Loss of the receptor tyrosine kinase Axl leads to enhanced inflammation in the CNS and delayed removal of myelin debris during Experimental Autoimmune Encephalomyelitis
© Weinger et al; licensee BioMed Central Ltd. 2011
Received: 22 December 2010
Accepted: 15 May 2011
Published: 15 May 2011
Axl, together with Tyro3 and Mer, constitute the TAM family of receptor tyrosine kinases. In the nervous system, Axl and its ligand Growth-arrest-specific protein 6 (Gas6) are expressed on multiple cell types. Axl functions in dampening the immune response, regulating cytokine secretion, clearing apoptotic cells and debris, and maintaining cell survival. Axl is upregulated in various disease states, such as in the cuprizone toxicity-induced model of demyelination and in multiple sclerosis (MS) lesions, suggesting that it plays a role in disease pathogenesis. To test for this, we studied the susceptibility of Axl-/- mice to experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis.
WT and Axl-/- mice were immunized with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide emulsified in complete Freund's adjuvant and injected with pertussis toxin on day 0 and day 2. Mice were monitored daily for clinical signs of disease and analyzed for pathology during the acute phase of disease. Immunological responses were monitored by flow cytometry, cytokine analysis and proliferation assays.
Axl-/- mice had a significantly more severe acute phase of EAE than WT mice. Axl-/- mice had more spinal cord lesions with larger inflammatory cuffs, more demyelination, and more axonal damage than WT mice during EAE. Strikingly, lesions in Axl-/- mice had more intense Oil-Red-O staining indicative of inefficient clearance of myelin debris. Fewer activated microglia/macrophages (Iba1+) were found in and/or surrounding lesions in Axl-/- mice relative to WT mice. In contrast, no significant differences were noted in immune cell responses between naïve and sensitized animals.
These data show that Axl alleviates EAE disease progression and suggests that in EAE Axl functions in the recruitment of microglia/macrophages and in the clearance of debris following demyelination. In addition, these data provide further support that administration of the Axl ligand Gas6 could be therapeutic for immune-mediated demyelinating diseases.
Axl, a member of the TAM family of receptor tyrosine kinases, has been shown to function in dampening the immune response, regulating cytokine secretion, clearing apoptotic cells and debris, and maintaining cell survival [1–5]. In the nervous system, Axl is widely expressed on microglia, oligodendrocytes, and neurons [4, 6–9], and its ligand Growth-arrest-specific protein 6 (Gas6) is expressed on neurons, endothelial cells, astrocytes, and in the CSF [5, 10–13]. Although Axl and Gas6 are highly expressed during development, Axl is expressed at low levels in the adult central nervous system (CNS), but is upregulated in disease states, such as in the cuprizone toxicity-induced model of demyelination and in multiple sclerosis (MS) lesions [5, 11, 14, 15].
In vitro, Axl signaling can reduce expression of the pro-inflammatory cytokine TNF⍺ by upregulating TWIST, resulting in TWIST binding the E-box of TNF⍺, thereby blocking NFκB-dependent transcription of TNF⍺ [16, 17]. Several cytokines and chemokines including TNF⍺, and the potent immune cell chemoattractant chemokines MCP1 (CCL2) and RANTES (CCL5) are upregulated in EAE and MS lesions, and contribute to inflammation and demyelination [18–24].
It is well established that inflammation in the CNS can block remyelination but clearance of debris by phagocytosis can allow for efficient remyelination (for review see ). Microglia, the resident CNS immune cells of monocyte lineage, are capable of phagocytosis, as well as secreting trophic factors that help repair damage [26–28]. Clearance of apoptotic cells and debris is a critical pathologic response to inflammation. If inflammation is left unchecked it can lead to tissue necrosis and further apoptosis and inflammation. Under pathologic situations, microglia become activated, migrate to the site of injury, surround damaged and dead cells, and clear tissue debris from the area . Axl signaling has a role in phagocytosis; in vitro studies have shown that deletion of Axl reduced phagocytosis by fifty percent . Thus, upregulation of Axl in MS lesions may reflect an attempt to protect resident CNS cells from apoptosis, to dampen inappropriate activation of the immune response, and to aid in clearing myelin debris .
In this study, we sought to characterize the role of Axl in the CNS following MOG35-55-induced EAE, an autoimmune disease of the CNS that shares several clinical and pathologic features with MS, including breakdown of the blood brain barrier (BBB), perivascular inflammation, demyelination, and axonal degeneration. We studied the acute phase of disease in EAE-induced WT and Axl-/- mice. We speculated that Axl is important in minimizing an immune-mediated CNS insult by dampening the inflammatory response, modulating microglia activation, and protecting against axonal damage and demyelination.
Axl-/- and Tyro3-/- mice were obtained from Dr. Greg Lemke at the Salk Institute and further backcrossed more than six generations onto C57/Bl6J mice (Jackson ImmunoResearch Laboratories, Bar Harbor, ME) in our select pathogen-free barrier facility. C57/Bl6J WT littermates were used as controls. All experiments were performed with age and sex-matched mice at 8 weeks of age. All protocols followed internationally recognized guidelines and were approved by the Animal Care and Use Committee at the Albert Einstein College of Medicine, Protocol Number 20080308; NIH OLAW/NIH Assurance number is A3312-01.
Mice were immunized with MOG peptide35-55 (3 mg/ml; Celtek Bioscience, Franklin, TN) emulsified in an equal volume of CFA composed of mycobacterium tuberculosis (10 mg/ml; Difco Laboratories, Detroit, MI) in incomplete Freund's adjuvant (Difco Laboratories). Mice were anaesthetized with isoflurane and 100 μl of emulsion was injected subcutaneously on each flank (200 μl total/mouse) on day 0. In addition, 200 μl of pertussis toxin (2.5 μg/ml; List Biological Laboratories, Campbell, CA) was injected into the tail vein on days 0 and 2. In our colony, this regimen produced a milder form of EAE than has been reported in the literature . Mice were monitored and graded daily for clinical signs of disease as follows: 0, no disease; 1, limp tail; 2, limp tail and hind limb weakness; 3, hind limb paralysis; 4, hind limb and front limb paralysis; 5, moribund. Mice that were considered borderline between scores were given a half score; 0.5, 1.5, 2.5, 3.5. Data analyses shown in all figures were performed during the acute phase of disease, days 14-19.
Spinal cord dissection and tissue preparation
Mice were anaesthetized with ethyl ether (Fisher Scientific, Pittsburgh, PA) and sacrificed by total body perfusion with 4% paraformaldehyde (Fisher Scientific), Trump's Fixative (4% gluteraldehyde (Polysciences Inc, Warrington, PA) + 2% paraformaldehyde), or ice cold 1× PBS, pH 7.3. Spinal cords were removed and dissected into cervical, thoracic, and lumbar regions. Trump fixed tissue was embedded in epon and one micron thick sections were stained with toluidine blue. For immunohistochemistry, paraformaldehyde fixed tissue was immersed in 30% sucrose overnight at 4°C prior to embedding in paraffin or OCT. Representative images shown are WT CI = 1.5 and Axl-/- CI = 2.5, equivalent to the average CI for each genotype at the peak of acute phase of disease. For protein or RNA isolation, the spinal cord was flushed out using an 18 gauge needle and 10 cc syringe with ice cold 1× PBS. For total protein homogenates, PBS-perfused tissue was sonicated on ice with a tissue master 125 sonicator (Omni International, Marietta, GA) in protein buffer (140 mM NaCl, 1 mM Tris, pH 7.4) containing 0.5% Triton X-100 and protease inhibitors [2 μg/mL leupeptin; 2 mM ethylene glycol-bis(β-aminoethylether)-N,N,N',N'-tetraacetic acid; 4 μg/mL pepstatin; 5 mM sodium pyrophosphate; 30 mM β-glycerophosphate; 30 mM sodium fluoride; 100 mM sodium orthovanadate; 100 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride]. The homogenates were cleared by centrifugation at 4°C at 7000 g for 10 min and aliquots were frozen at -80°C. For RNA isolation, lumbar sections of spinal cord were sonicated in Qiazol and isolated with an RNeasy Lipid Tissue Mini Kit (Qiagen Inc, Valencia, CA). The RNA samples were quantified on a nanodrop ND-1000 spectrophotometer (Thermo Scientific, West Palm Beach, FL) and aliquoted at 50 ng/μl.
Western blot analysis
Eighty μg of protein was loaded in 1× final concentration loading buffer containing 2% SDS, 0.017% bromophenol blue dye, and 0.28 M β-mercaptoethanol (loading dye), and separated in a 10% SDS-PAGE . Following electrophoresis, proteins were transferred to nitrocellulose , and incubated with 5% non-fat dry milk and 5% goat serum in 1× Tris buffered saline pH 7.4 (1× TBS) for 1 hr at room temperature . After blocking, membranes were incubated with respective primary antibodies followed by HRP-conjugated secondary antibodies. Primary monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) included: β-actin mAb (IgG2A, 1:5,000; Sigma) and Axl pAb (1:1000). Secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) included: goat ⍺-rabbit IgG (1:10,000), and goat ⍺-mouse IgG2A (1:10,000). Visualization of all secondary antibodies was by electrochemiluminescence (ECL) (GE Healthcare, Piscataway, NJ). Relative densitometric intensity (rdi) was determined for each band as previously described .
Ex vivo proliferation assay
Axl-/- and WT mice were sensitized with MOG35-55 as described above and lymphocytes were harvested from draining lymph nodes on day 16. Single cell suspensions were prepared and cultured in vitro with 20 μg/ml MOG35-55 peptide. After 3 days, 3H-thymidine was added to the cultures for an additional 48 h and assayed for thymidine uptake using a cell harvester .
The Taqman RNA-to-CT 1-Step kit (Applied Biosystems, Foster City, CA) was used with a total of 100 ng of RNA per reaction with TNF⍺, MCP1, RANTES, and GAPDH primers (Applied Biosystems). All results were normalized to GAPDH as an internal control. The reactions were aliquoted in triplicate in an optical 96 well-plate; RNase-free water was used as a blank. Reactions were run in the StepOnePlus real-time PCR machine (Applied Biosystems) with the following cycles: hold stage, 48°C for 15 min for reverse transcription, 95°C for 10 min to activate the DNA polymerase, followed by 40 cycles at 95°C for 15 seconds, and 60°C for 1 min. The real-time quantification was monitored directly by the StepOnePlus software and the comparative thresholds (CT) were identified for each gene with each RNA sample and calculated at the end of the measurements. After normalization of the (CT), the fold change was determined as fold change = 2-(ΔΔCT) where ΔΔCT is defined as the normalized change in CT between two groups. Statistics were performed on the normalized CT values.
Paraffin-embedded and frozen sections were prepared from the paraformaldehyde fixed tissue. Seven micron paraffin-embedded sections were deparaffinized, rehydrated and then incubated for 30 min with 1× TBS containing 0.25% Triton X-100 (0.01% Triton X-100 for Axl pAb) plus 3% hydrogen peroxide, followed by a 1 hr incubation in 5% goat serum and 5% nonfat dry milk in 1× TBS, and incubated with antibodies diluted in 5% nonfat dry milk in 1× TBS, overnight at 4°C, with the exception of CD45 and CD3, which were not incubated in 0.25% Triton X-100 plus 3% hydrogen peroxide. Primary antibodies consisted of mAb to MBP (SMI99; 1:1500; Covance, Inc, Emeryville, CA), mAb CD45 (1:20; BD Biosciences, San Jose, CA), or mAb Neurofilament H nonphosphorylated (SMI32; 1:30,000; Covance). Ten micron frozen sections were stained with the mAb MAC3 (1:20; BD Pharmingen), pAb Iba1 (1:400; Wako Chemicals, Richmond, VA) or pAb Axl (1:200; Amgen, Thousand Oaks, CA). Control staining consisted of omission of the primary antibody or for a polyclonal antibody control preimmune serum was used. Sections were washed 3× in 1× TBS and incubated with mouse monoclonal or rabbit polyclonal secondary antibodies followed by incubation with the appropriate Vecta staining kit (Vector Laboratories, Inc., Burlingame, CA) and visualized by 0.3 mg/ml DAB (Sigma) in 0.1 M TRIS pH 7.4 plus 0.009% hydrogen peroxide. Quantification was determined using an objective stage micrometer with a reticule (Nikon, Melville, NY). Quantification of CD45 by integrated density was performed in Image J and statistical analysis was evaluated in Prism.
Ten micron thick frozen sections were prepared from spinal cords from 4% paraformaldehyde perfused mice. Sections placed on Excell plus slides (Richard-Allan Scientific, Kalamazoo, MI) were dried by placing them at 37°C for 2 hr, or overnight at room temperature. Sections were washed in 1× TBS and blocked with 5% milk in 1× TBS plus 5% goat serum for 1 hr at room temperature. Double-label immunofluorescence was performed sequentially. Between all primary and secondary antibody incubation steps sections were washed 3× with 1× TBS. Primary antibody: CD3 hamster mAb (1:200, Novus Biological, Littleton, CO) overnight at 4°C, secondary antibody: goat anti-hamster Alexa-568 (1:1000, Invitrogen, Carlsbad, CA) for 1 hr at room temperature, followed by Iba1 pAb (1:400, Wako Chemicals) overnight at 4°C, secondary antibody: goat anti-rabbit Alexa-633 (1:1000, Invitrogen) for 1 hr at room temperature. All antibodies were diluted in 5% milk in 1× TBS. Hoechst stain (1:1000; Sigma), 10 min at room temperature, was used as a counterstain for nuclei. Sections were mounted with Aquamount (Biomeda, Foster City, CA). All samples were examined under an Olympus (Melville, NY) 1 × 70 with a 20× numerical aperture 1.0 or a 40× numerical aperture 1.0 Plan Apo optics inverted microscope with a Photometrics (Roper Scientific, Tucson, AZ) Censys-cooled CCD camera; images were collected with IPLab Spectrum software (Scanalytics, Fairfax, VA).
Frozen sections were incubated in distilled water for 1 min followed by a 2 min incubation in 100% propylene glycol (Polyscientific, Bayshore, NY) whereupon the sections were transferred to Oil-Red-O for 36 h at room temperature. Sections were incubated for 1 min in 85% propylene glycol, rinsed briefly with distilled water, lightly stained with hematoxylin and mounted with gelatin mounting medium.
Serum Protein Analysis
Individual serum samples from naïve wild-type (n = 6) and Axl-/- (n = 5) mice, as well as MOG-peptide induced EAE wild-type (n = 5) and Axl-/- (n = 9) mice, underwent quantitative proteomic analysis for 60 chemokines, growth factors and cytokines as previously described (Rules Based Medicine, Inc., Austin, TX) . Briefly, sera were analyzed in a blinded fashion at a previously optimized dilution in a Luminex-based platform. Each antigen assay was calibrated with standard curves, performed in duplicate, and intensity measurements were interpreted as protein concentrations within Rules Based Medicine's proprietary software.
CNS Preparation for Flow Cytometry and Analysis
All experimental data was acquired on an LSR II flow cytometer (BD Biosciences). Data analysis was performed using FlowJo software (Tree Star, Ashland, OR). Following dissection of brain and spinal cord, the organs were injected with 2 ml of RPMI1640 containing 0.3 Wünsch Units/ml Liberase TL (Roche Molecular Biochemicals, Indianapolis, IN) and 10 μg/ml DNAse I (Sigma, St. Louis, MO) and incubated at 37°C for 30 minutes. The organs were then mashed through a 70 micron cell strainer, washed twice with RPMI and centrifuged at 400 × g for 10 min at 4°C. The CNS pellets were then resuspended in 30% percoll and overlayed onto 70% percoll, followed by centrifugation at 600 × g for 25 min at room temperature. Cells at the interface were collected as the CNS mononuclear cell fraction, and analyzed by flow cytometry. Briefly, cells were resuspended in flow cytometry buffer (PBS + 2% Fetal Calf Serum + 0.05% NaN3) and blocked with rat anti-mouse FcγII/III (clone 2.4G2) and stained with a cocktail of the following antibodies; CD4 (clone GK1.5), CD8 (clone 53-6.7), CD3e (clone 145-2C11), CD11b (clone M1/70), CD11c (N418), Ly6G (clone 1A8) (BD Biosciences), CD45 (clone 30-F11) and NKp46 (clone 29A1.4) (eBioscience, San Diego, CA) for the evaluation of CNS infiltrating leukocyte populations. For regulatory T cell (T regs) analysis, CNS preparations were stained with BluVID, blocked with 2.4G2 and surface stained with anti-CD3, anti-CD4, and anti-CD25 (clone PC-61; BD Biosciences). Cells were then prepared for intracellular staining using a FoxP3 staining kit (eBioscience) according to the manufacturer's instructions and stained with anti-FoxP3 (clone FKJ-16S) (eBioscience).
Intracellular Cytokine Staining
For analysis of cytokine producing CD4+ T cells subsets, CNS mononuclear cells were prepared from WT and Axl-/- mice 2 weeks following MOG induction of EAE and re-stimulated with 10 μg/ml MOG35-55 peptide plus 1 μg/ml soluble anti-CD28 mAb (eBioscience). After incubation (37°C, 5% CO2) for 1 h, 10 μg/ml brefeldin A and 2 μM monensin (Sigma-Aldrich) was added for an additional 4 h. Cells were labeled with BluVID, blocked with mAb 2.4G2, and stained with antibodies against CD3e, CD4, CD11b, and CD45R/B220 (clone RA3-6B2) (BD Biosciences). Cells were fixed with 2% paraformaldehyde, washed with permeabilization buffer (1× PBS with 1 mM Ca2+, 1 mM Mg2+, 1 mM HEPES, 2% FCS, and 0.1% saponin), and then blocked in permeabilization buffer plus 5% normal mouse serum (Jackson ImmunoResearch Laboratories). Intracellular cytokines were detected with antibodies against IL-2 (clone JES6-5H4), IL-17A (clone eBio17B7) (eBioscience), IFN-γ (clone XMG1.2), and TNF⍺ (MP6-XT22) (BD Biosciences) .
Quantitative Procedures and Statistical Analysis
A student's t-test was performed for clinical indices of WT and Axl-/- mice derived from 3 independent experiments. Inflammatory cuffs were quantified using a minimum of 4 sections per cord from 3 WT and 4 Axl-/- mice. For toluidine-blue stained sections, lesions were measured (mm2) using a reticule and a micrometer bar inserted into the ocular of a Nikon microscope. Statistical analysis (student t-test) was performed using mice from the same experiment and time point. Iba1+ activated microglia were counted in and surrounding 10 lesions in multiple sections from WT and Axl-/- mice. SMI32+ neurofilaments were counted in the ventral and dorsal funiculi of 3 sections per mouse for 3 WT and 3 Axl-/- mice. Statistical analysis was obtained by a student's t-test.
Clinical expression of EAE in Axl-/- mice
Concurrent experiments using TAM family member Tyro3 knockout mice (Tyro3-/-) showed no significant difference in clinical scores between WT and Tyro3-/- mice during the acute or chronic phases of disease (data not shown). Studies in other receptor tyrosine kinase families have shown that related receptor tyrosine kinases may be upregulated to compensate for the loss of another receptor . Thus, we examined whether the lack of increased disease severity in Tyro3-/- was a result of compensation by an upregulation of the Axl receptor, using Western blot analysis of lumbar spinal cord from naïve and MOG-sensitized WT or Tyro3-/- mice (Figure 1b,c). In naïve WT and Tyro3-/- mice, there was no difference in the expression of either the immature (110 kDa) or mature form of Axl, which migrates at 140 kDa . In WT EAE mice, the 140 kDa Axl was increased 2.7-fold over naïve WT mice. However, the 140 kDa form of Axl was increased 8-fold in Tyro3-/- EAE mice over WT EAE mice (Figure 1c, p = 0.02). Furthermore, there was a visible decrease in the amount of the smaller immature form of Axl in spinal cord homogenates from Tyro3-/- EAE mice, presumably because it was converted to the mature 140 kDa form. From these data, we conclude that activation of Axl occurs in the CNS of animals with EAE and that upregulation of Axl in Tyro3-/- mice likely compensates for loss of this TAM family receptor.
Analysis of peripheral immunological parameters in Axl-/- mice sensitized for EAE
Although Axl is not expressed on T cells, to verify that the loss of Axl did not affect the peripheral immune response to antigen, cytokine profiles of sera from naïve and sensitized mice were examined, using an automated assay (see methods). No significant difference in values for 60 different factors (see methods) was detected in naïve or EAE-sensitized WT or Axl-/- mouse sera. In both WT and Axl-/- mice sensitized for EAE, increased expression over that found in naïve mice was noted for KC/Gro⍺, MCP-1, MMP-3, MIP1γ, MIP1⍺, M1P1β, MIP2, MIP3β, IL-4, IL-6, IL-7, IL-11, VEGF, LIF, OSM, and TNF⍺. To explore the possibility that there was differential activation of peripheral lymphocytes in the Axl-/- mice during EAE lymphocytes were isolated from draining lymph nodes on day 16 post-MOG injection. Following activation with 20 μg/ml MOG, the fold-increase for proliferating lymphocytes was 3.9 and 4.1 for WT and Axl-/- mice, respectively (data not shown). Thus, there was no indication that the loss of Axl affected the peripheral immune response to MOG in these mice.
Analysis of immunological parameters in the CNS of Axl-/- mice sensitized for EAE
Number and cellular-makeup of inflammatory infiltrates consistent between Axl-/- and WT mice
Pro-inflammatory cytokines are upregulated in Axl-/- mice during EAE
Pathological analysis of the spinal cord in mice with EAE
Distribution of infiltrating lymphocytes is altered in Axl-/- mice
Fewer microglia/macrophages are present at lesions in Axl-/- mice
Axl-/- mice have more myelin debris than WT mice
There is more axonal damage in Axl-/- mice
Summary of clinical and pathological data for the acute phase of EAE in WT and Axl-/- mice
Average size of lesions (p < 0.01)
0.13 ± 0.03
0.23 ± 0.02
Iba1+ microglia/macrophages per lesion (p < 0.01)
50.3 ± 4.7
24.2 ± 1.6
Ratio of CD3+ to Iba1+ cells per lesion (p < 0.01)
1.3 ± 0.07
3.7 ± 0.26
Extent of Oil-Red-O+ debris per lesion (p < 0.01)
Mean number SMI32+ axons
Ventral lumbar spinal cord (p < 0.05)
27.7 ± 6.3
90.1 ± 12.6
Dorsal lumbar spinal cord (p < 0.01)
3.9 ± 0.9
123.6 ± 22.6
EAE is an immune-mediated inflammatory disease of the CNS that results in myelin and CNS cell loss, and axonal damage. In this study, we used the MOG-induced mouse model of EAE to determine the contribution of Axl to inflammation in the CNS, including assessment of the role of Axl in cytokine/chemokine expression, microglia/macrophage migration and the efficiency with which these cells clear tissue debris. Based on the multiple functions of Axl pertinent to protection against EAE, results from our previous cuprizone study , and current data that showed Axl is upregulated in EAE, we predicted that Axl-/- mice would have a more severe clinical disease course and more severe pathology than WT mice. Consistent with this prediction, the results showed that although the day of disease onset was the same for Axl-/- and WT mice, Axl-/- mice had higher clinical indices at 14 days post-MOG-injection and that increased disease severity persisted in Axl-/- mice throughout the acute phase. In contrast to Axl-/- mice, the clinical course of MOG-induced EAE in Tyro3-/- and WT mice was the same, perhaps reflecting the upregulation of the mature 140 kDa form of Axl that compensated for the lack of Tyro3.
The acute phase of the disease is attributed to breakdown of the BBB, edema, and influx of inflammatory cells, resulting in myelin loss, and axonal damage in the spinal cord. The detection of higher clinical scores in the Axl-/- mice predicted there would be more damage in the spinal cord of Axl-/- mice than WT mice. As predicted, relative to WT mice the loss of Axl resulted in larger inflammatory cuffs comprised of CD45 and CD3 stained leukocytes but fewer lesion-associated Iba1+ microglia/macrophages. Spinal cord lesions in Axl-/- mice also displayed increased loss of myelin, increased accumulation of myelin debris, and significantly more SMI32+ axons. This increased axonal damage likely reflects increased loss of myelin, which renders axons more vulnerable to damage and axonal die-back [40, 41].
The severity of disease is mediated at least in part by the increase in pro-inflammatory cytokines and chemokines. For example, an increase in TNFa can lead to increased vascular permeability and expression of chemoattractant cytokines, such as MCP1 and RANTES, which together could result in greater tissue damage in the Axl-/- spinal cord [42–44]. Previous studies have shown that RANTES is activated at the onset of EAE and that expression correlates with leukocyte accumulation in the CNS . Activated astrocytes and cells of monocyte/macrophage lineage secrete MCP1, which binds to its ligand CCR2 to recruit other monocytes/macrophages to areas of damage. Naïve mice had very low levels of TNF⍺, MCP1, and RANTES mRNA and there was no difference in the lumbar spinal cord between Axl-/- and WT mice. However, during EAE, mRNA for these cytokines and chemokines were upregulated in the spinal cord with levels corresponding to the severity of disease. Flow cytometric analysis determined there was no difference in TNF⍺ expression by infiltrating T cells in the brain and spinal cord between Axl-/- and WT mice. Therefore, the observed differences in TNF⍺ mRNA in the lumbar spinal cord can likely be attributed to macrophages and/or resident CNS cells. Differences in cytokine expression in Axl-/- mice versus WT mice were most obvious in mice with lower CI's. At CI's ≤ 2 there was significantly more mRNA for TNF⍺ and RANTES in the spinal cord of Axl-/- mice. At CI's > 2 no difference in the ratio of TNF⍺, MCP1, and RANTES transcripts was detected suggesting that the cell machinery was maximally producing these transcripts. Despite the increase in MCP1 transcripts in Axl-/- spinal cord during EAE, Iba1+ microglia/macrophages were under-represented in lesions relative to spinal cord lesions of WT mice. This may reflect a role for Axl in cell migration.
The most striking difference in spinal cord pathology between Axl-/- mice and WT mice was noted in the distribution of activated microglia associated with lesions and the lack of clearance of Oil-Red-O+ debris. Normally, resting microglia are evenly distributed throughout the gray and white matter of the spinal cord where they are thought to function as monitors of tissue damage and the influx of foreign material . Once damage or a foreign antigen is detected microglia become activated and migrate to affected areas of the CNS . Several studies have implicated a role for Axl signaling in cell migration. In studies of gliomas, dominant-negative Axl lacking the intracellular domain interfered with cell migration , and in GN11 cells Gas6 signaling downstream of Axl was implicated in cell migration . We observed that although there was no difference in the number of microglia in the brain and spinal cord of mice with EAE, Axl-/- mice failed to display equivalent numbers of activated microglia/macrophages surrounding lesioned areas of the spinal cord when compared with WT counterparts. Furthermore, the microglia/macrophages recruited to lesioned areas of the cord in the WT mice appeared to form concentric rings around the lesions, suggesting that these cells acted to cordon off the damaged tissue. Failure to do this in the Axl-/- mice could account for the increase in lesion size noted in these mice. Failure to efficiently recruit phagocytic microglia has also been implicated in the reduced capacity of older animals to effectively remyelinate (reviewed in [25, 50]). The additional Oil-Red-O+ debris associated with lesions in Axl-/- relative to lesions in WT mice is indicative of the persistence of myelin debris not cleared by Iba1+ or Mac3+ inflammatory cells. Several possibilities might account for the persistent Oil-Red-O+ debris including the increased clinical severity and higher scores observed in the Axl-/- mice resulting in extensive demyelination, the inability of the Axl-/- microglia/macrophages to efficiently migrate to the site of injury and/or inefficient uptake of the Oil-Red-O+ myelin debris by the inflammatory cells. In collaboration with the laboratory of Dr. Dianne Cox at Albert Einstein College of Medicine, in vitro uptake assays were performed to determine whether macrophages isolated from WT and Axl-/- mice efficiently engulfed debris. Qualitatively, there was no difference in FcgR-mediated or CR3-mediated phagocytosis as determined by equivalent uptake of IgG-coated erythrocytes or zymosan particles, respectively, in WT and Axl-/- macrophages (data not shown) indicating that macrophages from Axl-/-mice were capable of efficient engulfment.
In most animal models of CNS demyelination, elimination of myelin debris is a necessary prerequisite for remyelination and repair. In young animals this is accomplished efficiently by phagocytic macrophages and microglia (reviewed in ). Axl, Tyro3 and Mer have all been shown to be required for efficient phagocytosis of apoptotic cells . Also, it is possible for myelin debris to persist in the lesion area as shown by extracytoplasmic Oil-Red-O staining observed in pathologic and necrotic tissue, a condition present in EAE lesions [51–53]. Thus, increased Oil-Red-O+ debris in Axl-/- mice during EAE likely reflects a failure of Iba1+ and/or MAC3+ inflammatory cells to efficiently migrate to the injury site in order to clear debris, leading to impaired remyelination and tissue damage.
Taken together, these data support the conclusion that exacerbation of acute EAE in Axl-/- mice reflects the role of Axl in regulating the activity of cells of the monocyte/macrophage series, whilst having little effect on the activity of lymphocytes. This would be consistent with the known distribution of Axl, and other TAM receptors, which are constitutively but variably expressed by all subpopulations of monocytes/macrophages, but are not expressed by most mature lymphocytes (reviewed in ). The data show that Axl is important for several aspects of tissue homeostasis in the spinal cord of mice with EAE including migration of macrophages/microglia to sites of tissue damage, the efficient walling off of the lesion and the clearance of tissue debris that in turn likely limits the extent of damage. These findings have significant relevance for our understanding of the pathogenesis of MS. Our previous work has shown that Axl and a deleterious soluble form of Axl are upregulated in MS lesions, and that the soluble form of Axl negatively correlates with expression of the TAM receptor ligand Gas6 . The data further suggest that increasing the availability of Gas6 in MS lesions may lead to enhanced Axl activation resulting in the arrest of lesion enlargement thus favoring remyelination and repair of tissue damage.
This study was funded by the National Multiple Sclerosis Society (NMSS) Research Grants RG3020 (BSZ) and RG4046 (BSZ), National Institutes of Health (NIH) grant NINDS 1R21NS061128-01A1 (BSZ), the Training Program in Cellular and Molecular Biology and Genetics grant, NIH NIGMS GM07491 (JGW). We thank Dr. Phyllis Novikoff for her help with the Oil-Red-O staining. We thank Dr. Greg Lemke and Dr. Qingxian Lu at the Salk Institute for Biological Studies for receipt of the Axl-/- and Tyro3-/- mice. We are grateful to Dr. Laura Santambrogio at Albert Einstein College of Medicine for her constructive comments and stimulating discussions. We thank Christina Liu at Albert Einstein College of Medicine for help with qRT-PCR.
- Camenisch TD, Koller BH, Earp HS, Matsushima GK: A novel receptor tyrosine kinase, Mer, inhibits TNF-alpha production and lipopolysaccharide-induced endotoxic shock. J Immunol. 1999, 162: 3498-3503.PubMed
- Lu Q, Lemke G: Homeostatic regulation of the immune system by receptor tyrosine kinases of the Tyro 3 family. Science. 2001, 293: 306-311. 10.1126/science.1061663.View ArticlePubMed
- Scott RS, McMahon EJ, Pop SM, Reap EA, Caricchio R, Cohen PL, Earp HS, Matsushima GK: Phagocytosis and clearance of apoptotic cells is mediated by MER. Nature. 2001, 411: 207-211. 10.1038/35075603.View ArticlePubMed
- Binder MD, Cate HS, Prieto AL, Kemper D, Butzkueven H, Gresle MM, Cipriani T, Jokubaitis VG, Carmeliet P, Kilpatrick TJ: Gas6 deficiency increases oligodendrocyte loss and microglial activation in response to cuprizone-induced demyelination. J Neurosci. 2008, 28: 5195-5206. 10.1523/JNEUROSCI.1180-08.2008.PubMed CentralView ArticlePubMed
- Weinger JG, Omari KM, Marsden K, Raine CS, Shafit-Zagardo B: Up-regulation of soluble Axl and Mer receptor tyrosine kinases negatively correlates with Gas6 in established multiple sclerosis lesions. Am J Pathol. 2009, 175: 283-293. 10.2353/ajpath.2009.080807.PubMed CentralView ArticlePubMed
- Janssen JW, Schulz AS, Steenvoorden AC, Schmidberger M, Strehl S, Ambros PF, Bartram CR: A novel putative tyrosine kinase receptor with oncogenic potential. Oncogene. 1991, 6: 2113-2120.PubMed
- O'Bryan JP, Frye RA, Cogswell PC, Neubauer A, Kitch B, Prokop C, Espinosa R, Le Beau MM, Earp HS, Liu ET: axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase. Mol Cell Biol. 1991, 11: 5016-5031.PubMed CentralView ArticlePubMed
- Graham DK, Dawson TL, Mullaney DL, Snodgrass HR, Earp HS: Cloning and mRNA expression analysis of a novel human protooncogene, c-mer. Cell Growth Differ. 1994, 5: 647-657.PubMed
- Jia R, Hanafusa H: The proto-oncogene of v-eyk (v-ryk) is a novel receptor-type protein tyrosine kinase with extracellular Ig/GN-III domains. J Biol Chem. 1994, 269: 1839-1844.PubMed
- Funakoshi H, Yonemasu T, Nakano T, Matumoto K, Nakamura T: Identification of Gas6, a putative ligand for Sky and Axl receptor tyrosine kinases, as a novel neurotrophic factor for hippocampal neurons. J Neurosci Res. 2002, 68: 150-160. 10.1002/jnr.10211.View ArticlePubMed
- Shankar SL, O'Guin K, Cammer M, McMorris FA, Stitt TN, Basch RS, Varnum B, Shafit-Zagardo B: The growth arrest-specific gene product Gas6 promotes the survival of human oligodendrocytes via a phosphatidylinositol 3-kinase-dependent pathway. J Neurosci. 2003, 23: 4208-4218.PubMed
- Sainaghi PP, Collimedaglia L, Alciato F, Leone MA, Puta E, Naldi P, Castello L, Monaco F, Avanzi GC: Elevation of Gas6 protein concentration in cerebrospinal fluid of patients with chronic inflammatory demyelinating polyneuropathy (CIDP). J Neurol Sci. 2008, 269: 138-142. 10.1016/j.jns.2008.01.005.View ArticlePubMed
- Manfioletti G, Brancolini C, Avanzi G, Schneider C: The protein encoded by a growth arrest-specific gene (gas6) is a new member of the vitamin K-dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade. Mol Cell Biol. 1993, 13: 4976-4985.PubMed CentralView ArticlePubMed
- Prieto AL, Weber JL, Tracy S, Heeb MJ, Lai C: Gas6, a ligand for the receptor protein-tyrosine kinase Tyro-3, is widely expressed in the central nervous system. Brain Res. 1999, 816: 646-661. 10.1016/S0006-8993(98)01159-7.View ArticlePubMed
- Hoehn HJ, Kress Y, Sohn A, Brosnan CF, Bourdon S, Shafit-Zagardo B: Axl-/- mice have delayed recovery and prolonged axonal damage following cuprizone toxicity. Brain Res. 2008, 1240: 1-11.View ArticlePubMed
- Heide I, Sokoll AC, Henz BM, Nagel S, Kreissig K, Grutzkau A, Grabbe J, Wittig B, Neubauer A: Regulation and possible function of axl expression in immature human mast cells. Ann Hematol. 1998, 77: 199-205. 10.1007/s002770050443.View ArticlePubMed
- Sharif MN, Sosic D, Rothlin CV, Kelly E, Lemke G, Olson EN, Ivashkiv LB: Twist mediates suppression of inflammation by type I IFNs and Axl. J Exp Med. 2006, 203: 1891-1901. 10.1084/jem.20051725.PubMed CentralView ArticlePubMed
- Van Der Voorn P, Tekstra J, Beelen RH, Tensen CP, Van Der Valk P, De Groot CJ: Expression of MCP-1 by reactive astrocytes in demyelinating multiple sclerosis lesions. Am J Pathol. 1999, 154: 45-51. 10.1016/S0002-9440(10)65249-2.PubMed CentralView ArticlePubMed
- Cannella B, Raine CS: The adhesion molecule and cytokine profile of multiple sclerosis lesions. Ann Neurol. 1995, 37: 424-435. 10.1002/ana.410370404.View ArticlePubMed
- Hofman FM, Hinton DR, Johnson K, Merrill JE: Tumor necrosis factor identified in multiple sclerosis brain. J Exp Med. 1989, 170: 607-612. 10.1084/jem.170.2.607.View ArticlePubMed
- Selmaj K, Raine CS, Cannella B, Brosnan CF: Identification of lymphotoxin and tumor necrosis factor in multiple sclerosis lesions. J Clin Invest. 1991, 87: 949-954. 10.1172/JCI115102.PubMed CentralView ArticlePubMed
- dos Santos AC, Barsante MM, Arantes RM, Bernard CC, Teixeira MM, Carvalho-Tavares J: CCL2 and CCL5 mediate leukocyte adhesion in experimental autoimmune encephalomyelitis--an intravital microscopy study. J Neuroimmunol. 2005, 162: 122-129. 10.1016/j.jneuroim.2005.01.020.View ArticlePubMed
- Rieckmann P, Albrecht M, Kitze B, Weber T, Tumani H, Broocks A, Luer W, Helwig A, Poser S: Tumor necrosis factor-alpha messenger RNA expression in patients with relapsing-remitting multiple sclerosis is associated with disease activity. Ann Neurol. 1995, 37: 82-88. 10.1002/ana.410370115.View ArticlePubMed
- Tanuma N, Abe S, Shin T, Kojima T, Ishihara Y, Arai Y, Toyoshima S, Matsumoto Y: Pretreatment with T cell receptor peptides using a conventional immunization protocol does not induce effective protection against autoimmune encephalomyelitis. Cell Immunol. 1996, 168: 85-90. 10.1006/cimm.1996.0052.View ArticlePubMed
- Neumann H, Kotter MR, Franklin RJ: Debris clearance by microglia: an essential link between degeneration and regeneration. Brain. 2009, 132: 288-295.PubMed CentralView ArticlePubMed
- Rappert A, Bechmann I, Pivneva T, Mahlo J, Biber K, Nolte C, Kovac AD, Gerard C, Boddeke HW, Nitsch R, Kettenmann H: CXCR3-dependent microglial recruitment is essential for dendrite loss after brain lesion. J Neurosci. 2004, 24: 8500-8509. 10.1523/JNEUROSCI.2451-04.2004.View ArticlePubMed
- Streit WJ, Conde JR, Fendrick SE, Flanary BE, Mariani CL: Role of microglia in the central nervous system's immune response. Neurol Res. 2005, 27: 685-691.PubMed
- Hanisch UK, Kettenmann H: Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nat Neurosci. 2007, 10: 1387-1394. 10.1038/nn1997.View ArticlePubMed
- Heneka MT, O'Banion MK, Terwel D, Kummer MP: Neuroinflammatory processes in Alzheimer's disease. J Neural Transm. 2010, 117: 919-947. 10.1007/s00702-010-0438-z.View ArticlePubMed
- Seitz HM, Camenisch TD, Lemke G, Earp HS, Matsushima GK: Macrophages and dendritic cells use different Axl/Mertk/Tyro3 receptors in clearance of apoptotic cells. J Immunol. 2007, 178: 5635-5642.View ArticlePubMed
- Pluchino S, Quattrini A, Brambilla E, Gritti A, Salani G, Dina G, Galli R, Del Carro U, Amadio S, Bergami A, Furlan R, Comi G, Cescovi AL, Martino G: Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature. 2003, 422: 688-694. 10.1038/nature01552.View ArticlePubMed
- Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970, 227: 680-685. 10.1038/227680a0.View ArticlePubMed
- Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA. 1979, 76: 4350-4354. 10.1073/pnas.76.9.4350.PubMed CentralView ArticlePubMed
- Zamora-Leon SP, Bresnick A, Backer JM, Shafit-Zagardo B: Fyn phosphorylates human MAP-2c on tyrosine 67. J Biol Chem. 2005, 280: 1962-1970.View ArticlePubMed
- Chen L, Brosnan CF: Exacerbation of experimental autoimmune encephalomyelitis in P2X7R-/- mice: evidence for loss of apoptotic activity in lymphocytes. J Immunol. 2006, 176: 3115-3126.View ArticlePubMed
- Bertenshaw GP, Yip P, Seshaiah P, Zhao J, Chen TH, Wiggins WS, Mapes JP, Mansfield BC: Multianalyte profiling of serum antigens and autoimmune and infectious disease molecules to identify biomarkers dysregulated in epithelial ovarian cancer. Cancer Epidemiol Biomarkers Prev. 2008, 17: 2872-2881. 10.1158/1055-9965.EPI-08-0464.View ArticlePubMed
- Venkataswamy MM, Baena A, Goldberg MF, Bricard G, Im JS, Chan J, Reddington F, Besra GS, Jacobs WR, Porcelli SA: Incorporation of NKT cell-activating glycolipids enhances immunogenicity and vaccine efficacy of Mycobacterium bovis bacillus Calmette-Guerin. J Immunol. 2009, 183: 1644-1656. 10.4049/jimmunol.0900858.PubMed CentralView ArticlePubMed
- Mueller KL, Hunter LA, Ethier SP, Boerner JL: Met and c-Src cooperate to compensate for loss of epidermal growth factor receptor kinase activity in breast cancer cells. Cancer Res. 2008, 68: 3314-3322. 10.1158/0008-5472.CAN-08-0132.View ArticlePubMed
- O'Bryan JP, Fridell YW, Koski R, Varnum B, Liu ET: The transforming receptor tyrosine kinase, Axl, is post-translationally regulated by proteolytic cleavage. J Biol Chem. 1995, 270: 551-557. 10.1074/jbc.270.2.551.View ArticlePubMed
- Kuhlmann T, Lingfeld G, Bitsch A, Schuchardt J, Bruck W: Acute axonal damage in multiple sclerosis is most extensive in early disease stages and decreases over time. Brain. 2002, 125: 2202-2212. 10.1093/brain/awf235.View ArticlePubMed
- Wujek JR, Bjartmar C, Richer E, Ransohoff RM, Yu M, Tuohy VK, Trapp BD: Axon loss in the spinal cord determines permanent neurological disability in an animal model of multiple sclerosis. J Neuropathol Exp Neurol. 2002, 61: 23-32.View ArticlePubMed
- Ferrero E, Zocchi MR, Magni E, Panzeri MC, Curnis F, Rugarli C, Ferrero ME, Corti A: Roles of tumor necrosis factor p55 and p75 receptors in TNF-alpha-induced vascular permeability. Am J Physiol Cell Physiol. 2001, 281: C1173-1179.View ArticlePubMed
- Wolf G, Aberle S, Thaiss F, Nelson PJ, Krensky AM, Neilson EG, Stahl RA: TNF alpha induces expression of the chemoattractant cytokine RANTES in cultured mouse mesangial cells. Kidney Int. 1993, 44: 795-804. 10.1038/ki.1993.314.View ArticlePubMed
- Chen YM, Chiang WC, Lin SL, Wu KD, Tsai TJ, Hsieh BS: Dual regulation of tumor necrosis factor-alpha-induced CCL2/monocyte chemoattractant protein-1 expression in vascular smooth muscle cells by nuclear factor-kappaB and activator protein-1: modulation by type III phosphodiesterase inhibition. J Pharmacol Exp Ther. 2004, 309: 978-986. 10.1124/jpet.103.062620.View ArticlePubMed
- Glabinski AR, Tuohy VK, Ransohoff RM: Expression of chemokines RANTES, MIP-1alpha and GRO-alpha correlates with inflammation in acute experimental autoimmune encephalomyelitis. Neuroimmunomodulation. 1998, 5: 166-171. 10.1159/000026333.View ArticlePubMed
- Kreutzberg GW: Microglia, the first line of defence in brain pathologies. Arzneimittelforschung. 1995, 45: 357-360.PubMed
- Gehrmann J, Matsumoto Y, Kreutzberg GW: Microglia: intrinsic immuneffector cell of the brain. Brain Res Brain Res Rev. 1995, 20: 269-287.View ArticlePubMed
- Vajkoczy P, Knyazev P, Kunkel A, Capelle HH, Behrndt S, von Tengg-Kobligk H, Kiessling F, Eichelsbacher U, Essig M, Read TA, Erber R, Ullrich A: Dominant-negative inhibition of the Axl receptor tyrosine kinase suppresses brain tumor cell growth and invasion and prolongs survival. Proc Natl Acad Sci USA. 2006, 103: 5799-5804. 10.1073/pnas.0510923103.PubMed CentralView ArticlePubMed
- Allen MP, Linseman DA, Udo H, Xu M, Schaack JB, Varnum B, Kandel ER, Heidenreich KA, Wierman ME: Novel mechanism for gonadotropin-releasing hormone neuronal migration involving Gas6/Ark signaling to p38 mitogen-activated protein kinase. Mol Cell Biol. 2002, 22: 599-613. 10.1128/MCB.22.2.599-613.2002.PubMed CentralView ArticlePubMed
- Franklin RJ, Kotter MR: The biology of CNS remyelination: the key to therapeutic advances. J Neurol. 2008, 255 (Suppl 1): 19-25.View ArticlePubMed
- Rémy C, Fouilhé N, Barba I, Sam-Lai E, Lahrech H, Cucurella MG, Izquierdo M, Moreno A, Ziegler A, Massarelli R, Décorps M, Arús C: Evidence that mobile lipids detected in rat brain glioma by 1H nuclear magnetic resonance correspond to lipid droplets. Cancer Res. 1997, 57: 407-414.PubMed
- Lahrech H, Zoula S, Farion R, Rémy C, Décorps M: In vivo measurement of the size of lipid droplets in an intracerebral glioma in the rat. Magn Reson Med. 2001, 45: 409-414. 10.1002/1522-2594(200103)45:3<409::AID-MRM1053>3.0.CO;2-O.View ArticlePubMed
- Rizk T, Montero-Menei C, Jollivet C, Benoit JP, Menei P: Pitfalls in the detection of lipid vectors in neural cell culture and in brain tissue. J Biomed Mater Res A. 2004, 68: 360-364.View ArticlePubMed
- Lemke G, Rothlin CV: Immunobiology of the TAM receptors. Nat Rev Immunol. 2008, 8: 327-336. 10.1038/nri2303.PubMed CentralView ArticlePubMed
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