Published online before print October 21, 2004
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* Division of Immunology and Allergy, Department of Internal Medecine and
Biology of Ageing Laboratory and Department of Geriatrics, University Hospital, Geneva, Switzerland
1 Correspondence: Division of Immunology and Allergy, University Hospital, 24 rue Micheli-du-Crest, 1211 Geneva 14, Switzerland. E-mail: pascale.roux-lombard{at}hcuge.ch
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Key Words: oxidative metabolism • lymphocyte activation • neutrophil
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(TNF-), which play an important part in chronic inflammatory diseases (reviewed in ref. [5
]). The identity of the surface molecule(s) involved in contact-mediated activation of monocyte-macrophages is still elusive. Some studies have shown that CD40–CD40 ligand (CD40L) might be involved to some extent in proinflammatory cytokine production [6
, 7
]. CD69 and ß2-integrins, i.e., lymphocyte function-associated antigen-1 (CD18/CD11a), membrane-activated complex-1 (CD18/CD11b), and p153,95 (CD18/CD11c), as well as intercellular adhesion molecule-1 (ICAM-1), have also been shown to participate in the cell contact-mediated activation of monocyte-macrophages by stimulated T cells [8
9
10
]. However, blocking antibodies to the latter molecules and to others (CD2, CD18, CD23, CD29, CD95), as well as specific inhibitors of inflammatory cytokines such as TNF-soluble receptors and IL-1 receptor antagonist, failed to abolish this activation, suggesting that one or several other factors expressed at the surface of stimulated T cells are primarily involved in monocyte-macrophage activation. The induction of cytokine production in monocyte-macrophages upon direct cell–cell contact with stimulated T cells was strongly inhibited by high-density lipoproteins (HDL), which are likely to interact through their major protein component, apolipoprotein A-I, with a ligand at the surface of T cells [11 ], thus inhibiting specifically the activation of monocyte-macrophages. This study shows that direct cell–cell contact with stimulated T cells induces the production of ROS in PMN and that this mechanism is inhibited by HDL and involves CD18 engagement.
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Monoclonal antibodies (mAb)
Dr. Peter E. Lipsky (National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD) provided anti-CD40L mAb (Clone 5C8). Mouse mAb to CD11a [immunoglobulin G (IgG)2a, Clone 38, ANC-158-020], CD11b (IgG1, Clone ICRF44, ANC 159-020), CD11c (IgG1, Clone 3.9, ANC-160-020), to CD18 (IgG2a, Clone IB4, ANC-167-020) and CD69 (IgG2a, Clone HP-4B3, ANC-221-020) were purchased from Alexis Corporation (Läufelfingen, Switzerland). None of the antibody preparations contained azide.
T cells
The human T cell line HUT-78, obtained from American Type Culture Collection (Manassas, VA), was maintained in RPMI-1640 medium supplemented with 10% heat-inactivated FCS, 50 µg/ml streptomycin, 50 U/ml penicillin, and 2 mM L-glutamine in a 5% CO2 air-humidified atmosphere at 37°C. T lymphocytes were isolated from human peripheral blood of healthy donors as described previously [8
]. Isolated T lymphocytes (4x106 cells/ml) were stimulated by 1 µg/ml PHA and 5 ng/ml PMA. After 48 h of culture in 5% CO2 air-humidified atmosphere at 37°C, stimulated T lymphocytes were washed in PBS, and their membranes (msT) were isolated as described previously [12
]. HUT-78 cells (1x106 cells/ml) were stimulated for 6 h with 1 µg/ml PHA and 5 ng/ml PMA and washed, and their membranes were isolated [13
]. Alternatively, HUT-78 cells were fixed with paraformaldehyde as described previously [8
]. Plasma membranes of stimulated T cells (msHUT) were used to activate PMN, considering that their activity in human monocytes was similar to that of living T cells (i.e., in cocultures) or fixed T cells as described previously [11
]. The involvement of a putative carryover of PMA and PHA by T cell membranes in target cell activation was previously ruled out [3
, 8
].
PMN preparation
PMN were isolated from heparinized blood collected from healthy donors as described previously [14
]. Cells (1x107 cells/ml) were resuspended into external solution (ES) without Ca2+, consisting of 10 mM Hepes, pH 7.2, containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 0.1% (10 mM) glucose, kept on melting ice, and used within 2-4 h.
HDL isolation
Human serum HDL were isolated by high-density ultracentrifugation as described previously [11
]. To optimize the inhibitory effect of HDL, they were consistently added together with the stimulus.
Chemiluminescence measurement
PMN (2x106 cells/200 µl/well, i.e., 1x107 cells/ml) were stimulated by the indicated stimulus in ES containing 2 mM CaCl2, 5 U/ml HRP type II, and 50 µM luminol [15
]. Chemiluminescence was determined on a Luminoskan Ascent (Labsystems, Franklin, MA), using 96-well white plastic plates (Cliniplate, Bioconcept, Switzerland). To assess intracellular production of ROS, chemiluminescence was tested in the presence of 200 U/ml SOD and 2000 U/ml catalase to scavenge extracellular ROS.
Detection of ROS by NBT
NBT test was performed as described previously [16
]. Briefly, PMN (1x107 cells/ml) were incubated at 37°C with fixed HUT-78 cells in ES containing 2 mM CaCl2 in the presence of 1.7 mg/ml NBT (yellow) for the indicated time. In the presence of oxidative metabolites, NBT is reduced to formazan, which forms a blue precipitate, detected by light microscopy.
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Figure 1. Induction of PMN respiratory burst by contact with stimulated T cells. PMN (10x106 cells/ml) were incubated at 37°C in the presence or absence of the indicated stimulus. The resulting chemiluminescence (CL) was measured as described in Materials and Methods. (A) Cell equivalent (2.5, 5, 10, 15, and 20x106/ml) msT or 5 x 106 cell equivalent/ml membranes of unstimulated T lymphocytes (muT). (B) Cell equivalent (2.5, 5, 10, and 15x106/ml) msHUT or 5 x 106 cell equivalent/ml membranes of unstimulated HUT-78 cells (muHUT). (C) Cell equivalent (5x106/ml) msHUT or muHUT cells, 50 µM fMLP, and 5 ng/ml PMA. rlu, Relative light units.
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PMN activation is a result of cell–cell surface contact with stimulated T cells
Considering that membrane preparations might expose intracellular components and contain fragments of intracellular structures, fixed, stimulated T cells were used to determine whether ROS production was induced in PMN by molecules at the surface of T cells. Fixed, stimulated, but not unstimulated, HUT-78 cells triggered respiratory burst in PMN in a similar way to membranes isolated from HUT-78 cells (Fig. 2A
). To ensure that contact with stimulated T cells was directly activating PMN, the latter cells were incubated in the presence of fixed HUT-78 cells and NBT to visualize ROS production in living cells simultaneously. As shown in Figure 2B
, stimulated and unstimulated HUT-78 cells were in close contact with PMN. ROS production occurred mainly with fixed, stimulated HUT-78 cells, resulting in a marked activation of 24% of total PMN, and in the presence of unstimulated HUT-78 cells, only 7% of PMN was slightly activated after 50 min. Furthermore, there was no engulfment of T cells by PMN after 50 min. This demonstrates that ROS production was related to specific molecules on the surface of stimulated T cells and not to soluble factors or nonspecific contact.
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Figure 2. Fixed, stimulated HUT-78 cells displayed similar activity to membranes of the latter cells. (A) PMN (10x106 cells/ml) were incubated at 37°C with fixed, stimulated (fsHUT) or unstimulated (fuHUT) HUT-78 cells (4x106 cells/ml) or membranes of the latter cells, i.e., msHUT and muHUT, respectively. Results are expressed as maximum chemiluminescence (Max CL; arbitrary units). (B) PMN (10x106 cells/ml) were incubated at 37°C with 5 x 106 cells/ml fuHUT or 5 x 106 cells/ml fsHUT cells in the presence of 1.7 mg/ml NBT. Pictures were taken after 50 min of incubation at various magnifications (upper panels, 40x original; lower panels, 100x original). Solid arrows indicate the formation of blue (dark) formazan upon yellow NBT reduction.
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Figure 3. Induction of ROS production in PMN by membranes of stimulated HUT-78 was partially a result of CD18. PMN (10x106 cells/ml) were incubated at 37°C with membranes of stimulated HUT-78 cells (1.25x106 cell equivalent/ml) in the presence of antibody to the indicated surface molecule or control IgG isotype; anti-CD11a, -b, or -c (10 µg/ml), anti-CD18 (1.125 or 2.25 µg/ml), anti-CD69 (10 µg/ml), anti-CD40L (10 µg/ml), IgG2a (10 µg/ml), and IgG1 (10 µg/ml). Results are expressed as percentage of maximum chemiluminescence (CL) observed in the absence of antibodies (medium), mean ± SD, n = 3.
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HDL inhibit PMN activation induced by T cell contact
As HDL specifically inhibited contact-mediated cytokine induction in monocytes [11
], the effect of HDL on ROS production by PMN was assessed. To determine whether HDL would inhibit PMN respiratory burst induced by T cell membranes, PMN were activated by msHUT in the presence of increasing concentrations of HDL or in its absence. At low concentrations (25 µg/ml), HDL inhibited by 50% the activation of PMN respiratory burst induced by msHUT. The inhibition of chemiluminescence increased proportionally to HDL concentrations and reached 100% at 200 µg/ml HDL (Fig. 4A
). The inhibitory effect of purified HDL was effective on msHUT and msT, as well as on fixed, stimulated HUT-78 cells (Fig. 4B)
. In contrast, HDL did not inhibit fMLP-induced ROS production, indicating that HDL did not exert a general inhibitory activity to ROS production by PMN. This suggests that HDL might preferentially interact with T cell membranes. Low-density lipoproteins or albumin used as controls had no inhibitory effect on PMN oxidative metabolism (data not shown).
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Figure 4. Purified HDL inhibited ROS production in PMN activated by membranes of stimulated T cells. (A) PMN (10x106 cells/ml) were incubated at 37°C with membranes of stimulated HUT-78 cells (5x10 6 cell equivalent/ml) in the presence or absence of increasing concentrations of purified HDL (50, 100, 150, and 200 µg/ml). (B) PMN (10x106 cells/ml) were incubated at 37°C with msHUT cells (12 µg/ml proteins, 5x106 cell equivalent/ml), msT cells (10 µg/ml proteins), fsHUT cells (5x106 cells/ml), and fMLP (50 µM) in the presence (shaded bars) or absence (open bars) of 200 µg/ml HDL. Results are expressed as maximum chemiluminescence (Max CL; arbitrary units), mean ± SD, n = 3.
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Figure 5. Contact with stimulated T cells mainly induced intracellular ROS in PMN (10x106 cells/ml), which were incubated at 37°C with the indicated stimulus in the presence or absence of SOD and catalase to measure intracellular (Intra) and total (Tot) ROS production in the presence (+) or absence (–) of 200 µg/ml HDL. msHUT were at 5 x 106 cell equivalent/ml and fMLP, at 50 µM. Results are expressed as maximum chemiluminescence (CL; arbitrary units), mean ± SD, n = 3.
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Activation by contact with T cell membranes induced mainly intracellular ROS production in PMN, as the levels of chemiluminescence measured in the presence or absence of SOD and catalase were similar. This might explain why in previous studies, we found only a slight, direct activation of PMN by cellular contact with stimulated T cells and essentially the priming of PMN to respond to fMLP [3
]. In our attempts at the time to detect chemiluminescence induced by ROS, we used lucigenin, which recently proved to be less sensitive than luminol in detecting intracellular ROS generation [17
]. However, in this former study, extracellular generation of ROS was measured by cytochrome c reduction upon contact with intact T cells. In contrast, in the present study, this extracellular probe failed to detect ROS production upon PMN stimulation by msHUT (data not shown), which may be a result of differences between fixed T cells and membrane preparations as far as the configuration of the molecules involved are concerned and consequently, their ability to stimulate PMN. It is also possible that soluble products (IFN-
or TNF) leaking from fixed T cells were responsible for the stimulation of the extracellular generation of ROS. The use of T cell membranes precludes interference with soluble products and is more appropriate for examining the mechanism of PMN oxidative metabolism activation upon T cell contact. This approach gave rise to results that strongly suggest that ROS generation is intracellular.
ROS generation is often related to phagocytosis and destruction of pathogens, which in this case, usually occur in phagolysosome [18 ]. However, particle-mediated cross-linking of CD18 on the neutrophil surface mediates intracellular production of ROS without phagosome formation [16 ]. Multiple ligands can bind to ß2-integrins and induce signals in PMN, including extracellular matrix proteins, receptors of the Ig superfamily such as ICAM-1 and ICAM-2, blood coagulation proteins, and microorganism products [19 ]. Although our results did not warrant a conclusion as to whether the CD18 molecules implicated in T cell contact-mediated activation of ROS production in PMN are located on T cells, PMN, or both, by analogy with the previous study [16 ], it is likely that a putative CD18 ligand at the surface of T cells initiates a signaling cascade, leading to the generation of intracellular ROS without extracellular release of ROS. In this regard, some studies suggest that oxygen species may also act as intracellular messengers by activating transcription factors and protein phosphorylation [20 21 22 ]. Whether the intracellular generation of ROS in response to activated T cell contact is part of a signal-transduction cascade deserves further investigation.
We demonstrated previously that the capacity of T cell clones to prime PMN oxidative metabolism correlates with their capacity to induce cytokine production in monocytic cells, suggesting that similar molecules at the surface of T cells are involved in the activation of PMN and monocytes [4 ]. The present data show that similar to contact-mediated activation of monocyte-macrophages, HDL indeed inhibits contact-mediated induction of PMN respiratory burst. This further suggests that analogous molecules at the surface of T cells are likely to be involved in the activation of monocyte-macrophages and PMN. However, there are differences in the involvement of other surface molecules, which might be considered coactivators. According to the present data, CD18 is involved in the induction of PMN respiratory burst by cellular contact with stimulated T cells. This is reminiscent of numerous other studies demonstrating the involvement of ß2-integrins in activation, adhesion, and transendothelial migration of PMN [16 , 23 24 25 26 27 28 29 ]. The involvement of CD18 in the induction of respiratory burst in nonadherent PMN upon contact with stimulated T cells differs from that observed with monocyte-macrophages, whose activation by contact with T cells was not affected by blocking antibodies to CD18 [8 , 9 ]. However, contact-mediated activation of monocyte-macrophages involves the engagement of ß2-integrins, as it is partially inhibited by antibodies to CD11a, CD11b, and CD11c [8 ], which did not affect PMN activation. Together, these results demonstrate that ß2-integrins are involved in T cell contact-mediated activation of PMN and monocyte-macrophages but that the use of ß2-integrin chains varies depending on the target cell type. Another difference between monocyte-macrophages and PMN is the involvement of CD69, which is not implicated in the activation of PMN but in that of monocyte-macrophages [9 ].
Similar to the induction of proinflammatory cytokines in monocyte-macrophages, HDL potently inhibited T cell contact-induced ROS production in PMN. HDL had no effect on ROS induction by fMLP, whether intra- or extracellular, confirming that HDL did not exert a general inhibitory activity to ROS production by PMN, but rather specifically interfered with cell–cell contact induction of ROS in PMN by interacting with stimulated T cell membranes. It is interesting that other authors have demonstrated that lipid-free apolipoprotein A-I, the main protein constituent of HDL, inhibits IgG-induced activation of PMN, i.e., degranulation and superoxide production [30 ]. However, the authors failed to detect an inhibitory effect of HDL, which is consistent with the hypothesis of a specific inhibitory activity of HDL to the contact-mediated activation of PMN.
Our observation suggests that the well-known antiatherogenic function of HDL [31 32 33 34 ], mainly attributed to their role in reverse cholesterol transport, may also be a result of the inhibition of immune cell activation (PMN and monocyte-macrophages) involved in atherosclerosis [35 , 36 ]. Indeed, it has recently been postulated that PMN play an important part in the pathogenesis of thrombosis and atherosclerosis as a result of the release of ROS, which in turn induces lipid oxidation and thus contributes to the production of tissue factor [11 , 37 ]. Taking into account the present data, the inhibition of ROS production by PMN upon activation by T lymphocytes may also contribute to the protective effect of HDL in atherosclerosis. This is sustained by clinical observations showing that the incidence of atherosclerosis is enhanced in patients with rheumatoid arthritis and systemic lupus erythematosus, whose serum HDL levels are decreased [1 , 38 39 40 ]. Therefore, the ability of T cells to activate PMN by cellular contact in a HDL ligand-dependent manner constitutes an additional link between inflammation and lipid metabolism.
In conclusion, by demonstrating the direct induction of PMN respiratory burst by cellular contact with stimulated T cells, the present study substantiates previous observations, pointing out that the direct cellular contact within the inflammatory site is a potent signal to stimulate the release of inflammatory mediators. The potency of contact-mediated induction of IL-1ß and TNF- production in monocyte-macrophages and its inhibition by HDL are well established [5
, 11
]. The triggering effect of stimulated T cells on PMN respiratory burst and its inhibition by HDL further demonstrate that similar molecules are involved in the activation of both target cells. However, various integrins or other molecules may function as coactivators depending on the target cell type. Furthermore, HDL might play a protective role overall by inhibiting the inappropriate activation of circulating monocytes and PMN by activated T lymphocytes in the bloodstream.
Received June 25, 2004; revised September 21, 2004; accepted September 23, 2004.
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and 1,25(OH)2D3 induce on THP-1 cells distinct patterns of cell surface antigen expression, cytokine production, and responsiveness to contact with activated T cells J. Immunol. 149,2040-2046[Abstract] production in rheumatoid arthritis Nat. Med. 3,189-195[CrossRef][Medline] by blocking contact-mediated activation of monocytes by T lymphocytes Blood 97,2381-2389-kallikrein does not directly activate blood neutrophils Thromb. Res. 55,109-119[CrossRef][Medline]This article has been cited by other articles:
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