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IMMUNOBIOLOGY
Tumor necrosis factor- Tumor necrosis factor- We hypothesized that, in analogy with other homeostatic systems,
natural inhibitors are likely to exist that interfere with the
lymphocyte/monocyte interaction. Such inhibitory activity should be
present in plasma because stimulated T lymphocytes in the bloodstream
of normal subjects or patients during inflammation show little evidence
of contact-mediated activation of monocytes. Indeed, the occasional
presence of TNF- Reagents
Antibodies
T cells and preparation of T-cell plasma membranes HUT-78, a human T cell line,21 was obtained from the ATCC. 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 (complete RPMI medium) in 5% CO2-air humidified atmosphere at 37°C. HUT-78 cells (1 × 106 cells/mL) were stimulated for 6 hours by PHA (1 µg/mL) and PMA (5 ng/mL). Stimulated HUT-78 cells were either fixed with 1% paraformaldehyde1,4 or their plasma membranes prepared as previously described.15 T lymphocytes were obtained from buffy coats of healthy donors as previously described,1 and contained 94% to 98% CD2+, 83% to 94% CD3+, and less than 2% CD14+ as assessed by flow cytometry. T lymphocytes were stimulated for 48 hours by PHA (1 µg/mL) and PMA (5 ng/mL), washed thoroughly, and fixed with 1% paraformaldehyde as previously described.1,4 Peripheral blood mononuclear cells (PBMC) were obtained from buffy coats of healthy donors by density centrifugation on Ficoll-Paques (Amersham-Pharmacia, Uppsala, Sweden).Monocytes and monocytic cells The human monocytic cell line THP-1, derived from a patient with acute monocytic leukemia,22 was obtained from the ATCC. Peripheral blood monocytes were obtained as described.13Protein concentration and N-terminal microsequencing The protein concentrations were determined by the method of Bradford. The purified inhibitory fraction was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membrane, and visualized by Coomassie blue staining. The Mr = 28-kd band was excised and N-terminal sequence analysis was performed on a Procise 494-HT protein sequencer (PerkinElmer, Foster City, CA).PBMC cultures The PBMC were cultured in 96-well culture plates at a density of 4 × 105 cells/200 µL/well in the presence of the indicated stimulus for 48 hours (cytokine production) or 72 hours (proliferation). For proliferation, 3H-thymidine was added 24 hours before cell harvesting.Contact-mediated activation of THP-1 cells and monocytes The THP-1 cells (5 × 104 cells/well) or monocytes (8 × 104 cells/well) were dispensed onto 96-well culture plates (Falcon, Becton Dickinson, Plymouth, England) and activated by the indicated stimulus in a total volume of 200 µL complete RPMI medium in the presence or absence of the indicated inhibitor. After 48 hours, culture supernatants were analyzed for their contents in TNF-
and IL-1 as previously described.1,4
Isolation of serum high-density lipoproteins Flow cytometry The HDL were labeled with fluorescein isothiocyanate (FITC-HDL) as described.24 The binding of FITC-HDL to cells was analyzed by direct flow cytometry on a flow cytometer (Epics, Coulter Electronics, Hialeah, FL) essentially as described previously.25 The mean fluorescence intensity was recorded on gating of living cells and expressed in arbitrary units in 4 decade logarithmic scale. The percentage of positive cells was based on the percentage of fluorescent events exceeding unconjugated FITC control.HDL delipidation The extraction of HDL apolipoproteins was performed as described.26 Briefly, 1 volume of HDL isolated by ultracentrifugation was slowly added to 12 volumes of ice-cold methanol on constant stirring. Then 28 volumes of ice-cold diethylether was added to the solution. After 10 minutes stirring on ice, the mixture was centrifuged at 500g for 5 minutes. The protein pellet was resuspended in 40 volumes of diethylether. After 10 minutes stirring on ice, the mixture was centrifuged as above. The pellet was recovered and dried under nitrogen flux. To minimize aggregation, the delipidated HDL lipoproteins were solubilized at 2 mg/mL proteins in 0.1 M Tris-HCl pH 7.4 containing 0.1M NaCl, 1 mM NaN3, 1 mM EDTA, and 2 M guanidinium chloride and then dialyzed in PBS.Treatment of HDL with proteinase K The HDL (100 µg proteins) were incubated in the presence or absence of 1 U proteinase K linked to agarose beads (Sigma Fine Chemicals, St Louis, MO) in a final volume of 200 µL PBS at 37°C for 1 hour. The proteolytic reaction was stopped by centrifugation and its efficacy assessed by SDS-PAGE.Electroelution of HDL proteins The HDL proteins were isolated by electroelution from gel slices essentially as previously described.27 Briefly, 1 mg protein of delipidated HDL was subjected to SDS-PAGE on unreduced conditions. The gel was stained with copper and the detected bands (Mr = 56 000-66 000, 50 000, 28 000, and 18 000) were cut, destained, and eletroeluted for 5 hours in 50 mM Tris-HCl and 384 mM glycine (pH 8.3) containing 5 mM EDTA and 0.1% SDS. SDS was discarded by precipitating proteins in acetone. Proteins were lyophilized and resuspended in 0.1 M Tris-HCl (pH 7.4) containing 0.1 M NaCl, 1 mM NaN3, 1 mM EDTA, and 2 M guanidinium chloride and then dialyzed in PBS. Alternatively, proteins from delipidated HDL were solubilized in 0.1 M Tris-HCl pH 7.4 containing 0.1 M NaCl, 1 mM NaN3, 1 mM EDTA, and 2 M guanidinium chloride and subjected to gel filtration on Superdex S75 (75 × 1.6 cm, Pharmacia) equilibrated in the same buffer. Fractions corresponding to Mr = 28 000 were pooled, concentrated and dialyzed in PBS for testing their inhibitory activity. Fractions were analyzed by Western blot for their content in apo A-I using mouse mAb from Calbiochem-Novabiochem.Messenger RNA analysis Total RNA was isolated from THP-1 cells and monocytes with TRIzol reagent (Life Technologies, Basel, Switzerland) according to the manufacturer's procedure. Two and 10 µg total RNA were used to quantify messenger RNAs (mRNAs) in monocytes and THP-1 cells, respectively, using a commercially available "RNase protection assay system" kit with hck2 template set (Pharmingen, San Diego, CA) to which an antisense riboprobe for TNF- was added. TNF- antisense riboprobe was obtained from TNF- complementary DNA (cDNA)
template prepared with SP6 RNA polymerase after linearizing pSP64/hTNF
plasmid provided by Dr C. V. Jongeneel (Ludwig Institute for
Cancer Research, Lausanne, Switzerland).
Human serum inhibited T-cell signaling of both monocytes and THP-1 cells To assess whether HS displayed an anti-inflammatory activity by inhibiting cytokine production, unseparated PBMC were stimulated by PHA in medium supplemented with either 10% FCS or HS. In the presence of HS, the production of both TNF- and IL-1 was inhibited in PBMC
cultured with HS as compared with FCS (Figure
1A), but cell proliferation was similar
in HS and FCS (Figure 1B). Because it was likely that cytokine
production by PBMC was induced in monocytes by direct contact with
stimulated T lymphocytes, the inhibitory effect of HS was ascertained
in several culture systems in which either fixed, stimulated T
lymphocytes or HUT-78 T cells, or plasma membranes from the latter
cells were added to peripheral blood monocytes or THP-1 monocytic
cells. The induced production of TNF- and IL-1 was measured
(Figure 2).
Stimulated T lymphocytes isolated from peripheral blood were fixed with
paraformaldehyde and added to THP-1 cells in complete RPMI medium
supplemented with either HS or FCS, that is, a final serum
concentration of 20%. In these conditions, T lymphocytes triggered the
production of IL-1 Isolation of HS factor inhibiting T-cell signaling of monocytes To identify the inhibitory factor(s), HS was fractionated by serial chromatography on Blue Sepharose fast flow, Q Sepharose fast flow, phenyl Sepharose 6 fast flow, and Superdex 200 (Pharmacia). The latter revealed one peak of inhibitory activity that was recovered in fractions 23 to 26 (Figure 3A), representing a Mr = 179 × 103 ± 46 × 103 (mean ± SD, n = 4 inhibitory fractions). On analysis by SDS-PAGE the inhibitory activity correlated with the enrichment of a 28-kd protein band (Figure 3A,B). According to N-terminal microsequencing this band was consistent with apo A-I, the main protein component of HDL. This accounted for the discrepancy in Mr analysis between gel filtration by which intact HDL were separated and SDS-PAGE in which HDL proteins were separated (Figure 3A,B). These data demonstrate that the inhibitor was associated with HDL particles.
To confirm that HDL displayed inhibitory activity, isolated
lipoproteins and serum proteins were tested for their inhibitory activity in THP-1 cells activated by membranes of stimulated HUT-78 cells. The inhibitory activity was recovered in HDL, whereas LDL and
serum proteins did not display inhibition (Figure
4). To determine whether the inhibition
was due to a proteic or a lipidic component, HDL were subjected to
either delipidation or proteolytic treatment with proteinase K. HDL
proteins obtained by diethylether/methanol treatment displayed a high
inhibitory activity, whereas HDL lipids obtained after proteolytic
digestion with proteinase K were no longer inhibitory (Figure 4). This
demonstrates that the production of both TNF-
Apo A-I is the HS inhibitor of T-cell signaling of monocytes To determine whether apo A-I would display the inhibitory activity, various preparations were tested. Commercially available apo A-I inhibited the production of IL-1 and to a lesser extent TNF-
HP-1 cells activated by membranes of HUT-78 cells in a dose-dependent manner (Figure 5A), suggesting that apo
A-I displayed the inhibitory activity. However, because apo A-I
preparation contained 3% unidentified contaminants (according to the
supplier) other preparations of apo A-I were required to confirm this
result. Proteins from delipidated HDL were subjected to preparative
SDS-PAGE. After copper-staining, bands
(Mr: 56 000-66 000, 50 000, 28 000,
and 18 000) were excised and electroeluted. The inhibitory activity
was recovered in 28 000 and 18 000 bands, which inhibited the
production of both IL-1 and TNF- (Figure 5B,C). All inhibitory
fractions contained apo A-I as demonstrated by Western blot analysis
(Figure 5E), establishing that apo A-I was the inhibitor of T-cell
signaling of monocytes. Indeed, it is very unlikely that another HDL
apolipoprotein should display the same characteristics as apo A-I in
terms of size of protein and proteolytic fragment (Figure 5E, lanes a
and b). Alternatively, proteins from delipidated HDL were subjected to
gel filtration on Superdex S75. Pooled fractions corresponding to
Mr = 28 000 ± 10 000 displayed the
inhibitory activity (Figure 5D). These fractions contained apo A-I as
determined by Western blot analysis (Figure 5E, lane c), providing
additional proof that apo A-I was the inhibitor.
HDL interact with stimulated T cells through apo A-I S To establish whether the inhibitory activity of HDL-associated apo A-I could be due to binding to stimulated T cells or THP-1 cells, either membranes isolated from stimulated HUT-78 cells or THP-1 cells were preincubated in the presence or absence of FCS, HS, or isolated HDL. After washing, the residual activating capacity of membranes from stimulated HUT-78 cells was assessed on THP-1 cells. The inhibition of TNF- and IL-1 production was observed only when membranes of
stimulated HUT-78 cells were incubated with HS or HDL but not when
THP-1 cells were incubated with either FCS, HS, or HDL (Figure
6A). Consequently the inhibitory activity of HS and HDL was mainly directed to the activating factor(s) expressed
at the surface of stimulated T cells.
To confirm that the inhibitory factor(s) interacted with surface factors on stimulated T cells, isolated HDL was labeled with FITC, and its binding to different cell types was assessed by flow cytometry. No binding of FITC-HDL was observed on THP-1 cells (Figure 6B), whereas fluorescence of monocytes was slightly enhanced when incubated with FITC-HDL as compared to unconjugated FITC control (Figure 6C). A low level of binding of FITC-HDL to unstimulated HUT-78 cells was observed, whereas stimulated HUT-78 cells bound FITC-HDL and displayed 2 fluorescent peaks suggesting the presence of at least 2 different HDL binding sites (Figure 6D,E). At a lower FITC-HDL concentration, a single fluorescent peak was observed. In the presence of anti-apo A-I antibodies, a shift toward lower fluorescence intensity was observed, demonstrating that HDL interacted with stimulated T cells via apo A-I-specific binding (Figure 6F). Together these results show that HDL interacted preferentially with stimulated T cells, implying that the inhibitory activity of apo A-I was directed to surface factors on T cells. Optimal inhibition of mRNA steady-state required the addition of apo A-I simultaneously or shortly after the stimulus To further elucidate the mechanism of action of apo A-I the inhibitory effect of isolated apo A-I on steady-state levels of TNF-
and IL-1 mRNA was assessed. THP-1 cells were stimulated with
membranes of stimulated HUT-78 cells in the presence of apo A-I added
at the indicated time. Apo A-I diminished the steady-state levels of
TNF- and IL-1 mRNA in THP-1 cells activated with membranes of
stimulated HUT-78 cells (Figure 7A,C).
The inhibition of TNF- mRNA was less pronounced than that of IL-1
mRNA, correlating with the data obtained at the level of protein
production. To achieve optimal inhibition of cytokine mRNA steady-state
levels apo A-I had to be added simultaneously or shortly after
activation by membranes. Similar results were observed with peripheral
blood monocytes activated by membranes of stimulated T lymphocytes
(Figure 7B,D) in which steady-state levels of both TNF- and IL-1
mRNAs were diminished by apo A-I, although mRNA induction set in more rapidly in monocytes than in THP-1 cells. On monocytes inhibition of
mRNA steady-state levels was also optimal when apo A-I was added
together with membranes or shortly after (Figure 7D). No inhibition was
observed when apo A-I was added after 30 minutes of activation. The
latter results demonstrate that apo A-I inhibited contact-mediated
activation of monocytes regardless of the cell type, confirming the
data in Figure 2.
Apo A-I inhibits TNF-
and IL-1 production was inhibited by either apo A-I or delipidated
HDL (Figure 8). TNF- production was
inhibited to a lesser extent than that of IL-1, confirming results
of Figure 1. This result demonstrates that (1) the inhibition of
cytokine production by unfractionated HS depicted in Figure 1 was due
to apo A-I, and (2) that apo A-I inhibits monocyte activation
regardless of the T-cell stimulus suggesting that antigen-specific and
mitogen stimulation induced similar activating factors on T
lymphocytes, although resulting in different levels of expression.
Furthermore, these results confirm that contact between monocytes and
stimulated T cells was required for the induction of TNF- and
IL-1 in PBMC.
The present study reveals a new anti-inflammatory activity
elicited by apo A-I, a known "negative" acute-phase protein. The inhibitory activity was absent in FCS and CBS that contain low amounts
of apo A-I.28 Although the mechanism of action of apo A-I
is not fully elucidated, the present results suggest that apo A-I
exerts most of its inhibitory activity by specifically blocking the
interaction between T cells and monocytes. The blockade or inhibition
of TNF- The inhibitory activity of apo A-I seems to be specifically directed to
T-cell signaling of monocytes because activation of THP-1 cells by
other stimuli (LPS and PMA) was not affected by HS. However, in
peripheral blood monocytes stimulated by LPS the inhibition of IL-1 The present data demonstrate that HDL interact with stimulated T cells
via binding of apo A-I. A specific HDL binding site on human
lymphocytes has been described but not identified.41 Together with the premise that apo A-I is a major player in the reverse
transport of cholesterol, this suggests that apo A-I may disturb the
lipid organization of a raft membrane microdomain containing the
activating factor(s),42,43 thus abolishing the activating
capacity of stimulated T cells. The latter hypothesis is, however, very
unlikely because contact-mediated activation of monocytes by
paraformaldehyde-fixed, stimulated T cells was efficiently inhibited by
HS. According to flow cytometry analyses (Figure 6), HDL also bind
monocytes in accordance with data showing the binding of HDL
(HDL3) to monocytes in PBMC.24 It is therefore possible that HDL also affect monocyte activation by directly modulating the level of activation of the latter cells. Indeed, when
added shortly after the stimulus apo A-I still decreased cytokine mRNA
steady-state levels, although to a lower extent than when added at
t = 0 (Figure 7). Although this might be due to the premise that
membranes of stimulated T cells deliver sustained activation signals,
it does not discard the possibility of a direct effect of the inhibitor
on monocytes/THP-1 cells. Despite the fact that HDL binding or
functional activity was not observed on resting THP-1 cells, the latter
may express HDL receptors once differentiated
(activated).44 Therefore, the present study demonstrates that HDL-associated apo A-I can inhibit the production of
proinflammatory cytokines on activation of monocytes by contact with
stimulated T cells through 2 different pathways: (1) the main pathway
consisting of the blockade of the binding of the T cell activating
factor to its receptor on monocytes, thus inhibiting the expression of both TNF- No interaction has been described between apo A-I or HDL and the different surface molecules that have been claimed to be involved in T-cell signaling of monocytes. It is unlikely that CD40L should be the apo A-I ligand because this molecule is not detected on either HUT-78 cells. Furthermore, blocking antibodies to CD40L (5C8, a kind gift from Dr P. E. Lipsky, University of Texas, Dallas, TX) failed to inhibit contact-mediated activation of monocytes, whereas in the same experiment apo A-I inhibited 85% to 90% of cytokine production (unpublished data, January 2000). An assumption is that apo A-I ligand belongs to a family of HDL plasma membrane receptors.45 However, because HDL receptor proteins such as SR-BI or CD36 are not specific for a particular apolipoprotein (ie, they interact with apo A-I, apo A-II, apo C, HDL, and LDL45,46) and because LDL do not display inhibitory activity toward T-cell signaling of monocytes, it is unlikely that apo A-I ligand(s) on stimulated T cells belong(s) to this HDL receptor family. On the other hand, specific HDL-binding proteins (HB1 and HB2) have been described that are expressed in rat liver plasma membrane and human blood monocytes.24 HB2 is homologous with ALCAM, a cell-adhesion molecule belonging to the immunoglobulin superfamily,45 whose function has not been clearly elucidated. Because apo A-I is involved in the binding of HDL to HB2 it is possible that the HDL receptor on stimulated T cells displays some homology with the HB2 protein family. Recently, an epithelial protein (cubilin) was shown to display high affinity for apo A-I.47 Whether the latter 460-kd protein and HB2 are expressed in stimulated T lymphocytes remains to be determined. The identification of HDL-associated apo A-I receptor(s) on stimulated T cells may lead to the elucidation of the mechanism of action of the inhibitor of T-cell signaling of monocytes. Apo A-I inhibits contact-mediated monocyte activation by stimulated T cells or T lymphocytes stimulated by various stimuli, eg, antigen-specific (TT), PHA, or a mixture of both PHA and PMA. This suggests that similar factors interacting with apo A-I are induced at the surface of stimulated T cells by either stimuli. Furthermore, it is likely that similar activating factors are expressed by different types of T cell, freshly isolated T lymphocytes, or HUT-78 cells, because apo A-I displays inhibitory activity in both systems. However, because the levels of activation of monocytes differ as a function of the stimulus,4,7,8 the expression levels of the activating factors may differ as a function of the type of T cells and their stimuli. The inhibition of T-cell signaling of monocytes might be important in maintaining a low level of monocyte activation within the bloodstream, although static conditions used in this study might not reflect shear stress induced by blood flow. However, variations of apo A-I concentration were observed in systemic lupus erythematosus, an inflammatory disease of autoimmune etiology,48 in which apo A-I plasma concentrations were diminished. This decrease was associated with the presence of anti-apo A-I antibodies in 32% of patients.49 Recently, it was shown that the inflammatory condition in juvenile rheumatoid arthritis was associated with hypo-high density lipoproteinemia50 and a significant decrease in apo A-I concentration in patient plasma. In rheumatoid arthritis, the levels of circulating apo A-I and HDL cholesterol in untreated patients are lower than in normal controls.51-53 In contrast, apo A-I is enhanced in synovial fluid of patients with rheumatoid arthritis,54 although its concentrations remained 10-fold lower in synovial fluid than in plasma. The elevation of apo A-I levels in synovial fluid of patients with rheumatoid arthritis was accompanied by an enhancement in cholesterol, suggesting an infiltration of HDL particles in the inflamed joint. This putative regulatory mechanism might, however, be overpowered by serum amyloid A (SAA), a positive acute-phase protein that is produced in the rheumatoid arthritis synovium.55,56 Indeed, SAA can displace apo A-I from HDL, and HDL-associated SAA displays proinflammatory activity.57,58 Increasing evidence strongly supports the contention that inflammatory responses are an integral part of atherosclerosis.59 Indeed, monocyte-macrophages and T lymphocytes are present at all stages of lesion development, and the earliest lesion (fatty streak) is composed predominantly of macrophages and T lymphocytes.60 Therefore, T lymphocyte-signaling of monocytes may occur in atherosclerosis. Gene transfer of apo A-I reduces atherosclerosis in several mouse models.61,62 This was usually attributed to the function of HDL-associated apo A-I in lipid metabolism and transport. However, our study suggests that HDL fulfill protective functions at several levels in atherosclerosis including the decrease in contact-mediated monocyte activation by T lymphocytes. Furthermore, the premise that the incidence of atherosclerotic heart disease is higher in patients with systemic lupus erythematosus and rheumatoid arthritis63 is in agreement with the inverse correlation of the concentration of HDL with the incidence of atherosclerosis. In conclusion, we have identified a novel anti-inflammatory function
for HDL-associated apo A-I. This may be a general mechanism of
protection against the activation of monocytes in inflammatory conditions when stimulated T lymphocytes are found in the bloodstream, as well as an important counterregulatory mechanism to macrophage activation by leakage of plasma proteins into the inflamed tissue. A
new concept emerging from the present results is the importance of
"negative" acute-phase proteins such as apo A-I as
anti-inflammatory molecules able to block T-cell signaling of
macrophages and thus to inhibit TNF-
We thank Mrs M.-T. Kaufmann and Dr Hsien-Sen Lu for skillful technical assistance, Drs R. Rezzonico, C. Chizzolini, and R. W. James for advice and discussions.
Division of Immunology and Allergy (Hans Wilsdorf Laboratory), Department of Internal Medicine, University Hospital, Genève, Switzerland; and Amgen, Thousand Oaks, CA.
Submitted August 25, 2000; accepted December 21, 2000.
Supported in part by a grant from the Swiss National Science Foundation (no. 31-50930-97), the Hans Wilsdorf Foundation, and a grant from the Swiss Society for Multiple Sclerosis.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Danielle Burger, Clinical Immunology Unit, University Hospital, 24 rue Micheli-du-Crest, CH-1211 Genève 14, Switzerland; e-mail: danielle.burger{at}hcuge.ch.
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and tumor necrosis factor-
by blocking contact-mediated activation
of monocytes by T lymphocytes
Top
Abstract
Introduction
) or presence of FCS (10%), HS
(10%), or HDL (0.32 mg/mL protein) for 45 minutes on ice; after
washing treated (hatched and black columns) and untreated (white
columns) THP-1 cells were cultured in the presence of treated (white
and black columns) or untreated (hatched columns) membranes of
stimulated HUT-78 cells; TNF-
in T
lymphocytes.