Patent Application
20110189194
The present invention refers to the use of an inhibitor of the CD95/CD95L system for the prevention and/or treatment of an inflammatory disorder or for the prevention and/or treatment of an inflammatory process in a neuronal disorder, particularly in a CNS disorder.
Death of neurons and oligodendrocytes is the ultimate cause of loss of function below the lesion site in spinal injured patients. Some of these cells actively switch on a death program for their own demise, apoptosis. The CD95Ligand (CD95L; FasUAPO1-L) is one of the best characterized triggers of apoptosis and its neutralization in spinal injured mice reduced the number of cells undergoing apoptosis. The achieved rescue of neurons and oligodendrocytes resulted in increased recovery of locomotor activity in the previously paralysed limbs. Improvement of motor function upon inhibition of CD95L was also observed in rats after contusion injury of the spinal cord and in spinal injured CD95-deficient MRU/pr mice (Ackery et al., Casha et al., Yoshino et al.). CD95L is a type II transmembrane protein poorly expressed in the naïve adult spinal cord. Upon injury it can be presented by resident spinal cord cells and infiltrating leukocytes. Identifying the source of detrimental-CD95L is crucial for the design of administration protocols for CD95L-neutralizing agents to treat spinal injuries.
There is increasing evidence that CD95L can be involved in processes other than apoptosis. In the CNS, we previously reported that CD95L increases the number of branches in developing neurons and the motility of malignant astrocytes (Kleber et al., 2008; Zuliani et al., 2006). Likewise, in dorsal root ganglion cells CD95L increases axonal growth (Desbarats et al., 2003). But also in the immune system CD95L can increase T cell proliferation (Kennedy et al., 1999).
CD95 (Fas, APO-1) has long been viewed as a death-inducing receptor. Triggering of CD95 by binding of its cognate ligand (CD95L, FasL, Apo-1L) leads to recruitment of the adaptor protein FADD to its death domain (DD) via homotypic interactions. Thereafter, interaction of the death-effector domain (DED) of FADD with procaspase-8 and -10 allows their recruitment and activation within the death-inducing signaling complex (DISC). These initiator caspases can activate downstream effector caspases finally committing the cell to death with or without further involvement of the mitochondrial pathway. However, the assumption of CD95 as an exclusive mediator of apoptosis has been put to rest. In the CNS, the CD95 system has been shown to increase branching of developing cells, axonal growth of dorsal root ganglion cells (DRGs) and increased migration of malignant glioma cells (Desbarats et al., 2003; Kleber et al., 2008; Zuliani et al., 2006). Whereas, in DRGs, the CD95 system is thought to mediate axonal growth via ERK activation, in malignant glioma cells, CD95 mediates migration via activation of the Src/PI3K/MMP pathway (Desbarats et al., 2003; Kleber et al., 2008). In the immune system, activation-induced cell death (AICD) was thoroughly described in activated cycling T-cells as a CD95-dependent process (Dhein et al., 1995; Krammer, 2000). In contrast, resting T-cells seem to be resistant to CD95-mediated apoptosis (Klas et al., 1993). However, further studies also showed a role for CD95L/CD95 in T cell proliferation by inducing the production of IL-2 (Kennedy et al., 1999). Solid evidence that the CD95L can also act as a proinflammatory mediator came from studies where tissue engineered to over-express CD95L was colonized by neutrophils (Kang et al., 1997; Seino et al., 1997). However, the molecular mechanism by which CD95 induces inflammation has remained elusive.
Injury to the spinal cord elicits an inflammatory response within the first hours after injury that lasts for several weeks. This response includes endothelial damage, release of proinflammatory mediators, changes in vascular permeability, infiltration of peripheral inflammatory cells and activation of astrocytes and microglia. Infiltrating inflammatory cells can on the one side promote wound healing events but, on the other side, release toxic factors that amplify tissue damage (Jones and Tuszynski, 2002; Rolls et al., 2009). Yet, the precise signals leading to leukocyte infiltration are still unknown.
Several studies illustrated an increased expression of CD95 in the injured spinal cord (Casha et al., 2001; Li et al., 2000; Matsushita et al., 2000; Sakurai et al., 1998; Zurita et al., 2001). Inhibition of CD95 signaling prevented death of motomeurons following spinal ischemia and axotomy of the facial nerve (Ugolini et al., 2003). Importantly, neutralization of CD95L significantly reduced death of neurons and oligodendrocytes and improved functional recovery of spinal injured animals (Demjen et al., 2004). These results were further confirmed in CD95-deficient mutant mice (lpr) (Casha et al., 2005; Yoshino et al., 2004) and in rats treated with a CD95-Fc (Ackery et al., 2006). However, the actual source of CD95L and the mechanism by which the CD95/CD95L system induces damage following injury had not been addressed yet.
According to the present invention, it was found that the CD95/CD95L system is involved in increasing migration of immune cells, particularly of neutrophils and/or macrophaages. Thus, inhibition of the CD95/CD95L system might be beneficial for the prevention and/or treatment of inflammatory disorders or for the prevention and/or treatment of inflammatory processes in neuronal disorders. The present invention is particularly suitable for use in human medicine.
A first aspect of the present invention refers to the treatment of inflammatory disorders. Specific examples of inflammatory disorders are chronic inflammatory bowel disease, e.g. Morbus Crohn or colitis ulcerosa, inflammatory rheumatoid disorders associated with increased macrophage activity, e.g. rheumatoid arthritis, chronic polyarthritis, ankylosating spondylitis (Morbus Bechterew), psoriatic arthritis, juvenile idiopathic arthritis as well as collagenoses, i.e. connective tissue disorders and vasculitides, i.e. inflammatory vasculatory disorders such as lupus erythematodes, sclerodermia, Sjögren-syndrome, polymyositis and dermatomyositis, mixed collagenose and Wegener-granulomatosis (Morbus Wegener).
In this embodiment of the invention, a CD95/CD95L inhibitor may be administered in a therapeutically effective dose and by a route suitable for the treatment of the above disorders. The administration may e.g. be locally or systemically, preferably by injection or infusion or by any other suitable route.
A second aspect of the present invention refers to the treatment of inflammatory processes in neuronal disorders. Specific examples of neuronal disorders are CNS disorders, such as cerebral or spinal cord injury, e.g. cerebral lesions or partial or complete spinal core lesions, e.g. stroke, particularly paraplegia. Although the use of CD95/CD95L inhibitors for the treatment of CNS disorders is already disclosed in WO 2004/071528, the present invention differs therefrom by referring to the prevention and/or treatment of inflammatory processes in such a disorder. Since inflammatory processes in CNS disorders are associated with migration of immune cells, e.g. neutrophils, the inhibitor is administered in a therapeutically effective dose and by a route to reduce or inhibit immune cell, e.g. neutrophil and/or macrophage migration. Preferably, the inhibitor is administered immediately after occurrence of CNS injury, e.g. immediately after the occurrence of the injury, e.g. up to 2 h, 4 h, 6 h or 8 h after the occurrence of the injury. Further, it is preferred that the composition is systemically administered, thereby reducing the activity of immune cells in the whole organism to be treated.
In a preferred embodiment of the invention, the inhibitor is a CD95-ligand (Fas ligand; APO1 ligand) inhibitor. For example, CD95-ligand inhibitors may be selected from
Preferred are inhibitory anti-CD95L-antibodies and antigen-binding fragments thereof and soluble CD95R molecules or CD95L-binding portions thereof. Examples of suitable inhibitory anti-CD95L antibodies are disclosed in EP-A-0 842 948, WO 96/29350, WO 95/13293 or as well as chimeric or humanized antibodies obtained therefrom, cf. e.g. WO 98/10070. Further preferred are soluble CD95 receptor molecules, e.g. a soluble CD95 receptor molecule without transmembrane domain as described in EP-A-0 595 659 and EP-A-0 965 637 or CD95R peptides as described in WO 99/65935, which are herein incorporated by reference.
Especially preferred is a CD95L inhibitor which comprises an extracellular domain of the CD95R molecule (particularly amino acids 1 to 172 (MLG . . . SRS) of the mature CD95 sequence according to U.S. Pat. No. 5,891,434) optionally fused to a heterologous polypeptide domain, particularly a Fc immunoglobulin molecule including the hinge region e.g. from the human IgG1 molecule. Particularly preferred fusion proteins comprising an extracellular CD95 domain and a human Fc domain are described in WO 95/27735 and PCT/EP2004/003239, which are herein incorporated by reference.
Further preferred inhibitors are multimeric CD95R fusion polypeptides comprising the CD95R extracellular domain or a fragment thereof and a multimerization domain, particularly a trimerization domain, e.g. bacteriophage T4 or RB69 foldon fusion polypeptides as described in WO 2008/025516, which is herein incorporated by reference.
The Fas ligand inhibitor FLINT or DcR3 or a fragment, e.g. a soluble fragment thereof, for example the extracellular domain optionally fused to a heterologous polypeptide, particularly a Fc immunoglobulin molecule is described in WO 99/14330, WO 99/50413 or Wroblewski et al., Biochem. Pharmacol. 65, 657-667 (2003), which are herein incorporated by reference. FLINT and DcR3 are proteins which are capable of binding the CD95 ligand and LIGHT, another member of the TNF family.
In a further embodiment of the present invention, the inhibitor is a CD95R inhibitor which may be selected from
Examples of suitable inhibitory anti-CD95R-antibodies and inhibitory CD95L fragments are described in EP-A-0 842 948 and EP-A-0 862 919 which are herein incorporated by reference.
In still a further embodiment of the present invention the inhibitor is a nucleic acid effector molecule. The nucleic acid effector molecule may be selected from antisense molecules, RNAi molecules and ribozymes which are capable of inhibiting the expression of the CD95R and/or CD95L gene.
In a still further embodiment the inhibitor may be directed against the intracellular CD95R signal transduction. Examples of such inhibitors are described in WO 95/27735 e.g. an inhibitor of the interleukin 1β converting enzyme (ICE), particularly 3,4-dichloroisocoumarin, YVAD-CHO, an ICE-specific tetrapeptide, CrmA or usurpin (WO 00/03023). Further, nucleic acid effector molecules directed against ICE may be used.
In still a further embodiment, the inhibitor may be directed against a metalloproteinase (MMP), particularly against MMP-2 and/or MMP-9.
The inhibitor or a combination of the above inhibitors is administered to a subject in need thereof, particularly a human patient, in a sufficient dose for the treatment of the specific condition by suitable means. For example, the inhibitor may be formulated as a pharmaceutical composition together with pharmaceutically acceptable carriers, diluents and/or adjuvants. Therapeutic efficacy and toxicity may be determined according to standard protocols. The pharmaceutical composition may be administered systemically, e.g. intraperitoneally, or intravenously, or locally, e.g. intrathecally or by lumbar puncture.
The dose of the inhibitor administered will of course, be dependent on the subject to be treated, on the subject's weight, the type and severity of the injury, the manner of administration and the judgement of the prescribing physician. For the administration of anti-CD95R or L-antibodies or soluble CD95R proteins, e.g. CD95-Fc fusion proteins, a daily dose of 0.001 to 100 mg/kg is suitable.
Further, the present invention is explained in more detail by the following Figures and Examples.
Alignment of the C-terminal sequences of bacteriophage T4 and bacteriophage RB69 fibritin (accession numbers CAA31379 and NP-861864). Identical amino acid residues are marked.
The amino acid sequence of the CD95-RB69 fusion protein is shown. The endogenous CD95 signal-peptide is underlined, and the CD95-ECD is printed in bold letters; whereas the RB69 fibritin-Foldon sequence is printed in red letters. The linker between the CD95-ECD as well as the flexible positioned strep-tag-II is printed in blue letters. Please note, that R17 is the first amino-acid of the secreted protein (marked by an additional number 1 in bold face) and that the R87S mutation refers to this enumeration. Arginine 87 is printed in bold-face and underlined.
After affinity purification, approximately 100 μg of CD95-RB69 (A) or CD95(R87S)-RB69 (B) in a final volume of 0.1 ml were separated on a Superdex200 10-300GL column (GE Healthcare, Germany) at a flow rate of 0.5 ml/min using PBS as running buffer. The CD95-RB69 fusion proteins elute within a symmetrical, well shaped peak from the column. Based on the calibration of the SEC-column, the peaks eluting after 11.21 (A) or 10.93 ml (B) correspond to apparent molecular weights of approx. 240 and 280 kDa.
SEC fractions A1-A14 (lane numbers 1 to 14; M=marker) of the CD95-RB69 (A) or CD95(R87S)-RB69 (B) elution profile were analysed by SDS-PAGE (silver-stain), performed under reducing conditions. A major protein band running between 30-40 kDa is detected in the peak fractions; shown by an arrowhead.
Mutation of R87S abrogates the ability of the CD95-RB69 protein to inhibit CD95L-mediated killing of Jurkat cells. Jurkat cells were incubated with 250 ng/ml of human (A) or mouse (B) CD95L-T4 in the presence of wild-type and mutant CD95-RB69 in duplicates for each concentration of the fusion proteins. Decreased cell death is represented by low DEVD-AFC cleavage rates.
A In a two chamber in vitro migration assay, CD95L-T4 induced migration of neutrophils. Data are representative of at least 3 independent experiments.
B CD95L-T4 induced MMP-9 expression in neutrophils. Data are representative of at least 2 independent experiments. C MMP-2/9 inhibitor blocked CD95L-T4 induced migration of neutrophils. D CD95L-T4 induced in vitro migration of macrophages. Data are representative of 5 independent experiments. E Neutralizing antibodies to CD95L (MFL3) blocked basal migration of macrophages. Data from 2 independent experiments were pooled and represented as % of migrating cells.
CD95L activity and their respective controls (A) Annexin V staining of neutrophils in the spinal cord 24 h after injury in animals treated with CD95-RB69 or CD95-(R87S)-RB69. (B) Annexin V staining in thioglycollate-elicited neutrophils 6 h after injection in CD95Lf/f;LysMcre and respective control animals. (C) Annexin V staining in thioglycollate-elicited neutrophils 6 h after injection in animals treated with CD95-RB69 or CD95-(R87S)-RB69. Data are presented as mean±SEM.
Bone marrow neutrophils were isolated from the femur of mice by flushing the bones with PBS/2 mM EDTA. Harvested bone marrow cells were resuspended in ACK buffer (150 mM NH4Cl, 10 mM KHCO3, 1 mM Na2EDTA, pH 7.3) and incubated for 1 min to lyse erythrocytes. Neutrophil selection was performed using MACS-positive selection by magnetic beads according to the manufacturer's protocol (Miltenyi, #130-092-332). Purity of neutrophils was assessed by FACS and reached >96%.
In vivo activated neutrophils were isolated by washing the peritoneal cavity of mice 6 h after the injection of 3% thioglycollate.
1.1.2. Cell Isolation of CD11b+ Cells
Bone marrow cells were isolated as previously described. CD11b selection was performed according to the manufacturer's protocol (Miltenyi #130-092-333).
To obtain bone marrow-derived macrophages (BMDM), femurs and tibias were harvested bilaterally and marrow cores were flushed using syringes filled with PBS/2 mM EDTA. Cells were triturated and RBCs were lysed (0.15 mol/L NH4CI, 10 mmol/L KHCO3, 0.1 mmol/L Na2EDTA; pH 7.4). After washing once in media, the cells were plated and cultured in RPMI 1640 supplemented with 1% penicillin/streptomycin, 0.001% a-mercaptoethanol, 10% FBS, 1% L-glutamine, 1% non essential amino-acids, 1% sodium pyruvate and 20% supernatant from macrophage colony stimulating factor secreting L929 cells. The sL929 drives bone marrow cells towards a macrophage phenotype (7-10 days). At day 1 non-adherent cells were collected and further cultivated. 4 days later fresh medium was added to boost the cell growth. At harvest, 95±0.7% of cells were macrophages (assessed by CD11b and F480 immunostaining). Supplemented culture media was replaced with RPMI/10% FBS on the day of stimulation so that stimulations were performed in the same media for all cell types.
At least 1×107 cells were treated with 10 (neutrophils) or 20 (macrophages) ng/ml of mCD95L-T4 for 5 minutes at 37° C. or left untreated, washed twice in PBS plus phosphatase inhibitors (NaF, NaN3, pNPP, NaPPi, β-Glycerolphosphate, 10 mM each and 1 mM orthovanadate), and subsequently lysed in buffer A [(20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, protease inhibitor cocktail (Roche), 1% Triton X-100 (Serva, Heidelberg, Germany), 10% glycerol, and phosphatase inhibitors (NaF, NaN3, pNPP, NaPPi, β-Glycerolphosphate, 10 mM each and 1 mM orthovanadate)]. Protein concentration was determined using BCA kit (Pierce). 500 μg of protein was immunoprecipitated overnight with either 5 μg anti-CD95 Ab Jo2 (BD #554255) and 40 μl protein-A Sepharose or the corresponding isotype control (BD #554709). Beads were washed 5 times with 20 volumes of lysis buffer. The immunoprecipitates were mixed with 50 μl of 2× Laemmli buffer and analyzed on 15% SDS-PAGE. Subsequently, the gels were transferred to Hybond nitrocellulose membrane (Amersham Pharmacia Biotech, Freiburg, Germany), blocked with 5% milk in PBS/Tween (PBS plus 0.05% Tween 20) for 1 hour, and incubated with the primary antibody in 5% milk in PBS/Tween at 4° C. overnight. Blots were developed with a chemoluminescence method following the manufacturer's protocol (PerkinElmer Life Sciences, Rodgan, Germany). The highly CD95L sensitive thymoma cells (E20) were included as a positive control for analysing FADD recruitment (anti-FADD mouse monoclonal Ab, clone 1F7, Millipore #05-486)
Protein extraction and immunoblotting was performed as previously described. Membranes were probed with the following antibodies: phosphorylated AKT (P-Ser473-AKT, 1:1000, Cell signalling #9271), total AKT (T-AKT, 1:1000, Cell Signaling #9272).
Migration of bone marrow derived neutrophils or macrophages was assessed in vitro in a two chamber migration assay. Transwell inserts [3 μm (BD #353096) or 8 μm (BD #353097) pore size for neutrophils or macrophages respectively] were coated with matrigel (50 μg/ml; BD #354234). 5×105 neutrophils or macrophages were plated in 500 μl medium onto the upper chamber. Cells were left untreated or treated with CD95L-T4 by adding 10, 20 and 40 ng/ml to the upper chamber. The number of migrated cells was counted 3 hours for neutrophils and 24 hours for macrophages after treatment. CD95L-induced migration of macrophages was analysed by blocking basal migration of macrophages by using neutralizing antibodies to CD95L (MFL3, 10 μg; BD #555290) or the appropriate isotype control (IgG, 10 μg; BD #554709).
The role of metalloproteinases on neutrophil recruitment was investigated by using selective inhibitors of MMP-2/9. Neutrophils were pre-incubated with MMP-2/9 inhibitor (50 μM; Calbiochem #444251) 30 minutes prior to CD95L-T4 treatment and migrating cells were calculated.
MMP activity in cell-free supernatants from neutrophils treated with different doses of CD95L-T4 was determined by gelatinase zymography as described previously. In brief, neutrophils were treated with CD95L-T4 (10 and 20 ng/ml) for 6 hours. After electrophoresis and washing the gel with Triton X-100 (2.5% v/v, twice for 30 min), the gel was incubated in MMP reaction buffer [50 mmol/L Tris-HCl (pH 7.8), 200 mmol/L NaCl, 5 mmol/L CaCl2] at 37° C. for 16 h. Gelatinolytic activity was detected as transparent bands on staining with Coomassie Brilliant Blue G-250 solution and incubation in destaining solution (10% acetic acid, 20% methanol).
For the analysis of the of CD95/CD95L-interaction, the extracellular domain of CD95 is commonly used in form of recombinant dimeric fusion proteins. Currently, all commercially available recombinant CD95 proteins exhibit a C-terminally fused Fc-part of human or mouse IgG1 (CD95-Fc), e.g. as described in WO 2004/085478, which is herein incorporated by reference. To avoid Fc-based effector functions interfering with the readout strategy of this study, we therefore decided to design a CD95L-Trap based on an different protein scaffold. Due to the proposed three receptor binding sites per CD95L-trimere, a trimeric CD95-fusion protein should be the ideal CD95-ligand-trap. We used a homologue of the T4-Foldon, derived from bacteriophage RB69 (
Indeed, secretory based expression of the CD95- or CD95(R87S)-RB69-Foldon fusion proteins resulted in the formation of a glycosylated, stable protein species. (
While we were using a human CD95 fusion protein in mouse, we had to analyse the binding of the R87S based control-protein for the human CD95/murine CD95L-interaction prior to the studies performed. We addressed this question by examining the ability of the CD95-fusion proteins to neutralise the apoptosis inducing capacity of either human or mouse CD95L on Jurkat cells in vitro. Whereas the human CD95-RB69 protein efficiently neutralises the apoptotic activity of human and mouse ligand in vitro, the R87S-control protein has no protective effect (
The RB69 derived fibritin foldon domain was fused C-terminally to the human CD95-ECD (M1-E168). Between the CD95-ECD and the RB69-Foldon (Tyr181-Ala205), a flexible linker element (Gly169-Ser180) was placed. For purification and analytical strategies, a streptag-II including a flexible linker element (Ser206-Lys223) was added C-terminally. The amino acid sequence of the fusion protein was backtranslated and its codon usage optimised for expression in mammalian cells. Gene synthesis was done by ENTELECHON GmbH (Regensburg, Germany). In the case of the CD95(R87S)-RB69-protein, the necessary codon exchange in the expression cassette was introduced by PCR-based mutagenesis. The sequence-verified expression cassettes were subcloned into pCDNA4-HisMax-backbone, using unique Hind-III- and Not-I-sites of the plasmid.
Macrophage recruitment to the site of the lesion can be driven by the previously recruited neutrophils. To uncouple the possible influence of neutrophils on macrophage infiltration, we separately studied CD95L-induced migration of neutrophils and macrophages in vitro in a two-chamber transmigration assay. Migration of bone marrow-derived neutrophils significantly increased upon treatment with CD95L (
How does CD95L increase migration? In malignant glioma cells we have recently reported increased migration upon CD95L(5). In these cells the Src family kinase Yes and the p85 subunit of Phosphatidylinositol-3-Kinase (PI3K) get recruited to CD95 and activated upon CD95L. Thereafter the AKT/βcatenin pathway becomes activated leading to the final induction of MMP-9 expression. To address if PI3K is also needed in CD95-induced migration of myeloid cells, bone marrow derived neutrophils and macrophages were stimulated with CD95L and phosphorylation of the PI3K target AKT assessed. Phosphorylation and thus, activation of AKT was induced upon CD95L in both neutrophils and macrophages. As previously described for glioblastoma cells, AKT activation by CD95L in macrophages exhibited a dose-bell shape. We were unable to detect recruitment of FADD to neutrophils' or macrophages' CD95 upon treatment with CD95L, whereas treatment with CD95L of the thymoma cell line E020 efficiently recruited FADD to CD95. Further confirmation of the lack of FADD recruitment to CD95 and thus, of CD95-induced apoptosis is given by the missing differences in the rate of spontaneous apoptosis between neutrophils lacking CD95 activity and their respective controls after thioglycollate activation and SCI.
At present, the only treatment that shows a modest therapeutic benefit in spinal injured patients is the potent anti-inflammatory drug, methylprednisolone sodium succinate (MPSS). Patients treated with MPSS within the first 8 h of injury had significantly improved motor and sensory function compared to patients receiving placebo, naloxone, or MPSS at later time points (9). The required immediate use after injury indicates its major role in modulating the acute inflammatory response. Accordingly, depletion of circulating neutrophils, inhibition of neutrophil-related proteolytic enzyme activities or inhibition of neutrophils adhesion resulted in improved motor recovery of spinal cord injured mice (10). It is however noteworthy, that neutrophils also play an important role in cleaning the injury site and limiting bacterial infection. Thus therapies should aim at creating an inflammatory response devoid of devastating effects, such as CD95L-induced cell death of bystander cells (Brown and Savill, 1999), while still providing the beneficial effects. We therefore believe that a controlled modulation of the CD95L effects should provide a beneficial inflammatory response after SCI.
Animals used are described in the table below. CD95L−/− were described previously (Karray et al., 2004) and C57BL/6J mice were purchased from Charles River Laboratories. CD95L floxed mice (Karray et al., 2004) were bred with LysM Cre mice (Jackson Laboratory) and LCK Cre mice (a kind gift from Gunter Hammerling) in order to deplete CD95L in myeloid cells or T cells, respectively. Mice that ubiquitously express an enhanced green fluorescent protein were a kind gift of Bernd Arnold. For experiments animals were age-matched and used at 12-14 weeks of age. All animal experiments were performed in accordance with institutional guidelines of the German Cancer Research Center and were approved by the Regierungspräsidium Karlsruhe, Germany.
SCI models: Transection injury of the spinal cord was performed as previously described (Demjen et al., 2004). For the crush injury model, forceps were held on the spinal cord for 15 seconds resulting in a lateral compression of the spinal cord (Plemel et al., 2008). Immediately following injury and for an additional week mice received antibiotics (Gentamycin, 5 ml/kg of a 0.2 mg/ml solution) to prevent infections. Post-operative care included housing of the animals at 27° C., food and water ad libitum, and manual expression of the bladders once daily.
All experiments on human blood were performed in accordance with institutional guidelines of the German Cancer Research Center and were approved by the Ethic Commission in Mainz. Once the blood of a patient and a respective healthy control was collected, erythrocyte lysis was performed followed by fixation with 4% PFA. All the time points belonging to one patient as well as 5-6 respective control samples were stained together. For this, NOK-2 (BD, Pharmingen) or the respective IgG2% isotype (Acris) were incubated 1 hour on ice followed by 30 minutes incubation with the secondary antibody (anti-mouse APC, BD). Thereafter, samples were analyzed for CD95L expression on the surface of human neutrophils and lymphocytes by flow cytometry. Neutrophils were either identified by CD66b positive cells or by their FSC/SSC.
Mice were treated intravenously 5 minutes after SCI or induction of thioglycolate-induced peritonitis with 50 μg (solved in 200 μl sterile PBS) of either CD95-RB69 or a mutated form, CD95-(R87S)-RB69, which is unable to bind CD95L.
All behavioral tests were performed by two independent observers in a double-blind manner weekly for 9-11 weeks after injury. The general locomotor performance of the animals was assessed using the Basso Mouse locomotor rating Scale (BMS) and the swimming test, assessed as previously described (Demjen et al., 2004). For the BMS, animals were additionally tested at the first day after injury. Any mouse showing a BMS score over 0.5 at day 1 was excluded from further studies.
All statistical summary data including the sample size and results of statistical evaluations are listed in the table below. For behavioral experiments, the overall improvement in mice compared to the control group was statistically analyzed by using the Koziol test (Koziol et al., 1981), a non-parametric test appropriate for longitudinal data which allows to analyze these data combined over time. Statistical analyzes of all other endpoints was performed by using the standard unpaired Student t-test. No formal test for normality was applied in view of the small sample sizes when Student's t test was applied. All data were presented as mean±standard error of the mean (SEM). Statistical significance was reported by the p-value of the statistical test procedures and assessed, significant *p<0.05; strongly **p<0.01 and highly ***p<0.001 significant. All statistical analyses were performed with the program package ADAM of the Biostatistics Unit of the German Cancer Research Center, DKFZ.
We have previously shown that systemic neutralization of CD95L improves functional recovery of spinal injured mice by reducing the number of neurons and oligodendrocytes undergoing apoptosis (Deetjen et al., 2004). Yet, the actual source of CD95L remained elusive. CD95L is poorly expressed in the naïve adult spinal cord and it can be presented by resident spinal cord cells and/or infiltrating leukocytes. To characterize the different populations of immune cells recruited to the injured spinal cord we generated eGFP-bone marrow (BM) chimeras (
To gain mechanistic insight into the role of CD95L on myeloid cells, we studied the response of myeloid cells to CD95L. The CD95 receptor has been well established as an inducer of apoptosis (Krammer, 2000). Induction of apoptosis via CD95 occurs through the recruitment of the adaptor protein FADD to the DD of the CD95, further leading to activation of caspases. Thus, we first examined FADD association to CD95 on primary macrophages. Yet, CD95L treatment of primary macrophages did not induce a detectable recruitment of FADD to CD95, whereas the same treatment induced efficient recruitment of FADD to CD95 in the CD95-apoptosis sensitive thymoma cell line E20 (
We next addressed the molecular determinants for PI3K and SFKs activation in immune cells. As the YXXL motif in CD95 was first described in primary neutrophils , we decided to investigate potential CD95 interactors by using an SH2 array (
We next studied CD95L-induced migration of neutrophils and macrophages in vitro in a two-chamber transmigration assay (
To address if CD95L is also involved in AKT activation in peripheral myeloid cells in vivo, we first analyzed the activation status of AKT after SCI in wt and CD95L-deficient mice. Injury to the spinal cord induced AKT phosphorylation in wt but not CD95L-deficient PBCs (
To address this issue we examined the infiltration of immune cells in an animal model of peritonitis induced by an intraperitoneally injection of thioglycolate (
We have demonstrated that CD95L on peripheral myeloid cells is used to facilitate their recruitment to the site of injury/inflammation. Yet, what are the long term consequences of exclusive neutralization of CD95L-induced inflammation? To address this issue, we examined the long term clinical outcome and pathology of spinal injured animals with or without CD95L expression in the immune cell compartment in general or in the myeloid compartment. First, we generated bone marrow transplanted mice (BMT mice) from CD95L-deficient (CD95L−/−) or as a control, from wild-type (wt) donor mice and lethally irradiated wt recipient mice (BMT-CD95L−/− or BMT-wt mice, respectively) (
Second, we performed SCI in mice with exclusive deletion of CD95L in neutrophils and macrophages (CD95Lf/f;LysMcre) and their control littermates. Importantly, after transection injury, spinal cord CD95L mRNA levels were highly reduced in CD95Lf/f;LysMcre mice 24 hours after injury, further demonstrating that infiltrating myeloid cells are the major source of CD95L (
Neutralization of CD95L reduces infiltration of neutrophils and macrophages into the injured spinal cord leading to a long term recovery of the locomotor function. Thus, regulation of inflammation upon neutralization of CD95L on myeloid cells creates a controlled inflammatory response that facilitates functional recovery of spinal injured animals. In order to characterize the molecular events regulated upon neutralization of CD95L on myeloid cells, we examined the gene signature of CD95Lf/f;LysMcre mice and their littermate counterparts in the spinal cord 24 hours after transection injury. Already at this early time point, regenerative processes including organogenesis, development and neurogenesis are switched on in CD95Lf/f;LysMcre mice (
To finally assess the contribution to tissue damage of CD95L-induced inflammation vs. direct CD95L-induced apoptosis, we examined caspase activity in mice deficient of CD95 in resident neural cells (CD95f/f;NesCre) and their littermate controls (CD95f/f). The extent of caspase-3 activity did not differ between the two groups (
Our results reveal a novel mechanism by which CD95L/CD95 on myeloid cells mediates their recruitment to the inflammatory site via the Syk/AKT/MMP pathway. We show that an injury to the CNS increases expression of the CD95L/CD95 system on myeloid cells in rodents and humans. This system is also involved in the recruitment of myeloid cells to the inflamed peritoneum after thioglycolate injection. Further, we show that neutralization of CD95L reduces the initial infiltration of inflammatory cells creating an inflammatory response that facilitates recovery of locomotor function after SCI.
Until the mid 90's the dogma that apoptosis does not induce inflammation was strongly anchored in the scientific community. It was generally believed that CD95L resolves inflammation by inducing activation-induced-cell-death (AICD) of T cells (Griffith et al., 1995; Griffith et al., 1996; Nagata, 1999). Along this line, constitutive expression of CD95L by cells in the eye and testis was thought to contribute to the immune-privileged status of these organs (Griffith et al., 1995; Griffith et al., 1996). It was further suggested that constitutive CD95L expression by a variety of tumor populations would lead to immune evasion (Hahne et al., 1996; O'Connell et al., 1996; Strand et al., 1996). Regarding these findings, researchers postulated that forced expression of CD95L might effectively protect allografts from rejection. Unexpectedly, most cell types and tissues genetically engineered to express CD95L undergo destruction through neutrophils (Allison et al., 1997; Kang et al., 1997; Seino et al., 1997). This data would indicate a role for CD95L as a chemoattractant. Alternatively, it is known that CD95L is quickly removed from the surface of the cell by metalloproteinases and the released CD95L to the blood can bind to CD95 on peripheral myeloid cells and trigger their recruitment—in this case the engineered tissue. Indirect evidence for a similar role of CD95L in autoimmune disease is given by the fact that the lpr mutation ameliorates disease signs in mice with experimental autoimmune encephalomyelitis and collagen-induced arthritis (Hoang et al., 2004; Ma et al., 2004; Sabelko et al., 1997). Accordingly, in the inflamed peritoneum the recruitment of macrophages was lower in lpr animals than in their control counterpart. However, the basal lymphoproliferative disease resulting from the lpr mutation hampers the study of inflammation on this strain and can only be addressed by the conditional ablation of the CD95/CD95L on specific subsets of inflammatory cells. Here we show that exclusive deletion of is CD95L on myeloid cells ameliorates the innate inflammatory response in an animal model of peritonitis and of spinal cord injury. Accordingly, proinflammatory cytokines and chemokines such as IL-1β, IL-6, CXCL10 and CCL6 were down-regulated in the injured spinal cord of mice lacking CD95L in myeloid cells as compared to their control counterparts. Most of the proinflammatory cytokines are reported to impair axonal conduction and to amplify the inflammatory response following injury, thus further inducing tissue damage (Schnell et al., 1999; Yang et al., 2004). Consistently, neutralization of IL-6, IL-1 or CXCL10 is reported to improve functional recovery after SCI (Akuzawa et al., 2008; Gonzalez et al., 2007; Okada et al., 2004).
Former strategies to study the role of circulating neutrophils, dealing with their depletion, inhibition of neutrophil-related proteolytic enzyme activities or inhibition of neutrophil adhesion did not lead to a full ablation of neutrophilic function and resulted in improved motor recovery of spinal cord injured mice (Trivedi et al., 2006). A recent study showing full depletion of neutrophils via the Ly6/Gr1 antibody prior to SCI reports increased levels of several proinflammatory cytokines including IL-6 and a worsened clinical outcome following SCI of depleted animals (Stirling et al., 2009). Thus, it seems that a complete abrogation of neutrophils amplifies the inflammatory response. It is noteworthy, that neutrophils and macrophages not only contribute to tissue damage but also play an important role in cleaning the injury site, limiting bacterial infection and promoting wound healing. In our study, neutralization of CD95L led to a reduction without complete abrogation of infiltrating neutrophils and macrophages. Whether the dose of resulting inflammation is beneficial or rather the fact of having inflammatory cells without CD95L remains subject of future studies. At least, since mice with exclusive deletion of CD95 in neural cells were not protected from apoptosis, it seems that CD95L on infiltrating inflammatory cells does not have an additional role on direct induction of apoptosis of CD95-bearing cells.
We have previously shown that CD95L triggers invasion in a glioblastoma model via the PI3K/β-catenin/MMP pathway (Kleber et al., 2008). In primary neutrophils and macrophages, CD95 stimulation led to phosphorylation of AKT, activation of MMP-9 and, ultimately, increased migration. Pharmacological inhibition of MMP-2 and MMP-9 blocked migration triggered by CD95L, demonstrating that MMPs are crucial for CD95L-induced migration. In primary macrophages blocking of CD95L by neutralizing antibodies led to a reduced basal migration, pointing out that CD95L is needed for migration of these cells. But how does CD95 induce PI3K activation? In 1996, Atkinson and colleagues reported for the first time a physical interaction between CD95 and a non-receptor tyrosine kinase, the Src family member Fyn in T cells (Atkinson et al., 1996). They further described the presence of a highly conserved tyrosine-containing YXXL motif located in the death domain of CD95 that resembles an Immunoreceptor-Tyrosine-Activation-Motif (ITAM). Six years later, Daigle and colleagues (Daigle et al., 2002) showed that stimulation of CD95 in primary neutrophils leads to phosphorylation of this motif, thus serving as docking sites for SH2-domain containing proteins. Phosphorylation of the receptor is thought to be driven by members of the Src family of nonreceptor tyrosine kinases (SFKs: Src, Fyn, Yes, Lck, Hck and Lyn) (Atkinson et al., 1996). Once the YXXL motif is phosphorylated, other SH2-containing protein kinases or phosphatases could potentially bind and initiate activation of downstream signaling pathways. Here, we show that CD95L stimulation of CD95 on myeloid cells activates Syk, further leading to PI3K/MMP signaling. Accordingly, blocking PI3K or Syk has been shown to inhibit migration of immune cells (Ali et al., 2004; Boulven et al., 2006; Frommhold et al., 2007; Schymeinsky et al., 2007). This finding may have broader implications. Syk is known as an important activator of inflammatory responses by ITAM-coupled activated receptors, the inflammatory response mediated by proinflammatory crystals and activation of the inflammasome (Gross et al., 2009; Schymeinsky et al., 2006). Recently, Syk inhibitors have shown beneficial clinical effects in inflammatory disorders, which might at least in part, involve the CD95 receptor (Pine et al., 2007; Weinblatt et al., 2008).
While regulation of cell death is one of the best known functions of CD95, it is also capable of activating signal transduction pathways leading to the induction of proinflammatory responses (Baud and Karin, 2001). Pre-apoptotic macrophages and neutrophils can release proinflammatory cytokines, like MCP-1 and IL-8, which participate in the induction of the inflammatory response. Hohlbaum and colleagues indicated that preapoptotic peritoneal macrophages produce MIP-2, IL-16, MIP-1α, MIP-1β, followed by neutrophil extravasation (Hohlbaum et al., 2001). However, after thioglycolate activation or SCI, the number of neutrophils undergoing apoptosis was similar in mice lacking CD95L activity and their respective controls. Furthermore, resident numbers of peritoneal macrophages were not changed between mice lacking CD95L in myeloid cells and their controls. Thus, CD95L activation of the innate immune response seems to be independent of CD95L-induced apoptosis.
Do neurons and oligodendrocytes die in the injured spinal cord due to direct CD95-induced death or rather to CD95-elicited inflammation? It has been shown that neutrophils can kill bystander cells in co-culture systems through the CD95 system (Brown and Savill, 1999; Serrao et al., 2001). Further, phagocytosis triggers macrophage release of CD95L and, thus, is able to induce cell death of bystander cells. In addition, a recent study from Michael Fehlings group demonstrated that CD95L is directly able to induce death of oligodendrocytes through both intrinsic and extrinsic pathways of the CD95-mediated apoptotic signaling (Austin and Fehlings, 2008). However, all these data have been provided by in vitro studies. To correctly address this question in vivo, we specifically deleted the CD95 receptor in the CNS resident neural cells during embryonic development and assessed caspase-3 activity after SCI. Interestingly, CD95 expression in the CNS compartment does not seem to influence the apoptosis levels in the injured spinal cord. In addition comparison of the gene signature of spinal injured animals with either pharmacological, ubiquitous or exclusive inhibition of CD95L in the myeloid compartment revealed a high degree of similarity that indicates that, at least within the acute phase following SCI, the main role of CD95L is induction of inflammation. Altogether, these data suggest that CD95L rather kills neurons and oligodendrocytes through an inflammation-induced mechanism and not as previously thought through a direct apoptosis mechanism. As a consequence, neutralizing agents to CD95L do not have to be administered locally in the CNS but can be systemically applied directly after injury by paramedics. Beyond this, neutralization of CD95/CD95L system appears as a candidate therapy for inflammatory diseases in general.
To detect significant differential expression of a gene between animals with or without CD95L inhibition across studies that used different modes of action, we applied a meta-analysis approach as described by Choi and colleagues (Choi et al., 2003). For each gene in every study i, the standardized mean difference between animals with CD95L inhibition and those of the control group was calculated as an effect size di=(Xai−Xci)/Spi, where Xai and Xci represent the means of the group of animals with CD95L inhibition or of the control group, respectively, and Spi is the pooled standard deviation. A test statistic Q was used to decide whether a fixed effects model (FEM) or a random effects model (REM) is more appropriate to combine the effect sizes of the different studies. A FEM assumes that the effect sizes (here, the standardized mean differences) observed in the different studies are samples of the same distribution. A REM explicitly accounts for differences between the studies by postulating that each effect size is drawn from a distribution with study-specific parameters. Under the assumption that the differences in the effect sizes between studies is due to sampling error alone, the values for Q are distributed according to a X2 distribution. Upon inspection of the distribution of Q, it was decided that a REM would be more appropriate (data not shown).
Study-specific effect sizes were then combined in order to estimate the average effect size as described by Choi and colleagues (Choi et al., 2003). Genes were chosen by comparing the effect size estimates with a given threshold and estimating the statistical significance with the concept of false discovery rate (FDR) based on empirical null distributions generated by random permutations (Choi et al., 2003).
We purchased RPMI 1640 medium (#21875-091), penicillin/streptomycin (#15140-163), L-Glutamin (#25030024) and 55 μM 1′-Mercaptoethanol (#31350) from Invitrogen, Karlsruhe, Germany. Fetal Calf Serum (FCS, #S0115) was purchased from Biochrom, Berlin, Germany.
The following antibodies were used for flow cytometry experiments: Fitc-conjugated anti-mouse Ly6G mAb (BD #551460), PE-conjugated anti-mouse F4/80 mAb (Caltag #MF48004), PE-conjugated rat IgG2a mAB (isotype control, BD #553930), PercP-Cy5-conjugated anti-mouse CD45.2 mAb (BD #552950), Fitc-conjugated anti-mouse CD45.1 mAb (BD #553775), PeCy7- or APC-conjugated anti-mouse CD3 mAb (BD, APC #553066, PeCy7 #552774), Alexa-680- or APC-conjugated anti-mouse CD11b mAb (BD, Alexa 680 #RM2829, APC #553312), APC-conjugated anti-mouse GR-1 mAb (BD #553129), APC-Cy7-conjugated anti-mouse CD19 mAb (BD #557655), biotin-conjugated anti-mouse CD95L mAb (BD #555292), biotin-conjugated hamster IgG mAb (isotype control, BD #553970), streptavidin-APC (BD #349024, 1:50), mouse anti-human CD95L (NOK-2, BD #556375), anti-mouse APC (BD #550826), mouse IgG2K (Acris #AM03096PU-N), Fitc-conjugated anti-human CD66b (BD #555724), PE-conjugated pAKT (BD #560378) and PE-conjugated IgG (BD #554680). Unless otherwise indicated, all antibodies from BD were used at a dilution of 1:100.
Recipient mice (4-6 week old) carrying the congenic marker CD45.1 were lethally irradiated with 450 rad 2 times at 3 h intervals in order to deplete their own bone marrow (BM). Bone marrow cells (BMCs) were isolated from the femur and tibia of either male mice that ubiquitously express an enhanced green fluorescent protein or wt and CD95L−/− female mice carrying the congenic marker CD45.2. Three hours after the last irradiation, recipient mice were injected in the tail vein with 4−6×106 cells. Mice were kept in a specific pathogen-free facility and were given drinking water containing amoxicillin (1 mg/ml) to prevent infections. Eight weeks after transplantation, bone marrow reconstitution was checked by flow cytometry using antibodies against CD45.1 and 2 as well as antibodies for the different immune cell populations. Mice with lower reconstitution than 90% were excluded from further studies.
Stainings were performed on cells derived from bone marrow, peritoneum, blood or spinal cord tissue. For preparation of mouse cells derived from spinal cord tissue, the animals were perfused with HBSS to remove blood from the organs. Then the spinal cord (1 cm around the lesion site) was isolated and lysed for 3 h in thermolysin (0.5 mg/ml, Sigma #T-7902) on a shaker at 37° C. Tissue was incubated for 10 more minutes in trypsin 0.5%-EDTA (Invitrogen #25300096) and finally homogenized by passing 10 times through a Pasteur pipette and through a 40 μm cell strainer (BD #352340).
The staining was performed on this homogenized fraction.
For all stainings, cells were resuspended in FACS buffer (PBS, 0.2% NaN3) and preincubated in Fc block for 10 minutes before stained with the respective antibodies 30 minutes on ice. For intracellular stainings blood samples were fixed with 4% PFA after Ery Lysis and permeabilized with methanol before the staining. Samples were run on a FACSCantoll flow cytometer (BD) and analyzed using FACSDiva (BD) software or FlowJo software. For all FACS analyses done on cells derived from spinal cord tissue 1,000,000 events were counted.
For all tissue analysis, neutrophils were identified as CD45 positive, GR-1 high-positive and their characteristic forward (FSC) and side scatter (SSC) profile. Macrophages were identified as CD45 high-positive, CD11b positive is and F4/80 positive. In the time kinetic analysis, all immune cell types were identified by the same marker as described in this paragraph. However, hematopoietic cells in the eGFP BMT mice were GFP positive and therefore, appeared in the FITC channel without any prior antibody staining contrary to all other studies in which they were followed by CD45 positivity. T cells were identified as CD3 positive. Resident microglia are also known to express CD45 at low levels. However, we could not find any sign of cre recombination in the microglia population of the LysMcre line (data not shown), indicating that this cell population would not primarily be affected. In addition, detection of CD45 by flow cytometry enabled the distinction between CNS-resident microglia (CD45 low) and infiltrating macrophages (CD45 high).
Concerning the cells derived from the bone marrow or from the thioglycolate-induced peritonitis, we used the Ly6G mAb to characterize neutrophils.
At the described time points after surgery, animals were deeply anesthetized with an overdose of Rompun and Ketanest intra-peritoneally (i.p.) and killed by transcardial perfusion with HBSS (for RNA and protein and tissue extraction) or HBSS and 4% PFA (for immune-histochemistry and fluorescence). Depending on the experiment, 0.5 cm (caspase-3 activity assay), 1 cm (infiltration assays) or 2.5 cm segments (microarrays) around the lesion site were extracted.
For thioglycolate-induced peritonitis, 1 ml of 3% thioglycolate broth (Fluka #70157) was injected i.p. in CD95Lf/f;LysMcre and CD95Lf/f mice or in wt mice acutely treated with CD95-RB69 or its respective control. In this model, neutrophils are known to start infiltrating the peritoneum within the first hours, whereas macrophage infiltration peaks at 72 h. At the indicated times, mice were sacrificed, blood samples collected and peritoneal cavities lavaged with 10 ml sterile Hanks balanced salt solution (HBSS; Invitrogen #14170-138) containing 0.25% bovine serum albumin (Roche #10735094001). Total cell counts were performed in a Neubauer hematocytometer (Brand), and differential cell counts were carried out by flow cytometry. Results are expressed as the absolute number of neutrophils or macrophages×105/cavity. For every experiment performed, blood immune cell populations were analyzed by the appropriate cell markers.
MMP activity in cell-free supernatants from neutrophils, dHL-60 or macrophages treated with different doses of CD95L-T4 was determined by gelatinase zymography as described previously. In brief, neutrophils were treated with CD95L-T4 (10 and 20 ng/ml) for 6 h, dHL-60 with CD95L-T4 (10, 20 and 40 ng/ml) for 6 h, and macrophages with CD95L-T4 (10, 20 and 40 ng/ml) for 24 h. After electrophoresis and washing the gel with Triton X-100 (2.5% v/v, twice for 30 minutes)(Sigma #X-100), the gel was incubated in MMP reaction buffer [50 mmol/L Tris-HCl (pH 7.8), 200 mmol/L NaCl, 5 mmol/L CaCl2] at 37° C. for 16 h. Gelatinolytic activity was detected as transparent bands on staining with Coomassie Brilliant Blue G-250 solution and incubation in destaining solution (10% acetic acid, 20% methanol). Data are representative of at least 2 independent experiments.
Annexin-V staining was performed on the neutrophil population either from the peritoneal exudates or from the injured spinal cord. After gating on the neutrophil population using appropriate markers and characteristic FSC and SSC, the percentage of annexin-V positive cells was determined by using a phycoerythrin-conjugated annexin-V according to the manufacturer's protocol (Calbiochem # CBA060).
Bone marrow neutrophils were isolated from the femur of mice by flushing the bones with PBS/2 mM EDTA. Harvested bone marrow cells were resuspended in ACK buffer (150 mM NH4Cl, 10 mM KHCO3, 1 mM Na2EDTA, pH 7.3) and incubated for 1 min to lyse erythrocytes. Neutrophil selection was performed using MACS-positive selection by magnetic beads according to the manufacturer's protocol (Miltenyi, #130-092-332). Neutrophils were given in culture medium and left for 2 h until used for further experiments (RPMI 1640 supplemented with 1% penicillin/streptomycin, 0.1% 55 μM β-mercaptoethanol, 10% FCS, 1% L-glutamine, 10 mM Hepes, 1% non-essential amino-acids, 1% sodium pyruvate). Purity of neutrophils was assessed by FACS and reached >96%. In vivo activated neutrophils were isolated by washing the peritoneal cavity of mice 6 h after the injection of 3% thioglycolate.
4.10 Cell Isolation of CD11b+ Cells
Bone marrow cells were isolated as previously described. CD11b selection was performed according to the manufacturer's protocol (Miltenyi #130-092-333).
To obtain bone marrow-derived macrophages (BMDM), femurs and tibias were harvested bilaterally and marrow cores were flushed using syringes filled with PBS/2 mM EDTA. Cells were triturated and red blood cells were lysed using the ACK buffer. After washing once in media, the cells were plated and cultured in RPMI 1640 supplemented with 1% penicillin/streptomycin, 0.1% 5.5 μM β-mercaptoethanol, 10% FCS, 1% L-glutamine, 1% non essential amino-acids, 1% sodium pyruvate and 20% supernatant from macrophage colony stimulating factor secreting L929 cells (sL929; a kind gift from Dr. Tobias Haas). The sL929 drives bone marrow cells towards a macrophage phenotype (7-10 days). At day 1 non-adherent cells were collected and further cultivated. 4 days later fresh medium was added to boost the cell growth. At harvest, 95±0.7% of cells were macrophages (assessed by CD11b and F4/80 immunostaining). Supplemented culture media was replaced with RPMI/10% FCS on the day of stimulation so that stimulations were performed in the same media for all cell types.
Transfection of primary macrophages was performed at day 8 in culture with lipofectamine (Invitrogen #11668019) according to the manufacturer's protocol. Briefly, macrophages were transfected with mouse 600 μmol Syk siRNA ON-TARGETplus SMARTpool siRNA or a non-targeting SMARTpool siRNA using Lipofectamine 2000. 48 h later Syk knockdown was assessed by Western Blot. At the same time, cells were stimulated with CD95L-T4 and analysed after 24 h for migration, MMP-activity or Western blots.
4.12 Cell Culture and Transfection of dHL-60 Cells
The human myeloid HL-60 cell line (ACC 3) was kindly provided by Dr. Lucie Darner. PMN-like differentiation of HL-60 cells and the electroporation protocol was described previously. Briefly, HL-60 cells were allowed to differentiate in presence of 1.3% DMSO for 6 days before used for protein analysis. Electroporation of dHL-60 cells was performed at day 4. For electroporation, a 400 μL aliquot of dHL-60 (1×107 cells/mL) in RPMI was transferred to a Gene Pulser cuvette with an 0.4-cm electrode (Bio-Rad, Hercules, Calif.) and mixed with 600 μmol Syk siRNA ON-TARGETplus SMARTpool siRNA or non-targeting SMARTpool siRNA. Cells were incubated for 10 minutes at room temperature (RT) and subjected to an electroporation pulse of 310 V and 1175 μFF (Gene Pulser Biorad, Munich, Germany). 48 h to 72 h after electroporation, Syk knockdown was assessed by Western Blot. At the same time, cells were stimulated with CD95L-T4 and analysed after 4 h for migration.
The Transsignal SH2 Domain Array (Panomics) was performed according to the manufacturers instructions. For hybridisation of whole cell lysates, cells were harvested as described above. Lysates were then incubated with 5 μg anti-CD95 antibody Jo2—biotin and subsequently hybridised to the SH2-array membrane. After washing the array was incubated with streptavidin-HRP and developed.
Protein extraction and immunoblotting was performed as previously described. Membranes were probed with the following antibodies: phosphorylated AKT (p-Ser473-AKT, 1:1000, Cell Signaling #9271), total AKT (t-AKT, 1:1000, Cell Signaling #9272), phosphorylated Src (p-Src Tyr 416, 1:1000, Cell Signaling #2101), total Src (1:1000, Cell Signaling #2108), phosphorylated Syk (pSyk Tyr 319/352, 1:1000, Cell Signaling #2701), total Syk (1:1000, Cell Signaling #2712).
At least 1×107 cells were treated with 10 (neutrophils) or 20 (macrophages) ng/ml of mCD95L-T4 for 5 minutes at 37° C. or left untreated, washed twice in PBS plus phosphatase inhibitors (NaF, NaN3, pNPP, NaPPi, II-Glycerolphosphate, 10 mM each and 1 mM orthovanadate), and subsequently lysed in buffer A [(20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, protease inhibitor cocktail (Roche #11836145001), 1% Triton X-100 (Sigma, X-100), 10% glycerol, and phosphatase inhibitors (NaF, NaN3, pNPP, NaPPi, β-Glycerolphosphate, 10 mM each and 1 mM orthovanadate)]. Protein concentration was determined using BCA kit (Pierce #23225). 500 μg of protein was immunoprecipitated overnight with either 5 μg anti-CD95 Ab Jo2 (BD #554255) and 40 μl protein-A Sepharose (Sigma #P3391) or the corresponding isotype control (BD #554709). Beads were washed 5 times with 20 volumes of lysis buffer. The immunoprecipitates were mixed with 50 μl of 2× Laemmli buffer and analyzed on 15% SDS-PAGE. Subsequently, the gels were transferred to Hybond nitrocellulose membrane (Amersham Pharmacia Biotech #RPN203D), blocked with 5% milk in PBS/Tween (PBS plus 0.05% Tween 20) for 1 h, and incubated with the primary antibody in 5% milk in PBS/Tween at 4° C. overnight. Blots were developed with a chemoluminescence method following the manufacturer's protocol (PerkinElmer Life Sciences, Rodgan, Germany). The highly CD95L-sensitive mouse thymoma cells (E20), kindly provided by Dr. Mareike Becker, were included as a positive control for analysing FADD recruitment (anti-FADD mouse monoclonal Ab, clone 1F7, Millipore #05-486).
Biotinylated peptides including CD95-tyrosine 283 in their phosphorylated and non-phosphorylated forms as well as scramble peptides were produced by the DKFZ Peptide Synthesis facility. Briefly, 50 μM peptides were incubated with 500 μg of total protein lysates overnight at 4° to allow displacement and binding by molarity competition with endogenous protein complexes. The formed peptide-protein complexes were precipitated with 40 μl monomeric avidin beads (Thermo Scientific, #20228) for 1-2 hours at 4° and washed five times with 1 ml IP lysis buffer. After washing, beads were resuspended in 40 μl of 2× Laemmli buffer and the precipitates were analysed by SDS-PAGE and Western blotting.
To determine caspase-3 activity after SCI, the spinal cord (0.5 cm around the lesion site) was dissected and homogenized in 10 times the volume of lysis buffer (250 mM HEPES, 50 mM MgCl2, 10 mM EGTA, 5% Triton-X-100, 100 mM DTT, 10 mM AEBSF, pH 7.5). Samples were centrifuged for 10 minutes at 12,000 g. Apoptosis is paralleled by an increased activity of caspase-3. Hence, cleavage of the specific caspase substrate Ac-DEVD-AFC (Biomol) was used to determine the extent of apoptosis. Ac-DEVD-AFC can be cleaved by several caspases, however, caspase-3, -7 and -8 display by far the strongest specificity for this substrate.
For the Caspase activity assay, 20 μl cell lysate were transferred to a black 96-well microtiterplate. After the addition of 80 μl buffer containing 50 mM HEPES, 1% Sucrose, 0.1% CHAPS, 50 μM Ac-DEVD-AFC, and 25 mM DTT, pH 7.5, the plate was transferred to a Tecan Infinite F500 microtiterplate reader and the increase in fluorescence intensity was monitored (excitation wavelength 400 nm, emission wavelength 505 nm). The substrate cleavage of the samples is quantitatively determined by using an AFC standard curve. The results are expressed in pmol/min/μg protein.
Migration of bone marrow derived neutrophils or macrophages was assessed in vitro in a two chamber migration assay. Transwell inserts [3 μm (BD #353096) or 8 μm (BD #353097) pore size for neutrophils or macrophages, respectively] were coated with matrigel (50 μg/ml; BD #354234). 5×105 neutrophils, 1×106 dHL60 or 2×105 macrophages were plated in 500 μl medium onto the upper chamber. Cells were left untreated or treated with CD95L-T4 (engineered Mus musculus CD95L (Kleber et al., 2008)) by adding 10, 20 and 40 ng/ml to the upper chamber. The number of migrated cells was counted 3 h for neutrophils, 4 h for dHL-60 and 24 h for macrophages after treatment by using a hemocytometer. CD95L-induced migration of macrophages was analyzed by blocking basal migration of macrophages by using neutralizing antibodies to CD95L (MFL3, 10 μg; BD #555290) or the appropriate isotype control (IgG, 10 μg; BD #554709). Data of the migration assays are representative of at least 4 independent experiments with 6 technical replicates per condition.
The role of metalloproteinases on neutrophil and macrophage recruitment was investigated by using selective inhibitors of MMP-2/9. Neutrophils, dHL-60 and macrophages were pre-incubated with MMP-2/9 inhibitors (50 μM; Calbiochem #444251) 30 minutes prior to CD95L-T4 treatment and the number of migrated cells was counted at the times indicated previously.
Depending on the experiment, mice were transcardially perfused 9-11 weeks following SCI using HBSS and 4% paraformaldehyde (PFA). Spinal cords were dissected, post-fixed overnight at 4° C. in 4% PFA and processed for paraffin embedding. Paraffin blocks were mounted on a microtome and cut into 8-10 μm transverse sections. For immunohistochemistry, sections were permeabilized with 0.2% Triton-X 100 at RT and blocking of unspecific binding was performed using serum. After staining, slides were coverslipped with Mowiol, dried overnight at RT and stored at 4° C. until they were analyzed with an Olympus microscope. In all immunohistochemistry stainings, one slide was used as a negative control to assess non-specific binding. For neuron and oligodendrocyte labeling, slides were incubated with the primary antibody at 4° C. overnight followed by a fluorescent labeled secondary antibody (1 h at RT). Primary antibodies used were anti-NeuN (mouse, 1:200; Chemicon #MAB377) and anti-CNPase (mouse, 1:200; Sigma #C5922), respectively. Secondary antibody used was donkey anti-mouse rhodamine X (1:200; Dianova #715-296-150). To label the nuclei, Dapi (Sigma #D9564) 1:3000 was used. In order to quantify neurons, images were taken at the epicenter of injury and every 350 μm until reaching 1500 μm rostral and caudal to the epicenter and NeuN positive cells were counted in mice 10-11 weeks after SCI. The mean of NeuN positive cells per slide is presented. In order to quantify oligodendrocytes, CNPase stainings of tissue sections taken every 350 μm rostral and caudal to the lesion site were analyzed. Analysis was performed by determining the distance between the lost CNPase signal rostral and the reappearance of the CNPase staining caudal to the lesion site in the dorsal funiculus of the spinal cord. The distance indicates the level of white matter sparing in the spinal cord. A shorter distance correlates with a higher white matter sparing.
For tissue, spinal cords were dissected out and RNA was extracted with the mirVana microRNA Extraction Kit essentially according to the manufacturer's protocol (Ambion #AM1560). mRNA of injured mice were represented as normalized to the respective uninjured animals. Cells from peritoneal exudates or bone-marrow derived cells were washed with PBS and taken up in RLT-buffer containing β-mercaptoethanol. RNA was extracted using the RNeasy Mini Kit (Qiagen, #74104).
In all cases, real-time quantitative PCR was carried out using Sybr Green core kits (Eurogentec) and Uracil-N-glycosylase (Eurogentec). Primers used for quantitative real-time PCR were designed using Primer 3 software (http://fokker.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Data were analysed using the 2ΔCt method.
4.21 Microarray analysis
.Cel files were generated using Affymetrix software and imported into Chipinspector. The data were analyzed by Genomatix Chipinspector as described by the manufacturer's guidelines (Genomatix GmbH, Munich, Germany, http://www.genomatix.de). dChip software was used for hierarchical clustering of datasets (http://biosuntharvard.edu/complab/dchip/). A 5% p-value was applied as a cut-off.
Gene expression profiling was performed for 3 different datasets: (1) genetic depletion of CD95L in the myeloid cell lineage (CD95Lf/f;LysMcre) and the control littermates (CD95Lf/f) and (2) mice treated with a neutralizing agent to CD95L (CD95-RB69) and vehicle-treated animals and (3) complete deletion of CD95L (CD95L˜1 and wt control mice. For the dataset 1 selected genes of apoptosis and immune response from gene-ontology categories were clustered using hierarchical clustering and a sub-tree, showing similar gene expression pattern, was selected and shown in
A 5% p-value was applied as a cut-off.
| Number | Date | Country | Kind |
|---|---|---|---|
| 08012685.7 | Jul 2008 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2009/005127 | 7/14/2009 | WO | 00 | 4/14/2011 |
