Mammalian responses to bacterial LPS (lipopolysaccharide) from the outer membrane of Gram-negative bacteria can lead to an uncontrolled inflammatory response that can be deadly for the host. It has been shown that the innate immune system employs at least three cell surface receptors, CD14, TLR4 (Toll-like receptor 4) and MD-2, in order to recognize bacterial LPS. In our previous work we have found that Hsps (heat-shock proteins) are also involved in the innate recognition of bacterial products. Their presence on the cell surface, as well as their involvement in the innate recognition process, are poorly understood. In the present study we have investigated the association of TLR4 with Hsp70 and Hsp90 following LPS stimulation, both on the cell surface and intracellularly. Our results show that Hsp70 and Hsp90 form a cluster with TLR4 within lipid microdomains following LPS stimulation. In addition, Hsp70 and Hsp90 seem to be involved in TLR4/LPS trafficking and targeting to the Golgi apparatus, since upon LPS stimulation we found that both Hsps are targeted to the Golgi along with TLR4. The present study sheds new light into the involvement of Hsps in the innate immune response.
Hsps (heat-shock proteins) are a family of highly conserved proteins found in cells. Hsps are constitutively expressed intracellularly, and act as molecular chaperones by binding nascent polypeptides in order to assist proper folding, assembly and intracellular trafficking. Although Hsps are found in different intracellular compartments, some have also been found to be expressed on the cell surface [1–4]. They have been detected on the cell surface of tumour cells  as well as on apoptotic cells . The origin of membrane-associated Hsps still remains elusive, although some have suggested that Hsps might be secreted by the cell before associating with the plasma membrane [7,8]. Membrane-associated Hsps have been shown to play a role in the immune response [7,9], and particularly in LPS (lipopolysaccharide) recognition [10–12], although this is not very well understood.
Recognition of LPS by the innate immune system leads to immediate cell activation and release of pro-inflammatory cytokines. This activation can lead to overproduction of cytokines that are harmful and, in most cases, deadly for the host. At least three cell-surface molecules have been recognized as components of the mammalian signalling receptor for LPS: CD14 , TLR4 (Toll-like receptor 4) [14–16] and the adaptor molecule MD2 [17–19]. The recent study by Visintin et al.  has shed new light on the initial events of LPS recognition, demonstrating that LPS binds to MD2, which in turn binds to TLR4 and induces aggregation and signal transduction. The possibility that additional receptor components such as Hsps [10,12], CXCR4 (chemokine receptor 4)  or CD55  have been suggested to be part of this activation cluster, possibly acting as additional LPS transfer molecules.
In the present study we demonstrate that Hsp70 and Hsp90 associate with the TLR4–MD2 cluster in response to LPS stimulation. This association takes place within lipid rafts. In addition, using confocal imaging we show that upon LPS stimulation TLR4 is targeted to the Golgi apparatus along with Hsp70 and Hsp90. Thus both Hsps seem to be involved not only in the binding and transfer of LPS to the TLR4–MD2 complex on the cell surface, but assist further in the trafficking and targeting of LPS to the Golgi apparatus.
Materials and methods
Hybridoma cells secreting 26ic (anti-CD14) and W6/32 secreting MHC class I-specific monoclonal antibody were obtained from the American Type Culture Collection (Vanassas, MD, U.S.A.). Hsp70 rabbit polyclonal serum was obtained from Dako (Cambridge, U.K.). Hsp90-specific rabbit polyclonal serum was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, U.S.A.). TLR4-specific monoclonal antibody HTA-125 was purchased from HyCult (Uden, The Netherlands), MyD88 polyclonal antibody was purchased from eBioscience (San Diego, CA, U.S.A.), MD2 polyclonal antibody was purchased from Imgenex (San Diego, CA, U.S.A.). Cy3 and Cy5 labelling kits were purchased from Amersham Biosciences (Little Chalfont, Bucks., U.K.).
Monocytes were isolated from human A+buffy coats. Adherent cell monolayers (1×105–2×105 monocytes/well) were cultured in 24-well plates in serum-free medium (Gibco) supplemented with 0.01% L-glutamine and 40 μg of gentamicin/ml.
HEK cells transfected with CD14 and YFP-TLR4 were kindly provided by Professor D. Golenbock (University of Massachusetts, MA, U.S.A.). Cells were maintained in Dulbecco's modified Eagle's medium containing 4.6 g/l glucose with 10% (v/v) fetal calf serum, 5 μg/ml puromycin and 0.5 mg/ml G418 sulphate.
FRET (fluorescence resonance energy transfer) measurements
Cells were imaged on a Carl Zeiss, Inc. LSM510 confocal microscope (with an Axiovert 200 fluorescent microscope) using a 1.4 numerical aperture 63× Zeiss objective. The images were analysed using LSM 2.5 image analysis software (Carl Zeiss, Inc.). For FRET imaging, Cy3 and Cy5 were detected using the appropriate filter sets. Using typical exposure times for image acquisition (less than 5 s), no fluorescence was observed from a Cy3-labelled specimen using the Cy5 filters; neither was Cy5 fluorescence detected using the Cy3 filter sets.
We have previously shown that Hsp70 and Hsp90 are involved in the innate recognition of LPS . In the present study, using FRET we provide evidence that the TLR4–MD2 complex is part of the LPS-activation cluster that we had identified previously (Figure 1A). Hsp70 and Hsp90 seem to associate with both TLR4 and MD2 following stimulation by bacterial LPS. This interaction seems to take place within lipid rafts (Figure 1B).
TLR4/MD-2 heterotypic associations in response to LPS
Furthermore, when we investigated the intracellular distribution of Hsp70 and Hsp90 in response to LPS stimulation, we found that both Hsps, similarly to TLR4, were targeted to the Golgi apparatus (Figure 2).
TLR4, Hsp70 and Hsp90 are localized in the Golgi following LPS stimulation
Since we had already found that LPS and its receptor molecules localize in lipid rafts in the initial binding steps, we investigated whether lipid raft-disrupting drugs such as nystatin , which had been tested and were not cytotoxic at the optimal concentrations of 25 μM, could inhibit TLR4/Hsp70/Hsp90 targeting to the Golgi apparatus. Fluorescence labelling revealed that treatment with nystatin prevented Hsp70, Hsp90 and TLR4 co-localization to the Golgi (Figure 3), leading us to believe that TLR4/Hsp entry and targeting to the Golgi apparatus is dependent on lipid raft integrity.
Intracellular distribution of TLR4 and Hsp70 or Hsp90 after lipid raft disruption
Although Hsps have been shown to be able to bind LPS  and to be involved in the innate recognition of bacterial products [10,12], the possible mechanism for their participation in innate immunity has not been elucidated yet. In the present study using fluorescence imaging techniques we demonstrate that Hsps associate with the TLR4–MD2 complex upon LPS stimulation within lipid rafts. Following stimulation the TLR4–MD2 complex is targeted to the Golgi along with Hsp70 and Hsp90. Overall, our data suggest that TLR4 and the Hsps follow the considerable fluidity of lipid rafts between the plasma membrane and the Golgi complex in order to gain access to the cell. Our findings are in agreement with Latz et al.  and Nichols et. al , who have demonstrated a novel rapid recycling pathway from the plasma membrane to the Golgi that is followed by LPS and lipid raft markers.
Another recent study by Broquet et al.  has confirmed the presence of Hsp70 within lipid rafts and has suggested that this might be the mechanism of Hsp70 delivery and release to the plasma membrane. The interaction of Hsp70 with lipids has been shown further by the studies of Arispe and co-workers [29,30], who have demonstrated that Hsp70 incorporates into artificial phospholipid membranes as well as liposomes. These studies demonstrate that Hsps are released through a lipid-raft-dependent pathway and, once on the plasma membrane, can interact with the lipid bilayer. Since they have an affinity for LPS, they could act as a transfer molecule within the lipid raft and deliver LPS to the TLR4–MD2 complex. It has been shown recently that the TLR4–MD-2–LPS complex traffics to and from the Golgi apparatus in a lipid-raft-dependent manner , Hsps might be assisting in the targeting of this complex to the Golgi apparatus. Thus Hsps seem to play an important role in the innate recognition of bacteria, not only as receptors on the cell membrane, but also as important molecules for internalization.
Heat Shock Proteins and Modulation of Cellular Function: Focused Meeting held at Guy's Hospital, London, U.K. Organized by C. Kelly (King's College London) and I. Dransfield (MRC Centre for Inflammatory Research, Edinburgh). Edited By C. Kelly.
This work was supported by Sport Aiding Research for Kids (SPARKS) and the Wellcome Trust. We thank Professor Doug Golenbock for providing us with his HEK transfectants.