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T cells respond to heat shock protein 60 via TLR2: activation of adhesion and inhibition of chemokine receptors¹

The Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel

²Correspondence: Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: irun.cohen{at}weizmann.ac.il

SPECIFIC AIMS

The mammalian 60 kDa heat shock protein (HSP60) is a molecule of many facets. Besides serving as a chaperone, HSP60 is expressed by cells exposed to stress or immune activation, is present in the blood during inflammation, and has been found to be a target of autoantibodies and autoimmune T cells in healthy individuals and, to a greater extent, in those suffering from autoimmune diseases. Recently, HSP60 was discovered to activate macrophages by way of the innate toll-like receptor 4 (TLR4) to induce enhanced production of NO and IL-12, IL-15, TNF-, IL-6, and other Th1-type cytokines. Thus, HSP60 would seem to be an important factor in inflammation generally. The present study was undertaken to test whether HSP60 might also exert a direct, innate effect on T cell physiology.

PRINCIPAL FINDINGS

1. HSP60 induces T cell adhesion to FN
We purified T cells from the peripheral blood of healthy human donors, fractioned the T cells into CD45RA⁺ and CD45RO⁺ subpopulations, incubated the T cells with various concentrations of human HSP60 for various times, and assayed adhesion of the T cells to immobilized ECM components: fibronectin (FN), laminin, or collagen type 1. We found that HSP60 did not affect cell viability but enhanced T cell adhesion specifically to FN; there was little or no effect on T cell adherence to laminin or collagen type 1. The nonactivated CD45RA⁺ population responded to relatively low concentrations of HSP60 (0.1–1.0 ng/mL). Adhesion decreased at concentrations of 10–100 ng/mL, then increased again at concentrations of 1000–5000 ng/mL. The nonactivated CD45RO⁺ population responded mostly to the higher concentrations (>100 ng/mL) of HSP60. Activation by IL-2, however, increased sensitivity to HSP60 of the CD45RO⁺ T cell population by 1000-fold. HSP60 was as effective as were optimal concentrations of the proinflammatory chemokines SDF-1 (100 ng/mL) or RANTES (100 ng/mL) or the cytokine IL-2 (100 IU/mL). Thus, HSP60 seems to induce T cell adhesion to FN to about the same degree as do other physiological signals for T cell adhesion.

2. HSP60 mediates adhesion via a TLR2 pathway
Figure 1 A shows that human T cells express TLR2 to a much greater extent than TLR4. Figure 1B shows that mAb anti-TLR4 did not affect HSP60-induced T cell adhesion to FN. To test whether TLR2 or TLR4 might be functionally involved in the activation of T cell adhesion by HSP60, we tested the effect of HSP60 on thymocytes taken from mice and Chinese hamsters. Mice of the C3H/HeJ strain harbor a point mutation in TLR4, rendering it nonfunctional, whereas the related C3HeB/FeJ strain has an intact TLR4 molecule. Figure 1C shows that mouse thymocytes of both strains responded to HSP60. Chinese hamsters carry a frameshift mutation in TLR2. Chinese hamster thymocytes adhered to FN in response to PMA and ConA (not shown), but showed no response to recombinant HSP60. These results are compatible with the conclusion that TLR2, and not TLR4, is important for T cell adhesion to FN induced by HSP60. Finally, Fig. 1D shows that the anti-TLR2 antibody specifically blocked HSP60-induced adherence to FN of purified human CD45RA⁺ T cells, but not adherence by SDF-1 or IL-2. Thus, the anti-TLR2 blocking was specific for HSP60-induced adherence.

View larger version (20K): [in this window] [in a new window]   Figure 1. TLR2, but not TLR4, is involved in HSP60-induced T cell adhesion. A) 5X106 CD45RA+ or CD45RO+ T cells, unseparated T cells, or PBMC were lysed and the level of TLR2 and TLR4 expression in the cells was determined by Western blot using specific mAb against TLR2 and TLR4. The SDS-PAGE profile shows a representative experiment of 3 that produced similar results. B) CD45RA+ T cells were pretreated with mAb anti-TLR2 or anti-TLR4 (20 µg/mL). Cells were treated with HSP60 (0.1 ng/mL) and percent adhesion to FN was determined. Mean ±SD of 4 independent experiments is shown. C) Thymocytes of wild-type C3HeB/FeJ, TLR4-mutated C3H/HeJ mice (>95% CD45RA+ T cells), or thymocytes of Chinese hamsters were radiolabeled, pretreated with HSP60, and seeded onto FN-coated microtiter wells. Mean ±SD of 3 different experiments is shown. D) Anti-TLR2 mAb treatment inhibits HSP60-induced adhesion of CD45RA+ T cells but not adhesion induced by SDF-1 or IL-2. T cells were treated with HSP60 (0.1 ng/mL), SDF-1 (100 ng/mL), or IL-2 (100 IU/mL) and tested for adhesion. Open bars: control mAb. Mean ±SD for 5 different experiments is shown.

3. HSP60-induced adhesion is not due to contaminating LPS
LPS is known to activate cells via both TLR4 and TLR2. However, our results using HSP60 could not be attributed to contaminating LPS: 1) LPS was not detectable in our preparation of human HSP60; 2) LPS itself activated T cell adhesion at µg/mL concentrations and was not active at the ng/mL concentrations we found effective for human HSP60; 3) polymyxin B interfered with LPS but not with HSP60, and boiling inhibited HSP60- but not LPS-induced adhesion; 4) a monoclonal antibody to HSP60 blocked HSP60- but not LPS-induced adhesion.

4. HSP60-induced T cell adhesion depends on ß1 integrins
HSP60-induced adhesion was significantly inhibited by monoclonal antibodies to VLA4, VLA5 (the FN-specific 4 and 5 chains of ß1 integrins), or CD29 (the common ß1 integrin chain); in contrast, control monoclonal antibodies to VLA2 (2ß1), VLA3 (3ß1), or CD44 did not affect HSP60-induced adhesion. HSP60-induced adhesion appears to involve PI-3 kinase and PKC signaling because it was inhibited by the compounds wortmannin and GF109203X, but not by pertussis toxin, which inhibits specifically G-protein-coupled signaling. A short treatment (15 min) of nonactivated CD45RA⁺ T cells with HSP60 (0.01–100 ng/mL) induced phosphorylation of Pyk-2. Thus, Pyk-2 phosphorylation accompanies the response of T cells to effective concentrations of HSP60.

5. HSP60 inhibits chemokine-induced T cell chemotaxis and actin polymerization
The ability of T cells to navigate through the ECM depends on combined signals mediated by proadhesive mediators like cytokines and chemoattractants associated with ECM glycoproteins. Using a 3-dimensional, ECM-like gel system designed to follow in real time the migration of individual leukocytes along chemotactic gradients in vitro, we found that treatment of T cells with a concentration of 0.1 ng/mL of HSP60 inhibited 66% of the T cells migrating toward SDF-1 or ELC (P<0.01). Thus, a concentration of HSP60 that induces adhesion also inhibits T cell migration toward SDF-1 within an ECM-like gel.

T cell adhesion to and migration through immobilized ECM ligands requires actin polymerization and subsequent cellular polarization. HSP60 inhibited actin polymerization triggered by SDF-1. Moreover, this inhibitory effect of HSP60 was itself inhibited by an anti-TLR2 mAb, but significantly less so by the isotype-matched anti-TLR4 monoclonal antibody. Hence, the effect of HSP60 on the rearrangement of the cytoskeleton and its associated signaling molecules also involves TLR2 signaling.

6. HSP60 inhibits CXCR4 and CCR7 expression
Inhibition of T cell migration to SDF-1 and ELC raised the question of the effect of HSP60 on the expression of chemokine receptors: CXCR4, which is involved in T cell chemotaxis toward SDF-1, and CCR7, which is involved in immune cell chemotaxis toward ELC. Accordingly, we incubated human T cells with various concentrations of HSP60 and assayed the expression of the chemokine receptors CXCR4 and CCR7 at various times. We found that 18 h of incubation sufficed to reduce specifically the expression of these receptors: 0.1 ng/mL of HSP60 down-regulated the expression of CXCR4 and CCR7 by 40–50% in CD45RA⁺ T cells. (Similar HSP60 treatment of CD45RA⁺ T cells did not affect the expression of LFA-1 or of L-selectin.) Down-regulation of CXCR4 on CD45RA⁺ T cells by HSP60 was blocked by mAb anti-TLR2, but not by anti-TLR4. The inhibitor of PKC signaling, GF109203X, also inhibited the effect of HSP60 on CXCR4 expression. Thus, the regulatory effect of HSP60 on CXCR4 requires TLR2 and PKC signaling.

DISCUSSION

TLR molecules are thought to constitute "pattern recognition receptors" directed to "pathogen-associated molecular patterns." Yet TLR4 and, as we find here, TLR2 are likely to be involved in signaling by self-HSP60. The present results, which relate to T cell behavior in vitro, call attention to the possibility that the innate T cell response to HSP60 might have functional consequences.

HSP60 seems to interact with diverse receptor molecules like TLR2 and TLR4. It has been shown that HSP70, gp96, and HSP90 interact with human macrophages via the CD91 and TLR4 molecules and thereby affect cytokine and chemokine secretion by these cells and their antigen presentation capacities. It is conceivable that molecules such as CD91, TLR2, and TLR4 are components of common signaling pathways activated by diverse ligands. The different ligands may bind directly to other molecules that trigger CD91 and TLR molecules further downstream. It appears that HSP60 requires CD14, TLR2, and TLR4 for signaling of macrophages, but HSP60 does not bind directly to these molecules. It is conceivable that HSP60 and other stress protein molecules activate TLR signaling by first forming complexes with other carrier molecules.

HSP60 expression is up-regulated during inflammation and HSP60 has been detected in soluble form in the blood in the ng–µg/mL range of concentrations; thus, HSP60 is accessible. Hence, soluble HSP60 may modulate the innate arm of the immune system by its effect on macrophages and, as we show here, by its direct effects on T cell physiology (Fig. 2 ). Based on the present results, low concentrations of HSP60 would be expected to impede the mobilization to an inflammatory site of "high-affinity" cells like the "naive" CD45RA⁺ T cell population. But low concentrations of HSP60 would not inhibit the chemotaxis of the nonactivated "memory" CD45RO⁺ T cell population. A high concentration of HSP60 at the inflammatory site might prevent the exit of the CD45RO⁺ T cells from the site by enhancing their adhesion to the ECM. Upon contact with T cell regulatory factors such as IL-2, the CD45RO⁺ T cells would acquire increased sensitivity to HSP60 signals. These possibilities are open for further study.

View larger version (52K): [in this window] [in a new window]   Figure 2. Schematic diagram.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1139fje; doi: 10.1096/fj.02-1139fje

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