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J Biol Chem, Vol. 274, Issue 47, 33455-33461, November 19, 1999

CD45-induced Tumor Necrosis Factor  Production in Monocytes Is Phosphatidylinositol 3-Kinase-dependent and Nuclear Factor-B-independent^*

A. Louise Hayes, Clive Smith, Brian M. J. Foxwell, and Fionula M. Brennan

From the Kennedy Institute of Rheumatology, Hammersmith, London W6 8LH, United Kingdom

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The pro-inflammatory cytokine tumor necrosis factor (TNF)- plays a pivotal role in the pathogenesis of rheumatoid arthritis. The mechanisms involved in regulating monocyte/macrophage TNF production are not yet fully understood but are thought to involve both soluble factors and cell/cell contact with other cell types. Ligation of certain cell surface receptors, namely CD45, CD44, and CD58, can induce the production of TNF in monocytes. In this paper, we investigate further the signaling pathways utilized by cell surface receptors (specifically CD45) to induce monocyte TNF and compare the common/unique pathways involved with that of lipopolysaccharide. The results indicate that monocyte TNF induced upon CD45 ligation or lipopolysaccharide stimulation is differentially modulated by phosphatidylinositol 3-kinase and nuclear factor-B but similarly regulated by p38 mitogen-activated protein kinase. These results demonstrate that both common and unique signaling pathways are utilized by different stimuli for the induction of TNF. These observations may have a major bearing on approaches to inhibiting TNF production in disease where the cytokine has a pathogenic role.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Lipopolysaccharide (LPS)¹ is one of the most potent activators of monocytes/macrophages, resulting in the triggering of a range of cellular responses and the secretion of pro- and anti-inflammatory cytokines, including TNF, interleukin-1 (IL-1) and IL-6 (1-4). LPS, following interaction with serum proteins, e.g. LPS-binding protein and the cell surface receptor, CD14 (5), activates a number of signaling pathways. These include various tyrosine kinases (6, 7), protein kinase C (PKC) (8), the mitogen-activated protein kinases (MAPK) including p38 (9), p44/42 (extracellular signal-regulated kinase) (10), and p54 (stress-activated protein kinase/JNK) (11).

Numerous studies have shown that direct contact between monocytes or monocytic cell lines and prestimulated T cells leads to production of cytokines including, IL-1, TNF, IL-12, and IL-10 (12-17). A variety of T cell-associated cell surface receptors/ligands including CD69, CD40L, CD11b, and CD2 are thought to be important in modulating this monocyte cytokine production (13-15). Furthermore, direct engagement of certain cell surface receptors, namely CD44, CD58, and CD45, on monocytes induce TNF production (18, 19), suggesting that receptor engagement may be important in the regulation of cytokines. Potential ligands for CD44 (osteopontin) (20) and CD58 (CD2) (21) are expressed by activated T cells, whereas the ligand for CD45 still remains to be fully clarified, although the B cell adhesion molecule, CD22 (22), and the -galactosidase-binding protein, galectin-1 (23), have been proposed to bind to specific isoforms of CD45.

CD45 is a membrane-anchored protein-tyrosine phosphatases found exclusively on all nucleated hemapoietic cells (24, 25). The role of CD45 in T cells has been the subject of much investigation and has been shown to play an important co-stimulatory role in intracellular signal transduction in T lymphocytes (26-31). While ligation of CD45 on monocytes has been shown to induce synthesis of cytokines, including TNF, IL-1, and macrophage-colony stimulating factor (M-CSF) (18, 19), the signaling mechanisms involved and the functional relevance of CD45 on monocyte/macrophages remain unclear.

We have investigated the signaling pathways utilized upon CD45 ligation on monocytes leading to TNF production and compared this with the conventional stimulus, LPS. We demonstrate that CD45 ligation (but not LPS) activates the phosphatidylinositol 3-kinase (PI3K) pathway and that inhibitors of PI3K activation block CD45- but not LPS-induced TNF synthesis. The differences in signaling also extended to nuclear factor-B (NF-B), which, unlike LPS, was not required by CD45-induced TNF synthesis. In contrast, CD45, like LPS, activated p38 MAPK.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents-- LPS, wortmannin, and LY294002 were purchased from Sigma (Sigma, Poole, Dorset, United Kingdom (UK)). Rapamycin and SB203580 were purchased from Calbiochem-Novabiochem Ltd (Nottingham, UK). Human recombinant M-CSF was a generous gift from the Genetics Institute Inc. Phosphatidylinositol (4,5)P₂ (PtdIns (4,5)P₂ and phosphatidylserine (PtdS) were purchased from Sigma, Poole, Dorset, UK). All reagents and medium used for monocyte culture were shown to contain <0.1 unit/ml endotoxin as measured using the Limulus amebocyte lysate assay (BioWhittaker).

Antibodies-- Rabbit antisera to p38 MAPK was provided by Prof. J Saklatvala (Kennedy Institute of Rheumatology, London, UK) (32) and the antibody to the p85 subunit of PI3K was kindly provided by Dr. D. Cantrell (ICRF, London, UK). The antibody to p70 S6K was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to phosphorylated protein kinase B (pPKB) and PKB were obtained from New England Biolabs (Hitchin, Herts, UK). Mouse IgG2a mAb HB196 (4B2 anti-CD45) and mouse isotype control IgG2a mAb OKT8 (anti-CD8) and OX12 were obtained as hybridomas from ATCC, and antibodies were subsequently purified using a protein-G Sepharose column (Millipore, Watford, Herts, UK).

Monocyte Purification-- Human peripheral blood monocytes were isolated from single donor platelet pheresis residues purchased from the North London Blood Transfusion service (Colindale, UK) as described previously (16). Briefly, mononuclear cells were isolated by Ficoll/Hypaque centrifugation (specific density 1.077 g/ml; Nycomed Pharma A.S., Oslo, Norway), prior to cell separation in a Beckman JE6 elutriator. Monocyte purity was assessed by flow cytometry using fluorochrome-conjugated anti-CD45 and anti-CD14 mAb (Becton Dickinson, Oxford, UK) and routinely consisted of >85% CD45- or CD14-expressing cells, respectively.

Monocyte Culture-- Monocytes were cultured in complete medium at 4 × 10⁶ cells/ml in flat-bottomed 96-well culture plates (Nunc Life Technologies Ltd., Paisley, Scotland). At the start of the culture period, cells were either left unstimulated or were cultured with the following reagents as indicated in the text: 10 ng/ml LPS, 10 µg/ml immobilized anti-CD45 mAb or immobilized isotype-matched controls OX12 (IgG2a) and OKT8 (IgG2a). In some experiments monocytes were pre-treated for 15 min with wortmannin or LY294002, or for 1 h with SB203580 or rapamycin at the indicated concentrations prior to stimulation. After 18 h in culture at 37 °C with 5% CO₂, supernatants (200 µl/well, 3 wells/condition) were harvested and stored at 20 °C until used. All experiments were performed at least three times, and the figures show representative examples of these experiments.

Analysis of Cell Viability-- Cell viability was routinely determined following incorporation of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide into cultured cells and absorbance read at 570 nm.

Measurement of TNF by Sandwich ELISA-- Reagents for the TNF ELISA were provided by Dr W. Buurman (Rijks Universiteit Limbury, Maastricht, The Netherlands). The ELISA was performed as described previously (33) using immobilized anti-TNF mAb 61E71 and developed using a rabbit polyclonal anti-TNF antibody. The rabbit polyclonal antibody was detected using a peroxidase-conjugated goat anti-rabbit IgG (H+L) (Jackson Immunoresearch Laboratories, Westgrove, PA) followed by an appropriate substrate. The range of the assay was 1.6-5000 pg/ml. Results are expressed as the mean concentration of cytokine ± S.D. per condition above the minimum sensitivity of the ELISA.

Western Blotting-- Western blotting for phosphorylated forms of p38 MAPK and PKB was performed according to the antibody manufacturer's instructions (New England Biolabs, Hitchin, Herts, UK).

Immunoprecipitation and in Vitro Kinase Assays-- Following stimulation, cells were lysed at a density of 5 × 10⁶ cells/ml in PI3K lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40), p70 S6K lysis buffer (10 mM potassium phosphate (pH 7.05), 0.5% Triton X-100, 1 mM EDTA, 5 mM EGTA), or p38 MAPK lysis buffer (1% Triton X-100, 20 mM HEPES (pH 7.4), 50 mM -glycerophosphate, 2 mM EDTA, 10% glycerol), supplemented with 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml pepstatin, and 10 µg/ml leupeptin. To the supernatants was added either monoclonal antibody (U5) directed against the p85, or anti-p70 S6K, or the p38 MAPK antibody. After 30 min on ice, 20 µl of protein G-Sepharose (p85 and p38 MAPK) or protein A-Sepharose (p70 S6K) was added and the lysates rotated at 4 °C for 2 h.

PI3K Assay-- Beads containing immunoprecipitates were washed three times in PI3K lysis buffer, once in PBS, twice in 500 mM lithium chloride, once in water and once in PI3K assay buffer (40 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1 mM EGTA). Immunoprecipitates were resuspended in 40 µl of PI3K assay buffer. 50 µl of lipid substrate mixture (1 mg of PtdIns (4,5)P₂ and 1 mg of PtdS made up in 2 ml of 25 mM HEPES, 1 mM EDTA and dispersed by sonication in three 15-s bursts at 4 °C) was added to the immunoprecipitates. The reaction was initiated by the addition of 5 µCi of [-³²P]ATP and 100 mM ATP. The samples were incubated at room temperature for 15 min and the reaction quenched using 100 µl of 1 M HCl and 200 µl of 1:1 chloroform:methanol. The resultant lipid layer was removed and dried in vacuo. The dried samples were resuspended in 50 µl of chloroform, applied to a 1% oxalate sprayed thin layer chromatography (TLC) plate, and developed in propan-1-ol, 2 M glacial acetic acid (65:35; v/v). Reaction products (i.e. phosphatidylinositol 3,4,5-phosphate, PtdIns (3,4,5)P₃) were visualized by autoradiography using Hyperfilm MP (Amersham Pharmacia Biotech).

p70 S6K Assay-- Beads containing immunoprecipitates were washed three times in p70 S6K lysis buffer and once in p70 S6 kinase assay buffer (50 mM MOPS (pH 7.2), 1 µM dithiothreitol, 30 µM ATP, 5 mM MgCl₂, 10 mM p-nitrophenyl phosphate). The precipitates were resuspended in 45 µl of kinase assay buffer containing 1 µl of a 250 µM solution of substrate peptide (KKRNRTLTK; Ref. 34), 1 µl of 5 µM protein kinase A inhibitor (Santa Cruz Biotechnology Inc.), and 5 µCi of [-³²P]ATP. The reactions were performed at 37 °C for 30 min, and the products were separated by gel electrophoresis in the presence of urea. Products were visualized by autoradiography using Hyperfilm MP (Amersham Pharmacia Biotech).

p38 MAPK Assay-- Beads containing immunoprecipitates were washed twice in RIPA buffer (1% Nonidet P-40, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM tetrasodium pyrophosphate, 1 mM EDTA, 25 mM -glycerophosphate, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS) and twice in MAPK assay buffer (25 mM HEPES (pH 7.4), 25 mM -glycerophosphate, 25 mM MgCl₂). Kinase reactions were performed at room temperature (under constant agitation) for 20 min following the addition of 20 µl of 100 µg/ml ATF-2 (as prepared by L. Rawlinson), 10 µl of 180 µM ATP, 5 µCi of [-³²P]ATP (Amersham Pharmacia Biotech). Reactions were stopped by the addition of 25 µl of 4× gel sample buffer and boiled for 5 min at 95 °C. Samples were fractionated on a 12.5% SDS-polyacrylamide gel. The gel was fixed in a mixture of water:methanol:acetic acid (5:4:1) and dried. Phosphorylated ATF-2 was detected by autoradiography using Hyperfilm MP (Amersham Pharmacia Biotech).

NF-B Band Shift Assay-- Nuclear extracts were isolated as described previously (35). Briefly, following stimulation 10⁷ cells were resuspended in hypotonic buffer (10 mM HEPES, 1.5 mM MgCl₂, 10 mM KCl (pH 7.9)) for 5 min at 4 °C. Nonidet P-40 was added from stock solution (10%) to a concentration of 0.25%, and samples were vortexed. Samples were microcentrifuged and the supernatant was removed. The nuclei were resuspended in hypertonic buffer (5 mM HEPES, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5M NaCl, 25% glycerol (pH 7)) and agitated (1 h, 4 °C). Band shift assays were performed according to manufacturer's specifications (Promega, UK).

Adenoviral Infection-- Adenoviral infection was performed using an adenovirus encoding porcine IB under the control of the cytomegalovirus promoter and a nuclear localization sequence (AdvIB) (36) or control adenovirus containing no insert (Adv0). Adenoviral infection of monocytes was performed as described previously (37). Briefly, freshly elutriated monocytes were cultured at 1 × 10⁶/ml for 2-3 days with 100 ng/ml M-CSF. Following culture, M-CSF-treated monocytes were washed once with PBS to remove non-adherent cells and the remaining adherent monocytes were incubated with 10 ml of cell dissociation solution (Sigma, UK) for 30-45 min to dissociate from the plastic. M-CSF-treated monocytes were resuspended to 2 × 10⁶ cell/ml prior to stimulation with either 10 µg/ml immobilized anti-CD45 or 10 ng/ml LPS as indicated in the text for 18 h. Supernatants were harvested and assayed for TNF production.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CD45 Induces Monocyte TNF in Peripheral Blood Monocytes in a Concentration-dependent Manner-- Fig. 1a illustrates TNF synthesis following CD45 ligation on monocytes stimulated by immobilized anti-CD45 antibody in a concentration-dependent manner. There was also synergy between CD45 ligation and stimulation with LPS (10 ng/ml), as TNF production was enhanced 4-6-fold (Fig. 1b) over that observed with LPS alone. In all experiments, immobilized isotype control antibodies did not induce TNF production over that of cells alone.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Engagement of CD45 induces monocyte TNF production and synergizes with LPS to enhance TNF production. 4 × 106/ml monocytes were cultured with immobilized anti-CD45 () or relevant isotype control () at various concentrations (a) or immobilized anti-CD45 (10 µg/ml) with LPS (10 ng/ml) (b) for 18 h. Supernatants were harvested and TNF levels determined by ELISA. Results are expressed as the mean of triplicate cultures ± S.D. This figure is representative of three experiments performed using different donors.

Inhibition of PI3K Differentially Modulates Anti-CD45- and LPS-induced Monocyte TNF Production-- The signaling pathways involved in monocyte TNF following CD45 ligation are unknown. In contrast, signaling pathways involved in LPS-induced TNF production have received much attention. We have investigated the signaling pathways utilized upon CD45 ligation and compared these to LPS. Initial investigations focused on PI3K, which is reported to be activated in monocytes upon LPS stimulation (38). Monocyte TNF induced by anti-CD45 antibody (10 µg/ml) was inhibited in a dose-dependent manner by the PI3K inhibitor wortmannin (Fig. 2a) with an IC₅₀ of ~0.07 nM. In contrast wortmannin was found to synergize with LPS (10 ng/ml) to enhance TNF production (Fig. 2b). To determine if the effects seen with wortmannin were due to inhibition of PI3K and not another signaling pathway, we studied the effects of another, structurally unrelated PI3K inhibitor, LY294002. LY294002, like wortmannin, was shown to inhibit anti-CD45 antibody (10 µg/ml)-induced monocyte TNF production (IC₅₀ ~0.07 µM), while having little effect on LPS (10 ng/ml)-induced monocyte TNF (Fig. 2, c and d).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Wortmannin and LY294002 differentially modulate LPS- and anti-CD45-induced monocyte TNF production. 4 × 106/ml monocytes were treated with wortmannin or LY294002 prior to culture with immobilized anti-CD45 (10 µg/ml) (a and c) or LPS (10 ng/ml) (b and d) for 18 h. Supernatants were harvested and TNF levels determined by ELISA. Results are expressed as the mean of triplicate cultures ± S.D. This figure is representative of five experiments performed using different donors. In all experiments immobilized relevant isotype control (10 µg/ml) did not induce TNF production (results not shown).

CD45 Induces PI3K Activity in Peripheral Blood Monocytes-- Due to the observed effects of the PI3K inhibitors, wortmannin and LY294002, we investigated PI3K activity. Engagement of CD45 on monocytes induced a transient increase in lipid kinase activity, maximal at 20 min and associated with immunoprecipitates of the anti-p85 subunit of PI3K (Fig. 3). Treatment of these monocytes with wortmannin prior to stimulation with anti-CD45 antibody totally inhibited kinase activity (Fig. 3). In contrast, only a weak activation of PI3K was observed following LPS stimulation and none at all in control immunoprecipitates of isotype-matched monoclonal antibodies. Similar experiments with LY294002 were not possible, because unlike wortmannin, this compound does not covalently bind to the enzyme and thus is removed during the assay procedure.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Engagement of CD45 induces PI3K activity in monocytes. 5 × 106 monocytes were cultured with immobilized anti-CD45 (10 µg/ml), relevant isotype control (IC) antibody (10 µg/ml) or LPS (10 ng/ml) for given times in the absence or presence of wortmannin (W, 50 nM). After cell lysis, the p85 subunit was immunoprecipitated, associated lipid kinase activity was assayed as described under "Experimental Procedures," and the 32P lipid product (PtdIns(3,4,5)P3) was separated by TLC and visualized by autoradiography.

Ligation of CD45 Phosphorylates and Activates Downstream Effectors, PKB and p70 S6K-- Recent studies suggest that PI3K-mediated events are transduced via protein kinase B (PKB) (39). Ligation of CD45 resulted in phosphorylation of PKB with similar kinetics to that seen for activation of PI3K, and found to be maximal at 20 min. Fig. 4 illustrates PKB phosphorylation in monocytes following CD45 ligation, which was inhibited by pre-incubation with wortmannin or LY294002. In contrast, LPS induced only a weak phosphorylation of PKB, similar to that seen with the isotype control antibody. We next investigated the involvement of another known downstream effector of PI3K, p70 S6K (40). Ligation of CD45 on monocytes also resulted in activation of p70 S6K (Fig. 5), which was maximal at 30 min and was inhibited by pre-treatment with rapamycin. Interestingly, however, the inclusion of rapamycin did not inhibit anti-CD45-induced monocyte TNF production (results not shown).

View larger version (50K): [in this window] [in a new window]   Fig. 4.   Wortmannin and LY294002 inhibit phosphorylation of PKB following ligation of CD45. a, 2 × 106 monocytes were stimulated with immobilized anti-CD45 (10 µg/ml), relevant isotype control (IC) (10 µg/ml) or LPS (10 ng/ml) for 20 min following prior incubation with wortmannin (W, 50 nM) or LY294002 (LY, 50 µM). Lysed samples were separated on a 10% SDS-polyacrylamide gel and phosphorylated proteins were detected using an antibody to phosphorylated PKB (pPKB) followed by anti-mouse horseradish peroxidase conjugate. b, the blot was acid-stripped and reprobed with an anti-PKB (PKB) antibody. Protein bands were visualized by autoradiography using Hyperfilm.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Ligation of CD45-induced p70 S6K activity in monocytes is rapamycin-sensitive. 5 × 106 monocytes were stimulated with immobilized anti-CD45 (10 µg/ml), relevant isotype control (IC) antibody (10 µg/ml), or LPS (10 ng/ml) for 30 min following preincubation with rapamycin (R, 10 µM) for 1 h. After cell lysis, p70 S6K activity was assayed as described under "Experimental Procedures," and the 32P-labeled substrate peptide was analyzed by 10% acrylamide-urea gel and visualized by autoradiography using Hyperfilm.

Anti-CD45-induced Monocyte TNF Production Is NF-B-independent-- After demonstrating that TNF production was differentially modulated by PI3K, we investigated the involvement of other factors known to regulate TNF gene expression. We focused upon the transcription factor NF-B, the activation of which has previously been shown to be important in TNF production following LPS stimulation (37). Furthermore, it has recently been reported that NF-B is activated by PI3K. Fig. 6a illustrates NF-B binding activity following 30 min stimulation with LPS (0.1-10 ng/ml). Virtually maximal activation was observed with 1 ng/ml LPS, whereas in contrast anti-CD45 antibody (10 µg/ml) resulted in only a weak activation of NF-B. It is unlikely that the difference in activation of NF-B between LPS and CD45 ligation was simply due to a weaker stimulation provided by anti-CD45, because similar amounts of TNF (750 pg/ml) were induced with anti-CD45 (10 µg/ml) and LPS (1 ng/ml) (Fig. 6b). These differences between LPS and CD45 ligation were further supported by the observation (Fig. 6c) that TNF synthesis in LPS- but not anti-CD45-stimulated monocytes was inhibited by >80% when monocytes were infected with an adenoviral vector expressing the inhibitor of NF-B (AdvIB).

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Anti-CD45-induced TNF production is NF-B-independent. a, 4 × 106 monocytes/ml were cultured with either immobilized anti-CD45 (10 µg/ml) or LPS (0.1-10 ng/ml) for 18 h. Supernatants were harvested and TNF levels determined by ELISA. b, 1 × 107 monocytes were stimulated with LPS (0.1-10 ng/ml), immobilized anti-CD45 or relevant control antibody (Ig) (10 µg/ml) and nuclear extracts were prepared as described under "Experimental Procedures." Nuclear protein (10 µg) was incubated with 32P-labeled oligonucleotides containing the consensus sequences for binding of NF-B protein. c, monocytes were cultured with M-CSF (100 ng/ml) for 2 days prior to adenoviral infection (uninfected ; AdvO, ; AdvIB, ) at a multiplicity of infection of 80:1. 2 × 106 monocytes/ml were stimulated with LPS (10 ng/ml), immobilized anti-CD45 (10 µg/ml). Culture supernatants were harvested after 18 h and assayed for TNF. Results are expressed as the mean of triplicate cultures ± S.D. All figures are representative of three experiments performed using different donors. In all experiments immobilized relevant isotype control (10 µg/ml) did not induce TNF production (results not shown).

Anti-CD45-induced TNF Production Is Regulated by the p38 MAPK Pathway-- We have demonstrated that TNF production in monocytes is differentially modulated by both PI3K and NF-B. Numerous studies have demonstrated the importance of MAPKs, in particular the p38 MAPK, in LPS-induced TNF production (9, 41). Therefore, we have investigated whether p38 MAPK is also involved in CD45-induced TNF production, using an inhibitor of p38 MAPK, SB203580. SB203580 was found to inhibit both anti-CD45- and LPS-induced monocyte TNF production, IC₅₀ values ~0.005 and 0.006 µM, respectively (Fig. 7, a and b). It has previously been demonstrated that LPS can activate p38 MAPK, with maximal stimulation seen at 10 min, followed by rapid loss of activation.² CD45 ligation also induced activation of p38 MAPK (maximal at 10 min) displaying similar kinetics to LPS (Fig. 8). Similarly, we have demonstrated that ligation of monocyte CD45 results in activation of p44/p42 MAPK with similar kinetics to LPS (results not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 7.   SB203580 inhibits both anti-CD45- and LPS-induced monocyte TNF production. 4 × 106/ml monocytes were treated with SB203580 for 30 min prior to culture with immobilized anti-CD45 (10 µg/ml) (a) or LPS (10 ng/ml) (b) for 18 h. Supernatants were harvested and TNF levels determined by ELISA. Results are expressed as the mean of triplicate cultures ± S.D. This figure is representative of five experiments performed using different donors. In all experiments immobilized relevant isotype control (10 µg/ml) did not induce TNF production (results not shown).

View larger version (38K): [in this window] [in a new window]   Fig. 8.   CD45 ligation and LPS stimulation of monocytes activates p38 MAPK. 5 × 106 monocytes were cultured with LPS (10 ng/ml), immobilized anti-CD45 (10 µg/ml), or isotype control (IC) antibodies (10 µg/ml) for given times. Postnuclear lysates were incubated with a suspension of protein G and anti-p38. p38 MAPK activity was assessed via [-32P]ATP incorporation into ATF-2. Phosphorylated products were visualized by autoradiography using Hyperfilm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this paper we investigated the signaling pathway(s) involved in monocyte TNF production following ligation of the cell surface receptor CD45 or LPS. Our results reveal the unexpected finding that CD45 ligation results in TNF production that is dependent upon the activation of PI3 kinase but independent of the transcription factor NF-B. In contrast, LPS-induced TNF production was dependent upon NF-B activation as previously reported (37) while PI3K-independent. These observations indicate that, while NF-B has previously been shown to be important in TNF production, it is not always necessary/required.

The importance of the cell surface receptor, CD45 in the activation of T and B cell antigen receptor-mediated signaling pathways and subsequent cellular responses has been well documented. Engagement of CD45 is known to regulate Src tyrosine kinases (p59^fyn, p56^lck, and p70^zap) phosphorylation (42, 43), phospholipase C1 regulation (44), inositol phosphate production (45), diacylglycerol production, PKC activation, and calcium mobilization (46). Ligation of CD45 has previously been shown to induce production of cytokines in monocytes (18, 19); however, the signaling pathways utilized upon CD45 ligation in monocytes have received little attention.

Ligation of monocyte CD45 results in activation of PI3K and the known downstream effectors PKB and p70 S6K. We have shown the anti-CD45-induced monocyte TNF production is inhibited by the PI3K inhibitors, wortmannin and LY294002. However the inhibitor of p70 S6K activation, rapamycin, did not inhibit anti-CD45-induced TNF production. These findings suggest that TNF production is p70 S6K-independent and other, as yet unidentified, downstream components of PI3K pathway are involved.

In contrast, wortmannin but not LY294002 enhanced LPS-induced monocyte TNF production, suggesting that the effects observed with wortmannin are not specific to PI3K activation. Wortmannin has other targets including PLA₂ (47), and we have shown that the PLA₂ inhibitor, AKTA, also enhances LPS-induced TNF production in monocytes,³ suggesting that the effect of wortmannin on LPS-induced TNF production may be due to PLA₂ inhibition. How PLA₂ negatively regulates TNF production is unclear, but this enzyme is required for synthesis of PGE₂, an inhibitor of TNF production (48). Wortmannin is known to stimulate the stress-activated protein kinase pathway (49), and this may also have a positive effect on TNF production. Furthermore, we observed only a weak increase in PI3K and p70 S6K activity following LPS stimulation, suggesting that neither of these pathways play a major role in LPS-mediated events in monocytes. These findings contradict with those performed by Herrera et al. (38), in which LPS was demonstrated to induce PI3K activity in monocytes, using similar methods to those described here. The reason for these apparently contradicting findings remain unclear. These studies have focused upon class 1_A PI3Ks, specifically those involving the p85 subunit and the involvement of other PI3K subclasses including those regulated by G-proteins and those which are wortmannin-insensitive have not been investigated.

p70 S6K and PKB are known downstream effectors of PI3K (39, 40, 50, 51); however, our studies indicate that CD45-induced TNF production in monocytes is p70 S6K-independent. Furthermore, while ligation of CD45 induces phosphorylation of PKB, the involvement of PKB in monocyte TNF production at this stage cannot be verified due to the lack of specific PKB inhibitors. These findings indicate that there must be a bifurcation of the signaling pathways downstream of PI3K that regulate TNF production. Several signaling molecules have been shown to directly and/or indirectly regulate PI3K, leading to the activation of transcription factors, e.g. atypical PKC and PKC. (52). Unfortunately, inhibitors of PKC were found to be toxic to monocytes and as such the involvement of PKC in anti-CD45-induced TNF production has not been assessed. Other potential downstream effectors include Rac, Rab5 (53, 59), Bruton's tyrosine kinase (55, 56), and JNK/stress-activated protein kinase (57, 58). The involvement of these molecules in PI3K-dependent TNF production still remains to be determined.

Several studies have suggested that LPS-induced TNF production in monocytes/macrophages is NF-B-dependent. Protease inhibitors, gliotoxin, and free radical scavengers have all been used to block NF-B activity; however, the lack of specificity of these reagents remains a constant problem. More recently, the over expression of IB following adenoviral infection (AdvIB) has been demonstrated to inhibit LPS-induced TNF production in monocytes (37). Curiously, we demonstrated that ligation of CD45 induced IB degradation (results not shown) but only a weak NF-B binding activity; the reasons for this remain unclear, although it suggests further complexity of the NF-B system. Overexpression of AdvIB did not inhibit anti-CD45-induced TNF production. These findings indicate that other, as yet unidentified, transcription factors are involved in anti-CD45-induced TNF production monocytes.

In T cells, induction of TNF gene expression is regulated by the nuclear factor of activated T cells (NFAT), not NF-B (59, 60, 61). NFAT binds to the 3 element of the TNF gene (located 97 and 88 nucleotides relative to the TNF start site), in association with ATF-2 and c-Jun proteins, which bind to the cyclic AMP response element site (62). NFAT DNA binding activity in activated T cells is prevented by the immunosuppressive drugs cyclosporin A (CsA), and FK506 (63, 64, 61). CsA and FK506 form complexes with their intracellular receptors (immunophilins), and inhibit the activity of calcineurin (protein phosphatase 2B), a ubiquitous calcium- and calmodulin-dependent phosphatase (reviewed in Ref. 65). Induction of TNF mRNA gene transcription in T cells can be blocked by CsA and FK506 (62), and expression of calcineurin is sufficient to activate a reporter gene whose transcription is driven by the TNF promoter (60). The involvement of NFAT in monocyte TNF production remains to be confirmed. However, CsA and FK506 failed to inhibit anti-CD45-induced TNF production in monocytes (results not shown), but this does not discount the involvement of CsA-insensitive NFAT in the regulation of monocyte TNF production. These findings suggest that NFAT, like NF-B, is not required for anti-CD45-induced TNF production.

LPS has previously been shown to activate the three major mammalian MAPK pathways, p42/44 (extracellular signal-regulated kinases 1/2), p38, and p54 MAPK (stress-activated protein kinase), in monocyte/macrophages (9, 10, 11). However, the relationship between the activation of these signaling molecules cytokine expression remains to be clarified. p38 MAPK is the only kinase that has been shown to play a pivotal role in the production of TNF (66). Previous studies have suggested that the post-transcriptional regulation of TNF is mediated through adenosine-uridine (AU)-rich elements present within the 3'-untranslated region of the TNF mRNA (67). Deletion of this region leads to the constitutive synthesis of TNF in cell lines (68) and transgenic animals (69). TNF reporter gene constructs that do not contain the 3'-AU-rich element regions lose their sensitivity to inhibition by the p38 inhibitor, SB203580, and it has been suggested that the p38 MAPK cascade is mediating the release of translational repression of TNF (66). The pyridinyl imidazole compound, SB203580, has been used to determine the involvement of p38 MAPK in the regulation of numerous pro-inflammatory cytokines including IL-1, IL-6, and TNF (9). Recently, SB203580 has been shown to inhibit TNF protein and mRNA induced by LPS, suggesting that TNF is being inhibited at the pre-translational level (70, 71). We have demonstrated that monocyte TNF production is regulated by distinct transcriptional mechanisms. Furthermore, we have demonstrated that both LPS- and anti-CD45-induced TNF production is regulated by p38 MAPK suggesting that both stimuli utilize similar translational mechanisms to regulate TNF production. We observed that ligation of CD45 resulted in activation of the MAPKs p38 and p42/p44 (results not shown) with similar kinetics to that observed with LPS. Furthermore, inhibitors of p38 MAPK (SB203580) and p42/44 MAPK (PD98059) (results not shown) were shown to block both anti-CD45 and LPS-induced TNF production. At higher concentrations SB203580 is known to inhibit the activity of JNK2 and JNK3 (72); however, the IC₅₀ values observed for SB203580 inhibition of anti-CD45- and LPS-induced monocyte TNF synthesis are consistent with its effects on p38 MAPK and not JNK, although the nonspecific actions of this drugs cannot be disregarded. These findings indicate that TNF production is regulated by distinct transcriptional signaling mechanisms, while the translational mechanisms appear to be identical.

In summary, this study demonstrates that TNF production in monocytes is regulated by multiple signaling pathways. The initiating signals for TNF production in inflammatory disorders such as rheumatoid arthritis are unknown. However, these findings suggest that engagement of specific cell surface receptors may be important in regulating TNF production via distinct signaling pathways and investigation of these mechanisms in both physiological and pathological systems is currently being investigated.³

	ACKNOWLEDGEMENT

We thank Dr. C. Ciesielski for assistance with adenoviral infection of monocytes.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Kennedy Inst. of Rheumatology, 1 Aspenlea Road, London W6 8LH, United Kingdom. Tel.: 44-181-383-4444; Fax: 44-181-383-4499; E-mail: f.brennan@cxwms.ac.uk.

² L. M. Williams and B. M. J. Foxwell, unpublished observation.

⁴ F. M. Brennan, A. L. Mayes, C. J. Ciesielski, P. Green, B. M. J. Foxwell, and M. Feldman, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: LPS, lipopolysaccharide; TNF, tumor necrosis factor ; IL, interleukin; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; JNK, Jun-N-terminal kinase; M-CSF, macrophage colony-stimulating factor; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; NF-B, nuclear factor-B; PLA₂, phospholipase A₂; IB, inhibitor of B; NFAT, nuclear factor of activated T cells; CsA, cyclosporin A; MOPS, 4-morpholinepropanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES