The fibrin-derived γ^377-395 peptide inhibits microglia activation and suppresses relapsing paralysis in central nervous system autoimmune disease

Fibrinogen directly activates microglia resulting in cytoskeletal rearrangements and increased phagocytosis

We first tested the effects of fibrinogen on pure primary microglia cells. Immobilized fibrinogen had a dramatic effect on microglia activation characterized by an increase in cell size (Fig. 1 A, top right) when compared with untreated controls (Fig. 1 A, top left). Quantitation revealed 85.5 ± 1.4% of activated microglia after fibrinogen treatment versus 8.2 ± 3.4% in untreated cells (Fig. 1 B; P < 0.001). In contrast to immobilized fibrinogen, soluble fibrinogen did not induce changes in microglia morphology (not depicted). LPS was used as a positive control (Fig. 1, A and B). Using an endotoxin determination assay, we verified that there was no LPS contamination in the fibrinogen-treated cultures. To assess the functional effect of this morphologic activation, we performed phagocytosis assays. Fibrinogen stimulation resulted in a 65.3% increase in phagocytosis as compared with untreated controls (P < 0.05), whereas LPS incubation, by comparison, increased phagocytosis by 39.1% (Fig. 1 C; P < 0.05). Previous studies have demonstrated that dynamic rearrangements of both the actin (26) and microtubule (27) networks are critical for phagocytosis. Deconvolution microscopy showed that fibrinogen stimulation resulted in dramatic rearrangement of the microglial cytoskeleton as determined by immunostaining of actin and β-tubulin (Fig. 1 D). Collectively, these results suggest that fibrinogen induces microglia differentiation into a phagocytic state.

Fibrinogen activates microglia via the Mac-1 integrin receptor

Mac-1 orchestrates the innate immune response by regulating phagocyte adhesion, migration, and engulfment of complement-opsonized particles (28). M1/70, an antibody that blocks binding of fibrinogen to CD11b (24, 29), inhibited fibrinogen-mediated microglia activation (Fig. 2 A). Quantitation showed 71 ± 4.9% of activated microglia in fibrinogen-treated cells versus 18 ± 0.7% in cells treated with fibrinogen in the presence of M1/70 (Fig. 2 B; P < 0.01). The inhibitory effect of M1/70 was specific for fibrinogen and did not affect LPS-mediated activation of microglia (Fig. 2 B). Moreover, blocking CD11b reduced phagocytosis of fibrinogen-treated cells by 40% (Fig. 2 C; P < 0.05). Previous studies have established that the Toll-like receptor (TLR)4 LPS receptor mediates LPS-induced microglia activation (30, 31). In contrast, blocking TLR-4 did not affect fibrinogen-mediated phagocytosis, suggesting that Mac-1 was the major receptor to induce fibrinogen-mediated phagocytosis (Fig. 2 C).

RhoA and phosphoinositide 3-kinase (PI3K) are the two major signaling pathways downstream of Mac-1 that mediate the cytoskeletal rearrangements for the induction of phagocytosis (28, 32, 33). Fibrinogen induced a 1.9-fold increase of active RhoA and a 24-fold increase in Akt phosphorylation in microglia (Fig. 2 D). Furthermore, blocking the PI3K signaling pathway using LY294002 inhibited the fibrinogen-induced increase of phagocytosis in microglia (Fig. 2 C; P < 0.01), suggesting that PI3K is downstream of fibrinogen–Mac-1 signaling in microglia cells. This is the first evidence suggesting that fibrinogen can induce Mac-1–dependent signaling in microglia.

Analysis of demyelinated spinal cords after the induction of proteolipid protein (PLP)_139-151 experimental autoimmune encephalomyelitis (EAE) in mice showed that CD11b⁺ microglia (Fig. 2 E, green) were surrounded by fibrin (Fig. 2 E, red). Similarly, analysis of acute demyelinating lesions of human MS showed that fibrin (Fig. 2 F, green) surrounded activated microglia cells (Fig. 2 F, red). Collectively, these results show that fibrinogen/CD11b signaling induces phagocytosis in microglia and demonstrate both in EAE and in human MS the presence of this ligand–receptor system on active microglia within inflammatory demyelinating lesions.

Fibrin depletion inhibits microglia activation in vivo and attenuates inflammatory demyelination

We further examined whether fibrin is required for the activation of Mac-1⁺ cells in vivo. We administered the anti-coagulant ancrod (34) in an established remitting-relapsing model of PLP_139-151 EAE (35) after the development of the first paralytic incidence. At the time of the first relapse, untreated mice show dramatic activation of CD11b⁺ cells characterized by thick processes (Fig. 3 A, left, green). In contrast, in ancrod-treated mice, CD11b⁺ cells appear with ramified morphology (Fig. 3 A, right, green) resembling CD11b⁺ cells in the normal spinal cord (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20061931/DC1). Single channel images are shown in Fig. S1. Untreated mice showed extensive inflammatory demyelinating lesions in the cerebellum (Fig. S2, asterisk) and spinal cord (Fig. S2, arrows). In contrast, in ancrod-treated mice, inflammatory demyelination was dramatically decreased (Fig. S2). Ancrod treatment resulted in decreased demyelination by sevenfold in the cerebellum and by twofold in the spinal cord. Splenocytes isolated from untreated and ancrod-treated mice showed no difference in proliferation upon stimulation with PLP_139-151 (Fig. S3)_, further suggesting that CD11b⁺ cells are the major cell target of fibrin in the nervous system. Fibrin-depleted mice recovered faster from the first paralytic incidence and in contrast to control mice never relapsed (Fig. 3 B). In a rotarod behavioral test performed before the first relapse of the untreated group, fibrin-depleted mice showed a threefold increase of motor strength and coordination when compared with the untreated group (Fig. 3 C). Control nonimmunized mice show values of ∼500 s on this assay. Collectively, these results suggest that fibrin is a major contributor to the local activation of CD11b⁺ cells in vivo. In addition, this is the first demonstration that an anti-coagulant may improve clinical symptoms and reduce inflammatory demyelination when administered after the onset of paralytic symptoms.

Fib^γ390-396A mice have reduced clinical scores and CNS inflammatory lesions

To examine the contribution of fibrinogen–Mac-1 signaling in vivo, we used mice with a knock-in mutation of seven amino acids at residues (N³⁹⁰RLSIGE³⁹⁶) to alanine residues (A³⁹⁰AAAAAA³⁹⁶) that abolishes fibrinogen binding to the Mac-1 receptor (15). These mice, termed Fib^γ390-396A, show decreased Mac-1–mediated neutrophil adhesion resulting in decreased bacterial clearance (15). Fib^γ390-396A and their age- and sex-matched littermate controls (Fib^γWT) were backcrossed six generations to C57BL/6 background (15) and immunized with myelin oligodendrocyte glycoprotein (MOG)_35-55 peptide (Fig. 4 A). Fib^γ390-396A mice (n = 29) showed an average clinical score of 2.1 ± 0.32 (ataxia) at day 20 after immunization, whereas the Fib^γWT mice (n = 28) developed clinical symptoms of paralysis, showing an average score of 3.3 ± 0.22 (hind limb paralysis; P < 0.01). On day 20, in the Fib^γWT mouse group, 74% of Fib^γWT mice (14/19) were paralyzed, in contrast to 35% of Fib^γ390-396A mice (7/20; clinical score > 3; Fig. 4 B), further demonstrating decreased severity of EAE. Fib^γ390-396A mice showed a 1.5-fold increase in motor skills when compared with Fib^γWT mice (Fib^γ390-396A, 269 ± 9 s vs. Fib^γWT, 169 ± 23 s; P < 0.05; Fig. 4 E). Inflammatory lesions were decreased threefold in Fib ^γ390-396A mice when compared with Fib ^γWT (Fig. 4 C; P < 0.05). Demyelination was reduced in Fib ^γ390-396A mice (1.57 ± 0.89%) when compared with Fib ^γWT (4.17 ± 0.83%; P < 0.04). In accordance, spinal cord sections showed a decrease of IsoB4⁺ cells in Fib ^γ390-396A mice when compared with Fib ^γWT controls (Fig. 4 F). Decrease in clinical severity was also observed at later time points after the immunization (Fig. 4, A and B). In addition, 25% of Fib ^γWT mice (7/28) died compared with only 3% of the Fib ^γ390-396A mice (1/29; Fig. 4 D; P < 0.03). Collectively, these results suggest that the γ^390-396 binding site of fibrinogen to Mac-1 regulates the severity of EAE.

The fibrin γ^377-395 peptide blocks microglia activation in vitro

Both the in vitro (Fig. 2) and in vivo experiments (Fig. 4) show that fibrinogen signaling through the Mac-1 receptor contributes to microglia activation and regulates the severity of EAE. Biochemical studies identified that the γ³⁷⁷YSMKKTTMKIIPFNRLTIG³⁹⁵ peptide blocks fibrin binding to Mac-1 (29). The γ^377-395 is a cryptic epitope in the fibrinogen molecule and is exposed only when fibrinogen is immobilized or converted to fibrin (25, 36). We used 200 μM of peptide, a concentration shown to inhibit adhesion of Mac-1–overexpressing cells to immobilized fibrinogen (37), to examine whether the γ^377-395 peptide could inhibit microglia activation. Fibrinogen treatment resulted in 71 ± 4.9% of activated microglia when compared with 38.3 ± 9.8% after the addition of the γ^377-395 peptide (Fig. 5, A and B; P < 0.05), suggesting that the γ^377-395 peptide reduced fibrin-mediated microglia activation. The γ^377-395 peptide did not affect the activation state of untreated microglia and did not inhibit LPS-mediated microglia activation (Fig. 5 B), further suggesting the specificity of γ^377-395 to block the activation of Mac-1 by fibrin. In accordance, examination of Akt phosphorylation showed that the γ^377-395 peptide could specifically inhibit fibrin-mediated and not LPS-mediated phosphorylation of Akt (Fig. 5 C). Overall, these results suggest that inhibition of fibrin–Mac-1 interactions by the γ^377-395 peptide inhibits both the morphological activation and the signaling cascade induced by fibrin-mediated activation of the Mac-1 microglia receptor.

Prophylactic or therapeutic administration of the fibrin γ^377-395 peptide suppresses EAE and inhibits microglia activation in vivo

Because genetic studies showed that the γ^377-395 binding epitope of fibrinogen to Mac-1 is not involved in coagulation (15) but is sufficient to inhibit fibrin-mediated microglia activation (Figs. 4 and 5), we examined whether blocking exclusively the inflammatory properties of fibrinogen in vivo using the γ^377-395 would be sufficient to ameliorate EAE. We therefore examined the effects of in vivo administration of the γ^377-395 fibrin peptide on microglia activation and clinical progression in an animal model for MS. We first assessed the effects of vaccination against γ^377-395 peptide. Vaccination with γ^377-395 peptide before the induction of EAE resulted in a significant reduction in disease penetrance and clinical symptoms. All control mice (15/15) developed clinical symptoms of EAE. In contrast, only 53% of the vaccinated mice (8/15) developed EAE. Moreover, mice vaccinated with the γ^377-395 peptide showed an average clinical score of 1.1, whereas the control group showed a score of 2.5 (Fig. 6 A; P < 0.01). In a rotarod test, vaccinated mice showed a 76% increase in motor strength and coordination when compared with the control mice. Quantitative histopathological analysis showed reduced inflammatory lesions in the cerebellum (index of 6.5 ± 5.9 vs. 12 ± 5.2; P < 0.05) and spinal cord (index of 1.5 ± 2 vs. 2.3 ± 1; P < 0.01) from γ^377-395 peptide–vaccinated mice as compared with control mice.

Because vaccination is a preventive treatment, we further assessed whether γ^377-395 peptide would be beneficial if administered after the onset of disease. We administered intranasally 30 μg γ^377-395 peptide daily after the first paralytic episode in remitting relapsing EAE (35). Intranasal delivery is a noninvasive delivery method that results in a higher degree of drug delivery to the nervous system and a lower degree of systemic drug delivery to tissues such as liver and lymph nodes when compared with intravenous delivery (38). Intranasal delivery has been previously shown to be effective as a method of drug delivery in EAE (39-41). γ^377-395 peptide–treated mice (n = 14) compared with control mice (n = 13) did not relapse (Fig. 6 B). Peptide-treated mice showed a 1.4-fold increase of motor functions when compared with the control mice after the first relapse on day 29. Administration of scramble peptide had no effect on the progression of EAE (Fig. S5, available at http://www.jem.org/cgi/content/full/jem.20061931/DC1).

Immunohistochemical analysis using Mac-3, a microglia/macrophage marker, revealed reduction of activated cells in the peptide-treated (Fig. 6 D) as compared with control animals (Fig. 6 C). High magnification images of Mac-3 immunostaining are shown in Fig. S6, which is available at http://www.jem.org/cgi/content/full/jem.20061931/DC1. In addition, inducible nitric oxide synthase (iNOS), a major product of activated CD11b⁺ microglia in EAE (42), was reduced in the γ^377-395 peptide–treated animals (Fig. 6 D, inset). Quantitation of the histopathological analysis shows reduction in both Mac-3 (P < 0.05) and iNOS (P < 0.01), whereas there are no major differences in T cell infiltrates (Fig. 6 E; P = 0.48). We further examined whether the γ^377-395 peptide affected the peripheral immune response. FACS analysis on the splenocytes of mice immunized with PLP_139-151 using six markers for peripheral immune cells, namely CD4 and CD8 T cells, CD11b macrophages, CD11c dendritic cells, and CD19 and B220 B cells, did not reveal any differences between untreated and γ^377-395 peptide–treated animals (Fig. S4), suggesting that similar to systemic fibrin depletion (Fig. S3), the γ^377-395 peptide does not affect the peripheral immune response. These results are in accordance with prior studies in which depletion of macrophages (43) or microglia (3) resulted in dramatic reduction of clinical symptoms in EAE even in the presence of functional T cells.

The fibrin γ^377-395 peptide does not affect the coagulation properties of fibrinogen

Several studies have demonstrated both in vivo and in vitro that fibrinogen interacts with different cellular receptors via non overlapping epitopes (for review see reference 16). Fibrinogen regulates blood coagulation by engaging the platelet α_IIbβ₃ integrin receptor via its γ^408-411 epitope, whereas it mediates inflammatory processes by engaging the Mac-1 receptor via its γ^377-395 epitope. As a result, fibrinogen knock-in mice, where the γ^390-396 Mac-1 binding site has been mutated, show normal coagulation properties, such as platelet aggregation, thrombus formation, and clotting time (15). To determine whether the γ^377-395 peptide interfered with blood coagulation, we examined its effects on the procoagulant properties of fibrinogen both in vivo and in vitro. As expected, the γ^377-395 peptide does not affect coagulation in mice (Fig. 7 A) or prothrombin time (Fig. 7 B). Moreover, in an in vitro fibrin polymerization assay, the γ^377-395 peptide did not alter the polymerization time of fibrin (Fig. 7 C). In contrast, the GPRP peptide, an established inhibitor of clot formation (44), inhibits fibrin polymerization (Fig. 7 C). Collectively, these results show that in vivo delivery of the γ^377-395 peptide reduces the progression and severity of EAE by specifically targeting microglia/macrophage activation in the CNS parenchyma without adverse hemorrhagic effects.

Animals.

6-wk-old female SJL/J and C57BL/6 mice were purchased from The Jackson Laboratory or Harlan Sprague Dawley. 6-wk-old female Fib ^γ390-396A mice were generated from double heterozygous matings to produce homozygous Fib ^γ390-396A mice (15) and Fib ^γWT littermate mice as controls. Fib ^γ390-396A mice were backcrossed six generations to C57BL/6 mice (15). All experiments were approved by the University of California, San Diego, Institutional Animal Care and Use Committee.

Induction of EAE.

EAE was induced in 6-wk-old female SJL/J or C57BL/6 mice by subcutaneous immunization with 150 μg PLP_139-151 (HSLGKWLGHPDKF; American Peptide Company and Azco Pharmchem) or 50 μg MOG_35-55 (MEVGWYRSPFSRVVHLYRDGK; Sigma-Aldrich) in complete Freund's adjuvant (Sigma-Aldrich) supplemented with 200 ng of heat-inactivated mycobacterium tuberculosis H37Ra (Difco Laboratories). Mice were injected intravenously with 200 ng pertussis toxin (Sigma-Aldrich) on days 0 and 2 of the immunization. Mice were scored daily as follows: 0, no symptoms; 1, loss of tail tone; 2, ataxia; 3, hindlimb paralysis; 4, hindlimb and forelimb paralysis; 5, moribund. Data are represented as the mean clinical score and are mean ± SEM, and the Mann-Whitney test was used for statistical analysis. For the survival curve, statistical analysis was performed with the log-rank test using PRISM software.

Systemic defibrinogenation.

Mice were depleted of fibrinogen as described previously (65). The pumps deliver 0.5 μl/hour; thus, the mice received 2.4 U ancrod/day. In control animals, buffer-filled mini-pumps were implanted.

Fibrinogen γ^377-395 peptide vaccination.

Vaccination was performed as described previously (66) with the following modifications. 5-wk-old female SJL/J mice were immunized with 200 μg fibrinogen γ^377-395 peptide (YSMKETTMKIIPFNRLSIG; Azco Pharmchem) emulsified with an equal volume of incomplete Freund's adjuvant (Sigma-Aldrich). Mice were immunized four times in alternating rear flanks over the course of 2 wk. Control animals were immunized with incomplete Freund's adjuvant.

Intranasal γ^377-395 peptide administration.

Fibrinogen γ^377-395 peptide (YSMKETTMKIIPFNRLSIG; Azco Pharmchem) or scramble peptide (KMMISYTFPIERTGLISNK; Azco Pharmchem) was resuspended in 0.9% NaCl at a concentration of 3 mg/ml. Peptides were aliquoted as single doses and stored at −20°C. Mice were administered 5 μl peptide or 0.9% NaCl as a control in each nare daily using a P10 micropipettor (Gilson) beginning after the peak of the first paralytic episode.

Histopathology.

Histopathologic analysis and quantitation of inflammation and demyelination in mouse tissue were performed on cryostat or paraffin sections as described previously (67). Sections were stained with hematoxylin/eosin and luxol fast blue/nuclear red. Spinal cord sections were fixed with 2% paraformaldehyde for 10 min at 4°C and immunostained with a sheep anti-fibrinogen antibody (1:200; US Biological), rat anti-CD11b (1:5; Chemicon), von Willebrand Factor (1:1,000; DakoCytomation), iNOS (polyclonal; 1:750; Chemicon), Mac-3 (rat anti–mouse; 1:200; BD Biosciences), and CNPase (monoclonal; 1:2,000; Sternberger Monoclonals). The inflammatory index is defined by the average number of inflammatory blood vessels per spinal cord cross section. 10–15 cross sections per animal were counted by two observers blinded to the genotypes of the mice. For the cerebellum, the number of cuffs was quantified in two cerebellar sections per animal. To additionally analyze the CNS lesions, we counted Luxol Fast Blue/nuclear red–stained sections, and the relative proportions of demyelinated areas (percentage with SEM) were determined using an ocular morphometric grid as described previously (67). For human MS, paraffin-embedded material was obtained from the Archives of the Center for Brain Research, Medical University of Vienna. Double immunofluorescence was performed with antibodies against CD68 and fibrin. Images were collected using an Axioplan 2 Zeiss microscope with an Axiocam HRc camera or were processed for confocal microscopy using Olympus and Zeiss confocal microscopes.

Rotarod behavior test.

The rotarod assay was performed on a TSE RotaRod apparatus (TSE Technical and Scientific Equipment GmbH) as described previously (68). Rotarod assays were conducted with a 350-s maximum time limit, and means were collected for at least three trials. Statistical calculations were made by using Student's t test.

Culture of primary microglia cells.

Microglia were isolated from cultures of mixed cortical cells as described previously (69). In brief, cortices from P1 mice were isolated and digested with trypsin (0.4%) for 20 min at 37°C. Cells from four pups were plated onto poly-D-lysine–coated 75 cm² tissue culture flasks. The culture medium (DMEM, 10% heat-inactivated FBS, and 1% Pen/Strep) was changed on day 2. After 2–3 wk in culture, the microglia were removed by the addition of 12 mM lidocaine (Sigma-Aldrich) and orbital shaking (180 rpm) for 20 min at 37°C. The cells were centrifuged (350 g), and the pellet was resuspended in complete medium and plated onto poly-D-lysine–coated tissue culture dishes. Microglia cell cultures were >95% pure as determined with three different markers, IsoB4, IBA-1, and CD11b. To immobilize fibrinogen, tissue culture dishes were incubated overnight with 50 μg/ml fibrinogen (Calbiochem) in 20 mM Tris, pH 7.5, and 0.1 M NaCl at 37°C for all fibrinogen treatments. To serve as positive controls, LPS (Sigma-Aldrich) was added to the media at 1 μg/ml.

Phagocytosis assay.

Phagocytosis assays were performed on both primary murine microglia and a murine microglia cell line (BV2). BV2 cells are an established cell line used to study microglia responses (47). BV2 cells were routinely passaged in DMEM, 20% heat-inactivated FBS, and 1% Pen/Strep. Microglia phagocytosis was assessed using the Vybrant phagocytosis assay kit (Invitrogen) according to the manufacturer's recommendations. Microglia were cultured in 96-well plates at 5,000 cells/well for 36 h at 37°C. Blocking experiments included the addition of 1 μM LY294002 (Cell Signaling), 10 μg/ml rat anti-CD11b (eBioscience), 10 μg/ml rat anti-TLR4 (eBioscience), or 10 μg/ml rat IgG (Jackson ImmunoResearch Laboratories) to the media. Results were obtained from four separate experiments performed in triplicates. Statistical calculations were made by using Student's t test.

Morphometry.

Primary microglia were used for all morphologic analysis. Cells were plated on coated fibrinogen or in the presence of LPS for 72 h. Microglia were fixed with methanol and immunostained with Isolectin B4 (1:300; Sigma-Aldrich). Blocking experiments involved the daily administration of 10 μg/ml rat anti-CD11b (eBioscience), 10 μg/ml rat IgG (Jackson ImmunoResearch Laboratories), 200 μM γ^377-395 peptide, or 200 μM of scramble peptide. Results were obtained from six separate experiments performed in duplicates. 250 cells per condition were counted for each individual experiment. Activated microglia were quantitated as those cells >2,000 μm². Images were collected using an Axioplan 2 Zeiss microscope with an Axiocam HRc camera and analyzed using the Axiovision 4.5 program (Carl Zeiss). Quantitation of cell area was performed using the Interactive Measurement feature of the Axiovision 4.5 program (Carl Zeiss MicroImaging, Inc.). Statistical calculations were made by using Student's t test.

Endotoxin detection assay.

Fibrinogen samples were tested for contaminating endotoxins by a Limulus Amebocyte Lysate assay (E-TOXATE kit; Sigma-Aldrich). Fibrinogen-treated samples had undetectable endotoxin levels (<0.5 endotoxin units).

Immunoblots.

Western blots were performed using standard protocols (65). Microglia were serum-starved overnight and plated on fibrinogen or with LPS for 6 h. Lysates were electrophoresed on 4–12% gradient SDS-PAGE gels and probed with phospho and total Akt antibodies (1:1,000; Cell Signaling). RhoA activation was performed as described previously (70). In brief, 2 × 10⁷ microglia were plated on fibrinogen for 6 h or with LPS for 10 min. Cell lysates were incubated with GST-Rhotekin agarose beads (provided by J. Heller Brown, University of California, San Diego, San Diego, CA) for 45 min at 4°C. The beads were washed, resuspended in Laemmli sample buffer, and electophoresed on a 15% SDS-PAGE gel. Activated and total Rho were detected with mouse anti-RhoA (1:500; Santa Cruz Biotechnology, Santa Cruz Biotechnology, Inc.). Densitometry was performed using the National Institutes of Health (NIH) Scion Image software using three blots from three separate experiments.

Deconvolution microscopy.

Fluorescent images were obtained as described previously (70). Primary microglia were stimulated with fibrinogen as described above. Cells were immunostained with antibodies to total actin (1:100; Sigma-Aldrich) or β-tubulin (1:100; Sigma-Aldrich).

Isolation of mouse splenocytes and T cell proliferation assay.

Spleens were removed from control and fibrin-depleted mice after the induction of PLP_139-151 EAE and washed in PBS. Spleens were mechanically dissociated with sterile blades and filtered through 70-μm nylon screens. Splenocytes were pelleted and washed with an erythrocyte lysing solution (Biolegend). Cells were washed twice with PBS, counted, and seeded at a density of 0.5 × 10⁶ cells/96 wells. Cells from both control and fibrin-depleted mice were either untreated or stimulated with 20 μg/ml PLP_139-151, and proliferation was assayed by BrdU incorporation (Roche Applied Sciences) according to the manufacturer's instructions.

FACS analysis.

Primary mouse splenocytes were isolated from control and γ^377-395 peptide–treated mice immunized with PLP_139-151 on day 29 after immunization. Cells were immunostained with antibodies to CD4, CD8, CD11b, CD11c, CD19, and B220 (1:100; Biolegend) and analyzed on a Becton Dickinson FACSCalibur flow cytometer.

Hematological analyses.

Citrated plasma was prepared from mice administered intranasally 30 μg or 90 μg γ^377-395 peptide or saline control daily for 7 d. Plasma clotting times were measured by combining 10 μl of citrated plasma with 10 μl of 2U/ml bovine thrombin (Enzyme Research Laboratories) and 40 mM CaCl₂ in a weigh boat floating in a 37°C water bath. Time to clot was determined by mixing with a toothpick. Plasma thrombin times were established as described previously (15). Fibrin polymerization was evaluated by standard turbidity assays using plasma. In brief, citrate plasma (diluted tenfold in 20 mM Hepes, pH 7.4, containing 0.15 M NaCl and 5 mM ɛ-amino caproic acid) was combined with bovine thrombin (final concentration, 0.2 U/ml; Enzyme Research Laboratories) and Ca²⁺ (10 mM), and OD₃₅₀ measurements were taken every 30 s.

Online supplemental material.

Fig. S1 shows low power images of CD11b and fibrinogen double immunofluorescence that demonstrates that fibrin depletion using ancrod decreases microglia activation in EAE. Fig. S2 shows Luxol Fast Blue staining that reveals that fibrin depletion using ancrod reduces demyelinating lesions in EAE. Fig. S3 shows that there is no difference in proliferation under untreated or PLP_139-151–stimulated conditions between control and ancrod-treated mice subjected to PLP_139-151 EAE. Fig. S4 shows FACS analysis of splenocytes using the markers CD4, CD8, CD11b, CD11c, CD19, and B220 that reveals that the γ^377-395 peptide has no significant effect on peripheral immune cells compared with control. Fig. S5 shows that a scramble γ^377-395 peptide has no effect on progression of EAE. Fig. S6 shows high power images of Mac3 immunostaining in the spinal cord of untreated and γ^377-395 peptide–treated mice after the induction of EAE. The online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20061931/DC1.