can act as an inhibitory messenger for T cells The

From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only.

Prepublished online May 29, 2008; doi:10.1182/blood-2008-02-138131

The cytoplasmic tail of CD45 is released from activated phagocytes and can act as an inhibitory messenger for T cells Stefanie Kirchberger, Otto Majdic, Stefan Bluml, Catharina Schrauf, Judith Leitner, Christopher Gerner, Wolfgang Paster, Nina Gundacker, Maria Sibilia and Johannes Stockl

Articles on similar topics can be found in the following Blood collections Immunobiology (5020 articles) Phagocytes (973 articles) Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml

Advance online articles have been peer reviewed and accepted for publication but have not yet appeared in the paper journal (edited, typeset versions may be posted when available prior to final publication). Advance online articles are citable and establish publication priority; they are indexed by PubMed from initial publication. Citations to Advance online articles must include the digital object identifier (DOIs) and date of initial publication.

Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.


Blood First Edition Paper, prepublished online May 29, 2008; DOI 10.1182/blood-2008-02-138131

The cytoplasmic tail of CD45 is released from activated phagocytes and can act as an inhibitory messenger for T cells

Short title: Alternative function of the cytoplasmic tail of CD45 Stefanie Kirchberger1, Otto Majdic1, Stefan Blüml1, Catharina Schrauf1, Judith Leitner1, Christopher Gerner2, Wolfgang Paster3, Nina Gundacker2, Maria Sibilia2 and Johannes Stöckl1 1

Institute of Immunology, Medical University of Vienna, Austria; 2Institute of

Cancer Research, Medical University of Vienna, Austria; 3Department of Molecular Immunology, Medical University of Vienna, Austria;

Corresponding author: Johannes Stöckl Institute of Immunology, Medical University of Vienna Borschkegasse 8a A-1090 Vienna, Austria e-mail: [email protected] phone: +431 4277 64951 fax: +43142779649

Category: Immunobiology

1 Copyright © 2008 American Society of Hematology


Abstract CD45 is the prototypic transmembrane protein tyrosine phosphatase (PTP), which is expressed on all nucleated hematopoietic cells and plays a central role in the integration of environmental signals into immune cell responses. Here we report an alternative function for the intracellular domain of CD45. We

discovered

that

CD45

is

sequentially

cleaved

by

serine/metalloproteinases and γ-secretases during activation of human monocytes and granulocytes by fungal stimuli or PMA but not by other microbial stimuli. Proteolytic processing of CD45 occurred upon activation of monocytes or granulocytes but not of T cells, B cells or dendritic cells and resulted in a 95 kDa fragment of the cytoplasmic tail of CD45 (ct-CD45). CtCD45 was released from monocytes and granulocytes upon activationinduced cell death. Binding studies with ct-CD45 revealed a counter-receptor on preactivated T cells. Moreover, T cell proliferation induced by dendritic cells or CD3 antibodies was inhibited in the presence of ct-CD45. Taken together, the results of our study demonstrate that fragments of the intracellular domain of CD45 from human phagocytes can function as intercellular regulators of T cell activation.

2


Introduction CD45 (PTPRC, leukocyte common antigen, B220, T200) was one of the first signaling molecules identified on leukocytes.

1-7

Humans with certain

mutations in CD45 or mice that are deficient in CD45 develop a servecombined immunodeficiency phenotype, systemic lupus erythematosus and many other diseases.8-13 CD45 is expressed on all nucleated hematopoetic cells and is therefore widely used as a pan-leukocyte marker molecule. CD45 is a prototypic transmembrane protein tyrosine phosphatase (PTP) and it is generally accepted today that it sets the threshold of positive and negative signaling events in leukocytes.12-15 Through dephosphorylation and activation of the src-family kinases Lck, Fyn and Lyn, CD45 is a positive regulator for signaling via the T cell and B cell receptors, respectively.16-18 On the other hand, through dephosphorylation of JAK, CD45 has been shown to negatively regulate cytokine receptor signaling and promotes viral infections.19 In addition CD45 is involved in regulating development, adhesion and apoptosis in leukocytes.12,13 CD45 is an abundant and large cell surface glycoprotein of 180-220 kDa, comprising up to 10 % of the leukocyte surface area.20 It exists in several isoforms generated by alternative splicing of exons 4,5 and 6, each with varying sized extracellular domains.13 These extracellular domains are poorly conserved among species and heavily glycosylated. In contrast, the 707 aa large cytoplasmic part of CD45 is conserved and contains two tandemly duplicated protein tyrosine phosphatase homology (PTP) domains, D1 and D2. Only D1 has phosphatase activity. The function of D2 is unclear and may contribute to stabilize D1. 21-25

3


A growing number of PTPs is now known to undergo post-translational proteolytic processing.26-30 For instance, the CD45-related receptor-type PTP (RPTP) LAR has been reported to be cleaved by the presenilin/γ-secretase complex.27 Cleavage of the intracellular parts of type-I cell surface receptors by presenilin/γ-secretase is typically executed after the receptors have undergone ecto-domain shedding.26,27,29,30 Interestingly, proteolysis by the presenilin/γ-secretase is not only a simple degradation process but frequently generates fragments of cell surface receptors with novel biological functions.29,30 Whether CD45 is proteolytically processed in leukocytes has not been investigated yet. Therefore we used a novel monoclonal antibody (mAb) 8-301, which is directed against the cytoplasmic tail of CD45, as a tool to analyze potential CD45 cleavage fragments in leukocytes and test their immunological function. We demonstrate in this study that the intracellular PTP-domain of CD45 (ctCD45) is proteolytically cleaved and released upon phagocyte activation by fungi or PMA and functions as an inhibitor of T cell activation.

4


Materials and methods Media, reagents, and chemicals. Cells were maintained in RPMI 1640 (Gibco Ltd., Paisley, Scotland), supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 10 % FCS (Sigma-Aldrich, St. Louis, MO). Recombinant human GM-CSF and IL-4 were kindly provided by Novartis Research Institute (Vienna, Austria). LPS from Eschericha coli (serotype 0127-B8; used at 1 μg/mL), zymosan A (100 μg/mL), β-glucan from Saccharomyces cerivisiae (100 μg/mL), peptidoglycan from Staphylococcus aureus (100 μg/mL), laminarin from Laminaria digitata (5 mg/mL), Poly I:C (20 μg/mL), PMA (100 nM), cycloheximide (2 and 10 μg/mL), propidium iodide, AEBSF (200 μM), aprotinin (20 μg/mL) and E64d (100 μM) were from SigmaAldrich. Pam3CSK4 (10 μg/mL) and CpG (ODN2006, used at 0,5 mM) were from Invivogen (San Diego, CA). Annexin V-FITC was from Caltag (Burlingame, CA). Candida albicans (ATCC 44374) was obtained from ATCC (Rockville, Maryland, USA). Apocynin (used at 1 mM), piceatannol (50 μM), EGTA (5 mM), calpeptin (100 μM), MG-132 (100 μM), pepstatin (2 μM), PTPase CD45 inhibitor (1 mM) and γ-secretase inhibitor X (5 μM) were from Calbiochem (Darmstadt, Germany). IL-10 was purchased from R&D Systems Inc. (Minneapolis, MN). Recombinant, human CD45 (aa 607-1304, 95 kDa), LAR (aa 1275-1613, 37 kDa) and RPTPγ (aa 801-1147, 66 kDa) were from Biomol Research Laboratories (Butler Pike, PA). Recombinant CD45 and human serum albumin (HSA) (Aventis-Pharma GmbH, Vienna, Austria) were labeled with Alexa-448 from Molecular Probes Inc. (Eugene, OR) according to the manufacturer´s instructions and used for staining at 20 μg/mL. Recombinant 5


S100A4 (metastasin) was kindly provided by N. Ambartsumian (Department of Molecular Cancer Biology, Copenhagen, Denmark). The CytoTox 96 assay for measuring lactate dehydrogenase (LDH) release was obtained from Promega (Madison, WI) and performed following the instructions of the manufacturer. The SensoLyte FDP protein phosphatase kit was from Anaspec (San Jose, CA). Antibodies and fusion-proteins. MAb 8-301 (IgG1) was generated in our laboratory by immunizing BALB/c mice with human T cell lysates. Murine negative control Ab VIAP (calf intestine alkaline phosphatase specific) was also generated in our laboratory. Anti-CD45 (MEM-28) was from Abcam (Cambridge, UK). The neutralizing polyclonal anti- IL-10 Ab (clone 25209) was obtained from R&D. Anti- Gp91phox clone 53 was from Becton Dickinson (Franklin Lakes, NJ). CD3-mAb (OKT3) was from Ortho Pharmaceutical Corporation (Raritan, NJ). The human CD45- human IgG-Fc fusion-proteins were produced in our laboratory in HEK293 cells: Ct-CD45-Ig represents the aa 598-1304 of CD45 (Swiss-Prot P08575), D1-Ig (aa 651-910), the phosphatase-dead D1 C851S-Ig (aa 651-910, cysteine 851 changed to serine) and D2-Ig (aa 942-1226). The ICOS-L-Ig covers the aa 1-101. The control-Ig fusion protein was previously described.31

Cell lines, cell isolation and stimulation. CD45+ and CD45-deficient (J45.01) Jurkat cells were obtained from ATCC. For isolation of leukocytes whole blood of healthy donors was separated by standard density gradient centrifugation with Lymphoprep (Nycomed, Oslo, Norway). Approval was obtained from the Medical University of Vienna institutional review board for

6


these studies. Monocytes and T cells were isolated from PBMCs by magnetic sorting using the MACS technique (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) as described previously.

32

DC were generated by

culturing purified blood monocytes for 7 days with a combination of GM-CSF (50 ng/mL) and IL-4 (100 U/mL). PMN were isolated from the pellet of the Lymphoprep gradient. Contaminating erythrocytes in PMN were removed with ammonium chloride lysis buffer (157 mM NH4Cl, 10 mM KHCO3, 0,1 mM EDTA). In the standard stimulation protocol 2x106 cells were incubated in 24well plates for 90 min (unless otherwise noted) in the presence of different stimuli in RPMI 1640 + 10 % FCS at 37°C. Candida albicans yeast for monocyte stimulation was grown freshly on Sabouraud agar plates, counted using a Buerker-Tuerk chamber and different numbers of C.albicans were seeded into 24-well plates in RPMI + 10 % FCS. For the generation of hyphae these plates were incubated at 37°C for 2 h, whereas yeast plates were kept on ice. C.albicans yeast and hyphae were used either alive or were inactivated in the plate for 1 h at 65°C. Subsequently 2 x 106 monocytes/ well were added and co-cultured for 90 min at 37°C. T cell proliferation assays. Stimulation of allogeneic, purified T cells with DC was performed as described recently.33 Recombinant ct-CD45, IL-10 and S100A4 were titrated into an allogeneic MLR with a fixed number of T cells (1 x 105) and DC (1 x 104). Experiments were performed in 96-well cell culture plates. Proliferation of T cells was monitored by measuring (methyl-3H)-TdR (ICN Pharmaceuticals Inc., Irvine, CA) incorporation on day 5 of culture. Cells were harvested 18 h later, and radioactivity was determined on a microplate scintillation counter (Packard, Meriden, CT). Assays were performed in

7


triplicates. For proliferation assays with Ig-fusion proteins, plates were coated with both anti-mouse IgG (Caltag; 3 μg/mL) and anti-human IgG, Fc specific (Jackson, West Grove, PA; 3 μg/mL) overnight at 4°C, washed, and then they were incubated with the respective

fusion-protein at the indicated

concentrations plus anti-CD3 (1 μg/mL). After another washing step, T cells (1 x 105/well) were added and proliferation was measured after 72 h as described above. Flow cytometric analysis. For membrane staining, cells (5 x 105) were incubated for 30 min at 4°C with unconjugated mAb. After washing, Oregon Green–conjugated goat anti-mouse-Ig from Molecular Probes Inc. (Eugene, OR) was used as a second-step reagent. For cytoplasmic staining, cells were harvested, fixed with FIX (An der Grub, Kaumberg, Austria) for 20 min, washed, and permiabilized for 20 min with PERM solution (An der Grub) in the presence of the primary mAb. Oregon Green–conjugated anti-mouse-Ig was used as second step reagent. Annexin V staining was performed as previously described.34 Flow cytometric analysis was performed using a BD FACScalibur flow cytometer.

Subcellular fractionation of monocytes. After washing in PBS, 1 x 108 monocytes were incubated in 1 mL of hypotonic buffer (42 mM KCl, 10 mM HEPES (pH 7.4), 5 mM MgCl2 and protease inhibitors) for 15 min at 4°C. Cells were mechanically disrupted by 20 strokes with a Glas-Col S20 tissue homogenizer (Terre Haute, IN, USA). Nuclei and intact cells were removed by centrifugation at 300 x g for 10 min at 4°C. The cleared postnuclear supernatant was subjected to ultracentrifugation at 100000 x g for 1 h at 4°C.

8


The resulting supernatant was collected as cytoplasmic fraction, and the membrane pellet was solubilized for 30 min at 4°C in RIPA buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 % NP-40, 0.5 % Sodium deoxycholate, 0.1 % SDS and protease inhibitors).

9


Immuno-affinity purification and Western blotting. For Western blot samples 2 x 106 cells were harvested after stimulation, and immediately re-suspended in 100 μL reducing SDS sample buffer, frozen in N2 and boiled at 95°C for 5 min. For mass-spectrometry immuno-precipitation with mAb 8-301 was performed from lysates produced by solubilization of 2 x 108 monocytes in 1 mL lysis buffer containing 1 % IGEPAL CA-630 (Sigma) supplemented with Complete Protease Inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). For Western Blot released ct-CD45 was precipitated from cell culture supernatants from 5 x 107 cells cultivated in 10 mL RPMI + 10 % FCS. For immuno-precipitation 100 μg (for MS) or 10 μg (for WB) mAb 8-301 were loaded onto 7 x 108 or 7 x 107 sheep anti-mouse IgG 2,8 μm Dynabeads (Dynal, Oslo, Norway) as described in detail elsewhere.35 After washing twice in PBS / 0,01 % BSA, beads were incubated with cell lysate or cell-culture supernatant for 24 h at 4°C on a rotator. Subsequently the beads were washed with PBS and bound protein was eluted into reducing sample buffer by boiling for 5 min. Western blot detection limit of precipitated ct-CD45 was at 5 ng. Western blotting was performed under standard conditions using mAbs at 1 μg/mL. Bound mAbs were detected using anti-mouse-Ig-HRP (DAKO, Glostrup, Denmark; 1/10,000) and chemiluminescence detection (SuperSignal; Pierce, Rockford, IL, USA). Phosphatase assays. SensoLyte phosphatase substrate was prepared following the manufacturer´s instructions. Magnetic beads with precipitated ctCD45 were directly incubated with SensoLyte phosphatase substrate for 60 min. Then the beads were removed using a Dynal MPC-2 magnetic particle

10


concentrator and fluorescence was measured with a VICTOR plate reader (Perkin-Elmer, Waltham, MA). Mass spectrometry. Lysates of monocytes treated with PMA for 90 min were precipitated with mAb 8-301 as described above. Gels were silverstained, the protein lanes were cut out and the gel pieces were treated with trypsin. For the identification of amino acid sequences, peptides were separated by nanoflow LC (1100 Series LC system, Agilent, Palo Alto, CA) using the HPLC-Chip technology (Agilent) equipped with a 40 nl Zorbax 300SB-C18 trapping column and a 75 µm x 150 mm Zorbax 300SB-C18 separation column at a flow rate of 400 nl/min, using a gradient from 0,2 % formic acid / 3 % ACN to 0,2 % formic acid / 50 % ACN over 60 min. Peptide identification was accomplished by MS/MS fragmentation analysis with an iontrap mass spectrometer (XCT-Ultra, Agilent) equipped with an orthogonal nanospray ion source. The MS/MS data, including peaklist-generation and search engine, were interpreted by the Spectrum Mill MS Proteomics Workbench software (Version A.03.02, Agilent) allowing for two missed cleavages and searched against the SwissProt Database for human proteins (Version 20061207 containing 15.265 entries) allowing for precursor mass deviation of 1,5 Da, a product mass tolerance of 0,7 Da and a minimum matched peak intensity (%SPI) of 70 %. The scores were essentially calculated from sequence tag lengths, but also consider mass deviations. To assess reliability of the peptide scores,

we

performed

searches

against

the

corresponding

reverse

database.36 5,4 % positive hits were found with peptides scoring >9,0, while 1,18 % positive hits were found with peptides scoring >13,0. Consequently,

11


we set the threshold for protein identification to at least one peptide scoring higher than 13,0. To identify the molecule recognized by mAb 8-301 the 200 kDa band was isolated

from

monocyte

lysates

by

immunoprecipitation

and

mass

spectrometric analysis was performed. From this band 28 different peptides of the digest were matched to the leukocyte common antigen precursor (CD45 antigen, P08575) covering 26 % (within amino acids 292-1254) of the total sequence of 1304 amino acids.

12


Results Molecular characterization of the mAb 8-301 defined antigen. mAb 8-301 was produced in our laboratory by immunization of Balb/c mice with human T cell homogenates. We could demonstrate by mass spectrometric analysis (for details see materials & methods) and by Western Blot that mAb 8-301 is directed against CD45 (Figure 1A). Remarkably, mAb 8-301, in contrast to the classical CD45 mAb MEM28, did not show surface recognition (Figure 1B) but had strong intracellular reactivity. To analyze whether mAb 8-301 binds to the cytoplasmic tail of CD45 we tested if mAb 8-301 recognizes a recombinant CD45 protein representing the cytoplasmic domain (Figure 1C). MAb 8-301 recognized this 95 kDa protein, but not the intracellular domains of other related phosphatases like RPTPγ and LAR. Thus, mAb 8-301 is directed against the cytoplasmic tail of CD45.

A 95 kDa fragment of CD45 is produced in phagocytes. The phorbol ester PMA, a potent PKC activator, was shown previously to induce cleavage of PTPs like LAR in different cell lines.26,37 Here we applied mAb 8-301 as a tool to identify proteolytically-produced fragments of CD45 in different human leukocyte populations upon PMA stimulation. MAb 8-301 recognized total CD45 in the range of 200 kDa in all tested leukocyte populations (Figure 2A). Furthermore, we could detect a potential CD45 cleavage fragment of 95 kDa in monocytes rapidly after PMA stimulation. The amount of this protein increased until 3 h and then declined. Expression of this protein was not restricted to monocytes but was also induced in neutrophil granulocytes after

13


1 h, whereas DC, B cells, T cells or the myeloid cell lines U937 and THP-1 did not express it (Figure 2A and data not shown). As the recombinant cytoplasmic CD45 and the potential CD45 fragment have the same size, we speculated that this 95 kDa protein is the cytoplasmic tail of CD45 (ct-CD45). We analyzed the protein sequence of the potential fragment by massspectrometry. The data presented in supplementary figure 1 shows that we could identify 11 peptides, which all aligned to the intracellular part of CD45. Furthermore we found that upon 3 h stimulation the 95 kDa protein is located in the cytoplasmic and not in the membrane fraction (Figure 2B). The ratio of intact versus cleaved CD45 molecules was determined by densitometric analysis and we found that 29 ± 6% and 9 ± 4% of the CD45 molecules are cleaved upon PMA- (n=8) and zymosan-stimulation (n=7) in monocytes, respectively, within 90 min. In contrast to mAb 8-301, mAb MEM-28 neither reacted with PMA-induced ct-CD45 nor with the recombinant ct-CD45 (Figure 2C). Thus, the 95 kDa protein detected by mAb 8-301 in the cytoplasm of activated phagocytes is ct-CD45.

Fungal stimuli induce ct-CD45 in human monocytes. Sensing of pathogens is the first, critical step in launching an immune response. As phagocytes are especially equipped with pattern recognition receptors we wondered whether ct-CD45 is also induced by microbial stimuli. Interestingly, ct-CD45 was produced upon treatment of monocytes with zymosan, the particulate cell wall extract of Saccharomyces cerivisiae, but not upon treatment with the TLR2 ligands Pam3Cys or peptidoglycan (Figure 3A). Furthermore, neither stimulation via TLR3, TLR4 nor TLR9 by PolyI:C, LPS or CpG induced ct-

14


CD45. We also tested whether ct-CD45 is produced by the induction of cell death. Inducers of cell death like cycloheximide or staurosporine or freeze/thaw cycles did not lead to ct-CD45 generation (data not shown). Figure 3B shows that zymosan and PMA but not LPS stimulation of monocytes is accompanied by a diminished reactivity of MEM-28 seen by flow cytometry, indicating the release of the extracellular domain. Ct-CD45 generation upon zymosan stimulation of monocytes shows the same kinetics as upon PMA stimulation (Figure 3C and 2A). Stimulation of monocytes with heat-inactivated (Figure 3D) as well as with vital (data not shown) Candida albicans yeast and hyphae led also to ct-CD45 expression. Phagocytes express and utilize a variety of cell surface receptors for sensing fungi and in particular the fungal cell wall components β-glucan and mannan. Beside TLR2,

mannose

receptor

(CD206)

and

complement

receptor

3

(CD11b/CD18), Dectin-1, the major β-glucan receptor on myeloid cells, was recently discovered to be an important player in controlling fungal infection.3841

As zymosan is mainly composed of β-glucan42 and the TLR2 stimuli

Pam3Cys did not induce ct-CD45 (Figure 3A), we tested whether ct-CD45 can be produced upon ligation of Dectin-1. Therefore we used a particulate βglucan preparation from S. cerivisiae, which induced a slight ct-CD45 band (Figure 3E). Furthermore we investigated whether induction of ct-CD45 by zymosan can be blocked by the soluble β-glucan laminarin from Laminaria digitata, which was shown to block activation by zymosan via Dectin-1.

38,40,43

We found that laminarin strongly inhibited zymosan-induced production of ctCD45 (Figure 3E). Ligation of Dectin-1 leads to activation of Syk tyrosine kinase, which is important for zymosan-induced respiratory burst in

15


phagocytes.44 We found that the generation of ct-CD45 by zymosan but not by PMA was blocked by the Syk kinase inhibitor piceatannol (Figure 3F). Thus, we concluded that ligation of Dectin-1 is critically involved in the induction of ct-CD45 by zymosan. As both, zymosan and PMA are able to induce an oxidative burst in phagocytes (data not shown) we tested whether oxidative burst formation is involved in ct-CD45 generation. Remarkably, apocynin, which blocks the oxidative burst by specifically inhibiting the assembly of the functional NADPH-oxidase complex, was indeed able to block ct-CD45 production by zymosan as well as by PMA (Figure 3F).

Ct-CD45 is generated by proteolytic cleavage. As we could detect ct-CD45 in the cytoplasmic fraction (Figure 2B) and found a diminished reactivity of MEM-28 upon stimulation suggesting the release of the CD45 ectodomain (Figure 3B) we hypothesized that CD45 could be processed by a mechanism including several cleavage steps. However in addition we also examined whether ct-CD45 is a so far unknown translational variant. We tested whether inhibition of de novo protein synthesis with cycloheximide could inhibit ctCD45 generation.45 However, as shown in Figure 4A, ct-CD45 was expressed during monocyte stimulation with zymosan or PMA in the presence of cycloheximide, which completely prevented the induction of typical monocyte activation markers such as sialoadhesin (CD169) analyzed in parallel (data not shown). As ct-CD45 is not de novo synthesized we further examined our hypothesis that ct-CD45 is generated by proteolysis from intact CD45 molecules. To test which protease could be involved in ct-CD45 cleavage we

16


stimulated monocytes in the presence of different protease inhibitors to block generation of ct-CD45. Results presented in Figure 4B demonstrate that induction of ct-CD45 expression was prevented in the presence of serine protease inhibitors AEBSF or aprotinin and the metalloprotease-inhibitor EGTA. The calpain inhibitor calpeptin, the proteasome inhibitor MG-132, the cysteine protease inhibitor E64d, and pepstatin, an aspartyl protease inhibitor, did not diminish the generation of ct-CD45. Therefore potential candidates for the extracellular cleavage step are serine and metallo-proteases. Since the molecular weight of the cellular ct-CD45 and the recombinant ct-CD45 covering the full intracellular part of CD45 (Figure 2A) was similar we hypothesized that cleavage of the intracellular part would occur near the cell membrane. Cleavage of the intracellular domains of type-I cell surface receptors is typically executed by the presinilin/γ-secretase complex after the receptors,

including

some

RPTPs,

have

undergone

ecto-domain

shedding.27,29,30 Thus, we analyzed whether cleavage of the extracellular part of CD45 is followed by intracellular cleavage of ct-CD45 by γ-secretases (Figure 4C). After 90 min stimulation of monocytes with PMA we found that some ct-CD45 molecules are still linked to the membrane (right panel), whereas others are already released into the cytoplasm (left panel). AEBSF did block generation of membrane-linked as well as cytoplasmic ct-CD45. The release of ct-CD45 from the membrane into the cytoplasm was prevented by the γ-secretase inhibitor X. Therefore these results demonstrate that sequential cleavage of CD45 molecules by serine- and metallo-proteases and the presenilin/γ-secretase pathway in the course of activation leads to the generation of ct-CD45 in phagocytes.

17


Ct-CD45 is released by phagocytes upon stimulation. Stimulation of phagocytes with zymosan or PMA is known to induce activation induced cell death.46,47 We indeed found that the number of annexin and propidium iodide positive cells increased upon PMA or zymosan treatment (Figure 5A and data not shown). We hypothesized that the observed decrease of ct-CD45 in cell lysates (Figure 2A) might be due to further degradation or release of ct-CD45 from dying cells into the supernatant. Results presented in Figure 5B demonstrate that ct-CD45 was indeed detectable in the supernatant of activated monocytes after 4 to 16h. We measured by densitometric analysis of Western Blots that 1 x 106 monocytes release 859 ± 496 pg and 180 ± 81 pg of ct-CD45 upon stimulation with PMA and zymosan, respectively (n=3 donors). Granulocytes (1 x 106) release even slightly more ct-CD45 (1.181 ± 308 pg, n=3 donors) upon stimulation with PMA. The appearance of ct-CD45 in the supernatant of phagocytes correlated with lactate dehydrogenase release, a cytoplasmic protein typically released upon membrane leakage due to cell death (Figure 5C). We found that ct-CD45 isolated from the supernatant of phagocytes retained its phosphatases activity (Figure 5D). Taken together, we concluded that ct-CD45 after stimulation-induced cleavage seems to be released from phagocytes mainly due to cell death.

Ct-CD45 inhibits T cell proliferation. Next we analyzed if soluble ct-CD45 is able to interact with other immune cells. Using fluorescein-labeled recombinant ct-CD45 molecules for binding studies, we found that ct-CD45 did not bind to resting T cells, monocytes and DC (data not shown) but clearly

18


interacted with preactivated T cells (Figure 6A). Binding of ct-CD45 to activated T cells seems to be mediated via a saturable (Figure 6B) and specific receptor, since it was inhibited in the presence of mAb 8-301 and in the presence of unlabeled ct-CD45. Pretreatment of ct-CD45 with a specific inhibitor of the PTPase domain of CD45 did not block binding to T cells (Figure 6A)48, indicating that the PTPase activity of ct-CD45 is not essential for binding to T cells. In order to examine whether binding of ct-CD45 to T cells has immuneregulatory consequences we added ct-CD45 proteins to an MLR. We observed that ct-CD45 reduced T cell proliferation induced by DC. This inhibitory effect of ct-CD45 was comparable to that found with IL-10, the classical inhibitory factor for T cells, and was not seen with recombinant S100A4 protein used for control (Figure 6C). We also observed this inhibitory function when we stimulated T cells with plate bound anti-CD3 in the presence of immobilized ct-CD45-Ig fusion proteins (Figure 6D). Inhibition of proliferation was independent of the phosphatase domain of CD45 as a phosphatase-dead D1-Ig protein induced the same effect as active protein. The D2-Ig showed a weaker inhibitory effect than D1-Ig especially at a lower dose. Figure 6E shows that inhibition of T cell proliferation by different ctCD45-Ig proteins was not due to cell death. Ct-CD45 thus represents a novel immune-regulatory factor for human T cells.

19


Discussion In this study we demonstrate an alternative function for CD45. We observed that cytoplasmic tails of CD45 molecules are cleaved and released upon activation of phagocytes and act as cytokine-like factors with negative immune-regulatory function on T cells. Thereby, ct-CD45 can act as an intercellular regulator between the innate and the adaptive immune system. We have discovered this phenomenon when we studied the existence of post-translationally processed CD45 molecules with a mAb directed against the cytoplasmic part. Ct-CD45 was found in monocytes and neutrophil granulocytes but not in leukocytes or DC upon activation with PMA, the fungal cell wall component zymosan or C.albicans. Ct-CD45 was not detectable after induction of cell death. Together with the finding that ct-CD45 molecules are also generated in the presence of cycloheximide, which blocks ribosomal translation, the rapid appearance of ct-CD45 within 1 h suggested that it might not be derived from newly synthesized, alternative splice variants of CD45. Alternative splicing of the extracellular part of CD45 is wellinvestigated and responsible for the generation of CD45 isoforms but has not been reported to occur within the conserved intracellular PTP-domains.13,15,45 We could show that cleavage of the cytoplasmic tail from intact CD45 molecules by proteases is responsible for the accumulation of ct-CD45. Proteolysis of other transmembrane and non-receptor type PTPs has been reported before in several studies.49-51 Cleavage of the intracellular domains of type-I cell surface receptors is typically executed by the presinilin/γsecretase complex after the receptors, including some RPTPs, have undergone ecto-domain shedding.26,27,29,30 For instance, the CD45-related

20


receptor-type PTP LAR has been reported to be cleaved by the γ-secretase pathway.27 The extracellular part of CD45 has been reported to be cleaved by trypsin digestion.52 Here we found that in the course of CD45 processing upon stimulation with PMA or zymosan, sequential cleavage of CD45 molecules by serine- and metallo-proteases and the presenilin/γ-secretase pathway leads to the generation of ct-CD45 in phagocytes. The reutilization of intracellular proteins for alternative functions is an emerging theme in cell biology. Release of cytoplasmic or nuclear proteins such as HMGB-1 from dying cells or by non-classical (i.e. Golgi-independent) release mechanisms has been shown to function as a “danger signal” leading to the activation of DC and consequently promote adaptive immunity.53-55 We also observed that ct-CD45 seems to be released, but not produced, mainly upon cell death during PMA and zymosan stimulation. Yet, non-classical release mechanisms may also be involved in the release of ct-CD45. In contrast to HMGB-1, ct-CD45 acted as an inhibitory factor. One could speculate that released ct-CD45 helps to prevent exaggerated immune responses executed by T cells. How ct-CD45 inhibits T cell proliferation remains to be determined. However, direct binding to preactivated T cells suggests that there might be a specific receptor for ct-CD45. CD45 and in particular its intracellular part, is known to interact with a number of proteins, including various protein kinases (e.g. Lck and Fyn), cytoskeletal proteins such as fodrin and intramembrane cell surface receptors such as CD2.13 Thus, on condition that it is expressed on the cell-surface, one of the already described binding partners for the intracellular part of CD45 could also serve as an extracellular counter-

21


receptor for ct-CD45 on activated T cells. Casein-kinase 2 (CK-2) is an intracellular binding partner of CD45 but has been reported to be expressed upon cell activation on the outside of cell membranes and functions as ectoprotein kinase.56-58 We have investigated whether CK-2 is expressed outside of activated T cells, but mAb 1AD9 against CK-2 showed no extracellular surface reactivity and did not inhibit binding of ct-CD45 to T cells. Thus, CK-2 may not act as a cell surface receptor for ct-CD45 on T cells (data not shown). Inhibition of T cell proliferation seems to be independent of the phosphatase activity of CD45 as we found it also with a phosphatase-dead fusion protein. Interestingly inhibition was not only seen upon stimulation with an Ig fusion protein covering the total intracellular part or the D1 domain but also with a D2-Ig protein. Still, the inhibitory activity of D2-Ig was weaker than D1-Ig, especially at lower concentrations. Although it is surprising that both PTP domains are able to act inhibitory these results are supported by data that the tertiary structures of D1 and D2 are very similar.25 Remarkably, ct-CD45 was only generated in phagocytes like monocytes and granulocytes, but not in T cells, B cells or in DC. Monocytes and granulocytes are capable to produce a strong respiratory burst. Considering that, together with our observation that ct-CD45 generation was blocked by an inhibitor of NADPH-oxidase, we can speculate that oxidative burst formation is an important factor in the generation of ct-CD45. Phagocytes and their ability to induce an oxidative burst play a central role in the first line of defense against fungi. Yet, recent studies also support the idea that phagocytes and factors derived from these innate immune cells such as

22


IL-10 are pivotal in controlling adaptive immune responses against fungi.59,60 These processes are considered as central mechanism to establish a commensal relationship between fungus and host.61 Our finding that the activation of human phagocytes with fungal stimuli induces the cleavage and release of ct-CD45 in vitro represents a novel mechanism in the understanding of the complex host-fungus interaction. In order to investigate the physiological relevance of ct-CD45 in vivo, we have started out to analyze ct-CD45 levels in sera from patients with severe infectious diseases including systemic candidosis by ELISA and to test whether ct-CD45 molecules can act immunosuppressive in a murine model of rheumatoid arthritis.

23


Acknowledgment The authors thank Petra Kohl, Christa Zangerle, Edith Bayer, Saro Künig and Claus Wenhardt for expert technical assistance. This work was supported by grants of the Austrian Science Fund (FWFSFB2307 “Vision Fund” and APP20266FW).

Authorship Contribution: S.K., J.S., S.B, W.P. performed experiments. C.S. and J.L. generated fusionproteins. O.M. produced antibodies. C.G. and N.G. were responsible for mass spectrometry. M.S. supported writing. S.K. and J.S. designed the research and wrote the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

24


Figure legends Figure 1. Molecular characterization of the mAb 8-301 defined antigen. (A) Lysates of CD45+ and CD45-deficient Jurkat cells were used in Western blots to validate the reactivity of mAb 8-301 with CD45 in parallel with the classical CD45 mAb MEM-28. (B) Flow cytometric analysis revealed that mAb 8-301, in contrast to MEM-28, shows intracellular reactivity with monocytes. The figure shows histogram profiles including isotype-matched control mAb VIAP (open) and mAb 8-301 or MEM-28 (filled). (C) Reactivity of mAb 8-301 with cytoplasmic tails of protein tyrosine phosphatases CD45, RPTPγ and LAR was tested by immuno-blot (left). Loading was controlled by Ponceau S staining of the membrane (right). Representative experiments of at least two independent experiments are shown.

Figure 2. A 95 kDa fragment of CD45 is produced in monocytes and granulocytes. (A) Assessment of potential CD45 cleavage fragments with the intracellular-reactive CD45 mAb 8-301. B cells, T cells, monocytes (Mo), DC and granulocytes (Gr) were stimulated for different time periods with PMA and lysates were analyzed by Western Blot. A representative experiment of three independently performed is shown. (B) Membrane and cytoplasmic fractions of resting monocytes and of monocytes, stimulated for 3 h with PMA, were isolated and analyzed in Western blots with mAb 8-301 for localization of the 95 kDa band. Isolation of membranes was controlled with a gp91phox antibody, isolation of cytoplasm with an actin antibody. One representative experiment of two independent experiments is shown. (C) MEM-28 does not react with the PMA-induced ct-CD45. A recombinant cytoplasmic CD45 25


protein covering the total intracellular part (lane 1), resting monocytes (lane 2) and monocytes stimulated for 90 min with PMA (lane 3) were analyzed in Western blots with mAb 8-301 and MEM-28.

Figure 3. Fungal stimuli induce ct-CD45 in human monocytes. (A) Human monocytes were stimulated with Pam3CSK4 (P3C), peptidoglycan (PG), zymosan (Zym), Poly I:C (PI:C), LPS, CpG or PMA for 90 min. Lysates were analyzed by Western blot with mAb 8-301. (B) Binding of MEM-28 to resting monocytes (dashed line) or monocytes treated for 90 min with PMA, zymosan or LPS (black line, respectively) were examined by flow cytometry. Isotypematched control mAb VIAP (dotted line) was used as non-binding control. (C) Monocytes treated with zymosan were harvested at different time points and analyzed by Western Blot with mAb 8-301. (D) Different numbers of C. albicans hyphae and yeast were heat-inactivated and co-cultured with monocytes. Lysates were analyzed by Western Blot with mAb 8-301 for generation of ct-CD45. (E) Stimulation of monocytes with the Dectin-1 ligand β-glucan (β-Glu) leads to ct-CD45 production. Blocking with laminarin (Lam) also inhibited zymosan-induced ct-CD45. (F) The syk inhibitor piceatannol (picea, 50µM) strongly inhibited ct-CD45 production by zymosan. NADPHoxidase inhibitor apocynin (apo, 1mM) blocked ct-CD45 induced by PMA and zymosan. Representative experiments of three independently performed are shown.

Figure 4. The cytoplasmic tail of CD45 is proteolytically cleaved and released into the cytoplasm. (A) Inhibition of de novo protein synthesis by

26


cycloheximide (2 and 10 μg/mL) did not block production of ct-CD45 upon monocyte stimulation with PMA or zymosan. (B) The activity of protease inhibitors AEBSF, aprotinin, EGTA, calpeptin, E64d, MG-132 and pepstatin on production of ct-CD45 in zymosan-stimulated monocytes was tested in Western blots. (C) Membrane and cytoplasmic fractions of resting monocytes and monocytes stimulated for 90 min with PMA were isolated and analyzed in Western blots with mAb 8-301. Presence of the serine protease inhibitor AEBSF inhibited production of membrane-bound ct-CD45. The gammasecretase inhibitor X blocked the release of membrane-bound ct-CD45 into the cytosol. Isolation of membranes was controlled with anti-gp91phox, isolation of cytoplasm with an actin antibody. Representative experiments of at least two independently performed are shown.

Figure 5. Ct-CD45 molecules are released into the supernatant upon stimulation of phagocytes with zymosan and PMA. (A) Monocytes stimulated with PMA for different time periods were stained with Annexin-V and PI. Percentages of positive cells are indicated. (B) ct-CD45 was isolated from cell culture supernatants of monocytes stimulated with PMA or zymosan by immuno-precipitation with mAb 8-301. As a positive control recombinant ct-CD45 diluted in the same amount of RPMI was precipitated, as a negative control precipitation was performed from RPMI (medium ctrl). Isolated ctCD45 was immuno-blotted and detected by mAb 8-301. As a loading control the light chain of mAb 8-301 used for precipitation, detected with sheep-antimouse-HRP, was used. Representative experiments of at least two independent experiments are shown. (C) Release of ct-CD45 was

27


accompanied by the release of lactate dehydrogenase (LDH). The experiment shows the LDH values of the supernatants used for immuno-precipitation in (B). (D) The phosphatase activity of released ct-CD45 was assessed by precipitation of ct-CD45 with mAb 8-301 from supernatants of granulocytes (7 x 107 cells) incubated +/- PMA for 4 h. The figure shows mean values of relative fluorescent units from four different donors out of two different experiments +/- SD. A paired Student´s t-test was performed (** P< 0,05).

Figure 6. Ct-CD45 has immuno-regulatory function. (A) Binding of ctCD45 to resting T cells and to T cells activated for 24 h with PMA/ionomycin was analyzed with Alexa-488 labeled recombinant ct-CD45 molecules and flow cytometry. Pretreatment of cells with unlabeled recombinant protein or mAb 8-301 but not with PTPase inhibitor blocked binding of fluorescent ctCD45 (filled histograms) to T cells. The figure shows overlay histogram profiles including Alexa-488-labeled human serum albumin used as a negative control (open histogram), which are representative of three independent experiments. (B) Binding of increasing amounts of Alexa-488 labeled recombinant ct-CD45 molecules analyzed by flow cytometry. The figure shows mean values +/- SEM of four experiments of mean fluorescence intensity. The values of non-specific staining with Alexa-labeled HSA were subtracted from the values obtained with Alexa-labeled ct-CD45. (C) Addition of recombinant ct-CD45 (filled triangles) inhibits T cell proliferation induced by allogeneic DC (open dot). The immuno-suppressive cytokine IL-10 (filled squares) and recombinant S100A4 proteins (filled dots) were used as positive and negative control, respectively. Data represent mean cpm ± SEM of 3

28


independent experiments. (D) T cells were cultured in the presence of immobilized anti-CD3 alone (mock, taken as 100 %), anti-CD3 plus control-Ig (co-Ig), or as positive control anti-CD3 plus ICOS-L-Ig or anti-CD3 plus different CD45-Ig-fusion proteins (all Ig-fusion proteins coated at 0,5 or 2 μg/mL) for 72 h and proliferation was measured by

3

H- thymidine

incorporation. Mean values and SD of three different experiments are shown. A paired Student´s t-test was performed (*** P< 0,01, ** P< 0,05). (E) Percentages of Annexin and PI positive T cells cultured as in (D) with 2 μg/mL Ig-fusion proteins coated. Mean values and SD of three different experiments are shown.

29


References 1. Grossman Z, Min B, Meier-Schellersheim M, Paul WE. Concomitant regulation of T-cell activation and homeostasis. Nat Rev Immunol. 2004;4:387-395. 2. Reth M, Brummer T. Feedback regulation of lymphocyte signalling. Nat Rev Immunol. 2004;4:269-277. 3.

Zinkernagel RM. Immunity 2000. Immunol Today. 2000;21:422-423.

4. Thomas ML, Barclay AN, Gagnon J, Williams AF. Evidence from cDNA clones that the rat leukocyte-common antigen (T200) spans the lipid bilayer and contains a cytoplasmic domain of 80,000 Mr. Cell. 1985;41:83-93. 5. Ledbetter JA, Tonks NK, Fischer EH, Clark EA. CD45 regulates signal transduction and lymphocyte activation by specific association with receptor molecules on T or B cells. Proc Natl Acad Sci U S A. 1988;85:8628-8632. 6. Tonks NK, Charbonneau H, Diltz CD, Fischer EH, Walsh KA. Demonstration that the leukocyte common antigen CD45 is a protein tyrosine phosphatase. Biochemistry. 1988;27:8695-8701. 7. Pingel JT, Thomas ML. Evidence that the leukocyte-common antigen is required for antigen-induced T lymphocyte proliferation. Cell. 1989;58:10551065. 8. Cale CM, Klein NJ, Novelli V, Veys P, Jones AM, Morgan G. Severe combined immunodeficiency with abnormalities in expression of the common leucocyte antigen, CD45. Arch Dis Child. 1997;76:163-164. 9. Kung C, Pingel JT, Heikinheimo M, et al. Mutations in the tyrosine phosphatase CD45 gene in a child with severe combined immunodeficiency disease. Nat Med. 2000;6:343-345. 10. Lynch KW, Weiss A. A CD45 polymorphism associated with multiple sclerosis disrupts an exonic splicing silencer. J Biol Chem. 2001;276:2434124347. 11. Tchilian EZ, Wallace DL, Wells RS, Flower DR, Morgan G, Beverley PC. A deletion in the gene encoding the CD45 antigen in a patient with SCID. J Immunol. 2001;166:1308-1313. 12. Penninger JM, Irie-Sasaki J, Sasaki T, Oliveira-dos-Santos AJ. CD45: new jobs for an old acquaintance. Nat Immunol. 2001;2:389-396. 13. Hermiston ML, Xu Z, Weiss A. CD45: a critical regulator of signaling thresholds in immune cells. Annu Rev Immunol. 2003;21:107-137. 14. Huntington ND, Tarlinton DM. CD45: direct and indirect government of immune regulation. Immunol Lett. 2004;94:167-174.

30


15. Mustelin T, Vang T, Bottini N. Protein tyrosine phosphatases and the immune response. Nat Rev Immunol. 2005;5:43-57. 16. Ostergaard HL, Shackelford DA, Hurley TR, et al. Expression of CD45 alters phosphorylation of the lck-encoded tyrosine protein kinase in murine lymphoma T-cell lines. Proc Natl Acad Sci U S A. 1989;86:8959-8963. 17. Koretzky GA, Picus J, Thomas ML, Weiss A. Tyrosine phosphatase CD45 is essential for coupling T-cell antigen receptor to the phosphatidyl inositol pathway. Nature. 1990;346:66-68. 18. Mustelin T, Burn P. Regulation of src family tyrosine kinases in lymphocytes. Trends Biochem Sci. 1993;18:215-220. 19. Irie-Sasaki J, Sasaki T, Matsumoto W, et al. CD45 is a JAK phosphatase and negatively regulates cytokine receptor signalling. Nature. 2001;409:349-354. 20. Thomas ML. The leukocyte common antigen family. Annu Rev Immunol. 1989;7:339-369. 21. Volarevic S, Niklinska BB, Burns CM, June CH, Weissman AM, Ashwell JD. Regulation of TCR signaling by CD45 lacking transmembrane and extracellular domains. Science. 1993;260:541-544. 22. Hovis RR, Donovan JA, Musci MA, et al. Rescue of signaling by a chimeric protein containing the cytoplasmic domain of CD45. Science. 1993;260:544-546. 23. Desai DM, Sap J, Silvennoinen O, Schlessinger J, Weiss A. The catalytic activity of the CD45 membrane-proximal phosphatase domain is required for TCR signaling and regulation. Embo J. 1994;13:4002-4010. 24. Felberg J, Johnson P. Characterization of recombinant CD45 cytoplasmic domain proteins. Evidence for intramolecular and intermolecular interactions. J Biol Chem. 1998;273:17839-17845. 25. Nam HJ, Poy F, Saito H, Frederick CA. Structural basis for the function and regulation of the receptor protein tyrosine phosphatase CD45. J Exp Med. 2005;201:441-452. 26. Aicher B, Lerch MM, Muller T, Schilling J, Ullrich A. Cellular redistribution of protein tyrosine phosphatases LAR and PTPsigma by inducible proteolytic processing. J Cell Biol. 1997;138:681-696. 27. Haapasalo A, Kim DY, Carey BW, Turunen MK, Pettingell WH, Kovacs DM. Presenilin/gamma-secretase-mediated cleavage regulates association of leukocyte-common antigen-related (LAR) receptor tyrosine phosphatase with beta-catenin. J Biol Chem. 2007;282:9063-9072.

31


28. Halle M, Liu YC, Hardy S, et al. Caspase-3 regulates catalytic activity and scaffolding functions of the protein tyrosine phosphatase PEST, a novel modulator of the apoptotic response. Mol Cell Biol. 2007;27:1172-1190. 29. Kopan R, Ilagan MX. Gamma-secretase: membrane? Nat Rev Mol Cell Biol. 2004;5:499-504.

proteasome

of

the

30. Parks AL, Curtis D. Presenilin diversifies its portfolio. Trends Genet. 2007;23:140-150. 31. Pfistershammer K, Klauser C, Pickl WF, et al. No evidence for dualism in function and receptors: PD-L2/B7-DC is an inhibitory regulator of human T cell activation. Eur J Immunol. 2006;36:1104-1113. 32. Stockl J, Vetr H, Majdic O, Zlabinger G, Kuechler E, Knapp W. Human major group rhinoviruses downmodulate the accessory function of monocytes by inducing IL-10. J Clin Invest. 1999;104:957-965. 33. Kirchberger S, Majdic O, Steinberger P, et al. Human rhinoviruses inhibit the accessory function of dendritic cells by inducing sialoadhesin and b7-h1 expression. J Immunol. 2005;175:1145-1152. 34. Selenko N, Maidic O, Draxier S, et al. CD20 antibody (C2B8)-induced apoptosis of lymphoma cells promotes phagocytosis by dendritic cells and cross-priming of CD8+ cytotoxic T cells. Leukemia. 2001;15:1619-1626. 35. Stockl J, Majdic O, Kohl P, Pickl WF, Menzel JE, Knapp W. Leukosialin (CD43)-major histocompatibility class I molecule interactions involved in spontaneous T cell conjugate formation. J Exp Med. 1996;184:1769-1779. 36. Moore RE, Young MK, Lee TD. Qscore: an algorithm for evaluating SEQUEST database search results. J Am Soc Mass Spectrom. 2002;13:378386. 37. Serra-Pages C, Saito H, Streuli M. Mutational analysis of proprotein processing, subunit association, and shedding of the LAR transmembrane protein tyrosine phosphatase. J Biol Chem. 1994;269:23632-23641. 38. Brown GD, Gordon S. Immune recognition. A new receptor for betaglucans. Nature. 2001;413:36-37. 39. Rogers NC, Slack EC, Edwards AD, et al. Syk-dependent cytokine induction by Dectin-1 reveals a novel pattern recognition pathway for C type lectins. Immunity. 2005;22:507-517. 40. Taylor PR, Tsoni SV, Willment JA, et al. Dectin-1 is required for betaglucan recognition and control of fungal infection. Nat Immunol. 2007;8:31-38. 41. Underhill DM. Collaboration between the innate immune receptors dectin-1, TLRs, and Nods. Immunol Rev. 2007;219:75-87.

32


42. Di Carlo FJ, Fiore JV. On the composition of zymosan. Science. 1958;127:756-757. 43. Goodridge HS, Simmons RM, Underhill DM. Dectin-1 stimulation by Candida albicans yeast or zymosan triggers NFAT activation in macrophages and dendritic cells. J Immunol. 2007;178:3107-3115. 44. Underhill DM, Rossnagle E, Lowell CA, Simmons RM. Dectin-1 activates Syk tyrosine kinase in a dynamic subset of macrophages for reactive oxygen production. Blood. 2005;106:2543-2550. 45. Lynch KW, Weiss A. A model system for activation-induced alternative splicing of CD45 pre-mRNA in T cells implicates protein kinase C and Ras. Mol Cell Biol. 2000;20:70-80. 46. Munn DH, Beall AC, Song D, Wrenn RW, Throckmorton DC. 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. 1995;181:127-136. 47. Gasparoto TH, Gaziri LC, Burger E, de Almeida RS, Felipe I. Apoptosis of phagocytic cells induced by Candida albicans and production of IL-10. FEMS Immunol Med Microbiol. 2004;42:219-224. 48. Urbanek RA, Suchard SJ, Steelman GB, et al. Potent reversible inhibitors of the protein tyrosine phosphatase CD45. J Med Chem. 2001;44:1777-1793. 49. Frangioni JV, Oda A, Smith M, Salzman EW, Neel BG. Calpaincatalyzed cleavage and subcellular relocation of protein phosphotyrosine phosphatase 1B (PTP-1B) in human platelets. Embo J. 1993;12:4843-4856. 50. Gil-Henn H, Volohonsky G, Toledano-Katchalski H, Gandre S, Elson A. Generation of novel cytoplasmic forms of protein tyrosine phosphatase epsilon by proteolytic processing and translational control. Oncogene. 2000;19:4375-4384. 51. Gil-Henn H, Volohonsky G, Elson A. Regulation of protein-tyrosine phosphatases alpha and epsilon by calpain-mediated proteolytic cleavage. J Biol Chem. 2001;276:31772-31779. 52. Sunderland CA, McMaster WR, Williams AF. Purification with monoclonal antibody of a predominant leukocyte-common antigen and glycoprotein from rat thymocytes. Eur J Immunol. 1979;9:155-159. 53. Gardella S, Andrei C, Ferrera D, et al. The nuclear protein HMGB1 is secreted by monocytes via a non-classical, vesicle-mediated secretory pathway. EMBO Rep. 2002;3:995-1001. 54. Bianchi ME. Significant (re)location: how to use chromatin and/or abundant proteins as messages of life and death. Trends Cell Biol. 2004;14:287-293. 33


55. Dumitriu IE, Baruah P, Valentinis B, et al. Release of High Mobility Group Box 1 by Dendritic Cells Controls T Cell Activation via the Receptor for Advanced Glycation End Products. J Immunol. 2005;174:7506-7515. 56. Walter J, Schnolzer M, Pyerin W, Kinzel V, Kubler D. Induced release of cell surface protein kinase yields CK1- and CK2-like enzymes in tandem. J Biol Chem. 1996;271:111-119. 57. Greer SF, Wang Y, Raman C, Justement LB. CD45 function is regulated by an acidic 19-amino acid insert in domain II that serves as a binding and phosphoacceptor site for casein kinase 2. J Immunol. 2001;166:7208-7218. 58. Kubler D. Ecto-protein kinase substrate p120 revealed as the cellsurface-expressed nucleolar phosphoprotein Nopp140: a candidate protein for extracellular Ca2+-sensing. Biochem J. 2001;360:579-587. 59. Romani L, Mencacci A, Cenci E, Del Sero G, Bistoni F, Puccetti P. An immunoregulatory role for neutrophils in CD4+ T helper subset selection in mice with candidiasis. J Immunol. 1997;158:2356-2362. 60. Mencacci A, Montagnoli C, Bacci A, et al. CD80+Gr-1+ myeloid cells inhibit development of antifungal Th1 immunity in mice with candidiasis. J Immunol. 2002;169:3180-3190. 61. Shoham S, Levitz SM. The immune response to fungal infections. Br J Haematol. 2005;129:569-582.

34

Kirchberger et al., Fig. 6

rest. T cells

B

act. T cells

mean fluorescence intensity

A

cell number

+ unlabelled ct-CD45

µg/ml

C + mAb 8-301

60000

cpm

50000 40000 30000 20000

+ CD45 PTPase inhibitor

10000 0

mean fluorescence intensity (log scale)

10 100 1000 10000

ng/ml

D

anti-CD3 mAb

anti-CD3 mAb

D2-Ig

0 D1-Ig

D2-Ig

D1 C851S-Ig

D1-Ig

ct-CD45-Ig

ICOS-L-Ig

co-Ig

mock

0

25

D1 C851S-Ig

50

50

ct-CD45-Ig

***

100

75

co-Ig

150

100

ICOS-L-Ig

Ig-FP 2 µg/mL Ig-FP 0,5 µg/mL

200

mock

**

Annexin/PI positive cells (%)

E

250

stimulation index (%)

1