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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 5, 2006 as doi:10.1096/fj.05-4540fje. |
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,1
Departments of
* Neurobiology and
Immunology, The Weizmann Institute of Science, Rehovot, Israel
2Correspondence: Department of Neurobiology, The Weizmann Institute of Science, 76100 Rehovot, Israel. E-mail: michal.schwartz{at}weizmann.ac.il
SPECIFIC AIMS
Chondroitin sulfate proteoglycans (CSPGs) are a matrix component expressed naturally in the central nervous system (CNS) and up-regulated after CNS injuries and during the course of chronic neurodegenerative disorders such as multiple sclerosis (MS), Alzheimer’s disease, and glaucoma. They are considered to be a major factor inhibiting regeneration in the CNS due to their growth inhibitory effect on neurons and their association with activation of immune components. Studies have shown that degradation of CSPGs in vivo, using the enzyme chondroitinase ABC, promote recovery. We postulated that a disaccharidic degradation product of these glycoproteins (CSPG-DS) participates in the modulation of the inflammatory response and might therefore have potential as a treatment for inflammation-associated neurodegenerative conditions in the CNS. The aim of this study was to test this hypothesis by examining the ability of CSPG-DS to promote recovery and neuronal survival in the models of experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune uveitis (EAU) and to test its effects on components of the immune system such as T cells and microglia/macrophages.
PRINCIPAL FINDINGS
1. CSPG-DS alleviates the clinical symptoms of EAE
We induced EAE in four groups of C57Bl/6J mice by immunization with the encephalitogenic peptide MOG 35-55 emulsified in CFA and pertussis toxin. To examine the effect of CSPG-DS on the clinical symptoms of the disease, we injected mice i.v. in three groups with CSPG-DS: mice in the first group were treated only on day 0, mice in the second group on days 0 and 7, and mice in the third group on days 0, 3, 5, and 7. Mice in the fourth group (control) were injected with PBS. We noted a dose-dependent decline in severity of the induced disease with increasing numbers of CSPG-DS injections. Repeated treatment with CSPG-DS also significantly delayed disease onset (day 15.5±0.7 after disease induction in the CSPG-DS treated mice vs. 12.5±1 in the control) and shortened its duration.
2. CSPG-DS protects against loss of retinal ganglion cells in EAU
To determine whether the effect of CSPG-DS found in EAE is applicable to other neuroinflammatory conditions and to accurately measure the ability of CSPG-DS to protect neurons, we used an animal model of inflammation-mediated neuropathology in the eye, known as EAU. In this model the inflammatory response in the eye causes loss of neurons (RGCs), which can be accurately quantified. Treatment with steroids, while alleviating the inflammatory response, causes further death of RGCs. Such a situation makes this model an ideal system to test the potential dual function of CSPG-DS in controlling inflammation and in neuronal rescue. EAU was induced in Lewis rats by active immunization with a pathogenic peptide (R16) derived from interphotoreceptor retinoid binding protein, IRBP, emulsified in CFA. Three groups of the EAU-induced rats were included. One group was injected i.v with the steroid drug, methylprednisolone (MP), the second group with CSPG-DS, and the third group with PBS only (control). Immunization of naive Lewis rats with R16 was found to cause RGC loss of 52 ± 2% (mean±SD) relative to normal rats. Treatment of R16-immunized rats with MP had no beneficial effect on neuronal survival and even caused further RGC loss (59±1.6%). In contrast, treatment of the R16-immunized rats with CSPG-DS reduced the RGC loss to 24 ± 9% (Fig. 1
A).
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3. CSPG-DS reduces the number of accumulated T cells and promotes microglial/macrophage activation in EAU-induced rats
To gain a better understanding of the neuroimmunomodelatory mechanism of CSPG-DS, we used immunohistochemistry to compare the numbers of T cells and microglia/macrophages in the eyes of rats induced with EAU that were treated with CSPG-DS, MP, or PBS. The numbers of T cells in the eyes of animals with EAU were reduced significantly by treatment with CSPG-DS (Fig. 1B, C
), and even more so in the steroid-treated rats. A more complicated picture was obtained with respect to the effect of CSPG-DS on microglia/macrophages. We previously reported that CSPG-DS affects the activation of microglia by inducing them to adopt a phenotype that is protective of neural tissue. Microglia activated by CSPG-DS express high levels of class II major histocompatibility complex proteins (MHC-II) (MHC-II expression was reported to correlate with activation of microglia to a neuroprotective phenotype in other studies as well). IB-4 is a general marker characterizing microglia/macrophages and is used to determine the presence of these cells regardless of their phenotype. We therefore compared the incidences of microglia expressing both IB-4 and MHC-II in the eyes of EAU-induced rats treated with CSPG-DS, MP, or PBS. Immunohistochemical labeling of IB-4 indicated no significant difference in numbers of microglia/macrophages between the examined groups. However, MHC-II staining showed that relative to the control, MHC-II expression on microglia/macrophages (double-labeled cells) was increased by CSPG-DS treatment but almost eliminated by treatment with the steroid (Fig. 1D, E
).
4. CSPG-DS attenuates the delayed-type hypersensitivity (DTH) response, further demonstrating its ability to reduce the activation and motility of T cells
The above results indicated that CSPG-DS reduces the numbers of T cells in the eyes of rats with EAU. To further examine the potential effect of CSPG-DS on T cells, we used the DTH model. In this model the ears of Balb/c mice were sensitized with oxazalone and a delayed-type hypersensitivity (DTH) response was elicited 5 days later by additional challenge with oxazalone. The DTH response was measured 
24 h after the antigen challenge. This model is generally applied to analyze the effects of a specific compound on T cell migration or activation. Activation of T cells and their recruitment into an area of inflammation are crucial steps in development of the DTH response. The DTH response in mice treated with CSPG-DS was significantly weaker than in untreated mice (40% reduction in DTH response at the most effective CSPG-DS concentration of 1 µg/mouse), indicating that CSPG-DS can control T cell-associated inflammatory responses.
5. CSPG-DS down-regulates T cell motility
To determine whether CSPG-DS has a direct effect on T cell motility, we assessed migration of T cells toward the chemoattractive agent SDF-1
in a trans-well migration apparatus. Isolated T cells treated for 2 h with CSPG-DS showed reduced migration toward SDF-1 relative to that of untreated cells (24±12% migration vs. control at 1 µg/mL of CSPG-DS). We further demonstrated the effects of CSPG-DS on T cell adhesion by examining the adhesion levels of radioactively labeled T cells to fibronectin-coated microtiter well plates after preincubation for 2 h with CSPG-DS and activation by SDF-1. We found that CSPG-DS significantly reduced T cell adhesion (34±3% adhesion relative to control at 1 µg/mL of CSPG-DS).
6. CSPG-DS activates the suppressor of cytokine signaling (SOCS-3) in T cells
T cell growth, differentiation, and chemotactic responses require coordinated action between cytokines or chemokines and their intracellular targets. We wanted to determine whether CSPG-DS can also affect an intracellular mechanism known to be associated with an attenuated response to chemokines. Members of the SOCS-3 family of proteins have been identified as feedback regulators of JAK/STAT activation through their binding to JAK kinases or cytokine receptors. Therefore, by down-regulating the chemokine-mediated activation signal these proteins reduce cell migration in several different contexts, both in vivo and in vitro. SOCS-3 specifically down-regulates signals associated with responses mediated through the SDF-1 receptor CXCR4. We examined, by Western blot analysis, levels of SOCS-3 in T cells after pretreatment with CSPG-DS for 2 h and subsequent activation with anti-CD3 antibodies. It appeared that CSPG-DS treatment resulted in an increase (3.7-fold at 1 µg/mL of CSPG-DS) in SOCS-3 expression relative to untreated T cells, suggesting that CSPG-DS suppresses the signaling pathway through which SDF-1 mediates its effects, and providing a possible explanation for the reduction in T cell motility.
7. CSPG-DS reduces secretion of TNF- and IFN-
by activated T cells
A decrease in secretion of the Th1-associated cytokines IFN-, TNF-, or both can also contribute to the observed weakening of the DTH response. We examined by ELISA the direct effects of CSPG-DS on the secretion of cytokines by T cells. Pretreatment of T cells for 2 h with CSPG-DS prior to their activation with anti-CD3 antibodies caused a significant reduction in their secretion of IFN- (2.2-fold at 1 µg/mL of CSPG-DS) and TNF- (2.8-fold at 1 µg/mL of CSPG-DS) relative to T cells that were not treated with CSPG-DS.
8. CSPG-DS attenuates NF-
B activation by anti-CD3
An intracellular pathway that might directly reduce the secretion of cytokines is the NF-B cascade. The activity of NF-B is governed by its translocation to the nucleus, where it controls the transcription of genes responsible for regulating cell proliferation, cell survival, and inflammation. We examined by Western blot the ability of CSPG-DS to regulate NF-B activity induced after T cell receptor (TCR) activation by anti-CD3 antibodies. CSPG-DS significantly reduced NF-B levels after activation (3-fold at 1 µg/mL of CSPG-DS).
CONCLUSIONS AND SIGNIFICANCE
Intact CSPGs, the physiological source of CSPG-DS, limit axonal growth and induce CNS inflammation. However, their degradation product, CSPG-DS, promotes axonal growth and can also modulate the inflammatory response within the CNS. We provide evidence that CSPG-DS attenuates T cell motility, reduces IFN- and TNF- secretion, and modulate microglial activation to a neuroprotective phenotype resulting in reduction of the clinical symptoms in EAE, and rescue of neurons in EAU. By comparing the effects of CSPG-DS to the effects of steroid treatment on inflammation-associated neuropathologies, we found that CSPG-DS supports a balanced immune response that allows the mechanism to benefit from the therapeutic potential of the immune response manifested here by neuroprotective phenotype of microglia/macrophages and modulation of T cell levels and activation.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4540fje;
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