Cytokine Release as a Mechanism Mediating IGIV Adverse Reactions

Investigators

Abstract

Inflammatory immune responses with clinically relevant consequences can be associated with TNF, IL-1, IL-6 and other cytokines. These cytokines are rapidly produced by bacteria and bacterial components, such as DNA and LPS, but can also be induced, under certain circumstances, by triggering of complement receptors. Manufacture of plasma derivatives involves steps which are not aseptic and which can lead to bacterial contamination. Intact bacteria are removed by sterile filtration, but this step does not remove LPS, bacterial DNA (bDNA), or other microbial constituents. Adverse events with IGIV administration include fever and hypotension; fever has been associated with elevated TNF levels in recipients, and hypotension is a known side effect of LPS. We sought to determine whether the pattern of cytokine secretion by human monocytes was similar after IGIV and microbial stimulation of cells, and whether low levels of LPS and bDNA present in IGIV were sufficient to stimulate cytokine release. Multiple lots of IGIV from different manufacturers were assessed to determine whether they could stimulate TNF or IL-1 release from human mononuclear cells at concentrations that are present in humans after IGIV infusion. Many lots stimulated significant TNF and IL-1 production, most consistently observed when non-heat inactivated human serum was used in the culture media. Addition of polymyxin B, which neutralizes LPS, did not always abrogate cytokine release. Although no IGIV lots had LPS contamination > 0.91 EU/ml (the industry standard), dose response experiments using LPS showed that amounts lower than 0.91 EU/ml can stimulate cytokine release from monocytes. Bacterial DNA was detected in IGIV using PCR primers which recognized broadly conserved bacterial DNA sequences; the levels detected were lower than those needed to stimulate monocyte cytokines in vitro. However, the presence of bDNA suggests that other bacterial constituents, such as cell wall components of gram positive bacteria, could also be present, some of which may not be detected by current lot release standards such as LPS testing and rabbit pyrogen testing. In a related project, we are determining in vivo correlates of stimulation by bacterial components in relation to triggering of dendritic cell migration and activation in animal models. Current work is focusing upon dissecting whether bacterial constituents, or immunoglobulin itself, stimulate cytokine release from cells cultured with non-heat-inactivated serum. We are currently confirming the role of complement in this system using complement-deficient serum and complement blocking manouvers. If the role of complement is confirmed, then the pathways used may be exploited to determine whether the inciting factor is bacterially derived (alternate pathway) or Ig-related (classical pathway), or both. These studies will add to understanding of how infusion-related IGIV adverse events are mediated, and may lead to manufacturing improvements which can abrogate such effects.