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Inhibitory control of TGF-ß1 on the activation of Rap1, CD11b, and transendothelial migration of leukocytes

Caroline Basoni^*,, Muriel Nobles, Andrew Grimshaw, Claude Desgranges^*, Derek Davies^||, Mauro Perretti^¶, IJsbrand M. Kramer^*,,1 and Elisabeth Genot^*,

^* U441 INSERM, Pessac, France;
Molecular and Cellular Biology Section, European Institute of Chemistry and Biology, Pessac, France;
Department of Medicine, Rayne Institute, University College London, London, UK;
Imperial College, Kennedy Institute, London, UK;
^|| Cancer Research UK, London, UK; and
^¶ Department of Biochemical Pharmacology, Barts and the London School of Medicine, London, UK

¹ Correspondence: European Institute of Chemistry and Biology, University of Bordeaux, C/o Avenue des Facultés, Talence 33405, France. E-mail: i.kramer{at}iecb.u-bordeaux.fr

SPECIFIC AIMS

The aims of the study were to assess 1) the role of TGF-ß1 in the regulation of expression and activation of the ß2 integrin CD11b (Mac-1) in differentiated U937 cells and in isolated human peripheral blood monocytes and 2) the effect of TGF-ß1 on CD11b-dependent transendothelial cell migration.

PRINCIPAL FINDINGS

This study focuses on the interrelationship between three molecules: CD11b, Rap1, and TGF-ß1. Each plays an important role in the regulation of trafficking of bloodborne cells (leukocytes). CD11b (also known as Mac-1 or CR3) is a member of the ß2-integrin family of cell surface adhesion molecules. It is composed of two subunits, and ß (Mß2), which are noncovalently linked. It plays an important role in the initiation of leukocyte adherence to and subsequent migration through vascular endothelial cells. In this process, CD11b has to be switched "on," an event that involves an increase in its affinity for its ligands. Activation is ensured by chemokines and certain bacterial products such as formyl-peptides or lipopolysaccharides (endotoxin). Activated CD11b binds ICAM-1, which is highly expressed on endothelial cells at sites of inflammation, and it binds to the tight junction protein JAM-3, thereby directing the process of transendothelial migration. It binds fibrinogen, thereby recruiting leukocytes into thrombi. Once in the tissues, CD11b takes a leading role in killing microorganisms because it binds complement factor C3bi, a vital event in the recognition of opsonized microorganisms. Moreover, its engagement facilitates the release of type-1 granules from neutrophils (containing, among others, elastase and myeloperoxidase). Its engagement also facilitates the activation of the respiratory burst, production of toxic oxygen metabolites, and regulates the survival time of these cells. Leukocytes lacking expression of the common ß2 chain fail to adhere to vascular endothelium and fail to fight bacterial infection (leukocyte adhesion deficiency-1 syndrome).

The monomeric GTPase Rap1, a close homologue of the transforming protein Ras, mediates activation of members of the ß2 family of integrins at the intracellular level. It follows that Rap1 plays a strategic role in the regulation of inflammation. A detailed study of what in turn regulates the activity of Rap1 could provide new insights into the pathways regulating the inflammatory response.

The third molecule, TGF-ß1, is a cytokine intimately linked with the regulation of inflammation. It is present in blood plasma in an active and inactive form (latent TGF-ß1). Only the active form is recognized by specific receptors. Lack of TGF-ß1 expression causes a lethal, tissue-wasting syndrome, as a consequence of increased expression of MHC-class II paralleled by an excessive infiltration of tissues by leukocytes. A link between aberrant leukocyte trafficking and TGF-ß1 has been provided by the observation that patients with advanced atherosclerosis, a disease characterized by the accumulation of lipid-loaded monocytes in the intimal layer of arteries, have a significantly reduced plasma level of active TGF-ß1.

In this study, we provide evidence that prolonged incubation of differentiated U937 cells or isolated human peripheral blood monocytes with TGF-ß1 causes a reduction in cell surface expression of CD11b and, more important, a reduction of its activation state, as measured by expression of the neo-activation epitope after treatment of the cells with phorbol ester (PMA) (Fig. 1 ). Among the other integrins tested (CD11a, CD11c, and the ß1 integrin VLA-4), neither expression nor activation was affected by TGF-ß1. With respect to U937 cells, an inhibitory effect was observed only when these cells were differentiated by a cocktail consisting of dihydroxy-VitD3, retinoic acid, and TGF-ß1.

View larger version (35K): [in this window] [in a new window]   Figure 1. A) Expression of ß1- and ß2-integrins and CD14 on differentiated U937 after further incubation in heat-inactivated human plasma in the presence of various concentrations of TGF-ß1. Cell surface expression of integrins and CD14 was measured using flow cytometry. Note a significant reduction in CD11b expression on cells cultured in medium containing 120 pM of TGF-ß1 (n=6, P<0.05). B) Expression of the activation neo-isotope of CD11b after a 10 min treatment with PMA on differentiated U937 that had been incubated for 3 days in medium containing heat-inactivated human plasma (8.8 pM TGF-ß baseline concentration) or incubated for 3 days in the same medium but supplemented with TGF-ß1 (120 pM final concentration). TGF-ß1 strongly reduces the expression of the activation neo-epitope (n=4, P<0.05).

Under conditions where an inhibition of the activation of CD11b was observed, TGF-ß1 prevented the activation of the monomeric GTPase Rap1 by PMA or by the chemokine MIP-1. Similar results were obtained with human peripheral blood monocytes incubated for 40 h in the absence or presence of TGF-ß1 and treated with chemokines MCP-1 or MIP-1. By ectopic expression of a Rap1-specific GTPase activating protein (Spa-1) or a constitutively active mutant of Rap1 (V12Rap1), we show that this monomeric GTPase is instrumental in the regulation of the activation state of CD11b in differentiated U937 cells; expression of Spa-1 prevents PMA or MIP-1 mediated activation, whereas expression of V12Rap1 causes activation of CD11b, without having an effect on the expression level of the adhesion molecule.

We demonstrate that this lack of activation is the consequence of a reduced functional expression of the cAMP-sensitive nucleotide exchange factor Epac and this in turn seems to be the consequence of a TGF-ß1-mediated reduction in expression of the Epac transcript (Fig. 2 ). We could not detect changes in the expression of Spa-1 or Rap1GAP, other regulators of GTP loading of the Rap1 GTPase.

View larger version (80K): [in this window] [in a new window]   Figure 2. Expression of Epac in U937 cells under different culture conditions. A)Expression levels were assessed indirectly by evaluation of 8-pCPT-2'-O-Me-cAMP (8pCPT) mediated activation of Rap1. Nondifferentiated cells show a weak presence, as indicated by a weak GTP loading of Rap1, differentiated cells kept in culture show a strong level which is lost if the medium is supplemented with TGF-ß1 (final concentration 120 pM) (n=4). B)Expression of Epac mRNA in U937 cells kept under different culture conditions. Expression was assayed in an RT-PCR protocol using Epac-specific primers. Sample cDNA content was verified by assaying the presence of the GAPDH transcript. Prolonged treatment with TGF-ß1 causes a loss of expression of the Epac transcript to levels of undifferentiated cells (n=3). C) Treatment of peripheral blood monocytes for 40 h with TGF-ß1 (final concentration 120 pM) causes a reduction in the functional expression of Epac as measured by 8-pCPT-2'-O-Me-cAMP-mediated GTP loading of Rap1. A reduction of expression of Rap1 is observed but the level of diminution is highly donor dependent.

Finally, inhibition of activation of CD11b and Rap1 was paralleled by a strong inhibition of CD11b-dependent transendothelial migration, of cells treated with chemokines, as tested in a porous filter insert protocol (Transwell).

CONCLUSIONS AND SIGNIFICANCE

We conclude that with respect to monocytes, TGF-ß1 exerts an inhibitory effect on the activation of Rap1 and on the expression and activation of the ß2 integrin CD11b and that this inhibition is accompanied by a reduction in transendothelial migration (Fig. 3 ). In other words, TGF-ß1 may exert a general protective influence against aberrant transendothelial migration and thereby hold in check the inflammatory response. In the context of atherosclerosis, our study provides evidence at the molecular level why aberrant low plasma levels of active TGF-ß1 (l<8 pM) might allow accumulation of monocytes in the intimal layer of arteries and hence favor the development of vascular lesions. Alternatively, sufficiently high levels of TGF-ß1 could exert a general protective action. This was observed in a mouse model of atherosclerosis where treatment with tamoxifen, which increases the fraction of active TGF-ß1, resulted in a reduction of fatty streak formation compared with nontreated mice. Once atherosclerotic lesions have been formed, TGF-ß1 may have an additional protective effect by preventing their transition into unstable plaques due to its immunosuppressive and profibrotic action.

View larger version (26K): [in this window] [in a new window]   Figure 3. Schematic diagram.

Given these observations, it would be interesting to relate the expression of Epac and of CD11b and its activation neo-epitope in monocytes with the severity of the atherosclerotic lesions (and the concentration of active TGF-ß1 in their plasma). The outcome of this study would provide additional evidence for a putative inhibitory role of TGF-ß1 in regulating the state of activation of this integrin and provide important clues for further studies concerning the abnormal accumulation of monocytes in the arterial intima.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3085fje;

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