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Originally published online as doi:10.1189/jlb.0807551 on January 25, 2008

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(Journal of Leukocyte Biology. 2008;83:921-927.)
© 2008 by Society for Leukocyte Biology

Stimulated T cells generate microparticles, which mimic cellular contact activation of human monocytes: differential regulation of pro- and anti-inflammatory cytokine production by high-density lipoproteins

Anna Scanu1, Nicolas Molnarfi, Karim J. Brandt, Lyssia Gruaz, Jean-Michel Dayer and Danielle Burger2

Division of Immunology and Allergy, Clinical Immunology Unit, Department of Internal Medicine, University Hospital and School of Medicine, Geneva, Switzerland

2 Correspondence: Clinical Immunology Unit, BB-4866, University Hospital, 24 rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland. E-mail: danielle.burger{at}hcuge.ch


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ABSTRACT
 
Imbalance in cytokine homeostasis plays an important part in the pathogenesis of chronic inflammatory diseases such as multiple sclerosis and rheumatoid arthritis. We demonstrated that T cells might exert a pathological effect through direct cellular contact with human monocytes/macrophages, inducing a massive up-regulation of the prototypical proinflammatory cytokines IL-1β and TNF. This mechanism that might be implicated in chronic inflammation is specifically inhibited by high-density lipoproteins (HDL). Like many other stimuli, besides proinflammatory cytokines, the contact-mediated activation of monocytes induces the production of cytokine inhibitors such as the secreted form of the IL-1 receptor antagonist (sIL-1Ra). The present study demonstrates that stimulated T cells generate microparticles (MP) that induce the production of TNF, IL-1β, and sIL-1Ra in human monocytes; the production of TNF and IL-1β but not that of sIL-1Ra is inhibited in the presence of HDL. The results were similar when monocytes were stimulated by whole membranes of T cells or soluble extracts of the latter. This suggests that MP carry similar monocyte-activating factors to cells from which they originate. Thus, by releasing MP, T cells might convey surface molecules similar to those involved in the activation of monocytes by cellular contact. By extension, MP might affect the activity of cells, which are usually not in direct contact with T cells at the inflammatory site. Furthermore, this study demonstrates that HDL exert an anti-inflammatory effect in nonseptic activation of human monocytes, not only by inhibiting the production of IL-1β and TNF but also, by leaving sIL-1Ra production unchanged.

Key Words: IL-1β • TNF • IL-1Ra • inflammation • acute-phase reactants


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INTRODUCTION
 
Imbalance in cytokine homeostasis plays an important part in the pathogenesis of chronic inflammatory diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA). This suggests that the mechanisms ruling the production of proinflammatory cytokines, including TNF and IL-1β and their inhibitors, i.e., soluble receptors and secreted IL-1 receptor antagonist (sIL-1Ra), escape normal controls. TNF and IL-1β are mainly produced upon activation of monocytes/macrophages. In immunoinflammatory diseases, in the absence of an infectious agent (i.e., in nonseptic conditions), the nature of the factors triggering TNF and IL-1β production is still elusive. We previously demonstrated that direct cellular contact with stimulated T cells induces the massive up-regulation of IL-1 and TNF in monocytes/macrophages [1 ]. Besides triggering proinflammatory cytokine production, contact-mediated activation of monocytes induced the production and/or shedding of cytokine inhibitors such as sIL-1Ra and soluble receptors of IL-1 and TNF [2 3 4 5 ]. Most T cell types, including T cell clones, freshly isolated T lymphocytes, and T cell lines such as HUT-78 cells, induce IL-1 and TNF in monocytes/macrophages [6 ]. Furthermore, depending on T cell type and T cell stimulus, direct cell–cell contact with stimulated T lymphocytes can induce different patterns of products in monocytes/macrophages, as reviewed in refs. [1 , 7 , 8 ], suggesting that multiple ligands and counter-ligands are involved in the contact-mediated activation of monocytes/macrophages. In some cases, an imbalance in the production of proinflammatory versus anti-inflammatory cytokines has been observed. Indeed, TH1 cell clones preferentially induce IL-1β rather than sIL-1Ra production, and cytokine-stimulated T lymphocytes induce TNF production and fail to trigger that of IL-10 [4 , 9 ].

The production of IL-1β and TNF by monocytes upon contact activation by stimulated T cells is inhibited by high-density lipoprotein (HDL)-associated apolipoprotein A-I (apo A-I) [10 ], a "negative", acute-phase protein. In contact-mediated activation of monocytes, HDL-associated apo A-I exerts most of its inhibitory activity by interfering with monocyte-activating factors at the surface of T cells, thus decreasing the production of IL-1β and TNF [10 ]. In vivo, apo A-I is retained in the perivascular regions of RA-inflamed synovium, i.e., in T cell-rich regions [11 ].

Upon chronic inflammation, after extravasation, most T cells remain in the perivascular region, and other infiltrating cells, such as monocytes/macrophages and neutrophils, have to cross the perivascular layer of T cells and in turn, make contact with the latter cells before penetrating further into the target tissue. Consequently, direct cell–cell contact with T cells is less frequent outside perivascular regions. However, cells can disseminate cell surface molecules by generating microparticles (MP) and thus, ensure "distant" cellular contact. MP are fragments (0.1–0.8 µm diameter) shed from the plasma membrane of stimulated or apoptotic cells. As MP lose membrane polarity, phosphatidylserine residues are exposed at their surface, which in turn, can bind Annexin V [12 ]. Having long been considered inert debris reflecting cellular activation or damage, MP are now acknowledged as cellular effectors involved in cell–cell crosstalk [13 , 14 ]. Indeed, they harbor membrane proteins as well as bioactive lipids implicated in a variety of fundamental processes and thus, constitute a disseminated pool of bioactive effectors [15 ]. MP are found in the circulation of healthy subjects [16 17 18 ], but their numbers are increased in various pathological conditions, such as thrombotic or infectious diseases [17 , 19 20 21 22 23 ]. Elevated MP have also been reported in chronic inflammatory diseases [12 , 24 , 25 ], including RA [26 27 28 29 ] and MS [25 , 30 31 32 33 ]. In MS, MP are present in patients’ plasma, although cerebrospinal fluid has, to our knowledge, not been investigated for the presence of MP. In RA synovial fluid, MP are abundant and modulate fibroblast-like synoviocyte activity in vitro [28 , 29 ]. As most cell types can generate MP [29 , 34 ], the latter process might represent a way for T cells to disseminate cellular contact from a distance. This study was undertaken to assess whether MP generated by stimulated T cells could induce cytokine production in human monocytes and whether the latter process would be modulated by HDL.


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MATERIALS AND METHODS
 
Materials
FCS, streptomycin, penicillin, L-glutamine, RPMI-1640, PBS free of Ca2+ and Mg2+, and TRIzolTM reagent (Gibco, Paisley, Scotland); purified PHA (EY Laboratories, San Marco, CA, USA); Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden); PMA, PMSF, polymyxin B sulfate, TriReagent, and BSA (Sigma Chemical Co., St. Louis, MO, USA); and Annexin V-FITC (BD Biosciences, San Jose, CA, USA) were purchased from the designated suppliers. Other reagents were of analytical grade or better.

Monocytes
Peripheral blood monocytes were isolated from buffy coats of blood of healthy volunteers as described previously [10 ]. To avoid activation by endotoxin, polymyxin B (2 µg/ml) was added to all solutions during the isolation procedure.

Isolation of peripheral blood T lymphocytes
Peripheral blood T lymphocytes were prepared as described previously [35 ]. T lymphocytes (4x106 cells/ml) were stimulated or not for 48 h with PHA (1 µg/ml) and PMA (5 ng/ml), as described [36 ]. MP were isolated from cell supernatants as described below. Proteins were determined by the method of Bradford [37 ].

T cell stimulation and membrane isolation
The human T cell line HUT-78 was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated FCS, 50 µg/ml streptomycin, 50 IU/ml penicillin, and 2 mM L-glutamine in a 5% CO2 air-humidified atmosphere at 37°C. HUT-78 cells (2x106 cells/ml) were stimulated for 6 h by PHA (1 µg/ml) and PMA (5 ng/ml). Plasma membranes of stimulated (msHUT) or unstimulated (musHUT) HUT-78 cells were prepared as described previously [38 ]. Isolated membranes were solubilized in 8 mM CHAPS as described previously [39 ]. CHAPS extracts of msHUT and musHUT were referred to as CEsHUT and CEusHUT, respectively. Proteins were measured by the method of Bradford [37 ].

Isolation of MP
MP were isolated from culture supernatants of HUT-78 cells and peripheral blood T lymphocytes. Briefly, cell supernatant was obtained after cell removal by centrifugation at 800 g for 5 min. Supernatant was then centrifuged at 7000 g for 5 min to discard large debris. After additional centrifugation at 20,000 g for 45 min, MP were washed twice in PBS, resuspended in PBS, and measured for protein content [37 ]. Annexin V-FITC binding and MP concentration (number) were determined by flow cytometry (FACSCalibur, BD Biosciences), according to ref. [12 ]. An event discrimination threshold was set on the side-scatter channel at the lowest channel allowed, and a size gate was set using 0.8 µm latex beads (Sigma Chemical Co.). RNA and DNA content in MP was estimated by measuring OD at 260 nm after isolation with TriReagent as described by the supplier [39 ]. When kept at 4°C upon sterile conditions in PBS, isolated MP were stable for at least 3 weeks in terms of their ability to induce cytokine production in human monocytes.

Isolation of HDL
Human serum HDL were isolated according to Havel et al. [40 ] as described previously [10 ]. Proteins were measured by the method of Bradford [37 ]. To optimize the inhibitory effect of HDL, they were always added together with the stimulus.

Cytokine production and measurement
Monocytes (5x104 cells/well/200 µl) were activated with the indicated stimulus in RPMI-1640 medium supplemented with 10% heat-inactivated FCS, 50 µg/ml streptomycin, 50 U/ml penicillin, 2 mM L-glutamine, and 5 µg/ml polymyxin B sulfate (medium) in 96-well plates and cultured for 48 h unless stated otherwise. The production of sIL-1Ra, IL-1β, and TNF was measured in culture supernatants by a commercially available enzyme immunoassay: IL-1β (Immunotech, Marseille, France), TNF, and sIL-1Ra (Quantikine, R&D Systems, Minneapolis, MN, USA).

mRNA quantification
Monocytes (2.1x106 cells/well/3 ml) were cultured in six-well plates with the indicated stimulus for 3 h. Total RNA was isolated and analyzed by real-time PCR as described previously [39 ].


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RESULTS
 
Characterization of MP generated by T cells
MP generated by HUT-78 cells and isolated from cell culture supernatant were subjected to flow cytometry analysis. MP from stimulated HUT-78 and unstimulated HUT-78 cells displayed similar size characteristics with diameters between 0.1 and 0.8 µm. MP bound Annexin V-FITC (Fig. 1 ), demonstrating that phosphatidylserine was exposed at their surface. Stimulated HUT-78 cells produced 11.5 ± 2.3 x 106 MP/106 cells, whereas unstimulated HUT-78 cells produced 0.07 ± 0.03 x 106 MP/106 cells; i.e., approximately 200-fold less MP were produced by unstimulated HUT-78 as compared with stimulated HUT-78 cells. Similar results were obtained with peripheral blood T lymphocytes. Indeed, stimulated T lymphocytes generated 1.6 ± 0.4 x 106 MP/106 cells, whereas unstimulated T lymphocytes generated 0.03 ± 0.02 x 106 MP/106 cells. Comparable amounts of proteins were measured in MP from stimulated HUT-78 and unstimulated HUT-78 cells and peripheral blood T lymphocytes reaching 19.6 ± 4.8 µg protein/106 MP. Total RNA in MP generated by stimulated HUT-78 cells reached 34.3 ± 15.5 µg/mg proteins, i.e., 0.7 ± 0.3 µg RNA/106 MP. This suggests that MP were indeed closed vesicles able to protect RNA from degradation by RNases. IL-1β and IL-1Ra were not detected in MP produced by stimulated HUT-78 or unstimulated HUT-78 cells. TNF, which is present in membranes isolated from HUT-78 cells [36 ], was detected in MP from stimulated HUT-78 and unstimulated HUT-78 cells, reaching 455 ± 37 and 267 ± 45 pg/mg proteins, respectively. Furthermore, DNA was below the detection limit, thus amounting to <3 ng/mg proteins in MP from stimulated HUT-78 and unstimulated HUT-78 cells. This demonstrates that only few apoptotic bodies were present amongst MP generated by stimulated T cells.


Figure 1
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  		Figure 1. Phosphatidylserine residues are exposed at the surface of MP generated by T cells. MP generated by msHUT-78 cells were labeled or not with Annexin V-FITC and analyzed by flow cytometry. Fluorescence intensity (x-axis) versus MP count (y-axis) is shown. Open histogram, MP that were not incubated with Annexin V-FITC; filled histogram, MP that bind Annexin V-FITC.

MP from stimulated T lymphocytes induce monocytic cytokine production that is modulated by HDL
To assess whether MP would induce cytokine production, human monocytes were activated by increasing doses of MP from stimulated and unstimulated T lymphocytes in the presence or absence of an optimal concentration of HDL. As shown in Figure 2 , MP generated by stimulated T cells induced the production of TNF, IL-1β, and sIL-1Ra in human monocytes in a dose-dependent manner. In contrast, MP generated by unstimulated T lymphocytes did not affect cytokine production. The production of IL-1β and TNF induced by MP from stimulated T lymphocytes was inhibited in the presence of HDL, whereas the production of sIL-1Ra remained unchanged. This suggests that MP carry activating factors that induce the production of cytokines in human monocytes. As the generation of MP by T lymphocytes from human peripheral blood in sufficient amounts to activate monocytes required a large number of T lymphocytes, the monocytic cell line HUT-78 cell was used to further study cytokine induction in human monocytes by MP from stimulated T cells.


Figure 2
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  		Figure 2. MP generated by stimulated peripheral blood T lymphocytes display a cytokine-inducing capacity that is differentially affected by HDL. Monocytes (5x104 cells/200 µl/well; 96-well plates) were activated for 48 h with increasing doses of MP isolated from stimulated (circles) or unstimulated (triangles) T lymphocyte culture supernatants. Cells were activated in the presence ({circ}) or absence (•) of 0.2 mg/ml HDL. TNF, IL-1β, and sIL-1Ra were measured in culture supernatants of triplicates and represented as mean ± SD. Results are representative of three different experiments.

To ascertain that the monocyte-activating capacity of MP would be similar to that of T cell plasma membranes, i.e., cellular contact, MP and plasma membranes were isolated from the same HUT-78 cells that had been stimulated for 6 h (i.e., a period that had previously been tested for optimal contact-activating capacity). As shown in Figure 3A , MP generated by stimulated HUT-78 cells induced the production of cytokines in human monocytes to a similar extent as MP from stimulated T lymphocytes. MP-induced production of sIL-1Ra was not affected in the presence of HDL, whereas that of IL-1β and TNF was inhibited (Fig. 3A) . Comparable results were obtained when stimulated HUT was used as a stimulus (Fig. 3B) . Here again, the production of sIL-1Ra was not affected by HDL, whereas that of IL-1β and TNF, serving as controls, was strongly inhibited. Similar levels of cytokines were induced by MP and msHUT, although the inhibition of TNF and IL-1β production by HDL was more efficient when monocytes were activated by msHUT than by MP (Fig. 3A and 3B) . This might be a result of the differential accessibility of activating factors in plasma membrane preparation and MP. Together, these results imply that similar monocyte-activating factors were present at the surface of MP and the cells by which they were generated.


Figure 3
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  		Figure 3. MP generated by stimulated HUT-78 cells display a similar cytokine-inducing capacity to whole plasma membrane preparation. Monocytes (5x104 cells/200 µl/well; 96-well plates) were activated for 24 h with increasing doses of MP isolated from stimulated HUT-78 cell culture supernatants (A) or membranes isolated from the same HUT-78 cells (B). Cells were activated in the presence ({circ}) or absence (•) of 0.2 mg/ml HDL. The production of sIL-1Ra, IL-1β, and TNF was measured in cell supernatants of triplicate cultures. Results are presented as percentage of cytokine production. (A and B) Representative experiments out of three are presented.

HDL did not inhibit contact-mediated induction of sIL-1Ra in monocytes
We previously demonstrated that HDL inhibit the production of proinflammatory cytokines induced by membranes isolated from stimulated T lymphocytes or msHUT-78 cells, but the effect of HDL on sIL-1Ra production has not been assessed so far. As shown in Figure 4 , msHUT but not musHUT significantly induced the production of TNF, IL-1β, and sIL-1Ra in isolated blood monocytes. The production of sIL-1Ra remained unchanged, whereas that of proinflammatory cytokines used as controls was inhibited by HDL (Fig. 4) . We previously demonstrated that the effect of membranes isolated from stimulated T lymphocytes or stimulated HUT-78 cells was not a result of their particulate form, as a similar cytokine-inducing ability was observed when soluble extract of membranes was used to activate monocytes. We thus assessed the effect of HDL on cytokine production induced by soluble extracts from membranes of HUT-78 cells. As shown in Figure 4 , solubilized membranes of HUT-78 (CEsHUT and CEusHUT) displayed similar activity to whole membranes, although CEsHUT tended to induce four- to tenfold more IL-1β and two- to threefold less sIL-1Ra than msHUT, depending on monocyte preparation, i.e., depending on individual blood donors. HDL did not inhibit sIL-1Ra production induced by CEsHUT on monocytes but decreased the production of TNF and IL-1β (Fig. 4) . These results demonstrate that HDL display anti-inflammatory activity by affecting pro- and anti-inflammatory cytokine balance.


Figure 4
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  		Figure 4. HDL do not inhibit sIL-1Ra production in human monocytes upon contact-mediated activation by stimulated T cells. Isolated human blood monocytes (5x104 cells/200 µl/well; 96-well plates) were activated for 24 h with msHUT (6 µg/ml), musHUT (6 µg/ml), CEsHUT (7 µg/ml), and CEusHUT (7 µg/ml), as indicated, in the presence (open bars) or absence (hatched bars) of HDL (0.2 mg/ml proteins). sIL-1Ra, IL-1β, and TNF were measured in culture supernatants of triplicates and represented as mean ± SD. Results are representative of three different experiments.

HDL do not inhibit the expression of sIL-1Ra mRNA in contact-activated monocytes
HDL inhibit T cell contact-mediated induction of IL-1β and TNF at the transcriptional level. As cytokine production is controlled at several levels, the effect of HDL on sIL-1Ra mRNA expression was assessed to rule out the possibility that a putative inhibition of sIL-1Ra expression was compensated by the release of intracellular pools of IL-1Ra. As shown in Figure 5A , HDL did not inhibit sIL-1Ra mRNA expression in CEsHUT-activated monocytes, whereas IL-1β and TNF mRNA expression was inhibited. That the lack of sIL-1Ra inhibition was not a result of the release of intracellular pools was further confirmed, as CEsHUT-induced, cell-associated IL-1Ra levels were not diminished in the presence of HDL. The measurement of IL-1β and TNF production (assessed as controls) showed that the induction of both cell-associated and secreted IL-1β and TNF was inhibited by HDL (Fig. 5B and 5C) . These results suggest that sIL-1Ra was induced in monocytes by factor(s) whose activity is not affected by HDL.


Figure 5
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  		Figure 5. Expression of sIL-1Ra mRNA is not affected by HDL. (A) Isolated monocytes (2x106 cells/well/3 ml; six-well plates) were activated or not by CEsHUT (7 µg/ml) in the presence or absence of 0.2 mg/ml HDL. After 3 h, cells were harvested, and total RNA was analyzed by real-time PCR (see Materials and Methods). Results are presented as mean ± SD of three different experiments, 100% being considered as the level of cytokine mRNA induced by CEsHUT in the absence of HDL. (B and C) Monocytes (5x104 cells/200 µl/well; 96-well plates) were activated by CEsHUT in the presence or absence of 0.2 mg/ml HDL. After 24 h, the supernatants were harvested and monocytes solubilized in 200 µl PBS containing 1% Nonidet P-40. Cyokines were measured in culture supernatants (B) and cell lysate (C) to determine the production of secreted and cell-associated cytokine, respectively. Results are presented as mean ± SD of three different experiments, 100% being considered as the level of cytokine induced by CEsHUT in the absence of HDL.


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DISCUSSION
 
The present study demonstrates that stimulated T cells generate MP, which mimic the activation of human monocytes mediated by cellular contact. The fact that HDL display similar effects on cytokine production when monocytes were activated by MP, msHUT, or CEsHUT—i.e., they inhibited the production of IL-1β and TNF without affecting that of sIL-1Ra—suggests that similar activating factors are present on T cell surface and the MP they generate. Furthermore, the fact that HDL inhibit proinflammatory cytokine production while leaving the production of sIL-1Ra unchanged stresses the anti-inflammatory properties of HDL [8 ].

Our previous studies have shown that direct contact with stimulated T lymphocytes is an important pathway to induce proinflammatory cytokines and their inhibitors in human monocytes and that this mechanism might account for the unbalanced production of cytokines that drives uncontrolled inflammation in chronic/sterile inflammatory diseases such as MS and RA [1 , 7 , 8 , 41 ]. MP from stimulated HUT-78 cells and peripheral blood T lymphocytes display comparable activity with isolated cell membranes, suggesting that similar molecules are present in MP and at the surface of stimulated T cells. Thus, in addition to adjacent monocytes/macrophages, stimulated T cells could make contact with distant monocytes/macrophages (e.g., macrophage-like synoviocytes in RA pannus-lining layer) through the release of MP. Furthermore, as contact with T cell plasma membranes or fixed, stimulated T cells triggers the production of pro- and anti-inflammatory products in fibroblasts (fibroblast-like synoviocytes and dermal fibroblasts), endothelial cells, and neutrophils [35 , 38 , 42 43 44 ], MP might remotely convey activating factors from T cells to such cells and participate in the pathogenic mechanisms of chronic inflammation. For instance, in RA, T cells are rare or absent from the lining layer of the pannus, where fibroblast-like synoviocytes are located. In the latter cells, direct contact with stimulated T cells or MP they generate induces an unbalanced production of matrix metalloproteases and their inhibitors [29 , 38 , 45 ]. In the absence of neighboring T cells, this activity might be conveyed by T cell MP. Similarly, in active, chronic MS lesions, where T cells are more abundant at the edge of lesions, unlike macrophages, which are more frequent at the lesion center [46 ], the generation of MP by activated T cells might ensure remote contact-mediated activation of monocytes/macrophages from the edge to the center of the lesion and in turn, maintain the activation stage of monocytes/macrophages.

We previously showed that HDL inhibit the production of IL-1β and TNF production by human monocytes and the monocytic cell line THP-1 by affecting the induction of cytokine transcription by msHUT [10 ]. This is further confirmed here by quantitative real-time PCR in monocytes activated by CEsHUT. In contrast, neither the production nor the mRNA expression of sIL-1Ra was affected by the presence of HDL, demonstrating that the ratio of sIL-1Ra to IL-1β production was enhanced by HDL, stressing even more their anti-inflammatory nature. These results also demonstrate that several types of monocyte-activating factors are present at the surface of stimulated T cells—HDL inhibiting those responsible for the production of proinflammatory cytokines such as IL-1β and TNF.

MP may display pro- or anti-inflammatory activities [14 ]. In this study, MP released by stimulated T cells displayed inflammatory effects on isolated monocytes by inducing not only the production of the prototypical proinflammatory cytokines, IL-1β and TNF, but also, the secretion of sIL-1Ra, a specific inhibitor of IL-1 [47 ]. Thus, similarly to membranes of T cells or fixed T cells, MP induced the production of pro- and anti-inflammatory mediators. This production is driven toward a less-inflammatory pattern in the presence of HDL. The generation of MP might also be a way for the cell to remove detrimental molecules from its surface. However, surface proteins carried by MP still display activity. This suggests that MP generation by T cells does not aim at removing surface factors from the cell surface but rather, at generating vectors likely to convey signals between distant cells.

In conclusion, this study demonstrates that MP generated by T cells carry similar molecules to the cells from which they originate, as they are able to activate the production of cytokines in human monocytes, and HDL affect this mechanism by inhibiting the production of proinflammatory cytokines IL-1β and TNF but not that of the anti-inflammatory cytokine sIL-1Ra. Consequently, through the generation of MP, stimulated T cells may ensure distant contact-mediated activation of cells, which are not located in their proximity at the inflammatory site.


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ACKNOWLEDGEMENTS
 
This work was supported by grants [#320000-116259 (D. B.) and #3200-068286.02 (J-M. D. and D. B.)] from the Swiss National Science Foundation, a grant to project #CT-2006-LSHG-037749 from the European Union (D. B.), and a grant from the Swiss Society for Multiple Sclerosis (D. B.).


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FOOTNOTES
 
1 Current address: Section of Rheumatology, Department of Clinical and Experimental Medicine, University of Padova, Via Giustiniani, 2, 35128 Padova, Italy. Back

Received August 16, 2007; revised December 11, 2007; accepted January 3, 2008.


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