A CD18-dependent protein kinase C ß-mediated alternative cell death pathway of activated monocytes

Jean-Gabriel Castaigne1, Wenyan Guo2, Claire Lévéille1, Dominique Charron1 and Reem Al-Daccak1,2

1 Unité INSERM U396, Institut de Recherches Biomédicales des Cordeliers, 15 rue de l’école de médicine, 75006 Paris, France and Laboratoire d‘Immunologie et d‘Histocompatibilité, Hôpital Sainte Louis AP-HP, 75475 Paris France 2 Centre de Recherche en Rhumatologie et Immunologie, CHUL, Université Laval, Québec G1V 4G2, Canada

Correspondence to: R. Al-Daccak, INSERM, U396, 15 rue de l’École de Médicine, 75006, Paris, France. E-mail: Reem.Al-Daccak{at}bhdc.jussieu.fr
Transmitting editor: S. Romagnani


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Activated monocytes become resistant to numerous death stimuli including death receptors. Given that the uncontrolled activation of monocytes/macrophages and their persistence can lead to severe inflammatory conditions, it is critical to define the pathways that control their elimination. We previously reported that ligation of HLA-DR molecules on peripheral blood-derived monocytes induces their death. To investigate the mechanisms of HLA-DR-mediated death in monocytes, we used the THP-1 monocytic cell line as a model. We show that while THP-1 are equally resistant to HLA-DR- and to Fas-mediated death, treatment of THP-1 with IFN-{gamma} renders them sensitive to HLA-DR- but not to Fas-mediated death. Both activation of the Src family protein tyrosine kinase and classical protein kinase C (PKC) occur through HLA-DR, but only PKC activation is involved in HLA-DR-mediated death of these cells. Moreover, HLA-DR-mediated cell death of activated monocytes implicates a regulatory loop between the HLA-DR/CD18 complex and the downstream activation of PKCß. Thus, our study identifies an alternative physiological signaling pathway of monocyte death, and further investigation on its regulation is likely to provide significant insights into the control of monocyte homeostasis and inflammation.

Keywords: apoptosis, integrins, macrophages, MHC class II, protein tyrosine kinase, signaling


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cells of the monocyte/macrophage lineage are now recognized as pivotal regulators of inflammation and immunity, and are principal effector cells against a broad class of intracellular pathogens. To exert their effects, monocytes have to be primed by proinflammatory cytokines, in particular the T cell-derived lymphokine IFN-{gamma} (1,2). Although IFN-{gamma}-primed monocytes are crucial for host defense, they may also be dangerous cells because of their production of a large array of inflammatory mediators. Thus, these cells must be subjected to strict regulatory control to avoid their aberrant activation and persistence in diseased tissues that can result in a state of chronic inflammation and/or autoimmunity. Before their activation/differentiation, circulating monocytes are susceptible to Fas-mediated cell death unless they receive permission to survive by inflammatory mediators or growth factors (3). In contrast, activated/differentiated monocytes/macrophages are resistant to numerous death stimuli including death receptor ligation (4,5) and, therefore, activation of alternative death pathways must take place to eliminate dangerous activated/differentiated monocytes. Although the mechanisms responsible for their resistance to apoptosis are starting to be revealed (4,6,7), the identification of alternative death pathway mechanisms that could lead to macrophage cell death are still poorly investigated. In this regard, a form of activation-induced macrophage apoptosis that is initiated after activation with zymosan and phorbol esters in IFN-{gamma}-treated monocytes has been described (8).

In addition to their antigen-presenting function, the MHC class II molecules on antigen-presenting cells (APC) mediate signal transduction. The consequences of MHC class II-mediated signals include cell–cell adhesion, proliferation and differentiation, cytokine production, and cell death (9). An elegant demonstration of the importance of MHC class II-mediated signals has been provided by the induction of tyrosine kinase activation in B cells by TCR engagement of MHC class II–peptide complexes during physiological response (10). Furthermore, during T cell–B cell interaction the HLA-DR-generated signals can also induce IgM production from B cells in association with Syk activation (11). In monocytes, we and others have demonstrated the expression and production of various inflammatory cytokines, including IL-1ß and tumor necrosis factor-{alpha}, by engagement of MHC class II with their ligand superantigens (1214), and more recently it has been shown that differential activation of monokines through MHC class II molecules contributes to T cell response patterns during monocyte–T cell interactions (15).

In addition to cell activation, MHC class II-mediated signals can also lead to cell death, as we and others have demonstrated in B cells, monocytes and dendritic cells (1619). Although the mechanisms underlying MHC class II-mediated death are not yet fully elucidated, studies in B cells showed that this form of death is different from the classic apoptosis mediated by death receptors and mitochondria, and occurs in a caspase-independent manner with the display of a combination of other apoptotic features (17,18). Since death via HLA-DR occurs in activated rather than in resting B cells, it has been postulated to play a role in the termination of the immune response on the side of APC. Recently, it has been shown that the susceptibility to HLA-DR-mediated death can discriminate differentiation stages of dendritic/monocytic APC where mature dendritic cells, and not immature, were highly susceptible to this form of death (16). Indeed, these studies lead to the notion that signals generated via HLA-DR, although they participate in APC activation during the immune response, also lead to the demise of mature professional APC, thereby providing means of limiting the immune response.

With regard to monocytes, our recent studies with peripheral blood-derived monocytes described their susceptibility to HLA-DR-mediated death, a susceptibility that is remarkably enhanced by IFN-{gamma} priming, and suggested this novel pathway of monocyte death as a possible alternative mechanism to eliminate activated monocytes (19). To further address this possibility and to investigate the signaling mechanisms involved in HLA-DR-mediated death, we undertook the human monocytic cell line THP-1 as a cell model. Our results confirmed that HLA-DR-mediated cell death occurs mainly in IFN-{gamma}-treated THP-1 cells, and is independent from death receptor and caspase apoptotic pathways. Despite the activation of protein kinase C (PKC) ß and Src kinases downstream of HLA-DR, only activation of PKCß is involved in HLA-DR-mediated death of IFN-{gamma}-primed THP-1 cells. In addition and since HLA-DR-mediated death of both B cells and monocytes is cell–cell contact dependent, we also provide evidence for a role of the ß2 integrin CD18 in HLA-DR-mediated death. Indeed, HLA-DR-mediated PKCß activation and cell death are two events that are significantly inhibited in the presence of anti-CD18 blocking antibodies and we also demonstrate that CD18 is physically associated with HLA-DR molecules on IFN-{gamma}-primed THP-1 cells. These studies identify CD18 and PKCß as playing major roles in HLA-DR-mediated cell death in activated monocytes, and may be a significant pathway in the elimination of activated macrophages during the course of immune responses.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Antibodies and reagents
Anti-CD29, -CD49e (SAM1), -CD49d (HP2/1), -CD18 (7E4) and -CD95 (CH-11, ZB4) were purchased from Immunotech (Coulter, Villepinte, France); anti-CD18 (MEM-14) and -CD54 (MEM-11) were given by Vaclav Horejsi (Czech Republic); anti-HLA-DR (L243), -DP (B7/21) and -CD18 (TS1/18) were affinity-purified from liquid ascites using Protein G columns. Neutralizing anti-hTRAIL and anti-hTNF-{alpha} were purchased from R & D Systems (Minneapolis, NE). IgG1 (MOPC-21) and IgG2a (UPC-10) antibodies used as isotype control were purchased from Sigma (St Louis, MO). The caspase-8 (IETD-fmk) and broad-spectrum caspase (zVAD-fmk) inhibitors, the PTK inhibitors PP2, herbimycine, AG18, and the PKC{alpha}/ß inhibitor dequalinium chloride (DECA) were purchased from Calbiochem (San Diego, CA). m-Iodobenzyl-guanidine (MIBG), 3-Aminobenzamide (3-ABA), L-1-chloro-3-(4-tosylamido)-7-amino- heptanone (TLCK), L-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone (TPCK), cytochalasin B, cycloheximide and actinomycin D, were purchased from Sigma.

Cell culture
The human monocytic cell line THP-1 obtained from ATCC (Rockville, MD) was maintained in RPMI medium (Life Tech nologies, Cergy Pontoise, France) supplemented with 10% FCS (Life Technologies), 100 µg/ml streptomycin, 100 U/ml penicillin, 2 mM L-glutamine and 0.1% of ß-mercaptoethanol (Life Technologies) at 37°C in 5% CO2. For IFN-{gamma} pre-treatment, THP-1 cells were cultured with 50 U/ml of IFN-{gamma} (R & D Systems) for 48 h and the effect of IFN-{gamma} priming was assessed by flow cytometric analysis of HLA class II expression.

Flow cytometry analysis
Cell surface expression of HLA class II molecules as well as other cell surface receptors was analyzed by indirect immunofluorescence. Briefly, cells were incubated in PBS containing 10% human serum for 20 min at 4°C prior staining to saturate Fc receptors. After washing, cells were incubated with antibodies against tested molecules or their isotype-matched control for 30 min at 4°C, washed then incubated with FITC-conjugated Fab'2 goat anti-mouse IgG for another 30 min at 4°C. Cells were then analyzed by flow cytometry (FACScan; Becton Dickinson Biosciences, Mountain View, CA) and the CellQuest program.

Biotin labeling of cells
Cells (107/ml) were washed 3 times in ice-cold PBS and labeled with 200 µg/ml sulfo-NHS-LC-Biotin (Pierce, Rockford, IL) at 4°C for 30 min. They were then washed 3 times in ice-cold RPMI and finally once in ice-cold PBS.

Immunoprecipitation and Western blotting
Biotin-labeled or unlabeled cells were lysed in cold lysis buffer (25mM Tris–HCl, pH 7.6, 150 mM NaCl, 1% CHAPS) containing 1mM PMSF (Sigma), 1 mM EDTA (Sigma), 2 µg/ml aprotinin, 2 µg/ml leupeptin (Euromedex, Mundolsheim, France), 1 µM pepstatin (Euromedex), 10 µg/ml TLCK (Sigma) and 10 µg/ml TPCK (Sigma) for 60 min at 4°C. Insoluble material was removed by centrifugation at 16,000 g for 10 min at 4°C. Soluble lysates were first pre-cleared by constant rotation with goat IgG–agarose (Sigma) and then immunoprecipitated with mAb plus agarose beads coated with goat anti-mouse IgG. After washing the beads 4 times with lysis buffer, the bound material was eluted in an equivalent volume of Laemmli sample buffer and boiled for 5 min. The samples were analyzed by SDS–PAGE on either 9 or 10% gels. Gels were electroblotted to PVDF membranes using a semi-dry transfer apparatus (Biorad, Mississauga, Canada). The membranes were blocked for 1 h at room temperature in 1% BSA in TBST (Tris–HCl 20 mM, NaCl 150 mM and Tween 20 0.05%). The membranes were then either incubated with anti-HLA-DR {alpha} chain DA6.147 (unlabeled cells) or with ExtrAvidin–horseradish peroxidase (Sigma) (biotin-labeled cells) in blocking buffer for 1 h at room temperature. Bound mAb were detected with horseradish peroxidase-conjugated goat anti-mouse IgG or ExtrAvidin–horseradish peroxidase and visualized using the enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, Uppsala, Sweden).

Cell death assay
Cell death was evaluated by measuring the percentage of Annexin V+/propidium iodide (PI)+ cells. Briefly, after stimulation with soluble anti-HLA-DR L243 or with other cell death stimuli as indicated, cells were resuspended in PBS and stained with Annexin V–FITC (membrane marker of apoptotic cells), and then incubated with PI (which binds to DNA and is actively excluded by live cells) using the R & D Systems apoptosis detection kit. Cells were then analyzed by flow cytometry gating for Annexin V+/PI+ cells. In some experiments, the percentage of dead cells was determined by PI uptake where stimulated cells were resuspended in PBS, incubated with PI on ice and then analyzed by flow cytometry. Dead cells appeared as a well-defined cell population with decreased FSC (equating to decreased cell size) and increased PI uptake (reflecting changes in membrane permeability).

The percentage of specific cell death was calculated according to the following formula: 100 x [(% of dead test cells – % of dead isotype control cells)/(100 – % of dead isotype control cells)]. Percentage of specific inhibition was calculated using the following formula: 100 x [1 – (% of specific inhibitor-treated cell death/% of specific untreated cell death)].

DNA fragmentation was assessed after extraction of total DNA using TRIzol reagent (Life Technologies), according to the manufacturer’s instructions, and electrophoresis migration on a 0.4% agarose gel containing ethidium bromide.

RT-PCR
Total RNA was extracted with TRIzol reagent (Life Technologies), following the manufacturer’s instructions. Total RNA (1 µg) was reverse transcribed using a reverse transcription system kit (Promega, Madison, WI). PCR was performed with the Taq polymerase from Pharmacia Biotech, with amplification cycles of: 94°C, 2 min, one cycle; 94°C, 1 min, 58°C, 1 min, 72°C, 1 min, 30 cycles; 72°C, 4 min, one cycle. Amplification for a 695-bp cDNA fragment of TNF-{alpha} was performed with hTNF-{alpha}-specific primers: forward, 5'-GCA-ATG-ATC-CCA-AAG-TAG-ACC-TGC-CC-3'; reverse, 5'-ATG-AGC-ACT-GAA-AGC-ATG-ATC-CGG-3'. As a control, ß-actin levels were also determined with specific primers which amplify a 525-bp fragment: forward, 5'-AGC-CAT-GCC-AAT-CCT-ATC-TTG-T-3'; reverse, 5'-ACG-GCT-GCT-TCC-AGC-TCC-TC-3'.

PKC activity assay
Following stimulation, cells were suspended in ice-cold buffer A containing 25mM Tris, pH 7.4, 10 mM EDTA, 2 mM EGTA and a freshly prepared cocktail of inhibitors, and then sonicated for 5 s at a power of 2.5 W by a 550 Sonic Dismembrator (Fisher Scientific, St Louis, MO). Triton X-100 was added to a final concentration of 0.5% and lysates were left on ice for 30 min. Lysates were then centrifuged at 900 g for 10 min, and then supernatants were collected and mixed with 2 volumes of ice-cold buffer A. PKCß or {alpha} were specifically immunoprecipitated with 3 µg anti-PKCß or {alpha} and 20 µl Protein A/G. The beads were washed 3 times with ice-cold buffer A, and resuspended with PBS supplemented with 200 mM NaCl and 10 mM 2-mercaptoethanol. The non-radioactive PepTag PKC activity kit (Promega) was used to evaluate the PKC activity according to the manufacturer’s protocol with modification. Briefly, 27 µl samples were added to buffer containing 10 µl PepTag PKC 5 x reaction buffer, 1 µl PepTag C1 peptide (0.4 µg/µl), 10 µl sonicated PKC 5 x activator solution and 2 µl peptide protection solution then was incubated at 37°C for 1 h. Then 1 µl 80% glycerol and 1 µl 10% Triton X-100 were added to the samples, and samples were sonicated for 10 s at a power of 2.5 W. Samples (20 µl) were then loaded onto 0.75% of agarose gel and run at 100 V for 30 min. The gel was visualized under UV and photographed.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Treatment of THP-1 cells with IFN-{gamma} sensitizes them to MHC class II-mediated death
It is known that activated monocytes/macrophages are resistant to apoptosis induced by different apoptotic stimuli. In this regard, treatment of peripheral blood-derived monocytes with IFN-{gamma} induces their resistance to Fas-mediated death (5,8,20), a major pathway in the modulation of peripheral tolerance. However, we have previously shown that a similar treatment increases by at least 2-fold the sensitivity of these cells to HLA-DR-mediated death and suggested this cell death as an alternative mechanism to limit subsequent persistence of activated cells (19). To further characterize HLA-DR-mediated cell death in activated monocytes, we investigated the susceptibility of the human monocytic cell line THP-1, which expresses the same level of Fas and HLA-DR molecules as normal monocytes, to HLA-DR-mediated death. Cells were treated with soluble anti-HLA-DR L243 (10 µg/ml), anti-Fas 7C11 (100 ng/ml) or their isotype controls for increasing time periods (2–24 h), and then the percentage of dead cells was evaluated by dual Annexin V–FITC staining and PI uptake. The vast majority of anti-HLA-DR-treated or anti-Fas-treated THP-1 cells were Annexin V/PI(Fig. 1A), even when anti-HLA-DR was cross-linked with an anti-IgG or when saturating levels of anti-Fas (1 µg/ml) were used (data not shown). However, when THP-1 cells were treated for 48 h with IFN-{gamma}, then similarly stimulated with anti-HLA-DR or anti-Fas, up to 50% of anti-HLA-DR-treated cells were Annexin V+/PI+ after 4–6 h, whereas the majority (>90%) of anti-Fas-treated cells remained Annexin V/PI even after 24 h of treatment (Fig. 1B). A similar significant percentage (30–50%) of Annexin V+/PI+ cells was observed when IFN-{gamma}-treated THP-1 cells were stimulated with concentrations ranging from 2.5 to 10 µg/ml of soluble anti-HLA-DR L243 (data not shown). The absence of HLA-DR-mediated death in non-treated THP-1 cells was not due to the inability of HLA-DR molecules to mediate signals as evidenced by the induction of TNF-{alpha} gene expression upon stimulation of these cells with anti-HLA-DR L243 (Fig. 2A). In addition, the resistance to Fas-mediated apoptosis is not due to a change in the level of Fas expression. In fact, the expression of both HLA-DR and Fas molecules is significantly increased following IFN-{gamma} treatment (Fig. 2B). While primed THP-1 cells remain resistant to Fas-mediated death, they undergo cell death following HLA-DR ligation, indicating that this pathway can be important in the regulation of monocyte/macrophage homeostasis.



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Fig. 1. Treatment with IFN-{gamma} sensitizes THP-1 cells to HLA-DR-mediated cell death. Non-treated (A) or IFN-{gamma}-treated (B) THP-1 cells were left unstimulated or stimulated for different period of times with anti-HLA-DR L243 (10 µg/ml), anti-Fas 7C11 (100 ng/ml) or isotype control (10 µg/ml) antibodies. Cell death was determined by Annexin V/PI staining and FACS analysis as exemplified in the upper panel of (B). The results are expressed as percentage of Annexin V+/PI+ cells and are means of three independent experiments each one done in triplicates.

 


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Fig. 2. (A) Cross-linking of HLA-DR induces TNF-{alpha} gene expression in non-primed THP-1 cells. The cells were stimulated with control IgG antibodies or with increasing amounts of anti-HLA-DR antibody L243 cross-linked (L243-XL) or not for 1 h. As a control, the cells were also treated with lipopolysaccharide. Total cellular RNA was isolated and TNF-{alpha} gene expression was analyzed by RT-PCR as described in Methods. (B) Treatment of THP-1 cells with IFN-{gamma} increases expression of both Fas and HLA-DR molecules. THP-1 was left unprimed or primed with IFN-{gamma} (50 U/ml) for 48 h. The expression of Fas and HLA-DR was determined by indirect staining with specific antibodies and by FACS analysis. The results are representative of three different experiments.

 
Characterization of HLA-DR-mediated cell death in activated THP-1
The HLA-DR-mediated cell death in B lymphocytes has been shown to be different from the classical apoptosis that involves caspases downstream of death receptors or the mitochondria (17,18). Thus, we sought to determine whether in monocytes, HLA-DR-mediated death is similar to that described in B lymphocytes or if it falls in the category of classical apoptosis. Previously, we have shown that in peripheral monocytes, HLA-DR mediates a form of cell death that displays characteristic changes associated with apoptosis in these cells such as chromatin condensation, cytoplasmic vacuolization (honeycomb pattern of peripheral vacuolization) and phosphatidylserine exposure, and is independent from TNF and Fas (19). Based on inhibition studies, we also found that in THP-1, HLA-DR-mediated cell death is independent from Fas and TNF, and is independent from TRAIL/DR4/DR5 apoptotic pathways as well. Indeed, pre-incubation of IFN-{gamma}-treated THP-1 with blocking anti-Fas (ZB-4) or with neutralizing anti-hTNF-{alpha} or anti-hTRAIL antibodies did not have any significant effect on HLA-DR-mediated death of these cells (Fig. 3A). In addition, HLA-DR-mediated death of IFN-{gamma}-treated THP-1 does not implicate cleavage of the caspase substrate PARP (data not shown) and does not lead to oligosomal DNA fragmentation (Fig. 3B). Furthermore, the summarized data in Table 1 indicate that HLA-DR-mediated death of these cells (i) is not affected by irreversible inhibitors of caspases, the broad spectrum zVAD and a caspase-8-specific IETD, whereas similar concentration of zVAD significantly inhibited staursporin-induced apoptosis of these cells (65% specific inhibition in the presence of 50 µM of zVAD) (Fig. 3C); and (ii) does not require de novo protein synthesis (absence of inhibition in the presence of cycloheximide) and implicates serine proteases (60–65% specific inhibition in the presence of TLCK and TPCK) and mono- and poly-ADP-ribosyl transferases (69–74% of specific inhibition in the presence of MIBG and 3-ABA respectively). Together, these results indicate that HLA-DR-mediated cell death in monocytes is similar to that previously described in B lymphocytes (17,18). It is caspase-independent, independent from cell death receptors and display features of apoptosis-like programmed cell death (PCD) (21).



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Fig. 3. HLA-DR-mediated death is independent of the death receptor pathway and is not associated with DNA fragmentation. (A) IFN-{gamma}-treated THP-1 were left untreated or treated with anti-HLA-DR L243 for 6 h in the presence or absence of blocking antibodies (10 µg/ml) against TRAIL and TNF cytokines and Fas receptor. Cell death was then determined by PI uptake, and the results are expressed as percent of specific death according to the formula described in Methods and are means of three independent experiments each one done in triplicates. (B) IFN-{gamma}-treated THP-1 cells were treated with anti-HLA-DR L243, anti-CD32 (Iv.3) antibodies or left untreated, or as a control treated with staurosporin for 6 h. The cells were lysed, and the genomic DNA was isolated and analyzed by agarose gel as described under Methods. The results are representative of three independent experiments. (C) IFN-{gamma}-treated THP-1 cells were left untreated or treated with 50 µM zVAD and then were stimulated with anti-HLA-DR L243 or with staurosporin. Cell death was determined by Annexin V/PI staining, and the results are expressed as percentage of Annexin V+/PI+ cells and are means of three independent experiments each one done in triplicate.

 

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Table 1. Effects of different agents on HLA-DR-mediated cell death of IFN-{gamma}-treated THP-1 cells
 
HLA-DR-mediated death of IFN-{gamma}-treated THP-1 implicates PKC{alpha}
The above results demonstrating that HLA-DR-mediated death can be an alternative cell death pathway that regulates monocytes homeostasis prompted us to further investigate its underlying mechanisms. It is well established that ligation of HLA-DR induces the activation of PKC, in particular PKC{alpha} and PKCßII isoforms (22), and the phosphorylation of protein tyrosine kinases (PTK) (23), in particular the Src family of PTK (24). Thus, we were interested to analyze whether these pathways are implicated in the signaling cascade triggered through HLA-DR and leading to cell death of IFN-{gamma}-treated THP-1. To this end, we examined the effects of specific PKC{alpha}/ß and Src kinases inhibitors on this response. Treatment of the cells with DECA, a selective PKC inhibitor that binds to the catalytic and regulatory domains and irreversibly inhibits PKC{alpha} and ß (25), with concentrations ranging between 6 and 14 µM resulted in a dose-dependent inhibition of anti-HLA-DR-induced cell death. Up to 50% of cell death inhibition is observed in cells treated with DECA in concentrations between 11 and 14 µM of DECA (Fig. 4A), indicating that treatment of IFN-{gamma}-treated THP-1 with PKC{alpha}/ß-specific inhibitor significantly blocks their HLA-DR-mediated cell death. We then tested whether HLA-DR ligation induces PKC{alpha} activity in IFN-{gamma}-treated THP-1 cells. The results in Fig. 4(B) indicate that cross-linking of HLA-DR results in a significant induction of PKCß activity that is completely inhibited by pretreating the cells with the DECA inhibitor. Similarly, PKC{alpha} is also activated downstream of HLA-DR, although to a lesser extent (data not shown). That HLA-DR-mediated death is significantly inhibited by specific inhibition of PKC{alpha}/ß and that activation of PKCß occurs in a time period that precedes the induction of cell death strongly indicate that activation of cPKC is involved in HLA-DR-mediated death.



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Fig. 4. Activation of PKCß is involved in HLA-DR-mediated death of IFN-{gamma}-treated THP-1 cells. The cells were pre-incubated in the absence or in the presence of increasing concentrations of cPKC inhibitor DECA (A) or with tyrosine phosphorylation inhibitors [AG18 (75 µM), herbimycin A (1 µM) and PP2 (25 µM)] (C) for 1 h and then stimulated or not with anti-HLA-DR L243 for 6 h. Cell death was determined by the Annexin V/PI method and results are expressed as percent of specific cell death as described under Methods and are means of four three independent experiments each done in triplicate. (B) HLA-DR cross-linking induces activation of PKCß. The cells were stimulated with anti-HLA-DR L243 or with isotype control IgG for 30 min and then cross-linked for another 30 min, and PKC ß activity was evaluated as described in Methods. The histogram represents the densitometric quantification of the presented gel. (D) Ligation of HLA-DR induces tyrosine phosphorylation of several proteins. The cells were stimulated with anti-HLA-DR L243 or with isotype control IgG for 15 min and the profile of tyrosine phosphorylation was determined as described in Methods. The effects of DECA on PKC activity and PP2 on Src kinase activity were also determined. The results are representative of three independent experiments.

 
In contrast, treatment of IFN-{gamma}-treated THP-1 with PTK inhibitors AG18, a broad range inhibitor, herbimycin A, reported to inhibit c-Src kinases, or with PP2, a selective inhibitor of Src family of PTK, did not have any significant effect on L243-induced cell death (Fig. 4C). Since the inhibitors of PTK did not demonstrate any pronounced effects on HLA-DR-mediated cell death and it has been reported that HLA-DR stimulation in monocytes induces phosphorylation of PTK, we verified whether this pathway is activated downstream of HLA-DR in IFN-{gamma}-treated THP-1 cells and whether it can be blocked by PTK inhibitors. The results in Fig. 4(D) indicate that cross-linking of HLA-DR molecules on IFN-{gamma}-treated THP-1 cells induces tyrosine phosphorylation of several proteins, an event that is significantly inhibited with PP2 treatment. Similarly, AG18 and herbimycin A also inhibit HLA-DR-induced PTK phosphorylation (data not shown). Thus, the PTK and, in particular, Src kinase pathway is indeed activated downstream of HLA-DR in these cells, but does not appear to be involved in the induction of cell death.

HLA-DR-mediated death of IFN-{gamma}-treated THP-1 implicates CD18 that is physically associated with HLA-DR on THP-1 cells
Several cell surface molecules, such as integrins, have been reported to be implicated in the regulation of monocytes homeostasis (2629). In addition, we have previously reported that HLA-DR-mediated death of B cells is cell–cell contact dependent (18) and that of peripheral blood-derived monocytes is CD18 dependent (19). Pretreatment of IFN-{gamma}-treated THP-1 with 20 µM cytochalasin B caused a 72% of inhibition of specific cell death induced by anti-HLA-DR L243 (Table 1), indicating that the integrity of the cytoskeleton is required and therefore suggests the possible implication of cell–cell contact molecules in this form of death. Accordingly, we examined the effects of different blocking antibodies against ß1 and ß2 integrins, in particular the {alpha}4ß1, {alpha}5ß1, {alpha}Mß2 and {alpha}Lß2 that are expressed on monocytes and were implicated in monocytes cell death or survival (19,28,29). IFN-{gamma}-treated THP-1 cells were pre-incubated with saturating concentration (15–20 µg/ml) of anti-CD29 1), anti-CD49d ({alpha}4), anti-CD49e ({alpha}5) or anti-CD18 (7E4) and were then stimulated with anti-HLA-DR L243. None of these antibodies demonstrated any significant effect on HLA-DR-mediated death except for anti-CD18 antibody (7E4) that specifically inhibited (49.6% specific inhibition) cell death (Fig. 5A). It is noteworthy that pre-incubation with the blocking antibodies did not affect the fixation of anti-HLA-DR antibody as determined by flow cytometry analysis (data not shown). The inhibition of HLA-DR-mediated death with two other anti-CD18 mAb, MEM-14 and TS1/18 (Fig. 5B), confirms that the observed inhibition effect is not antibody specific and that, similar to peripheral blood-derived monocytes, CD18 is implicated in HLA-DR-mediated death of IFN-{gamma}-treated THP-1. Since PKCß is involved in HLA-DR-mediated cell death, we sought to determine whether the activation of PKCß downstream of HLA-DR is also dependent on CD18. Thus, we tested the ability of anti-CD18 blocking antibodies to block anti-HLA-DR-induced PKCß activity in IFN-{gamma}-treated THP-1 cells. The results in Fig. 5(C) indicate that pretreatment of the cells with anti-CD18 (TS1/18) inhibits L243-induced PKCß activity, indicating that activation of PKCß downstream of HLA-DR is also CD18 dependent.



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Fig. 5. HLA-DR-mediated cell death and PKCß activity are CD18 dependent. (A and B) The cells were pre-incubated or not with different anti-integrin blocking antibodies or with control IgG for 1 h and then stimulated with anti-HLA-DR L243 for 4 h. Cell death was determined as described and the results are expressed as percent of specific cell death (A) or percent of specific inhibition (B) as described in Methods, and are means of three independent experiments each done in triplicate. (C) Activation of PKCß via HLA-DR is inhibited by blocking anti-CD18 antibodies. The cells were pre-incubated with control isotype or with anti-CD18 antibodies for 1 h and then stimulated or not with anti-HLA-DR L243 for 1 h. PKCß activity was then determined as described. The histogram represents the densitometric quantification of the presented gel. (D) CD18 and HLA-DR are physically associated in THP-1 cells. Immunoprecipitates from biotin-labeled (upper panel) or unlabeled (lower panel) cells were prepared from CHAPS cell lysates with anti-CD18, anti-HLA-DR, anti-HLA-DP or with IgG isotype control antibodies, and analyzed by SDS–PAGE and immunoblotting with ExtrAvidin–horseradish peroxidase (upper panel) or with anti-HLA-DR {alpha} chain antibody DA6.147 (lower panel).

 
The quite well-established regulation of HLA-DR-mediated events by other cell surface molecules (30), the known absence of signaling motifs from their cytoplasmic domain (9) and the description of several multimolecular HLA-DR complexes on B cells that have been suggested to have an important role in their signal transducing function (3133) prompted us to investigate the possible physical association of HLA-DR with CD18 on THP-1 cells. THP-1 cells untreated or treated with IFN-{gamma} were biotin labeled and lysed in buffer containing 1% CHAPS, a detergent known to preserve multireceptor complexes. CD18 and HLA-DR were then specifically immunoprecipitated form cells lysates and immunoprecipitates were analyzed by immunoblot with ExtrAvidin–horseradish peroxidase (Fig. 5D).

Analysis of CD18 immunoprecipitates revealed the expected 90–95-kDa band of the CD18 molecule and the 165–170-kDa band of its associated CD11b (34), a band of 55 kDa, and two bands of 34 and 29 kDa. Analysis of HLA-DR immunoprecipitates revealed the characteristic 34- and 29-kDa bands of HLA-DR molecule (31), a band of 55 kDa, and a band of ~95 kDa. None of these protein bands were detected in isotype-matched control mAb immunoprecipitates. The intensity of the CD18 and HLA-DR co-immunoprecipitated bands was remarkably weaker in untreated THP-1 cells due to a low level of HLA-DR expression on untreated cells. The protein band at 90–95 kDa that co-immunoprecipitated with HLA-DR co-migrated with the characteristic 90–95-kDa band of CD18 precipitated from these cells with specific anti-CD18 antibody and the two protein bands that co-immunoprecipitated with CD18 showed a migration pattern similar to that of {alpha} and ß chains of HLA-DR molecules precipitated from these cells with anti-HLA-DR L243. The protein band of ~55 kDa that co-immunoprecipitated with both HLA-DR and CD18 molecules might represent a member of the tetraspanin family, probably CD63 previously reported as associated with both molecules (34,35), and studies are in progress to identify this protein. Nevertheless, these results strongly suggest a constitutive physical association between HLA-DR and CD18 molecules on THP-1 cells.

To confirm that HLA-DR associates with CD18 on THP-1 cells, CHAPS lysates from unlabeled IFN-{gamma}-treated THP-1 cells were subjected to immunoprecipitation with anti-CD18, anti-HLA-DR or IgG isotype control antibodies and immunoprecipitates were analyzed by immunoblot using anti-HLA-DR DA6.147 antibody that recognizes the HLA-DR {alpha} chain. The results in Fig. 5(D) show that the HLA-DR {alpha} chain (34 kDa) is present in CD18 immunoprecipitates, but not in the isotype-matched control immunoprecipitates, confirming the association between the two molecules and the expression of the HLA-DR–CD18 complex on THP-1 cells.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The fact that activated/differentiated monocytes become resistant to most apoptotic stimuli, including death receptor ligation, emphasizes the need for a pathway that can be induced under physiological conditions to terminate their biological responses. Previously, we have reported that peripheral blood-derived monocytes from normal donors differ remarkably in their susceptibility to HLA-DR-mediated death, but this is significantly enhanced after priming the cells with IFN-{gamma} (19). Herein, we confirmed these previous observations in the monocytic THP-1 cell line and we revealed some of the mechanisms that underlay HLA-DR-mediated death in IFN-{gamma}-primed THP-1 cells. We found that similarly to B cells (17,18), HLA-DR-mediated cell death is independent from activation of classic caspases and is not associated with oligonucloesomal fragmentation of DNA. Currently, growing evidence indicates the remarkable plasticity of the cellular death program and the diversification of the apoptosis program in higher eukaryotes with respect to the necessity and role of these caspases (21). Namely, apoptosis-like cell death mainly occurs without the activation of effector caspases and without affecting the efficient removal of dying cells. Indeed, the apoptosis-like PCD describes forms of PCD with chromatin condensation and with the display of phagocytosis-recognition molecules, the phosphatidylserines, along with any degree and combination of other apoptotic features, and most reported forms of caspase-independent apoptosis (3640) fall into this class. Although not fully elucidated, non-caspase proteases such as cathepsins and serine proteases are implicated in bringing about the apoptotic features of these forms of PCD (21,39). Thus, our data herein and those we previously reported (19) indicate that HLA-DR molecules on monocytes mediate an apoptosis-like pattern of PCD that might have evolved as an alternative caspase-independent death pathway of monocytes to fulfill a safe and non-inflammatory removal of activated cells.

Indeed, while THP-1 are resistant to both Fas- and HLA-DR-mediated cell death, treatment of THP-1 with IFN-{gamma} renders them susceptible to HLA-DR- but not to Fas-mediated death, indicating the HLA-DR signaling pathway as an important alternative death pathway in activated monocytes. Two non-exclusive mechanisms can account for the effects of IFN-{gamma} on HLA-DR-mediated cell death. Although the expression levels of HLA-DR molecules on non-treated THP-1 are sufficient to transduce cell signals, as evidenced by TNF-{alpha} gene expression following their ligation, treatment of THP-1 with IFN-{gamma} increases the expression levels of HLA-DR molecules (2) and therefore this can account for the induction of cell death via HLA-DR. The second mechanism by which IFN-{gamma} can sensitize THP-1 to HLA-DR-mediated death is by modulating negatively the signaling pathways involved in cell survival. In this regard, inhibition of the constitutive activity of NF-{kappa}B and phosphatidylinsitol-3 kinase/AKT survival pathways in macrophages induces their death through down-regulation of A1 and Mcl-1, two anti-apoptotic proteins of the Bcl-2 family, involved in mitochondria homeostasis (7,41). Whether phosphatidylinsitol-3 kinase/AKT and NF-{kappa}B survival pathways, and mitochondria homeostasis are involved in HLA-DR-mediated cell death, and whether they might be modulated by IFN-{gamma} treatment is unknown, and these possibilities are now under investigation.

The role of PKC implied by our observations is of particular interest. PKC is a family of serine/threonine kinase isotypes that have been subdivided in three subgroups: the classical PKC comprise {alpha}, ß and {gamma}; the novel PKC include isotypes {delta}, {epsilon}, {theta} and {eta}; and the atypical group is represented by {zeta}, {iota} and {lambda} isotypes (42). Increasing evidence indicates that the outcome of PKC activation vis-à-vis cellular death as well as contributions of distinct PKC isoforms to protection from apoptosis are likely to be stimulus and cell-type specific. In this context, the nPKC {delta} has been implicated in apoptosis of different cell types, including Fas-mediated spontaneous neutrophil and T cells apoptosis (43,44), whereas activation of cPKC {alpha} and ß has been shown to inhibit Fas-mediated apoptosis in Jurkat T cells (45,46). Moreover, the bufalin-induced differentiation and apoptosis of THP-1 are regulated by distinct PKC isozymes (47). Our inhibition studies showed that induction of cell death via HLA-DR in IFN-{gamma}-primed THP-1 cells is a process that depends on the activation of cPKC, specifically {alpha} and ß, rather than activation of the Src family of kinases or mitogen-activated protein kinases, despite their possible activation upon ligation of HLA-DR in monocytes (15) since specific inhibition of these kinases, using specific inhibitors of p38, ERK and MEK, did not have any effect on HLA-DR-mediated death of these cells (data not shown). This PKC implication is in agreement with previous findings. Indeed, it has been shown that PKCß is involved in bufalin-induced macrophage apoptosis (47) and similar implication of cPKC has been reported in zymosan plus phorbol myristate acetate-induced macrophage death (8). Interestingly, this form of monocyte death is also dependent on IFN-{gamma} priming and the feature of this described activation-induced death of monocytes highly resembles those of HLA-DR-mediated death, i.e. it occurs only in IFN-{gamma}-primed monocytes, it is rapid (within 2–4 h of activation) with ultrastructural changes of apoptosis and the absence of oligonucleosomal fragmentation, and it implicates PKC activation (8). In addition, the activation of PKC is among the mechanisms that can lead to the translocation of phospatidylserine in cells undergoing caspase-independent death (38,48). Thus, together, these observations indicate that PKCß may be an important transducer of alternative cell death in macrophages and since macrophage cell death seems to occur only in IFN-{gamma}-primed cells, the HLA-DR signaling death pathway could be important in the regulation of monocyte/macrophage homeostasis.

How PKC{alpha}/ß initiates HLA-DR-mediated death is not yet clear. However, it can be directly mediating the signaling cascade leading to cell death and/or suppressing survival machinery. In this regard, it has been demonstrated that in the human U397 myeloid cell line TPA targets stress-activated protein kinase to mitochondria by a mechanism dependent on activation of PKCß where this translocation is associated with release of cytochrome c and induction of apoptosis (49).

Our results showed that similar to peripheral blood-derived monocytes, HLA-DR mediated death in THP-1 is also dependent on the CD18 integrin. Together with the remarkable inhibition of this cell death in the presence of cytochalasin B, it is quite clear that cell–cell adhesion is a prerequisite of HLA-DR-mediated death. We did not observe any significant effect on HLA-DR-mediated death upon blocking ß1 (CD29) integrins {alpha}4ß1 and {alpha}5ß1 that are expressed on these cells, and have been reported to be implicated in gene expression and apoptosis of monocytes respectively (28,29). However, the fact that a significant (up to 50%) but still partial inhibition was observed in the presence of anti-CD18 might suggest the implication of other cell surface molecules in HLA-DR-mediated death. In addition, we also provide evidence that CD18 is involved in the activation of PKCß following HLA-DR stimulation, a signaling pathway involved in HLA-DR-mediated death. Whether CD18 is only mediating cell–cell contact required for HLA-DR-mediated death and PKCß activation or it has a role in the signaling pathway cannot be concluded herein. Nevertheless, the constitutive physical association that we described between CD18 and HLA-DR molecules on IFN-{gamma}-primed THP-1 strongly suggests a possible role for this integrin in the signaling pathway. Indeed, structure–function studies with mutated or truncated {alpha} and ß chains of HLA-DR indicated that the cytoplasmic tail of the ß chain is required for the translocation of PKCß following engagement of HLA-DR, although the cytoplasmic tails of MHC class II molecules are short and do not contain any signaling motifs (9,22). From these studies it has been suggested that other receptors or membrane-associated molecules act as bridges to effectively couple MHC class II to one or more signaling pathway. Thus, CD18 might act as a bridge to couple HLA-DR to the PKC signaling pathway.

In addition, it is becoming evident that CD18, as well as mediating adhesion, also mediates signal transduction in several types of leukocytes (50). Indeed, several signaling pathways are activated downstream of CD18, including the activation of PKC. In this regard, activation of PKC in polymorphonucleophils by anti-CD18 antibodies has been proposed as a mechanism to terminate the CD18-induced calcium signal and tyrosine phosphorylation (50). In our cell model, we have observed that tyrosine phosphorylation activity is not required for HLA-DR-induced cell death, but is involved in the induction of cytokine gene expression in response to HLA-DR stimulation, whereas PKC activity is involved in cell death and not in cytokine gene expression (manuscript in preparation). Together these results might suggest that in activated monocytes, induction of PKCß activity via CD18 is likely to be involved in the termination of monocytes activation by inducing cell death.

The role of CD18 in cell death has been described in several types of cells and in response to different apoptotic stimuli. Indeed, in T cell clones, engagement of LFA-1 synergizes with TCR in the induction of activation-induced cell death and plays a central role in T cell apoptosis induced following IL-2 withdrawal (5153). In neutrophils, ligation of CD18 has been shown to promote apoptosis (5456) and in mice deficient in CD11b/CD18, it has been shown that ß2 integrins play an important role in the control of inflammation, by accelerating the elimination of extravasated neutrophils by apoptosis (57,58).

Physiologically, IFN-{gamma}-primed monocytes express high levels of HLA-DR molecules that can be engaged with several of their natural ligands such as TCR and CD4 molecules. Thus, in conclusion, we describe a possible physiological alternative death pathway of activated monocytes/macrophages and demonstrate that it is mediated by a regulatory loop between the CD18–HLA-DR complex and the downstream activation of PKCß. The physiological importance of our described cell death is emphasized by the observation that mature dendritic cells undergo cell death after interaction with antigen-specific T cell clones (59). Further investigation into the mechanisms involved in HLA-DR-mediated cell death in monocytes and macrophages will provide significant insights into how signaling via MHC class II molecules can contribute to the control of monocyte homeostasis and inflammatory diseases.


    Acknowledgements
 
We would like to thank Dr Fawzi Aoudjit (CHUL, Québec, Canada) for helpful discussion and Dr V. Horejsi for the anti-CD18 MEM-14. This work was supported by grants from Arthritis Society of Canada, INSERM (Paris, France) and EC grant DNA VACATOPY. R. Al-D. is a recipient of a scholarship award from Fond de Recherche en Santé du Québec, J.-G. C. is a recipient of doctoral fellowship from Ministère de l’Education Nationale de la Recherche et de la Technologie (MENRT) (Paris, France). C. L. is a recipient of post-doctoral fellowship from Fondation de la Recherche Médicale (Paris, France).


    Abbreviations
 
3-ABA—3-aminobenzamide

APC—antigen-presenting cell

DECA—dequalinium chloride

MIBG—m-iodobenzyl-guanidine

PCD—programmed cell death

PI—propidium iodide

PKC—protein kinase C

PTK—protein tyrosine kinase

TLCK—L-1-chloro-3-(4-tosylamido)-7-amino-heptanone

TNF—tumor necrosis factor

TPCK—L-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Farrar, M. A. and Schreiber, R. D. 1993. The molecular cell biology of interferon-{gamma} and its receptor. Annu. Rev. Immunol. 11:571.[ISI][Medline]
  2. Boehm, U., Klamp, T., Groot, M. and Howard, J. C. 1997. Cellular responses to interferon-gamma. Annu. Rev. Immunol. 15:749.[ISI][Medline]
  3. Kiener, P. A., Davis, P. M., Starling, G. C., Mehlin, C., Klebanoff, S. J., Ledbetter, J. A. and Liles, W. C. 1997. Differential induction of apoptosis by Fas–Fas ligand interactions in human monocytes and macrophages. J. Exp. Med. 185:1511.[Abstract/Free Full Text]
  4. Perlman, H., Pagliari, L. J., Georganas, C., Mano, T., Walsh, K. and Pope, R. M. 1999. FLICE-inhibitory protein expression during macrophage differentiation confers resistance to fas-mediated apoptosis. J. Exp. Med. 190:1679.[Abstract/Free Full Text]
  5. Kikuchi, H., Iizuka, R., Sugiyama, S., Gon, G., Mori, H., Arai, M., Mizumoto, K. and Imajoh-Ohmi, S. 1996. Monocytic differentiation modulates apoptotic response to cytotoxic anti-Fas antibody and tumor necrosis factor alpha in human monoblast U937 cells. J. Leukoc. Biol. 60:778.[Abstract]
  6. Perlman, H., Pagliari, L. J., Nguyen, N., Bradley, K., Liu, H. and Pope, R. M. 2001. The Fas–FasL death receptor and PI3K pathways independently regulate monocyte homeostasis. Eur. J. Immunol. 31:2421.[ISI][Medline]
  7. Pagliari, L. J., Perlman, H., Liu, H. and Pope, R. M. 2000. Macrophages require constitutive NF-kappaB activation to maintain A1 expression and mitochondrial homeostasis. Mol. Cell. Biol. 20:8855.[Abstract/Free Full Text]
  8. Munn, D. H., Beall, A. C., Song, D., Wrenn, R. W. and Throckmorton, D. C. 1995. Activation-induced apoptosis in human macrophages: developmental regulation of a novel cell death pathway by macrophage colony-stimulating factor and interferon gamma. J. Exp. Med. 181:127.[Abstract]
  9. Watt, T. H. 1997. Signalling via MHC molecules. In Harnete, M. M. and Rigley, K. P., eds, Lymphocyte Signalling: Mechanisms, Subversion and Manipulation, p. 141. Wiley, New York.
  10. Lang, P., Stolpa, J. C., Freiberg, B. A., Crawford, F., Kappler, J., Kupfer, A. and Cambier, J. C. 2001. TCR-induced transmembrane signaling by peptide/MHC class II via associated Ig-{alpha}/ß dimers. Science 291:1537.[Abstract/Free Full Text]
  11. Tabata, H., Matsuoka, T., Endo, F., Nishimura, Y. and Matsushita, S. 2000. Ligation of HLA-DR molecules on B cells induces enhanced expression of IgM heavy chain genes in association with Syk activation. J. Biol. Chem. 275:34998.[Abstract/Free Full Text]
  12. Al-Daccak, R., Mehindate, K., Poubelle, P. E. and Mourad, W. 1994. Signalling via MHC class II molecules selectively induces IL-1 beta over IL-1 receptor antagonist gene expression. Biochem. Biophys. Res. Commun. 201:855.[ISI][Medline]
  13. Al-Daccak, R., Mehindate, K., Damdoumi, F., Etongue-Mayer, P., Nilsson, H., Antonsson, P., Sundstrom, M., Dohlsten, M., Sekaly, R. P. and Mourad, W. 1998. Staphylococcal enterotoxin D is a promiscuous superantigen offering multiple modes of interactions with the MHC class II receptors. J. Immunol. 160:225.[Abstract/Free Full Text]
  14. Scholl, P. R., Trede, N., Chatila, T. A. and Geha, R. S. 1992. Role of protein tyrosine phosphorylation in monokine induction by the staphylococcal superantigen toxic shock syndrome toxin-1. J. Immunol. 148:2237.[Abstract/Free Full Text]
  15. Matsuoka, T., Tabata, H. and Matsushita, S. 2001. Monocytes are differentially activated through HLA-DR, -DQ, and -DP molecules via mitogen-activated protein kinases. J. Immunol. 166:2202.[Abstract/Free Full Text]
  16. Bertho, N., Drenou, B., Laupeze, B., Berre, C. L., Amiot, L., Grosset, J. M., Fardel, O., Charron, D., Mooney, N. and Fauchet, R. 2000. HLA-DR-mediated apoptosis susceptibility discriminates differentiation stages of dendritic/monocytic APC. J. Immunol. 164:2379.[Abstract/Free Full Text]
  17. Drenou, B., Blancheteau, V., Burgess, D. H., Fauchet, R., Charron, D. J. and Mooney, N. A. 1999. A caspase-independent pathway of MHC class II antigen-mediated apoptosis of human B lymphocytes. J. Immunol. 163:4115.[Abstract/Free Full Text]
  18. Leveille, C., Zekki, H., Al-Daccak, R. and Mourad, W. 1999. CD40- and HLA-DR-mediated cell death pathways share a lot of similarities but differ in their use of ADP-ribosyltransferase activities. Int. Immunol. 11:719.[Abstract/Free Full Text]
  19. Thibeault, A., Zekki, H., Mourad, W., Charron, D. and Al-Daccak, R. 1999. Triggering HLA-DR molecules on human peripheral monocytes induces their death. Cell. Immunol. 192:79.[ISI][Medline]
  20. Mangan, D. F. and Wahl, S. M. 1991. Differential regulation of human monocyte programmed cell death (apoptosis) by chemotactic factors and pro-inflammatory cytokines. J. Immunol. 147:3408.[Abstract/Free Full Text]
  21. Leist, M. and Jaattela, M. 2001. Four deaths and a funeral: from caspases to alternative mechanisms. Nat. Rev. Mol. Cell. Biol. 2:589.[ISI][Medline]
  22. Rich, T., Lawler, S. E., Lord, J. M., Blancheteau, V. M., Charron, D. J. and Mooney, N. A. 1997. HLA class II-induced translocation of PKC alpha and PKC beta II isoforms is abrogated following truncation of DR beta cytoplasmic domains. J. Immunol. 159:3792.[Abstract]
  23. Al-Daccak, R., Mehindate, K., Hebert, J., Rink, L., Mecheri, S. and Mourad, W. 1994. Mycoplasma arthritidis-derived superantigen induces proinflammatory monokine gene expression in the THP-1 human monocytic cell line. Infect. Immun. 62:2409.[Abstract]
  24. Morio, T., Geha, R. S. and Chatila, T. A. 1994. Engagement of MHC class II molecules by staphylococcal superantigens activates src-type protein tyrosine kinases. Eur. J. Immunol. 24:651.[ISI][Medline]
  25. Rotenberg, S. A. and Sun, X. G. 1998. Photoinduced inactivation of protein kinase C by dequalinium identifies the RACK-1-binding domain as a recognition site. J. Biol. Chem. 273:2390.[Abstract/Free Full Text]
  26. de Fougerolles, A. R., Chi-Rosso, G., Bajardi, A., Gotwals, P., Green, C. D. and Koteliansky, V. E. 2000. Global expression analysis of extracellular matrix–integrin interactions in monocytes. Immunity 13:749.[ISI][Medline]
  27. Terui, Y., Furukawa, Y., Sakai, T., Kikuchi, J., Sugahara, H., Kanakura, Y., Kitagawa, S. and Miura, Y. 1996. Up-regulation of VLA-5 expression during monocytic differentiation and its role in negative control of the survival of peripheral blood monocytes. J. Immunol. 156:1981.[Abstract]
  28. McGilvray, I. D., Lu, Z., Bitar, R., Dackiw, A. P., Davreux, C. J. and Rotstein, O. D. 1997. VLA-4 integrin cross-linking on human monocytic THP-1 cells induces tissue factor expression by a mechanism involving mitogen-activated protein kinase. J. Biol. Chem. 272:10287.[Abstract/Free Full Text]
  29. Sugahara, H., Kanakura, Y., Furitsu, T., Ishihara, K., Oritani, K., Ikeda, H., Kitayama, H., Ishikawa, J., Hashimoto, K., Kanayama, Y., et al. 1994. Induction of programmed cell death in human hematopoietic cell lines by fibronectin via its interaction with very late antigen 5. J. Exp. Med. 179:1757.[Abstract]
  30. Bobbitt, K. R. and Justement, L. B. 2000. Regulation of MHC class II signal transduction by the B cell coreceptors CD19 and CD22. J. Immunol. 165:5588.[Abstract/Free Full Text]
  31. Leveille, C., Chandad, F., Al-Daccak, R. and Mourad, W. 1999. CD40 associates with the MHC class II molecules on human B cells. Eur. J. Immunol. 29:3516.[ISI][Medline]
  32. Leveille, C., Al-Daccak, R. and Mourad, W. 1999. CD20 is physically and functionally coupled to MHC class II and CD40 on human B cell lines. Eur. J. Immunol. 29:65.[ISI][Medline]
  33. Angelisova, P., Hilgert, I. and Horejsi, V. 1994. Association of four antigens of the tetraspans family (CD37, CD53, TAPA-1, and R2/C33) with MHC class II glycoproteins. Immunogenetics 39:249.[ISI][Medline]
  34. Skubitz, K. M., Campbell, K. D., Iida, J. and Skubitz, A. P. 1996. CD63 associates with tyrosine kinase activity and CD11/CD18, and transmits an activation signal in neutrophils. J. Immunol. 157:3617.[Abstract]
  35. Skubitz, K. M., Campbell, K. D. and Skubitz, A. P. 2000. CD63 associates with CD11/CD18 in large detergent-resistant complexes after translocation to the cell surface in human neutrophils. FEBS Lett. 469:52.[ISI][Medline]
  36. Pettersen, R. D., Bernard, G., Olafsen, M. K., Pourtein, M. and Lie, S. O. 2001. CD99 signals caspase-independent T cell death. J. Immunol. 166:4931.[Abstract/Free Full Text]
  37. Mateo, V., Lagneaux, L., Bron, D., Biron, G., Armant, M., Delespesse, G. and Sarfati, M. 1999. CD47 ligation induces caspase-independent cell death in chronic lymphocytic leukemia. Nat. Med. 5:1277.[ISI][Medline]
  38. Hirt, U. A., Gantner, F. and Leist, M. 2000. Phagocytosis of nonapoptotic cells dying by caspase-independent mechanisms. J. Immunol. 164:6520.[Abstract/Free Full Text]
  39. Foghsgaard, L., Wissing, D., Mauch, D., Lademann, U., Bastholm, L., Boes, M., Elling, F., Leist, M. and Jaattela, M. 2001. Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor. J. Cell Biol. 153:999.[Abstract/Free Full Text]
  40. Crawford, K. W. and Bowen, W. D. 2002. Sigma-2 receptor agonists activate a novel apoptotic pathway and potentiate antineoplastic drugs in breast tumor cell lines. Cancer Res. 62:313.[Abstract/Free Full Text]
  41. Liu, H., Perlman, H., Pagliari, L. J. and Pope, R. M. 2001. Constitutively activated Akt-1 is vital for the survival of human monocyte-differentiated macrophages. Role of Mcl-1, independent of nuclear factor (NF-)-kappaB, Bad, or caspase activation. J. Exp. Med. 194:113.[Abstract/Free Full Text]
  42. Parekh, D. B., Ziegler, W. and Parker, P. J. 2000. Multiple pathways control protein kinase C phosphorylation. EMBO J. 19:496.[Free Full Text]
  43. Pongracz, J., Webb, P., Wang, K., Deacon, E., Lunn, O. J. and Lord, J. M. 1999. Spontaneous neutrophil apoptosis involves caspase 3-mediated activation of protein kinase C-delta. J. Biol. Chem. 274:37329.[Abstract/Free Full Text]
  44. Scheel-Toellner, D., Pilling, D., Akbar, A. N., Hardie, D., Lombardi, G., Salmon, M. and Lord, J. M. 1999. Inhibition of T cell apoptosis by IFN-beta rapidly reverses nuclear translocation of protein kinase C-delta. Eur. J. Immunol. 29:2603.[ISI][Medline]
  45. Ruiz-Ruiz, C., Robledo, G., Font, J., Izquierdo, M. and Lopez-Rivas, A. 1999. Protein kinase C inhibits CD95 (Fas/APO-1)-mediated apoptosis by at least two different mechanisms in Jurkat T cells. J. Immunol. 163:4737.[Abstract/Free Full Text]
  46. Gomez-Angelats, M. and Cidlowski, J. A. 2001. PKC regulates FADD recruitment and death-inducing signaling complex formation in FAS/CD95-induced apoptosis. J. Biol. Chem. 1:1.[Free Full Text]
  47. Kurosawa, M., Tani, Y., Nishimura, S., Numazawa, S. and Yoshida, T. 2001. Distinct PKC isozymes regulate bufalin-induced differentiation and apoptosis in human monocytic cells. Am. J. Physiol. Cell Physiol. 280:C459.
  48. Volbracht, C., Leist, M., Kolb, S. A. and Nicotera, P. 2001. Apoptosis in caspase-inhibited neurons. Mol. Med. 7:36.[ISI][Medline]
  49. Ito, Y., Mishra, N. C., Yoshida, K., Kharbanda, S., Saxena, S. and Kufe, D. 2001. Mitochondrial targeting of JNK/SAPK in the phorbol ester response of myeloid leukemia cells. Cell Death Different. 8:794.[ISI][Medline]
  50. Dib, K. 2000. Beta 2 integrin signaling in leukocytes. Front. Biosci. 5:D438.
  51. Ropke, C., Gladstone, P., Nielsen, M., Borregaard, N., Ledbetter, J. A., Svejgaard, A. and Odum, N. 1996. Apoptosis following interleukin-2 withdrawal from T cells: evidence for a regulatory role of CD18 (beta 2-integrin) molecules. Tissue Antigens 48:127.[ISI][Medline]
  52. Damle, N. K., Leytze, G., Klussman, K. and Ledbetter, J. A. 1993. Activation with superantigens induces programmed death in antigen-primed CD4+ class II+ major histocompatibility complex T lymphocytes via a CD11a/CD18-dependent mechanism. Eur. J. Immunol. 23:1513.[ISI][Medline]
  53. Wu, M. X., Ao, Z., Hegen, M., Morimoto, C. and Schlossman, S. F. 1996. Requirement of Fas(CD95), CD45, and CD11a/CD18 in monocyte-dependent apoptosis of human T cells. J. Immunol. 157:707.[Abstract]
  54. Coxon, A., Rieu, P., Barkalow, F. J., Askari, S., Sharpe, A. H., von Andrian, U. H., Arnaout, M. A. and Mayadas, T. N. 1996. A novel role for the beta 2 integrin CD11b/CD18 in neutrophil apoptosis: a homeostatic mechanism in inflammation. Immunity 5:653.[ISI][Medline]
  55. Larsson, J., Serrander, L., Stendahl, O. and Lundqvist-Gustafsson, H. 2000. Involvement of the beta2-integrin CD18 in apoptosis signal transduction in human neutrophils. Inflamm. Res. 49:452.[ISI][Medline]
  56. Walzog, B., Jeblonski, F., Zakrzewicz, A. and Gaehtgens, P. 1997. Beta2 integrins (CD11/CD18) promote apoptosis of human neutrophils. FASEB J. 11:1177.[Abstract/Free Full Text]
  57. Walzog, B. and Gaehtgens, P. 2000. Adhesion molecules: the path to a new understanding of acute inflammation. News Physiol. Sci. 15:107.[Abstract/Free Full Text]
  58. Walzog, B., Scharffetter-Kochanek, K. and Gaehtgens, P. 1999. Impairment of neutrophil emigration in CD18-null mice. Am. J. Physiol. 276:G1125.
  59. Matsue, H., Edelbaum, D., Hartmann, A. C., Morita, A., Bergstresser, P. R., Yagita, H., Okumura, K. and Takashima, A. 1999. Dendritic cells undergo rapid apoptosis in vitro during antigen-specific interaction with CD4+ T cells. J. Immunol. 162:5287.[Abstract/Free Full Text]




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Articles by Castaigne, J.-G.
Articles by Al-Daccak, R.
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Articles by Castaigne, J.-G.
Articles by Al-Daccak, R.