Published online before print March 25, 2008
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National Centre for Cell Science, Ganeshkhind, Pune, India
1Correspondence: National Centre for Cell Science, Ganeshkhind, Pune 411007, India. E-mail: sahab{at}nccs.res.in
ABSTRACT
APCs express MHC-II molecules. Binding of enterotoxins to MHC-II generates a signal resulting in the production of TNF-
that mediates toxic shock syndrome. However, the signaling events that lead to TNF- production in macrophages are not well understood. We, for the first time, demonstrate that binding of staphylococcal enterotoxin B to MHC-II results in activation of TNF--converting enzyme, epidermal growth factor receptor, p38MAPK, and NF-
B inducing biphasic TNF- production. Paraformaldehyde-fixed, peptide-specific T cells also activate MHC-II signaling and TNF- induction in peptide-pulsed macrophages. Our results reveal a novel MHC-II signaling and bidirectional macrophage-T cell interaction regulating macrophage functions. This knowledge may help to develop novel, macrophage-directed, therapeutic strategies.
Key Words: MHC class-II signaling • staphylococcal enterotoxin B • superantigen • APC-T cell interaction
INTRODUCTION
APCs express MHC-II molecules, which select the antigenic determinants that are to be presented to T cells. The MHC-II-peptide complex is recognized by the antigen-specific TCR triggering signals in T cells leading to their differentiation and acquisition of effector functions [1
]. Likewise, MHC-II chain-bound superantigen (SAg) engages a specific Vβ chain of the TCR, Vβ2 (in human) or Vβ8 (in mouse), for staphylococcal enterotoxins B (SEB), resulting in T cell activation [2
]. These observations imply a unidirectional signaling through a MHC-II-SAg (or a peptide)-TCR ternary complex to the T cells. In contrast, purified monocytes or macrophages responded to staphylococcal SAg such as SEB and toxic shock syndrome toxin-1, resulting in the phosphorylation of tyrosine kinases [3
, 4
] and the production of inflammatory cytokines such as IL-1β and TNF- [5
, 6
]. In B cells, anti-MHC-II antibody triggers protein kinase C (PKC) activation and translocation to plasma membrane [7
, 8
] and increases intracellular concentration of Ca++ [9
, 10
]. Similar observations were reported in dendritic cells (DC) as well [11
, 12
]. These observations suggested that in B cells, DC, and macrophages, SAg binding to MHC-II induces MHC-II signaling, despite lack of signaling motifs in its short cytoplasmic tail. MHC-II needs adaptor molecules such as CD38 and CD9, which form the signaling complex with it to signal in APCs [13
].
Herein, we report that SEB induces MHC-II signaling, causing biphasic TNF- productions. Although the first phase of TNF- production is regulated by TNF--converting enzyme (TACE) activation and peaks within 30 min post-SEB stimulation, the second phase is transcriptionally regulated through epidermal growth factor receptor (EGFR)-triggered, p38MAPK-dependent NF-B activation. Preventing the initial signaling events blocks the SEB-induced, downstream events. Thus, the reported observations elucidate the MHC-II signaling and suggest that interference with the MHC-II signaling pathway can be a rationale for immunotherapy of many diseases.
MATERIALS AND METHODS
Mice, cell culture, reagents, and ELISA
Thioglycolate-elicited macrophages [14
] from BALB/c mice (Jackson Laboratory, Bar Harbor, ME, USA) were stimulated with SEB (Sigma Chemical Co., St. Louis, MO, USA; Toxin Technology, Hialea, FL, USA). Cycloheximide, a protein translation inhibitor [15
]; cytochalasin B, a microtubule polymerization inhibitor [16
]; TACE-specific inhibitor 2 (TAPI-2) and GM6001, the inhibitors of TACE [17
, 18
]; PD153035, the inhibitor of EGFR [19
]; and SB203580, the inhibitor of p38MAPK [20
], were purchased from Calbiochem (San Diego, CA, USA); caffeic acid phenethyl ester (CAPE), an inhibitor of NF-B [21
], was from Sigma Chemical Co. TNF- in culture supernatants was assayed using ELISA kits (BD PharMingen, San Diego, CA, USA), and anti-TNF--FITC was purchased from BD PharMingen. Anti-EGFR, anti-p38MAPK, and anti-NF-B antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). We followed Committee for the Purpose of Control and Supervision of Experiments on Animals-provided guidelines and Institutional Animal Care and Use Committee-approved protocols for the animal experimentation.
RT-PCR
The general protocol for RT-PCR was followed as described earlier [14
]. Macrophages were incubated for 2 h with the indicated inhibitors, followed by SEB treatment for 3 h. RNA was extracted, and cDNA was prepared. PCR (35 cycles) was done using specific forward (f) and reverse (r) primers: β-actin, (f) 5'-TGGAATCCTGTGGCATCCA-3', (r) 5'-TAACAGTCCGCCTAGAAGCA-3'; TNF-, (f) 5'-GCGAGGTGGAACTGGCAGAAG-3', (r) 5'-GGTACAACCCATCGGCTGGCA-3'; CD25, (f) 5'-CAGACATGCAGAAGCCAACAC-3', (r) 5'-GGTGAGCCCGCTCAGGCGAGGA-3'.
FACS
Mice were injected with thioglycolate (3%), i.e., 2 ml/mouse. Cells were harvested after 5 days. Cells were equally plated in six-well plates and cultured for 12 h in RPMI 1640 for adherence. Cells were preincubated with cycloheximide (100 µM) for 2 h, followed by stimulation with SEB for 30 min. Cells were then stained with directly labeled anti-TNF--FITC or corresponding isotype antibody for 30 min and washed three times with FACS buffer (1xPBS+5% FCS) at 1200 rpm and 4°C. The cells were acquired in a FACSVantage and analyzed by CellQuest Pro software.
Western blotting
Mice were injected with thioglycollate (3%), i.e., 2 ml/mouse. Cells were harvested after 5 days. Cells were equally plated in six-well plates and cultured for 12 h in RPMI 1640 to let them adhere. Cells were stimulated for different time-points and different doses of SEB and preincubated with different inhibitors; cells were then lysed with Triton X-100 buffer (150 mM NaCl+20 mM+1% Triton X-100), then run on SDS-PAGE (10% for p38MAPK and 7% for EGFR), and transferred onto a nitrocellulose membrane (300 mAmp for 3 h). Membrane was incubated with 5% milk for 1 h and washed thrice with TBS-T 10 min each. Blot was then incubated with respective antibodies for 3 h and then washed with TBS-T three times, 10 min each. Blot was now incubated with the corresponding secondary antibody conjugated with HRP for 1 h. After, 1 h, blot was washed again three times with TBS-T for 10 min each. Blots were then developed using ECL reagent [14
].
IB blots
For cytoplasmic extracts, cells were scraped and centrifuged at 11,500 rpm for 2 min. The pellet was resuspended in 3 vol cytoplasmic buffer [20 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM NaV, 1 mM EDTA, 10% glycerol, 1 mM DTT, 1 mM PMSF, supplemented with protease inhibitor cocktail], left on ice for 10 min, lysed using a Dounce homogenizer, and checked microscopically for cell lysis. The lysates were centrifuged again at 11,500 rpm for 5 min, and the supernatant containing cytoplasmic extracts was collected. The pellet was washed once with 3 vol cytoplasmic buffer and resuspended in 2 vol nuclear buffer [420 mM NaCl, 20% glycerol, 20 mM HEPES (pH 7.9), 10 mM KCl, 1 mM EDTA, 0.1 mM NaV, 1 mM PMSF, 1 mM DTT, supplemented with protease inhibitor cocktail], incubated on ice for 20 min, and centrifuged again at 12,500 rpm for 10 min. Cytoplasmic extracts were analyzed for IB degradation by Western blotting as described above, and nuclear extracts were incubated with labeled probe for 10 min.
Coculture experiments
The OVA-specific T cell clone was kindly supplied by the Late Professor Charles Janeway Jr. (Yale University, New Haven, CT, USA). The cells were fixed with 1.5% paraformaldehyde for 15 min. The fixed cells did not proliferate in response to anti-CD3 + anti-CD28 or Con A (Calbiochem) and also did not show p38MAPK activation. Macrophages were pulsed with OVA for the indicated time (2–16 h) and were cocultured with the fixed T cells for 30 min. Western blotting for the indicated signaling intermediates and ELISA for TNF- were performed with cell lysates and culture supernatants, respectively.
Statistical analyses
The in vitro cultures were assayed in triplicates. Experiments were reproduced a minimum of three times, and the representative data from one individual experiment are shown. Wherever shown, the error bars signify mean ± SD. The significance of differences between the means of control versus experimental groups or between two experimental groups, as indicated in the figures, was deduced by Student’s t-test. P value less than 0.05 was considered significant.
RESULTS
SEB-induced MHC-II signaling results in biphasic TNF- production by two different mechanisms
We examined the kinetics of SEB-induced TNF- production from macrophages. SEB induced biphasic TNF- productions (Fig. 1A
) from macrophages. The first peak occurred 30 min after SEB stimulation, whereas the second peak appeared 12 h post-SEB stimulation. To check the mode of generation of the first peak, macrophages were preincubated with cycloheximide (translation inhibitor) and cytochalasin B (microtubule inhibitor). Figure 1B
showed that the first phase of TNF-a production was independent of TNF- transcription and was independent of exocytosis, unlike IFN-
and IL-12, suggesting two different mechanisms for each phase. Alternatively, as the TACE, an intracellular metalloproteinase [22
], can cleave a TNF- precursor on the membrane [23
], we tested if SEB-activated TACE can induce TNF-. Indeed, TAPI-2 prevented TNF- production (Fig. 1C)
, suggesting SEB-induced TACE activation in macrophages. To further test the role of TACE, macrophages were preincubated with cycloheximide for 2 h and then stimulated with SEB for 30 min. Cells were then stained with anti-TNF--PE antibody and analyzed by FACS; staining was reduced from 55% to 5%, and cycloheximide, on the other hand, did not have any effect on TACE-mediated cleavage of pro-TNF-, which was almost equal to unstained, further implying the involvement of TACE in SEB-induced signaling in macrophages (Fig. 1D)
. In contrast, SEB-induced reduction in TNF- staining on macrophages was prevented by TAPI-2, a TACE inhibitor (Fig. 1E)
, only TAPI-2 did not have any effect on TNF- expression on these macrophages (Fig. 1E)
. These data suggest that TACE-mediated cleavage of membrane pro-TNF- results in the SEB-induced production of TNF-.
 View larger version (52K): [in a new window] |
Figure 1. SEB induces biphasic TNF- productions by activating TACE. (A) The thioglycollate-elicited peritoneal macrophages were stimulated with SEB (2 µg/ml) for the indicated time period. Cell culture supernatants and the RNA extracted from the cells were assayed for TNF- expression (upper panel). In some experiments, as shown in the right panel, the macrophages were stimulated with SEB (2 µg/ml) for 3 h, followed by RT-PCR for TNF- expression (lower panel). (B) Macrophages were preincubated with cycloheximide (100 µM) and cytochalasin B (10 µM) for 2 h and stimulated with SEB for 30 min. Supernatants were analyzed for TNF-. The early phase of TNF- production (30 min post-SEB) was cycloheximide-insensitive and cytochalasin B-insensitive [medium (Med) vs. SEB, P<0.05; SEB vs. SEB+A, P>0.1; SEB vs. SEB+B, P>0.1]. (C) Macrophages were preincubated with TAPI-2 (20 µM, 30 µM, and 40 µM) for 2 h and stimulated with SEB (2 µg/ml) for 30 min, and 3 h for ELISA and RT-PCR, respectively. TACE inhibitor TAPI-2 inhibits early and late phases of SEB-induced TNF- production [Medium vs. SEB, P=0.008; SEB vs. TAPI2 (20 µM), P=0.05; SEB vs. TAPI2 (30 µM), P<0.05; SEB vs. TAPI2 (40 µM), P=0.01]. (D) Peritoneal macrophages were stimulated with SEB and stained with anti-TNF-. SEB stimulation reduced the staining of TNF- from 55% to 6%. The data (mean±SD) represent one of three experiments. (E) Macrophages were preincubated with TAPI-2 (30 µm) for 2 h and then stimulated with SEB (2 µg/ml for 30 min). Cells were stained with anti-TNF--FITC antibody and analyzed by FACSCalibur using CellQuest Pro software.
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production and is shown to be produced by macrophages [25
]. To test the role of TACE and TGF- in the production of SEB-induced TNF- in macrophages, we first tested whether SEB can induce TGF- in macrophages and if it is mediated by TACE. Macrophages were preincubated with different doses of a specific inhibitor of TACE (TAPI-2) and then stimulated with SEB. We observed the dose-dependent inhibition of TGF- production (Fig. 2A
), suggesting the role of TGF- in SEB-induced TNF- production. Macrophages were then incubated with rhTGF- (5 ng/ml) for 3 h to further test the hypothesis. RT-PCR for TNF- was performed. We found that rhTGF- also induces TNF- (Fig. 2B)
in macrophages, and neutralizing TGF- inhibited the SEB-induced TNF- in macrophages (Fig. 2C)
, which suggests the possible role of EGFR. Further macrophages were preincubated with specific EGFR inhibitor (PD153035) for 2 h and then stimulated with SEB for 3 h. Cells were lysed by trizol, and RT-PCR for TNF- was performed. TNF- expression was inhibited in a dose-dependent manner, indicating the involvement of TGF- and EGFR in SEB-induced TNF- production (Fig. 2D
, left panel). To further confirm this, we incubated the macrophages with neutralizing antibody to EGFR and stimulated with SEB for 3 h. Dose-dependent inhibition was observed (Fig. 2D
, right panel). Further, direct activation of EGFR was evaluated to prove it conclusively. Macrophages were stimulated with rhTGF- or SEB for 5 min and then evaluated for phosphorylation of EGFR. We found that rhTGF- and SEB induced the phosphorylation of EGFR (Fig. 2E
/i), and inhibition of TACE inhibited the EGFR activation in a dose-related manner (Fig. 2E
/ii).
 View larger version (51K): [in a new window] |
Figure 2. EGFR mediates the SEB-induced TNF-. (A) Macrophages were preincubated with TAPI-2 (20 µM, 30 µM, and 40 µM) and then stimulated with SEB (2 µg/ml) for 30 min. ELISA was done from supernatant. TAPI-2 inhibited the SEB-induced TGF- [Med vs. SEB, P=0.01; SEB vs. TAPI2 (20 µM), P=0.09; SEB vs. TAPI2 (30 µM), P<0.05; SEB vs. TAPI2 (40 µM), P<0.01]. (B) Macrophages were stimulated with recombinant human (rh)TGF- for 3 h, and RT-PCR for TNF- was performed. rhTGF- induced TNF- in macrophages. (C) Macrophages were incubated with anti-TGF- and stimulated with SEB for 3 h, and RT-PCR for TNF- was performed. (D) Macrophages were preincubated with PD153035 (20 nM, 30 nM, and 40 nM) for 2 and then stimulated with SEB for 3 h; PD153035 inhibited the SEB-induced TNF-. (E) Macrophages were preincubated with anti-EGFR for 1 h and then stimulated with SEB for 3 h. Neutralization of EGFR inhibited the SEB-induced TNF-. (i) Macrophages were stimulated with rhTGF- and SEB for 5 min, Western blot for phosphorylated (p)EGFR and EGFR was done, and rhTGF- and SEB induced the EGFR phosphorylation. (ii) Macrophages were preincubated with TAPI-2 (20 µM, 30 µM, and 40 µM) and then stimulated with SEB (2 µg/ml) for 5 min, and Western blot for pEGFR and EGFR was done. TAPI-2 inhibited the SEB-induced EGFR phosphorylation.
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is mediated by p38MAPK and SEB, and we found that SEB and rhTGF- activated p38MAPK (Fig. 3B
/i). To further evaluate the involvement of TGF- in SEB-induced signaling in macrophages, cells were stimulated with SEB and cultured with anti-TGF- for 15 min. Again, we found dose-dependent inhibition of p38MAPK activation (Fig. 3B
/ii). As TGF- is a ligand for EGFR, we preincubated the macrophages with PD153035 (EGFR inhibitor) for 2 h and stimulated with SEB for 15 min. Dose-dependent inhibition of p38MAPK activation was observed (Fig. 3B
/iii). To further confirm the involvement of p38MAPK in SEB-induced events in macrophages, we preincubated the macrophages with SB203580 for 2 h and then stimulated with SEB for 15 min. We observed dose-dependent inhibition of p38MAPK phosphorylation (Fig. 3C)
and TNF- production (Fig. 3D)
. This confirms that SEB-induced TNF- production in macrophages is mediated by TACE, EGFR, and p38MAPK.
 View larger version (30K): [in a new window] |
Figure 3. SEB-induced p38MAPK is mediated by TACE and EGFR. (A) Macrophages were preincubated with TAPI-2 (20 µM, 30 µM, and 40 µM) and then stimulated with SEB (2 µg/ml) for 15 min. SEB-induced p38MAPK activation is inhibited by TAPI-2 (T-inh). (B/i) Macrophages were stimulated with rhTGF- or SEB for 15 min; SEB and rhTGF- induced p38MAPK phosphorylation. (B/ii) Macrophages were incubated with anti-TGF- and stimulated with SEB for 15 min, and p38MAPK phosphorylation was inhibited by TGF- neutralization. (B/iii) Macrophages were preincubated with PD153035 (20 nM, 30 nM, and 40 nM) for 2 h and then stimulated with SEB for 15 min (SEB+E inh). PD153035 inhibited the p38MAPK phosphorylation. (C) Macrophages were preincubated with SB203580 for 2 h and stimulated with SEB for 15 min. SB203580 inhibited the p38MAPK phosphorylation (SEB+p38-inh). (D) Macrophages were preincubated with SB203580 for 2 h and stimulated with SEB for 3 h (lower panel) and 12 h (upper panel; P-inh). RT-PCR (3 h after stimulation) and ELISA (12 h after stimulation) for TNF- were performed from cells and supernatant, respectively. SB203580 inhibited the TNF- in a dose-related manner [SEB vs. SB203580 (8 µM), P<0.001]. One representative set of data from three individual experiments was shown.
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is mediated by NF-BB translocation to the nucleus leads to SEB-induced IL-12 induction in macrophages [27
]. We tested whether NF-B is involved in the SEB-induced TNF- production in macrophages. We observed that the inhibitors of TACE and EGFR augmented cytoplasmic IB accumulation (Fig. 4A
and 4B
) but reduced SEB-induced nuclear translocation of NF-B (Fig. 4C)
. CAPE, a NF-B inhibitor [21
], reduced the SEB-induced TNF- production (Fig. 4D)
. These findings identify the details of MHC-II signaling and the mechanism of MHC-II-induced effector functions.
 View larger version (29K): [in a new window] |
Figure 4. SEB-induced TNF- is mediated by NF-B. (A) Peritoneal macrophages were preincubated with TAPI-2 (20 µM, 30 µM, and 40 µM), followed by stimulation with SEB (2 µg/ml) for 45 min. Cytoplasmic extracts were blotted for IB. TAPI-2 led to accumulation of IB in cytoplasm. (B) Macrophages were preincubated with PD153035 (20 nM, 30 nM, and 40 nM), followed by stimulation with SEB (2 µg/ml) for 45 min. Cytoplasmic extracts were blotted for IB; PD153035 led to accumulation of IB in cytoplasm. (C) Similarly, SEB stimulation of macrophages was found to increase NF-B in the nuclear extract, but the indicated inhibitors reduced the nuclear NF-B. T-inh, TACE inhibitor; E-inh, EGFR inhibitor. (D) Peritoneal macrophages were preincubated with CAPE (25 nM, 50 nM, 100 nM) for 2 h and then stimulated with SEB for 3 h or 12 h for RT-PCR and ELISA, respectively. We observed dose-dependent inhibition of TNF- by RT-PCR and ELISA (SEB vs. SEB+CAPE (100 nM); P<0.002). The experiments were performed thrice, of which one set of representative data is shown.
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production and p38MAPK activation. We observed TNF- in the supernatant and p38MAPK phosphorylation only when the macrophages were pulsed with the peptide (Fig. 5A
). The absence of p38MAPK activation in unrelated peptide-pulsed macrophage-T cell coculture suggests that macrophage activation works through the MHC-II-peptide-TCR complex and that the observed signaling was not a result of any other ligand-receptor interactions. The control cultures—macrophages with OVA alone, with nonspecific T cells, or with unrelated peptide pulsing—failed to induce any detectable TNF- (data not shown). To confirm the similarity in signaling in macrophages induced by SEB and TCR, macrophages were pulsed with OVA peptides for 4 h, and TAPI-2 was added after 2 h of peptide pulsing and continued for 2 h more to complete 4 h of peptide pulsing. These macrophages were cocultured with the peptide-specific T cells (Fig. 5B)
. We found dose-dependent inhibition of TNF- and p38MAPK by ELISA and Western blotting, respectively. The results confirm that SEB and TCR induce similar signaling in macrophages, which is mediated by TACE.
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Figure 5. Normal antigen presentation also induces similar events in macrophages. (A) The macrophages were pulsed with the OVA for 2, 4, 8, or 16 h (indicated in parentheses) before adding to the OVA-specific T cells (fixed) in culture. Thirty minutes later, cell lysates were assessed for p38MAPK activation (lower panel), and the culture supernatants were assayed for TNF- [macrophage (M) vs. M+Ova (8 h)+T cells, P=0.0004; M vs. M+Ova (4 h)+T cells, P=0.006; upper panel]. (B) Peritoneal macrophages were pulsed with OVA for 4 h and 2 h after OVA TAPI-2 (20 µM, 30 µM, and 40 µM) was added. Cells were then stimulated with SEB (2 µg/ml) at the end of 4 h for 30 min. We observed dose-related inhibition of TNF- [M vs. M+T cells, P=0.46; M vs. M+OVA+T cells, P=0.03; M+OVA+T cells vs. M+OVA+T cells+TAPI2 (20 µM), P=0.168; M+OVA+T cells vs. M+OVA+T cells+TAPI2 (30 µM), P=0.0512; M+OVA+T cells vs. M+OVA+T cells+TAPI2 (40 µM), P=0.03; upper panel] and p38MAPK (lower panel), respectively. The experiment was performed thrice. One set of representative data is shown. M , Macrophage.
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MHC-II is known to signal in macrophages, despite a lack of signaling motifs in their cytoplasmic domain. MHC-II molecules trigger a variety of intracellular signals that regulate APC function [28
, 29
]. MHC-II signaling has been well-characterized, explaining how PKC isoforms and lipid rafts control the downstream events in APCs [30
]. However, how it governs the production of TNF- in macrophages is not well understood. In this study, using various inhibitors (Table 1
), we have examined the SEB-induced signaling required for the regulation of this cytokine.
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Table 1. Inhibitors Used in the Study
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production induced by SEB. TACE has been implicated in endotoxic shock and many autoimmune diseases, such as diabetes and arthritis [32
33
34
]. Roche Diagnostics and Wyeth (Switzerland) have developed TACE inhibitors for treatment of arthritis (TACE generates TNF-, an important proinflammatory cytokine in this disease). TACE activity results in the generation of the first wave of TNF- and also the release of TGF-; the latter, being the ligand for EGFR, triggers another set of signals from EGFR, activating a p38MAPK-dependent, second wave of TNF- by transcriptional up-regulation. TACE is known to induce the shedding of growth factors, cytokines, and cytokine receptors, but in this report, we are showing for the first time that it also induces the expression of TNF-. TACE-induced first phase of TNF- has an important physiological function: This wave of TNF- prepares T cells by increasing the CD25 expression as shown by FACS (Supplementary Fig. 1
). p38MAPK activation is impaired in Leishmania infection [35
] and so is MHC-II expression [36
], which could account for the evasion mechanism of pathogens. Our study could help in designing the therapy to prevent infection. Possibly MHC-II ligand along with p38MAPK activator can prevent infection more efficiently. Blockade of this signaling pathway at the initial steps prevents the downstream events, which is evident from the fact that inhibition of p38MAPK but not ERK in macrophages modulates IL-2 production and T cell proliferation (Supplementary Fig. 2A). Interestingly, the first phase does not affect the second phase, as neutralization of TNF- did not inhibit the TNF- expression (Supplementary Fig. 2B). As MHC-II is the central molecule in shaping the acquired immune system against any pathogen, our study opens new insight in this aspect. We can potentially modulate the outcome of the acquired immune system by modulating the MHC-II signaling in macrophages or APCs.
ACKNOWLEDGEMENTS
Life Sciences Research Board (LSRB), Council of Scientific and Industrial Research (CSIR), and Department of Biotechnology (DBT), Government of India, provided the financial assistance.
Received October 11, 2007; revised February 20, 2008; accepted February 25, 2008.
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