In vitro studies have indicated the importance of Toll-like receptor (TLR) signaling in response to the fungal pathogens Candida albicans and Aspergillus fumigatus. However, the functional consequences of the complex interplay between fungal morphogenesis and TLR signaling in vivo remain largely undefined. In this study we evaluate the impact of the IL-1R/TLR/myeloid differentiation primary response gene 88 (MyD88)-dependent signaling pathway on the innate and adaptive Th immunities to C. albicans and A. fumigatus in vivo. It was found that 1) the MyD88-dependent pathway is required for resistance to both fungi; 2) the involvement of the MyD88 adapter may occur through signaling by distinct members of the IL-1R/TLR superfamily, including IL-1R, TLR2, TLR4, and TLR9, with the proportional role of the individual receptors varying depending on fungal species, fungal morphotypes, and route of infection; 3) individual TLRs and IL-1R activate specialized antifungal effector functions on neutrophils, which correlates with susceptibility to infection; and 4) MyD88-dependent signaling on dendritic cells is crucial for priming antifungal Th1 responses. Thus, the finding that the innate and adaptive immunities to C. albicans and A. fumigatus require the coordinated action of distinct members of the IL-1R/TLR superfamily acting through MyD88 makes TLR manipulation amenable to the induction of host resistance to fungi.

Heat shock protein (HSP)70 can be released from tumor cells and stimulate a potent antitumor immune response. However, HSP70 does not contain a consensus secretory signal and thus cannot traverse the plasma membrane by conventional mechanisms. We have observed HSP70 release from intact human prostate carcinoma cell lines (PC-3 and LNCaP) by a mechanism independent of de novo HSP70 synthesis or cell death. This pathway is similar to one used by the leaderless protein IL-1β. Our studies show that HSP70 release involves transit though an endolysosomal compartment and is inhibited by lysosomotropic compounds. In addition, the rate of HSP70 secretion correlates well with the appearance of the lysosomal marker LAMP1 on the cell surface, further suggesting the role for endolysosomes. The entry of HSP70 into this secretory compartment appears to involve the ABC family transporter proteins and ABC transporter inhibitor glibenclamide antagonizes secretion. Although the cell signals involved in triggering stress induced HSP70 release though this lysosomal pathway are largely unknown, our experiments suggest a regulatory role for extracellular ATP. These mechanisms appear to be shared by IL-1β secretion. Following release, we observed the binding of extracellular HSP70 to the cell surface of the prostate carcinoma cells. These findings suggest that secreted HSP70 can take part in paracrine or autocrine interactions with adjacent cell surfaces. Our experiments therefore suggest a mechanism for HSP70 secretion and binding to the surface of other cells that may be involved in recognition of the tumor cells by the immune system.

Extracellular stress proteins including heat shock proteins (Hsp) and glucose regulated proteins (Grp) are emerging as important mediators of intercellular signaling and transport. Release of such proteins from cells is triggered by physical trauma and behavioral stress as well as exposure to immunological ''danger signals''. Stress protein release occurs both through physiological secretion mechanisms and during cell death by necrosis. After release into the extracellular fluid, Hsp or Grp may then bind to the surfaces of adjacent cells and initiate signal transduction cascades as well as the transport of cargo molecules such as antigenic peptides. In addition Hsp60 and hsp70 are able to enter the bloodstream and may possess the ability to act at distant sites in the body. Many of the effects of extracellular stress proteins are mediated through cell surface receptors. Such receptors include Toll Like Receptors 2 and 4, CD40, CD91, CCR5 and members of the scavenger receptor family such as LOX-1 and SREC-1. The possession of a wide range of receptors for the Hsp and Grp family permits binding to a diverse range of cells and the performance of complex multicellular functions particularly in immune cells and neurones.

No abstract

Extracellular stress proteins including heat shock proteins (Hsps) and glucose-regulated proteins (Grps) are emerging as important mediators of intercellular signaling and transport. Release of such proteins from cells is triggered by physical trauma and behavioral stress as well as exposure to immunological "danger signals." Stress protein release occurs both through physiological secretion mechanisms and during cell death by necrosis. After release into the extracellular fluid, Hsp or Grp may then bind to the surfaces of adjacent cells and initiate signal transduction cascades as well as the transport of cargo molecules, such as antigenic peptides. In addition, Hsp60 and Hsp70 are able to enter the bloodstream and may possess the ability to act at distant sites in the body. Many of the effects of extracellular stress proteins are mediated through cell-surface receptors. Such receptors include toll- like receptors (TLRs) 2 and 4, CD40, CD91, CCR5, and members of the scavenger receptor family, such as LOX-1 and SREC-1. The possession of a wide range of receptors for the Hsp and Grp family permits binding to a diverse range of cells and the performance of complex multicellular functions particularly in immune cells and neurons.

No abstract

Extracellular HSP70 has been found to participate in both innate and adaptive immune responses. However, little is known about the molecular mechanisms that mediate this process. Previous reports suggest that HSP70 interacts with antigen presenting cells (APC) through a plethora of surface receptors. In this study, we have examined the relative binding of potential HSP70 receptors and found high affinity binding to LOX-1 but not other structures with a role in HSP70-APC interactions such as LRP/CD91, CD40, TLR2, TLR4 or another c-type lectin family member (DC-SIGN) closely related to LOX-1. In addition to APC, HSP70 can avidly bind to non-APC cell lines, especially those from epithelial or endothelial background.

Amphotericin B is the most effective drug for treating many life-threatening fungal infections. Amphotericin B administration is limited by infusion-related toxicity, including fever and chills, an effect postulated to result from proinflammatory cytokine production by innate immune cells. Because amphotericin B is a microbial product, we hypothesized that it stimulates immune cells via Toll-like receptors (TLRs) and CD14. We show here that amphotericin B induces signal transduction and inflammatory cytokine release from cells expressing TLR2 and CD14. Primary murine macrophages and human cell lines expressing TLR2, CD14, and the adapter protein MyD88 responded to amphotericin B with NF-B-dependent reporter activity and cytokine release, whereas cells deficient in any of these failed to respond. Cells mutated in TLR4 were less responsive to amphotericin B stimulation than cells expressing normal TLR4. These data demonstrate that TLR2 and CD14 are required for amphotericin B-dependent inflammatory stimulation of innate immune cells and that TLR4 may also provide stimulation of these cells. Our results provide a putative molecular basis for inflammatory responses elicited by amphotericin B and suggest strategies to eliminate the acute toxicity of this drug.