T-cell regulation through a basic suppressive mechanism targeting low-density lipoprotein receptor-related protein 1.

Cell adhesion is generally considered to depend on positive regulation through ligation of integrins and cytokine receptors. However, here we show that T-cell adhesion, and notably also T-cell receptor (TCR) -induced activation, are subject to constant suppression through shedding of low-density lipoprotein receptor-related protein 1 (LRP1). The broad-spectrum metalloprotease inhibitor GM6001 abrogated shedding, so inducing prominent cell surface expression of LRP1 while enhancing TCR-induced activation and adhesion to ß1 and ß2 integrin ligands, hence arresting the cells. Integrin ligands also inhibited shedding but the effect was less potent than that of GM6001. Unlike GM6001, integrin ligands also induced cell surface expression of full-length thrombospondin-1 (TSP170) and TSP130, which associated with LRP1, and TSP110, which did not associate with LRP1. Cell surface expression of LRP1 and TSP130 were induced exclusively in adhering cells, expression of TSP110 preferentially in non-adhering cells and expression of TSP170 correlated with T-cell motility. The pro-adhesive chemokine CXCL12 also inhibited LRP1 shedding and induced surface expression of TSP170 and TSP130 while inhibiting TSP110. Exogenous TSP-1 and ligation of CD28 inhibited shedding although less effectively than GM6001, and the inhibition through CD28 was independent of TSP-1. Small interfering RNA silencing experiments confirmed involvement of LRP1 and TSP-1 in integrin-dependent adhesion and TCR-induced activation. Hence, the poor LRP1 expression in T cells depends on shedding. Integrin ligands and CXCL12 antagonize shedding through a TSP-1-dependent pathway and ligation of CD28 antagonizes shedding independent of TSP-1. The disappearance of LRP1 from the cell surface may provide basic immunosuppression at the T-cell level.

Methotrexate and its therapeutic antagonists caffeine and theophylline, target a motogenic T-cell mechanism driven by thrombospondin-1 (TSP-1).

Methotrexate (MTX) is a widely used treatment for inflammatory diseases such as rheumatoid arthritis and psoriasis, based on the concept that it is immunosuppressive. Its mechanism of action, however, remains unclear, although it is thought to depend on adenosine. Caffeine and theophylline, which have several targets including adenosine receptors, have been shown to suppress the beneficial clinical effects of MTX. Here we show that MTX and caffeine and theophylline differentially affect a motogenic T-cell mechanism driven by endogenous thrombospondin-1 (TSP-1) and its receptor, low density lipoprotein receptor-related protein 1 (LRP1). MTX stimulated TSP-1 expression and the motogenic TSP-1/TSP-1 receptor mechanism in primary human T cells, hence mimicking IL-2 and CXCL12, which similar to MTX, dampen inflammatory disease. SiRNA-mediated gene silencing of TSP-1 and LRP1 inhibited this stimulatory effect. Caffeine and theophylline inhibited the TSP-1/TSP-1 receptor mechanism by inhibiting LRP1 expression. These results indicate that the effect of MTX on T cells is immunoregulatory rather than immunosuppressive, and suggest a pathway dependent on TSP-1/TSP-1 receptor interactions for the regulation of immune responses.

Antigen-induced regulation of T-cell motility, interaction with antigen-presenting cells and activation through endogenous thrombospondin-1 and its receptors.

Antigen recognition reduces T-cell motility, and induces prolonged contact with antigen-presenting cells and activation through mechanisms that remain unclear. Here we show that the T-cell receptor (TCR) and CD28 regulate T-cell motility, contact with antigen-presenting cells and activation through endogenous thrombospondin-1 (TSP-1) and its receptors low-density lipoprotein receptor-related protein 1 (LRP1), calreticulin and CD47. Antigen stimulation induced a prominent up-regulation of TSP-1 expression, and transiently increased and subsequently decreased LRP1 expression whereas calreticulin was unaffected. This antigen-induced TSP-1/LRP1 response down-regulated a motogenic mechanism directed by LRP1-mediated processing of TSP-1 in cis within the same plasma membrane while promoting contact with antigen-presenting cells and activation through cis interaction of the C-terminal domain of TSP-1 with CD47 in response to N-terminal TSP-1 triggering by calreticulin. The antigen-induced TSP-1/LRP1 response maintained a reduced but significant motility level in activated cells. Blocking CD28 co-stimulation abrogated LRP1 and TSP-1 expression and motility. TCR/CD3 ligation alone enhanced TSP-1 expression whereas CD28 ligation alone enhanced LRP1 expression. Silencing of TSP-1 inhibited T-cell conjugation to antigen-presenting cells and T helper type 1 (Th1) and Th2 cytokine responses. The Th1 response enhanced motility and increased TSP-1 expression through interleukin-2, whereas the Th2 response weakened motility and reduced LRP1 expression through interleukin-4. Ligation of the TCR and CD28 therefore elicits a TSP-1/LRP1 response that stimulates prolonged contact with antigen-presenting cells and, although down-regulating motility, maintains a significant motility level to allow serial contacts and activation. Th1 and Th2 cytokine responses differentially regulate T-cell expression of TSP-1 and LRP1 and motility.

Regulation of T-lymphocyte motility, adhesion and de-adhesion by a cell surface mechanism directed by low density lipoprotein receptor-related protein 1 and endogenous thrombospondin-1.

T lymphocytes are highly motile and constantly reposition themselves between a free-floating vascular state, transient adhesion and migration in tissues. The regulation behind this unique dynamic behaviour remains unclear. Here we show that T cells have a cell surface mechanism for integrated regulation of motility and adhesion and that integrin ligands and CXCL12/SDF-1 influence motility and adhesion through this mechanism. Targeting cell surface-expressed low-density lipoprotein receptor-related protein 1 (LRP1) with an antibody, or blocking transport of LRP1 to the cell surface, perturbed the cell surface distribution of endogenous thrombospondin-1 (TSP-1) while inhibiting motility and potentiating cytoplasmic spreading on intercellular adhesion molecule 1 (ICAM-1) and fibronectin. Integrin ligands and CXCL12 stimulated motility and enhanced cell surface expression of LRP1, intact TSP-1 and a 130,000 MW TSP-1 fragment while preventing formation of a de-adhesion-coupled 110 000 MW TSP-1 fragment. The appearance of the 130 000 MW TSP-1 fragment was inhibited by the antibody that targeted LRP1 expression, inhibited motility and enhanced spreading. The TSP-1 binding site in the LRP1-associated protein, calreticulin, stimulated adhesion to ICAM-1 through intact TSP-1 and CD47. Shear flow enhanced cell surface expression of intact TSP-1. Hence, chemokines and integrin ligands up-regulate a dominant motogenic pathway through LRP1 and TSP-1 cleavage and activate an associated adhesion pathway through the LRP1-calreticulin complex, intact TSP-1 and CD47. This regulation of T-cell motility and adhesion makes pro-adhesive stimuli favour motile responses, which may explain why T cells prioritize movement before permanent adhesion.

A cytokine-controlled mechanism for integrated regulation of T-lymphocyte motility, adhesion and activation.

The co-ordination of T-cell motility, adhesion and activation remains poorly understood. It is also unclear how these functions are co-ordinated with external stimuli. Here we unveil a series of molecular interactions in cis at the surface of T lymphocytes with potent effects on motility and adhesion in these cells, and communicating with proliferative responses. These interactions were controlled by the signature cytokines of T helper subsets interleukin-2 (IL-2) and IL-4. Low-density lipoprotein receptor-related protein 1 (LRP1) was found to play a key role for T-cell motility by promoting development of polarized cell shape and cell movement. Endogenous thrombospondin-1 (TSP-1) enhanced cell surface expression of LRP1 through CD47. Cell surface expressed LRP1 induced motility and processing of TSP-1 while inhibiting adhesion to intercellular adhesion molecule 1 and fibronectin. Interleukin-2, but not IL-4, stimulated synthesis of TSP-1 and motility through TSP-1 and LRP1. Stimulation of the T-cell receptor (TCR)/CD3 complex inhibited TSP-1 expression. Inhibitor studies indicated that LRP1 regulated TSP-1 expression and promoted motility through JAK signalling. This LRP1-mediated motogenic signalling was connected to CD47/Gi protein signalling and IL-2-induced signalling through TSP-1. The motogenic TSP-1/LRP1 mechanism antagonized TCR/CD3-induced T-cell proliferation. These results indicate that LRP1 in collaboration with TSP-1 directs a counter-adhesive and counter-proliferative motogenic cascade. T cells seem programmed to prioritize movement before adhesion through this cascade. In conclusion, vital decision-making in T lymphocytes regulating motility, adhesive interactions and proliferation, are integrated through a molecular mechanism connecting different cell surface receptors and their signalling pathways.