Soluble 60 kDa heat shock protein (HSP60) activates macrophages via TLR4. We now report that soluble HSP60 activates T cells via the innate receptor TLR2. HSP60 activated T cell adhesion to fibronectin to a degree similar to other activators: IL-2, SDF-1alpha, and RANTES. T cell type and state of activation was important; nonactivated CD45RA+ and IL-2-activated CD45RO+ T cells responded optimally (1 h) at low concentrations (0.1-1 ng/ml), but nonactivated CD45RO+ T cells required higher concentrations (approximately 1 microg/ml) of HSP60. T cell HSP60 signaling was inhibited specifically by monoclonal antibodies (mAb) to TLR2 but not by a mAb to TLR4. Indeed, T cells from mice with mutated TLR4 could still respond to HSP60, whereas Chinese hamster T cells with mutated TLR2 did not respond. The human T cell response to soluble HSP60 depended on phosphatidylinositol 3-kinase and protein kinase C signaling and involved the phosphorylation of Pyk-2. Soluble HSP60 also inhibited actin polymerization and T cell chemotaxis through extracellular matrix-like gels toward the chemokines SDF-1alpha (CXCL12) or ELC (CCL19). Exposure to HSP60 for longer times (18 h) down-regulated chemokine receptor expression: CXCR4 and CCR7. These results suggest that soluble HSP60, through TLR2-dependent interactions, can regulate T cell behavior in inflammation.

The migration of immunocytes within the extracellular matrix (ECM) is influenced by the activation state of the incoming cell and its responses to the presence of chemokines and cytokines. We studied the regulatory role of TGF‐β1 on T cell homing to secondary lymphatic organs, such as the spleen, and chemotaxis within an ECM‐like environment in using an ECM‐like 3‐dimensional gel system designed to follow the migration of individual leukocytes along chemokine gradients in real time. The numbers of migrating naive, but not memory T cells toward SDF‐1α markedly increased after pre‐incubating the cells with TGF‐β1 (0.25 ng / ml) for 24 h. The mechanisms underlying TGFβ1‐modulated migration involve the up‐regulation of the expression of the SDF‐1α receptor CXCR4, the enhancement of the SDF‐1α‐induced actin polymerization, and increased phosphorylation of Pyk2, a focal adhesion kinase involved in integrin‐mediated lymphocyte migration, adhesion and interactions with ECM. Interestingly, priming of naive human T cells with TGF‐β1 increased homing of these cells to the spleen of NOD / SCID mice in a CXCR4‐dependent manner. We propose that theeffect of TGF‐β1 on the chemotaxis of naive T cells may be important in the locomotion of naive T cells toward SDF‐1α‐rich niches.

SUMMARYAlthough the involvement of transforming growth factor-b (TGF-b) in in¯ammatory reactions has been extensively studied, its mode of action in the context of the extracellular matrix (ECM) is still not fully understood. We undertook this study in an attempt to reveal the putative roles of TGF-b in T-cell adhesion and migration. We found that a 60-min treatment of T cells with TGF-b regulates T-cell adhesion to ®bronectin (FN), a prototype cell adhesion protein of the ECM, depending on the presence of other activators. At 5 pg/ml to 1 ng/ml, TGF-b alone induced T-cell adhesion to FN in an integrin a 4 /b 1 -and integrin a 5 /b 1 -dependent manner. TGF-b also attenuated T-cell migration on the stromal cell-derived factor (SDF)-1a gradients. These effects of TGF-b were not accompanied by alteration in the expression of very-late activation antigen type 4 (VLA-4) and VLA-5, nor were they mediated by the cyclo-oxygenase pathway. The cellular mechanism underlying the adhesion-regulating activities of TGF-b involves adhesion-associated cytoskeletal elements. TGF-b induced the phosphorylation of focal adhesion kinase Pyk2, but not extracellular signal-regulated kinase (ERK), and this effect was markedly increased in the presence of immobilized FN, suggesting a collaborative role for FN-speci®c integrins. Indeed, TGF-b-induced Pyk2 phosphorylation was inhibited by monoclonal antibodies against VLA-4, VLA-5 and CD29. Thus, TGF-b, which may appear at extravascular sites during in¯ammation, affects the adhesion of T cells to ECM glycoproteins and their migration by its ability to differentially induce or inhibit the phosphorylation of Pyk2.

On their extravasation from the vascular system into inflamed tissues, leukocytes must maneuver through a complex insoluble network of molecules termed the extracellular matrix (ECM). Leukocytes navigate toward their target sites by adhering to ECM glycoproteins and secreting degradative enzymes, while constantly orienting themselves in response to specific signals in their surroundings. Cytokines and chemokines are key biological mediators that provide such signals for cell navigation. Although the individual effects of various cytokines have been well characterized, it is becoming increasingly evident that the mixture of cytokines encountered in the ECM provides important combinatorial signals that influence cell behavior. Herein, we present an overview of previous and ongoing studies that have examined how leukocytes integrate signals from different combinations of cytokines that they encounter either simultaneously or sequentially within the ECM, to dynamically alter their navigational activities. For example, we describe our findings that tumor necrosis factor (TNF)-α acts as an adhesion-strengthening and stop signal for T cells migrating toward stromal cell-derived factor-1α, while transforming growth factor-β down-regulates TNF-α-induced matrix metalloproteinase-9 secretion by monocytes. These findings indicate the importance of how one cytokine, such as TNF-α, can transmit diverse signals to different subsets of leukocytes, depending on its combination with other cytokines, its concentration, and its time and sequence of exposure. The combinatorial effects of multiple cytokines thus affect leukocytes in a step-by-step manner, whereby cells react to cytokine signals in their immediate vicinity by altering their adhesiveness, directional movement, and remodeling of the ECM.