Induction of heat shock proteins in B-cell exosomes

Abstract

Exosomes are nanometer-sized vesicles secreted by a diverse range of live cells that probably have physiological roles in modulating cellular immunity. The extracellular factors that regulate the quantity and phenotype of exosomes produced are poorly understood, and the properties of exosomes that dictate their immune functions are not yet clear. We investigated the effect of cellular stress on the exosomes produced by B-lymphoblastoid cell lines. Under steady-state conditions, the exosomes were positive for hsp27, hsc70, hsp70 and hsp90, and other recognised exosome markers such as MHC class I, CD81, and LAMP-2. Exposing cells to heat stress (42°C for up to 3 hours), resulted in a marked increase in these heat shock proteins (hsps), while the expression of other stress proteins such as hsp60 and gp96 remained negative, and other exosome markers remained unchanged. Stress also triggered a small increase in the quantity of exosomes produced [with a ratio of 1.245±0.07 to 1 (mean±s.e.m., n=20) of 3-hour-stress-exosomes to control-exosomes]. Flow-cytometric analysis of exosome-coated beads and immuno-precipitation of intact exosomes demonstrated that hsps were located within the exosome lumen, and not present at the exosome-surface, suggesting that such exosomes may not interact with target cells through cell-surface hsp-receptors. Functional studies further supported this finding, in that exosomes from control or heat-stressed B cells did not trigger dendritic cell maturation, assessed by analysis of dendritic-cell-surface phenotype, and cytokine secretion profile. Our findings demonstrate that specific alterations in exosome phenotype are a hitherto unknown component of the cellular response to environmental stress and their extracellular function does not involve the direct activation of dendritic cells.

Increased heat shock protein 70 levels in induced sputum and plasma correlate with severity of asthma patients

“…These findings suggest that neutrophils may be an important source of HSP70, and there may exist a positive feedback loop between HSP70 and neutrophils, an area we intend to explore in future studies. It was reported that HSP70 has been shown to be released by B cells which is consistent with our finding that the count and percentage of lymphocyte correlated positively with HSP70 levels in induced sputum from asthma patients (Clayton et al 2005). One study suggests the mast cells may also be a potential source of HSP70 (Mortaz et al 2006).…”

“…However, in this study, we did not find significant differences between noneosinophilic asthma and eosinophilic asthma patients, a finding that needs further investigation. One reason for this may be that there were different sources of HSP70 such as from mast cells, lymphocyte, and neutrophils (Asea 2007;Clayton et al 2005;Hunter-Lavin et al 2004).…”

“…Intracellularly, HSPs function to stabilize the cell and maintain cellular homeostasis (Hartl 1996). However, during the response to stress or injury like temperature, exercise, and infection, HSP-70 may be released from dying cells that have lysed, as well as from live cells via receptor-mediated exocytosis (Asea 2007;Clayton et al 2005;Hunter-Lavin et al 2004). When HSPs are released into the extracellular compartment, they are able to deliver a partial maturation signal to dendritic cells, which may play a crucial role in pathogenesis of asthma and activate the NF-kappaB pathway, and can also stimulate pro-inflammatory cytokines production in vitro (Asea et al 2000;Basu et al 2000).…”

Potential immunological functions of salivary Hsp70 in mucosal and periodontal defense mechanisms

“…Lipid rafts (also termed detergent-insoluble glycolipid fractions or detergent-resistant membranes) are specialized plasma membrane microdomains enriched in cholesterol and sphingolipids and which form a distinct, liquid-ordered phase in the lipid bilayer of membranes [18]. Exosomes are small (40-90 nm) membrane vesicles that are secreted by numerous cell types [20,43]. The release of Hsp70 through exosomes is independent of the common secretory pathway, because brefeldin-A, an inhibitor of the classical protein transport, did not block Hsp70 release in human peripheral blood mononuclear cells [43].…”

Exosome isolation from hemolymph of white-spotted flower chafer, Protaetia brevitarsis (Kolbe) (Coleoptera: Scarabaeidae).

“…RNA, microRNA, non-coding RNA, lipids, and proteins from their cells of origin (Sato-Kuwabara et al, 2015;Zhang et al, 2015; (Lai et al, 2015;Skogberg et al, 2015;Song et al, 2014;Nolte et al, 2009;Bhatnagar et al, 2007;Clayton et al, 2005;Thery et al, 2002). Also they can be found in body fluid such as blood, urine, saliva, and breast milk (Mathivanan et al, 2010;Miranda et al, 2010;Simons et al, 2009).…”