Rescue from acute neuroinflammation by pharmacological chemokine-mediated deviation of leukocytes
© Berghmans et al.; licensee BioMed Central Ltd. 2012
Received: 15 May 2012
Accepted: 11 October 2012
Published: 25 October 2012
Neutrophil influx is an important sign of hyperacute neuroinflammation, whereas the entry of activated lymphocytes into the brain parenchyma is a hallmark of chronic inflammatory processes, as observed in multiple sclerosis (MS) and its animal models of experimental autoimmune encephalomyelitis (EAE). Clinically approved or experimental therapies for neuroinflammation act by blocking leukocyte penetration of the blood brain barrier. However, in view of unsatisfactory results and severe side effects, complementary therapies are needed. We have examined the effect of chlorite-oxidized oxyamylose (COAM), a potent antiviral polycarboxylic acid on EAE.
EAE was induced in SJL/J mice by immunization with spinal cord homogenate (SCH) or in IFN-γ-deficient BALB/c (KO) mice with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Mice were treated intraperitoneally (i.p.) with COAM or saline at different time points after immunization. Clinical disease and histopathology were compared between both groups. IFN expression was analyzed in COAM-treated MEF cell cultures and in sera and peritoneal fluids of COAM-treated animals by quantitative PCR, ELISA and a bioassay on L929 cells. Populations of immune cell subsets in the periphery and the central nervous system (CNS) were quantified at different stages of disease development by flow cytometry and differential cell count analysis. Expression levels of selected chemokine genes in the CNS were determined by quantitative PCR.
We discovered that COAM (2 mg i.p. per mouse on days 0 and 7) protects significantly against hyperacute SCH-induced EAE in SJL/J mice and MOG35-55-induced EAE in IFN-γ KO mice. COAM deviated leukocyte trafficking from the CNS into the periphery. In the CNS, COAM reduced four-fold the expression levels of the neutrophil CXC chemokines KC/CXCL1 and MIP-2/CXCL2. Whereas the effects of COAM on circulating blood and splenic leukocytes were limited, significant alterations were observed at the COAM injection site.
These results demonstrate novel actions of COAM as an anti-inflammatory agent with beneficial effects on EAE through cell deviation. Sequestration of leukocytes in the non-CNS periphery or draining of leukocytes out of the CNS with the use of the chemokine system may thus complement existing treatment options for acute and chronic neuroinflammatory diseases.
KeywordsEncephalitis Leukocytes Chemokines Central nervous system
Neuroinflammation is a common denominator in a wide variety of diseases of the central nervous system (CNS), ranging from various forms of acute infectious or vascular meningoencephalitis to chronic inflammation associated with multiple sclerosis (MS) or neurodegenerative diseases. Hyperacute experimental autoimmune encephalomyelitis (EAE), henceforth named as such to distinguish it from T cell-mediated forms of EAE, is induced by immunization of mice with spinal cord homogenates, has an important neutrophil component  and is an appropriate model for the study of neuroinflammation in acute encephalitis. By contrast, chronic forms of EAE are often induced with CNS peptide antigens or by adoptive transfer of neuroantigen-specific T cell clones or T cells from sensitized animals, and are widely used to study disease mechanisms and new therapeutic approaches for MS .
Various aspects of recent research point to a role of the blood–brain barrier (BBB) as a crucial structure to prevent neuroinflammation [3, 4]. Neuroinflammation occurs upon migration of leukocytes through the endothelial and parenchymal layers of the BBB in order to gain access to the CNS parenchyma  and as a result of chemokine-governed attraction of specific leukocytes to the CNS . Current ways to prevent leukocyte entry into the CNS include treatment with IFN-α/β or anti-adhesive agents that prevent binding of leukocytes to CNS endothelium [7, 8].
IFN-β has been proven effective for the treatment of relapsing remitting MS : IFN-β decreases the relapse rate, ameliorates disease activity and reduces the number of inflammatory CNS lesions . Many studies have shown that IFN-β exerts beneficial effects on the development of EAE and on the stability of the BBB [10–12]. Therefore, induction of endogenous IFN-β is an alternative approach to substitution therapy with IFN-β. In early studies, such stimulation was typically achieved by induction of IFN-β with viral double-stranded RNAs or therapeutically by polyanions, including polyI:C and polyacrylates [13, 14]. PolyI:C has been reported to suppress disease in a murine model of relapsing EAE by inducing endogenous IFN-β but has high toxicity . Chlorite-oxidized oxyamylose (COAM), a synthetic polyanion with a therapeutic index of 300 to 500, is a potent antiviral agent , which we recently showed to result in almost complete protection against acute infection with the neurotropic mengovirus. Similarly, COAM has no cytotoxic effects in vitro[17, 18]. In this animal model of infection, COAM was shown to induce myeloid cell chemotaxis, in part through binding and activity of the chemokine granulocyte chemotactic protein-2/CXCL6 . In view of the supposed IFN-inducing capacity of COAM, we started a series of studies to determine its effects in EAE models. COAM was shown to suppress neuroinflammation significantly by interfering with the chemokine system and by causing retention of immune cells at the peritoneal injection site, thus affecting cell fluxes to the brain. These results suggest that diverting leukocyte chemotaxis from the brain into the non-CNS periphery may represent a novel and pharmacologically attainable treatment option that complements existing therapies for various forms of acute neuroinflammation.
SJL/J and IFN-γ KO mice were bred under conventional conditions in the Experimental Animal Breeding Facility of the University of Leuven, Belgium. The generation and basic characterization of IFN-γ-deficient mice of the 129 x BALB/c strain have been described previously . These mice were backcrossed for eight generations to the parental BALB/c strain. EAE experiments were carried out with 8 to 10 week old male and female mice. During the experiments, mice were kept under conventional housing conditions. They received a regular diet and acidified drinking water without antibiotics. All procedures were conducted in accordance with protocols approved by the local Ethics Committee (Licence number LA1210243, Belgium).
Mycobacterium tuberculosis strain H37Ra, Incomplete Freund’s Adjuvant (IFA) and Complete Freund’s Adjuvant (CFA) were purchased from Difco Laboratories (Detroit, MI, USA). Pertussis toxin was purchased from List Biological Laboratories (Campbell, CA, USA). COAM was prepared as described . It was free of endotoxin (<13.3 pg/mg COAM, assayed in the Limulus amoebocyte lysate assay) and devoid of contaminating proteins (assayed by protein staining) . Myelin oligodendrocyte glycoprotein peptide (MOG35-55) was produced by Fmoc (fluorenylmethoxycarbonyl) solid phase peptide synthesis, purified by reversed phase chromatography and peptide mass was confirmed by electrospray ion trap mass spectrometry .
Induction and clinical evaluation of EAE and treatment with COAM
For induction of hyperacute EAE in SJL/J mice, an emulsion was prepared consisting of 100 mg/ml of lyophilized SJL/J mouse spinal cord homogenate (SCH) in PBS and 4 mg/ml M. tuberculosis (strain H37Ra) in CFA. Chronic EAE was induced in IFN-γ KO BALB/c mice by injecting 50 μg of MOG35-55 peptide (1 mg/ml in saline) emulsified in IFA containing 4 mg/ml of M. tuberculosis. On day 0, mice were injected subcutaneously in each of the two hind footpads with 50 μl of the emulsion. Immediately thereafter and on day 2, 100 ng of pertussis toxin in 50 μl saline was intravenously (i.v.) administered in the tail vein. Animals were anaesthetized for injections.
Mice were evaluated daily for signs of clinical disease. Disease severity was recorded as follows: grade 0, normal; grade 0.5, floppy tail; grade 1, tail paralysis and mild impaired righting reflex; grade 2, mild hind limb weakness and impaired righting reflex; grade 3, moderate to severe hind limb paresis and/or mild forelimb weakness; grade 4, complete hind limb paralysis and/or moderate to severe forelimb weakness; grade 5, quadriplegia or moribund; grade 6, death.
COAM is hydrophilic and was dissolved in pyrogen-free 0.9% NaCl. Mice were treated with an intraperitoneal (i.p.) injection of COAM (2 mg in 0.2 ml 0.9% NaCl) on days 0 and/or 7 after EAE immunization. Control mice received an equivalent volume of saline (0.9% NaCl).
Isolation and induction of mouse embryonic cells
Mouse embryonic fibroblasts (MEF) were isolated from SJL/J mouse embryos around 17 days of gestation. The uterine horns were dissected and placed in a petri dish containing MEM (Invitrogen, Paisly, Scotland) supplemented with penicillin (500 U/ml; Continental Pharma, Brussels, Belgium) and streptomycin (500 μg/ml; Continental Pharma). Each embryo was separated from its placenta and surrounding membranes and washed three times with MEM. Subsequent procedures were according to standard conditions: incubation of embryo fragments in 50 ml trypsin-ethylenediaminetetraacetic acid (EDTA), centrifugation of cell suspension at 135 g for 15 minutes, two washings of the cell pellets with MEM growth medium containing 10% heat-inactivated FCS, 200 mM L-glutamine and 0.1% sodium bicarbonate (Invitrogen) and culture of adherent cells to confluency in growth medium in flat-bottomed flasks (75 cm2, TPP, Zurich, Switzerland) for four days.
For the induction of IFN-β in MEF, 1 x 106 cells in a total volume of 2 ml growth medium were seeded in six-well plates (TPP). After incubation for 24 hours, cells were stimulated with different concentrations of COAM in MEM with 2% FCS for 72 hours. Supernatants were collected for detection of IFN-β with a biological antiviral assay on IFN-sensitive fibroblastoid L929 cells and with ELISA for IFN-γ determination. The MEF cells were harvested after 72 hours and used for quantitative PCR (qPCR) analysis of cytokine and chemokine mRNAs.
Relative quantitation of cytokine, chemokine and chemokine receptor mRNAs by qPCR
Total RNA was purified from cells or tissues (RNeasy Mini Kit, Qiagen, Venlo, The Netherlands) and transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). qPCR reactions were carried out in an ABI Prism 7000 Sequence Detection System (Applied Biosystems) in a total volume of 30 μl, containing 50 ng of extracted RNA, 15 μl of TaqMan Gene Expression Master Mix (Applied Biosystems) and 1.5 μl of primer/probe mix for the appropriate cytokine, chemokine or chemokine receptor (TaqMan Gene Expression Inventoried Assays, Applied Biosystems). The qPCR conditions consisted of an initial step at 50°C for 2 minutes, an activation step at 95°C for 10 minutes followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. 18S or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Applied Biosystems) were included as endogenous reference genes for normalization of target mRNA transcripts. The fold change in gene expression normalized to the endogenous reference and relative to the untreated control was determined according to the comparative ΔΔCt method .
Bioassay on L929 cells for detection of IFN activity
IFN assays were carried out as described . Briefly, mouse L929 cells were seeded in flat-bottom 96-well plates at a density of 6 x 104 cells per well in MEM growth medium. In each assay a laboratory mouse IFN standard (consisting of mouse L929 cell-derived IFN-αβ, induced with Newcastle disease virus) was included and 0.5 log10 dilutions of the samples were made in growth medium. After 24 hours of incubation at 37°C, the cultures were challenged with 50 μl of mengovirus (multiplicity of infection, 0.01 plaque-forming units per cell). Cell controls received growth medium only. Plates were incubated at 37°C for 24 hours and cells were colored with crystal violet. The detection limit of this biological IFN assay was 3.16 units/ml.
Detection of cytokines and chemokines by ELISA
Mouse IFN-γ concentrations were determined by the sandwich ELISA described previously . Briefly, samples were incubated in microtiter plates coated with mouse anti-rat IFN-γ-specific mAb as capturing antibody (DB1; gift from Dr. P. van der Meide, Cytokine Biology Unit, Central Laboratory Animal Institute, Utrecht University, Utrecht, The Netherlands). The bound cytokine was detected by incubation in turn with rat anti-mouse IFN-γ-specific mAb (F1), used as primary detection antibody, and goat anti-rat immunoglobulin-peroxidase conjugate (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) as secondary detection antibody.
IFN-β protein concentrations in sera of IFN-γ KO mice were quantified using the VeriKine Mouse Interferon Beta ELISA kit (PBL InterferonSource, Piscataway, NJ, USA). GCP-2 was detected by an ELISA developed in our laboratory, as described . IL-17, KC and MIP-2 levels were measured by sandwich ELISA using paired antibodies according to the manufacturer’s recommendations (DuoSet ELISA Development System, R&D Systems, Abingdon, UK).
Cell preparation from various organs
Brain and spinal cord
Mice were sacrificed and gently perfused through the left cardiac ventricle with 50 ml ice-cold PBS to eliminate intravascular contaminating blood cells in the CNS. Spinal cords were removed by flushing the spinal column with sterile PBS and brains were dissected. Both tissues were homogenized and filtered through a cell strainer (Becton Dickinson Labware, Franklin Lakes, NJ, USA). After centrifugation (10 minutes, 300 g), the cells were resuspended in 40% Percoll and underlayed with 72% Percoll. The gradient was centrifuged at 500 g for 20 minutes at 10°C. Before staining analysis, the interphase cells were collected and extensively washed in PBS supplemented with 2% FCS.
Peripheral blood samples were taken at the orbital sinus, using heparin as an anticoagulant. Leukocytes were obtained by lysis of red blood cells by two incubations (5 and 3 minutes, 37°C) in NH4Cl solution (0.83% w/v in 0.01M Tris/HCl; pH 7.2). Remaining cells were washed two times with ice-cold PBS, supplemented with 2% FCS, and then analyzed.
Spleens were isolated, cut into small pieces and passed through cell strainers, to obtain single cell suspensions. Red blood cells were lysed by two incubations (5 and 3 minutes at 37°C) of the splenocyte suspension in NH4Cl solution (0.83% w/v in 0.01M Tris/HCl; pH 7.2). Remaining cells were washed two times with ice-cold PBS containing 2% FCS.
Peritoneal lavage fluid was collected following killing of the mice. Five ml of ice-cold PBS containing 2% FCS was injected i.p. and the abdominal space was gently massaged. The lavage fluid was collected and centrifuged at 300 g for 10 minutes.
Flow cytometry analysis
Single cell suspensions (0.5 x 106 cells) were incubated for 15 minutes with the Fc-receptor-blocking antibodies anti-CD16/anti-CD32 (BD Biosciences Pharmingen, San Diego, CA, USA), washed with PBS supplemented with 2% FCS and then stained for 30 minutes with the indicated fluorescein isothiocyanate (FITC)-conjugated and phycoerythrin (PE)-conjugated antibodies. Cells were washed twice and fixed with 0.37% formaldehyde in PBS. FITC-conjugated anti-CD8, PE-conjugated anti-CD4, FITC-conjugated anti-CD11b, PE-conjugated anti-Gr-1 and PE-conjugated anti-F4/80 were purchased from eBioscience (San Diego, CA, USA). Cells were analyzed by a FACSCalibur flow cytometer and data were processed with the CellQuest software (Becton Dickinson).
Differential cell counting and histopathology analysis
Cells from spinal cord and brain and from other anatomical compartments were applied to slides by centrifugation at a density of approximately 105 cells/slide using a Shandon Cytospin 2. Cytospin preparations were stained with Hemacolor (Merck, Darmstadt, Germany) and leukocytes were identified on the basis of morphology. Five series of 100 cells from each slide were counted and the results were expressed as a percentage of the total cell count.
Spinal cords and brains were fixed in 4% formalin. Four micron thick paraffin sections were stained with H & E and scored for signs of neuroinflammation by two independent observers.
Differences in the clinical course of EAE were analyzed by Wilcoxon’s non-parametric test or, where appropriate, by the Chi-square test with Yates’ correction. Significant differences between groups were evaluated using a non-parametric Mann Whitney test. All P values of 0.05 or less were considered significant.
COAM protects against hyperacute and chronic EAE without inducing interferon-β
Hyperacute EAE development in SJL/J mice treated with COAM
Day 0 + 7
Mean maximum disease score ± SEM
5.6 ± 0.25
3.8 ± 0.78*
3.6 ± 0.75
4.6 ± 0.62
5.75 ± 0.24
2.72 ± 0.79*
Mean day of onset ± SEM
12.3 ± 0.2
13.1 ± 0.37
13 ± 0.56
12.2 ± 0.14
11.8 ± 0.28
12.8 ± 0.37
After administration of COAM or other polyanions in viral infection models, low levels of IFN are detectable in the serum during the first 24 to 48 hours, with peak IFN levels after 18 hours . However, more recent antiviral studies have suggested that COAM does not induce IFN-β in vitro or in vivo. Hence, it is not clear whether COAM can induce IFN or whether virus challenge is the main inducer of IFN. To distinguish between these possibilities it is necessary to examine whether COAM can induce IFN-β in virus-free conditions. Therefore, we tested extensively whether IFN-β which protects against EAE , is upregulated in COAM-treated hyperacute EAE animals or cell cultures.
First, groups of SJL/J mice induced for EAE were bled 6, 18 and 24 hours after each COAM administration (2 mg i.p. on days 0 and 7 after immunization). Sera were tested for IFN bio-activity on L929 cells with the use of a viral cytopathogenic reduction assay. This analysis revealed no IFN-like activity (detection limit <3.16 units/ml) in any of the test samples, whereas standard IFN-α/β preparations yielded the expected titers between 2.0 to 2.5 log10 units/ml. In addition, no IFN-γ was detectable by a specific ELISA in any of the titrated sera samples (detection level was 1.25 units/ml). Similarly, peritoneal fluids from saline- and COAM-treated EAE mice, collected at days 5, 9 and 16 after immunization, did not contain any IFN-like activity. Sera from EAE-induced IFN-γ KO mice (collected at 0, 6, 18 and 24 hours after each COAM injection) were tested in an IFN-β ELISA (PBL Interferon Source). All samples were below the detection limit of 15.6 pg/ml, ruling out the possibility that the observed reduction in EAE with COAM in these mice is due to high IFN-β levels.
COAM does not induce IFN-β
1.17 ± 0.20
0.91 ± 0.31
1.00 ± 0.12
1.33 ± 0.35
1.07 ± 0.11
0.98 ± 0.18
1.62 ± 0.46
1.00 ± 0.25
1.12 ± 0.13
0.90 ± 0.15
0.19 ± 0.06
0.69 ± 0.37
0.17 ± 0.03
1.32 ± 0.65
1.00 ± 0.29
1.11 ± 0.19
0.52 ± 0.09
1.92 ± 0.05
1.44 ± 0.25
1.55 ± 0.66
0.65 ± 0.39
1.00 ± 0.22
1.28 ± 0.26
0.47 ± 0.14
1.43 ± 0.06
1.38 ± 0.38
1.5 ± 0.62
1.86 ± 0.59
1.00 ± 0.41
COAM reduces cell infiltration into the CNS
COAM alters expression of inflammatory chemokines and their receptors in the CNS of hyperacute EAE mice
Effects of COAM on blood cells and splenocytes
COAM drastically alters leukocyte populations at the injection site
In conclusion, COAM induced significant leukocyte recruitment at the peripheral injection site. Specifically, neutrophils as fast contributors to hyperacute neuroinflammation and also monocytes and lymphocytes as slower effector cells in acute inflammatory reactions were recruited to the periphery by COAM injection.
The present study provides a novel approach with therapeutic potential for the treatment of hyperacute CNS inflammation, namely by deviation of leukocyte trafficking from the CNS to peripheral body compartments. Furthermore, we provide an example of a pharmacological agent to attain this goal in vivo. Two rationales prompted us to evaluate COAM in EAE: (i) the suggestion  that COAM induces endogenous IFN-β with known therapeutic effects in MS and EAE and (ii) the fact that COAM protected against immunopathologies observed in acute lethal virus infection models [17, 19]. Our data provide no evidence for the first possibility and stimulated research into alternative explanations of mechanisms of action. Indeed, by a number of experimental approaches, including IFN bioassays, ELISAs and mRNA titrations in hyperacute EAE animals and on MEF cell cultures, we corroborated and extended recent data that COAM does not induce IFN-β in vitro and in vivo, but instead affects the chemokine system. Our present data are in line with findings in infection and tumor animal models [18, 19, 32].
One effect of COAM in the CNS is the reduction of KC/CXCL1 expression in brain and spinal cord and of MIP-2/CXCL2 and RANTES/CCL5 expression in the spinal cord. Reduction of specific chemokine expression levels may contribute to the observed significantly reduced leukocyte counts in the CNS. After treatment with COAM, reduced neuroinflammation was paralleled by significant reduction in hyperacute EAE disease scores. Further analyses were done to determine how COAM may affect trafficking of inflammatory cells. COAM treatment increased circulating and peritoneal neutrophil counts, in line with less influx into the CNS and compartmental deviation of neutrophils out of the CNS towards the COAM injection site. The latter was also observed in another study , in which it was found that i.p. COAM bound GCP-2/CXCL6 leading to enhanced chemotaxis of myeloid cells into the peritoneal cavity. The present study thus provides a practical example for the thesis that modulation of the chemokine system may be exploited therapeutically to reduce leukocyte infiltration into the CNS. Blocking of chemokine activity by binding to glycosaminoglycans (GAGs) was proposed as an anti-inflammatory modus [33, 34]. However, with COAM a different action of GAG mimicry is proposed: one that does not block but instead enhances chemokine activities in specific body compartments. Collectively, our findings lead to the suggestion that at the injection site COAM captures endogenous chemokines from the environment and creates a potent chemotactic gradient and thus lures inflammatory cells from the whole body into a specific compartment. Furthermore, these findings are complemented by the fact that COAM reduces CNS expression of specific chemokines with significant reduction of leukocytes in the CNS. We coined the term ‘cell deviation therapy’ for this concept.
Another way to decrease CNS infiltration by leukocytes is with the adhesion inhibitor Natalizumab/Tysabri, but this treatment increases the risk of polyomavirus infections [35–37]. COAM, being an immunomodulating agent with potent antiviral activity, might be an alternative treatment option for patients infected with such viruses, but this remains to be proven. Finally, the combination of COAM and Tysabri with complementary modes of therapeutic and antiviral actions constitutes another future approach to combat various forms of neuroinflammation.
Treatment of SCH-induced hyperacute neutrophilic EAE in SJL/J mice and of MOG-peptide-induced EAE in IFN-γ-deficient BALB/c mice with COAM resulted in significant amelioration of disease symptoms. This study provides a novel mechanism and an interesting molecular probe to study leukocyte deviation. Our data might have implications for the treatment of inflammatory and autoimmune diseases such as EAE and MS.
Complete Freund’s adjuvant
Central nervous system
Experimental autoimmune encephalomyelitis
Enzyme-linked immunosorbent assay
Fetal calf serum
- H & E:
Haematoxylin and eosin
Incomplete Freund’s adjuvant
Mouse embryonic fibroblasts
Modified Eagle’s medium
Myelin oligodendrocyte glycoprotein
Spinal cord homogenate
Quantitative polymerase chain reaction.
This work was supported by the “Geconcerteerde OnderzoeksActies” [GOA-2007/015 and GOA-2012/017], the EF/05/15 (Doctoral Fellowship to SL), the “InterUniversitaire AttractiePolen” (IUAP) and the Belgian Charcot Foundation (Postdoctoral Fellowship to NB) and the Fund for Scientific Research Flanders (FWO Vlaanderen). SL is recipient of a Postdoctoral Fellowship and a Travel Grant from the University of Leuven. We thank Prof. Paul Proost for the synthesis of the MOG peptide. We thank Chris Dillen for help with experiments and members of the Laboratories of Molecular Immunology and Immunobiology for helpful discussions.
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