Dendritic Cell Stimulation by Mycobacterial Hsp70 Is Mediated Through CCR5

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Science  20 Oct 2006:
Vol. 314, Issue 5798, pp. 454-458
DOI: 10.1126/science.1133515


An effective host immune response to mycobacterial infection must control pathogen dissemination without inducing immunopathology. Constitutive overexpression of mycobacterial heat shock protein (myHsp70) is associated with impaired bacterial persistence, but the immune-mediated mechanisms are unknown. We found that myHsp70, in addition to enhancing antigen delivery to human dendritic cells, signaled through the CCR5 chemokine receptor, promoting dendritic cell aggregation, immune synapse formation between dendritic cells and T cells, and the generation of effector immune responses. Thus, CCR5 acts as a pattern-recognition receptor for myHsp70, which may have implications for both the pathophysiology of tuberculosis and the use of myHsps in tumor-directed immunotherapy.

Mycobacterium tuberculosis infects one-third of the world's population and is responsible for two million deaths annually. A tightly controlled T cell response to M. tuberculosis results in granuloma formation, which limits mycobacterial replication and controls the immunopathological consequences of infection (1). The immune response and granuloma formation are regulated by host and mycobacterial factors (2); one of these, mycobacterial heat shock protein 70 (myHsp70), stimulates dendritic cells (DCs) to release proinflammatory mediators (3). Hsps are conserved between microorganisms and mammalian cells and have a well-characterized role in protein assembly (4). Their ability to bind and deliver short polypeptides to immature dendritic cells (iDCs) is being exploited in tumor-directed DC immunotherapy (5). Picomolar concentrations of peptide bound to myHsp70 can generate antigen-specific cytotoxic T lymphocyte (CTL) responses through efficient peptide delivery and direct iDC stimulation by myHsp70 (6). A calcium-dependent intracellular signaling cascade triggered by myHsp70, but not by Toll-like receptor (TLR) agonists (6), is critical for the generation of effector immune responses, but neither the surface receptor nor the cellular mechanisms involved are known.

To further characterize the myHsp70-mediated calcium signal in iDCs, we demonstrated that pertussis toxin, an inhibitor of heterotrimeric G-protein–coupled receptor (GPCR) transduction, inhibited signaling of both myHsp70 and the control GPCR-mediated agonist, n-formyl-Met-Leu-Phe (fMLP; Fig. 1A). Analysis of potential GPCRs expressed on iDCs, using a heterologous expression system, revealed that myHsp70 signaled through a CC chemokine receptor. We examined glioma cells expressing different chemokine receptors and found that myHsp70 triggered a calcium response in cells expressing CCR5, and to a much lesser extent CCR1 (Fig. 1B). Alexa488-conjugated myHsp70 showed specific binding to CCR5-expressing but not control CXCR4-expressing cells (Fig. 1C) and was inhibited by competition with unlabeled myHsp70 (Fig. 1C). CCR5-dependent signaling also occurred in primary human DCs, because the CCR5 antagonist TAK-779 (7) inhibited calcium signaling in iDCs triggered by myHsp70 and the endogenous CCR5 ligand, MIP-1β, but not calcium signaling by fMLP (Fig. 1D).

Fig. 1.

myHsp70 triggers intracellular signaling in human iDCs through CCR5. (A) The myHsp70 receptor on human DCs is G-protein–coupled. Calcium signaling in iDCs, preincubated with (gray) or without (black) pertussis toxin (PTX, 1 μg/ml for 12 hours), after stimulation with myHsp70 (200 nM, top), fMLP (1 μM, bottom), or subsequently thapsigargin (TG, 1 μM) is shown. (B) Calcium signaling in chemokine receptor–expressing glioma cell lines after myHsp70 stimulation (200 nM, arrows). (C) myHsp70 binds to CCR5. (Top) Alexa488-labeled myHsp70 (220 nM) staining of CCR5-expressing (black) versus control CXCR4-expressing (gray) cells. (Bottom) Labeled myHsp70 (220 nM)–specific binding was inhibited by increasing doses of unlabeled myHsp70. (D) TAK779 inhibits myHsp70 calcium signaling. Calcium signaling in iDCs, pretreated with TAK-779 (100 nM, gray), or vehicle alone (black) after the addition of myHsp70 (200 nM, top), MIP-1β (100 nM), or fMLP (1 μM, bottom) is shown. (E) The CCR5Δ32 mutation confers resistance to myHsp70. Calcium responses in iDCs homozygous for wild-type (wt) CCR5 (black) or CCR5Δ32 (gray) after stimulation with myHsp70 (30 nM, top) or MIP-1β (200 nM, bottom), followed by fMLP (1μM), are shown. (Lower inset) CCR5 expression on CCR5 wild-type (black) or CCR5Δ32 (gray) iDCs (iDC isotype: gray fill). (F) Dose response of myHsp70 (black) versus MIP-1β (gray) calcium signaling in iDCs. (G) M. bovis lysates trigger calcium responses through CCR5 in iDCs. Calcium responses in untreated (black) or TAK-779–pretreated (gray) iDCs after treatment with M. bovis BCG lysate (arrow) are shown. (H) myHsp70 does not signal via murine CCR5. Calcium signaling in mouse peritoneal macrophages from CCR5+/+ (black) and CCR5–/– (gray) mice after the addition of myHsp70 (top) or mouse MIP-1β (bottom) is shown.

The requirement for CCR5 in myHsp70 signaling was confirmed by examining calcium responses in iDCs from a donor homozygous for the CCR5Δ32 allele, the naturally occurring 32-nucleotide deletion that prevents CCR5 surface expression (8) (Fig. 1E and fig. S1). The absence of myHsp70- or MIP-1β–mediated calcium signaling in CCR5Δ32 iDCs confirmed an absolute requirement for CCR5 in this pathway (Fig. 1E). A dose-response comparison of calcium signals in iDCs showed MIP-1β to be five times more potent than myHsp70 (Fig. 1F). A lysate from M. bovis bacille Calmette Guérin (BCG) also triggered a TAK-779–inhibitable calcium signal, suggesting that sufficient myHsp70 is present to promote CCR5-dependent signaling (Fig. 1G). Furthermore, CCR5-dependent myHsp70 signaling was species-specific, because no myHsp70 signal was seen in CCR5-expressing mouse macrophages (Fig. 1H), myHsp70 did not involve TLR signaling pathways [as assessed by microarray analysis (fig. S2A and table S1) and assays of nuclear factor κB activity (fig. S2, B and C)], and myHsp70 induces a calcium-dependent phenotypic maturation of DCs (fig. S3).

The immediate morphological consequences of CCR5-mediated Hsp70 calcium signaling were further investigated by live cell imaging of iDCs (Fig. 2, A to C, and video S1). The addition of myHsp70 triggered calcium oscillations, immediately followed by rapid cell membrane ruffling and pseudopodia projection (Fig. 2C). Correlation of intracellular calcium oscillations with cell movement, by means of simultaneous fluorescence differential interference contrast (DIC) imaging, suggested that a rise in intracellular calcium was required, but not sufficient, to trigger membrane changes and cell aggregation. Membrane changes and cell aggregation were inhibited by chelation of intracellular calcium and could not be induced by calcium rises induced by adenosine triphosphate (ATP) or phorbol myristate acetate/ionomycin (Fig. 2D and fig. S4). These morphological changes were also mediated through CCR5. They were inhibited by TAK-779, were not seen in DCs from CCR5Δ32 homozygotes (Fig. 2D), and were not induced by TLR agonists (Fig. 2D). Thus, myHsp70 stimulation of human DCs results in rapid CCR5-mediated, calcium-dependent, nonrandom migration of iDCs and the projection of plasma membrane extensions.

Fig. 2.

myHsp70 signaling triggers rapid calcium-dependent iDC aggregation. (A) myHsp70 induces changes in iDC morphology and trafficking. Simultaneous fluorescence (top) and DIC (middle) (×100) imaging of Fluo3-labeled iDCs after the addition of myHsp70 (200 nM) is shown. DIC images were analyzed with edge-detection analysis software (bottom) to enhance the visualization of membrane ruffling. (B) Merged fluorescence and DIC images from the experiment shown in (A) 450 s after the addition of myHsp70, at lower (×40) magnification. (C) Quantification of membrane ruffling after myHsp70 addition. The fold increase in the edge signal derived from image analysis is shown for three representative cells. The arrow indicates addition of myHsp70. (D) myHsp70 calcium responses are required for iDC aggregation. The correlation of intracellular calcium concentration (F/F0) with cell movement [determined by x-y displacement (analyzed every 5 s)] in iDCs treated with myHsp70 (200 nM), myHsp70 after preincubation with the intracellular calcium chelator BAPTA-AM, ATP (100 μM), LPS (100 ng/ml), TAK-779 (1 μM), or iDCs from CCR5Δ32 homozygotes treated with myHsp70 is shown.

To examine how myHsp70 affected interactions between DCs and T cells, we stimulated iDCs and autologous T cells with myHsp70. Autologous T cells rapidly clustered around the DCs (Fig. 3A). More T cells (up to nine) associated with each DC after stimulation with myHsp70 than with bovine serum albumin (BSA) or lipopolysaccharide (LPS), and this enhanced interaction was inhibited by 1,2-bis(o-aminophenoxy)ethane-N,N,N,′N′-tetraacetic acid (BAPTA) preloading (Fig. 3B). The trapping of T cells by DCs was observed as early as 30 s after stimulation of iDCs (Fig. 3C and videos S2 and S3), was facilitated by the formation of a network of DC membrane extensions (Fig. 3C), and was seen with both autologous and allogeneic CD4 and CD8 T cells (Fig. 3C). The formation of the membrane extensions and T cell clusters was dependent on functional CCR5 receptors on DCs but not on T cells (Fig. 3C and video S4). The rapid clustering of DCs and associated T cells suggested an integrin-mediated mechanism. The addition of myHsp70 led to a rapid increase in β2-integrin–specific iDC binding to immobilized intercellular adhesion molecule–2 (ICAM-2), ICAM-3, and (to a lesser extent) ICAM-1 (Fig. 4A) (9). This rapid T cell trapping by DCs stimulated with myHsp70 was followed at later time points (after 30 min) by T cell polarization of talin and LFA-1 toward the point of contact with the DC (Fig. 4B), suggesting the formation of antigen-independent immune synapses (10, 11). The ability of myHsp70 to promote iDC–T cell interactions may explain why T cells are required for myHsp70 to stimulate pro-inflammatory cytokine release from DCs (fig. S5) (6).

Fig. 3.

myHsp70 signaling through CCR5 promotes DC–T cell clustering. (A) iDCs and CD8 T cells were stimulated with myHsp70 (200 nM) or control BSA (200 nM) on glass coverslips at 37°C for 30 min. After the removal of unbound cells, iDCs were labeled with antibody to major histocompatibility complex II (anti–HLA-DR) (red) and CD8 T cells were labeled with anti-CD3 (green); cells were then examined by confocal microscopy. (B) T cells associated with each iDC were quantified 30 min after the addition of myHsp70, myHsp70 after preincubation with BAPTA-AM, LPS (100 ng/ml), or BSA control. (C) myHsp70-induced formation of DC–T cell contacts was analyzed by simultaneous fluorescence and DIC imaging of Fluo3-labeled iDCs with CD4 or CD8 T cells (unlabeled) after the addition of myHsp70 (200 nM), using cells from CCR5 wild-type or CCR5Δ32 homozygotes. Contact between myHsp70-stimulated iDCs and T cells was reduced in the absence of functional CCR5 on iDCs.

Fig. 4.

myHsp70 signaling through CCR5 promotes functional DC–T cell immune synapse formation. (A) myHsp70 up-regulates β2 integrin activity. β2-integrin–dependent adherence of iDCs to substrate coated with ICAM-1, -2, or -3 measured 10 min after treatment with myHsp70 or vehicle is shown. (B) myHsp70 induces DC–T cell immune synapse formation. Thirty minutes after stimulation with myHsp70 (200 nM), T cells (green) and iDCs [labeled with anti–HLA-DR (red)] were examined for polarization of talin (left) and LFA-1 (right) by confocal microscopy. (C) CCR5Δ32 iDCs show impaired IL-6 release after myHsp70 treatment. IL-6 release from CCR5 wild-type (black bars) or CCR5Δ32 (gray bars) iDCs 72 hours after stimulation with myHsp70 (200 nM), LPS (0.5 ug/ml), or RANTES (400 nM) is shown. (D) TAK-779 and pertussis toxin block the generation of peptide-specific CTLs by myHsp70. myHsp70-peptide–pulsed iDCs were used to generate antigen-specific CTLs (6). The effect of pretreating iDCs with increasing concentrations of pertussis toxin (left) or TAK-779 (right) on the induction of peptide-specific CTLs by Hsp70 was determined. Results are shown as percent inhibition of specific lysis, where inhibition is plotted as a percentage of the maximal response (n = 3 replicates ± SEM). (E) The effect of TAK-779 on intracellular mycobacteria replication. iDCs from immune individuals were incubated for 2 hours with M. bovis BCG-lux, washed to remove noninternalized mycobacteria, and cocultured with autologous CD4+ and CD8+ T cells in the absence (blue) or presence (red) of TAK-779. Cell-associated luminescence (correlating with viable mycobacteria) was determined in triplicate samples before and 24 hours after the addition of T cells. Results are representative of three separate experiments.

CCR5 mediated a number of functional consequences of myHsp70 signaling. myHsp70 and the CCR5 agonist RANTES induced interleukin-6 (IL-6) release from wild-type but not CCR5Δ32 iDCs (Fig. 4C). Furthermore, pretreatment of iDCs with pertussis toxin or TAK-779 inhibited the ability of peptide-loaded myHsp70 to stimulate DCs to generate influenza peptide–specific CTLs (Fig. 4D). To examine the effect of CCR5-mediated signaling in mycobacterial infection, we used the model pathogen M. bovis BCG-lux (12). As reported for murine DCs (13), human DCs from immune people were unable to kill internalized mycobacteria, even in the presence of autologous T cells. TAK-779 inhibition of CCR5 led to a dose-dependent enhancement of intracellular mycobacterial replication (Fig. 4E and fig. S6A) at all concentrations of mycobacteria tested (fig. S6B), suggesting an important role for this receptor in controlling mycobacterial infection.

The identification of CCR5 as the critical receptor for myHsp70-mediated DC stimulation has implications for both mycobacterial infection and the therapeutic use of myHsp70. CCR5 is important in immune cell cross talk. Interaction with its naturally occurring ligand MIP-1β promotes the recruitment of cells to sites of inflammation (14), facilitates immune synapse formation (15), orchestrates T cell interactions within lymph nodes (16), and controls the activation and differentiation of T cells (17). Our finding that a mycobacterial lysate, as well as purified myHsp70, stimulated a CCR5-dependent calcium response indicates a further connection between the innate and adaptive immune responses during mycobacterial infection. The cellular aggregation induced by myHsp70 signaling through CCR5 may play an important role in the formation of granulomas, the hallmark of mycobacterial infection.

Microbial-induced DC responses need to be highly regulated, reflecting a balance between a rapid and appropriate response to invading microbes and the inducement of immunopathology (2)—a particular problem in mycobacterial infection. An increasing number of human pathogens, including HIV (18), toxoplasma (19), and M. tuberculosis (as described here), target the CCR5 receptor. This role of CCR5 as a pattern-recognition receptor for myHsp70 may, at least in part, be responsible for the maintenance of the high CCR5Δ32 allele frequency (10 to 15%) in Northern European populations (20) and may alter the pattern of disease seen in people with the CCR5Δ32 allele.

Supporting Online Material

Materials and Methods

Figs. S1 to S7

Table S1

Videos S1 to S4


References and Notes

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