists. Finally, we investigated how various forms of IL-6 signaling influenced the activation of intracellular signaling pathways (i.e. mediator phosphorylation status) in resting and activated (CD3/CD28 receptor ligation) peripheral blood CD4+ and CD8+ T cells derived from allotransplant recipients 90 days posttransplant. We used the two designer cytokines hyper-IL-6 and sgp130-FC that allows for both isolated IL-6 trans-signaling stimulation and blockage. We observed that IL-6 signaling potentiated the phosphorylation/activation of STAT3, AKT and mTor; these effects were observed especially after activation of circulating CD4+ cells derived from patients with previous acute GVHD (aGVHD). Taken together, our results suggest that IL-6 family cytokines are important for the regulationof inflammation and immunity in allogeneic stem cell transplant recipients. However, the influence of IL-6 and IL-6 family cytokines is only one of several factors that contribute to the final clinical outcome after allotransplantation, and the heterogeneity among both donors and recipients with regard to IL-6 family levels/activity suggests that the impact of these cytokines differs between patients.