Study No. 2

Title: The Role of SDF1-∝ and CXCR4 In the Pathogenesis of Lyme Disease of the Central And Peripheral Nervous System

Background and Significance

Lyme borreliosis, caused by infection of the spirochete Borrelia bugdorferi, is a multisystem disorder usually contracted by a tick bite. The disease has three typical stages. First constitutional upset and rash (erthema migrans), second, carditis, arthritis, a meningopolyneuritis, chronic skin (acrodermatitis chronica atrophica), arthritic and neurological symptoms. In the final stage mild distal sensorimotor neuropathy and carpal tunnel syndrome are frequently present. Manifestations in peripheral nerves vary in pattern. These may include cranial (facial) nerve involvement, plexopathy, multifocal neuropathy and, rarely, a severe generalized neuropathy similar to Guillain-Barre Syndrome.

The pathogenesis of Lyme disease affecting the central and peripheral nervous system is unclear. For example, perivascular infiltrates of lymphocytes and plasma cells surrounding endoneurial, perineurial and epineurial blood vessels have been present in nerve biopsies. Spirochetes have not been identified in peripheral nerve, lymphocytes and plasma cells have presented as infiltrating ganglia upon post mortem examination, and endateritis obliterans is occasionally seen without necrotizing vasculitis.

SDF-1α, also known as CXCL12, is a ligand of the chemokine receptor CXCR4, also known as fusin. Knockout mouse experiments demonstrated that CXCR4 and SDF1-α are required for normal embryonic development of the nervous, gastro-intestinal, hematopoietic, and cardiovascular systems. For example, SDF1-α is chemotactic for CD34 progenitor cells, induces proliferation of B-cell progenitors, and regulates leukocyte and endothelial precursor migration.

We hypothesize that the upregulation of SDF-1α in pathological tissues of the central and peripheral nervous system (CNS, PNS) tissue is responsible for the attraction of CXCR4 inflammatory cells, thus causing damage. CD4+ lymphocytes have been implicated in the pathogenesis of Lyme Disease. It has been demonstrated that CD4+ T-cells specifically responding to the Borrelia burgdorferi lysate have been present in the CSF of patients with acute Lyme encephalitis. Therefore, we intend to investigate the CD4+ cells in our implication. We hypothesize that the molecular basis for this recruitment is the SDF-1α/CXCR4 pathway.

Specific Aims

Specific Aim I. To investigate the expression of SDF-1α in cerebrospinal fluid (CSF) and blood of patients with Lyme disease, (early, midterm and late stages of Lyme disease.)

Specific Aim II. To investigate the expression of SDF-1 α and CXCR4 in pathological specimens of nerve, skeletal muscle, and if available, brain biopsies. Specific Aim III. To test the effect of SDF-1α on migration of CD4+ T cells in vitro using the Boyden chamber assay.

 Preliminary Data

We have validated the SDF-1α ELISA test, immunohistochemical analysis for SDF-1 α and CXCR4 in tissues as well as in our in vitro migration assays. These in vitro and in vivo assays are well suited for dissecting the events implicated in the pathogenesis of Lyme disease affecting the CNS and the PNS. Our proposed studies should provide fundamental knowledge on these processes by i) testing the levels of SDF-1 α in CSF and peripheral blood; ii) evaluating SDF-1α and CXCR4 expression in nerve, muscle, and CNS tissue and iii) testing the effect of an SDF-1 α gradient on the migration of CD4+ T-cells.

Experimental Design

Specific Aim I-ELISA

Blood and CSF in patients with different stages of Lyme Disease will be tested for the presence of SDF-1α using the Human SDF-1α Quantikine ELISA kit. Reagents, samples and standards will be prepared as per the manufacturer's instructions. The sample will be read at 450nm within 30min correction 540 or 570 nm.

Specific AIM II- Immunohistochemistry

Whenever a muscle, nerve, or CNS biopsy is performed, samples will be stained for SDF-1α and CXCR4. Immunohistochemistry for SDF-1α and CXCR4 positive cells and SDF-1 expressing structures, double label immunohistochemistry will be done as previously described. Control procedures will include isotype-matched murine monoclonal antibodies for SDF-1α and CXCR4.

Specific Aim III- In Vitro Migration Studies

We will test the effect of SDF-1α migration of CD4+ T-cells in vitro using the Boyden chamber. Cells and reagents. CD4+ T-cells will be cultured in 5% CO2 at 37çC in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). 1% penicillin and streptomycin, and 2mM glutamine (Gibco BRL, Grand Island, NY) as described. Migration assay. BD Biocoat chambers (BD Biosciences Doscovery Labware, Bedford, MA) with 8um pore size polystyrene filter inserts will be used according to the manufacturer's instructions and as described. Briefly, cells in 400 ul of DMEM with 10% FBS will be seeded onto the upper compartment of each chamber and placed into wells containing 600 ul of complete medium in the lower chamber in the absence or presence of SDF-1α (0ng/ml or 100ng/ml), in the absence or presence of 100nM of AMD3100. The migration chambers will be incubated for 24h in normoxic or hypoxic conditions at 37çC. Following incubation, the insets will be fixed and stained and the number of migrating cells will be counted as described. Two independent experiments done in triplicate will be performed. Western blot analysis. CD4+ T-cells will be seeded in 10cm dishes in complete growth medium. After 48 h, the medium will be changed to DMEM containing 1% FBS and 50mM HEPES (pH 7.4). Cells will be exposed to normoxic or hypoxic conditions for 24 h. Cells will be lysed in RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 1% deoxycholate, 0.1% SDS, 10mM Tris-HCl, pH 8.0 1mM EDTA, pH 8.0) supplemented with protease inhibitors. Protein quantification and electrophoresis will be carried out as previously described. Western blot analysis will be performed with the following antibodies: rabbit anti-CXCR4 polyclonal antibody (IMG-537 Imgenex), Sheep anti-mouse and donkey anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies will be used at 1:2500 dilutions. Immunodetection will be carried out with the Supersignal West Pico Chemiluminescent substrate ECL detection system (Pierce Biotechnology Inc., Rockford, IL) followed by visualization and densitometry using NIH Image software. Two independent experiments will be performed. Flow cytometry. Cells will be collected from the culture dishes and incubated with a rabbit polyclonal anti-human SDF-1α antibody (Peprotech, Inc) diluted 1:500 for 1 h at 4çC. After washing, cells will be incubated for 30 min at 4çC with a secondary anti-rabbit FITC-conjugated antibody (Jackson Immunoresearch), diluted 1:1000. Cells will be examined using a LSRII flow cytometer (Becton Dickinson Biosciences, San Jose, CA) and data will be analyzed by FlowJo software (Tree Star Inc, Ashland, OR). Three independent experiments will be performed.

Western blot analysis. CD4+ T-cells will be seeded in 10cm dishes in complete growth medium. After 48 h, the medium will be changed to DMEM containing 1% FBS and 50mM HEPES (pH 7.4). Cells will be exposed to normoxic or hypoxic conditions for 24 h. Cells will be lysed in RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 1% deoxycholate, 0.1% SDS, 10mM Tris-HCl, pH 8.0 1mM EDTA, pH 8.0) supplemented with protease inhibitors. Protein quantification and electrophoresis will be carried out as previously described. Western blot analysis will be performed with the following antibodies: rabbit anti-CXCR4 polyclonal antibody (IMG-537 Imgenex), Sheep anti-mouse and donkey anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies will be used at 1:2500 dilutions. Immunodetection will be carried out with the Supersignal West Pico Chemiluminescent substrate ECL detection system (Pierce Biotechnology Inc., Rockford, IL) followed by visualization and densitometry using NIH Image software. Two independent experiments will be performed. Flow cytometry. Cells will be collected from the culture dishes and incubated with a rabbit polyclonal anti-human SDF-1α antibody (Peprotech, Inc) diluted 1:500 for 1 h at 4çC. After washing, cells will be incubated for 30 min at 4çC with a secondary anti-rabbit FITC-conjugated antibody (Jackson Immunoresearch), diluted 1:1000. Cells will be examined using a LSRII flow cytometer (Becton Dickinson Biosciences, San Jose, CA) and data will be analyzed by FlowJo software (Tree Star Inc, Ashland, OR). Three independent experiments will be performed.