Journal of Neuroimmunology 162 (2005) 122 – 129 www.elsevier.com/locate/jneuroim
CCL2 and CCL5 mediate leukocyte adhesion in experimental autoimmune encephalomyelitis—an intravital microscopy study Adriana Carvalho dos Santosa, Michele Mendes Barsanteb, Rosa Maria Esteves Arantesc, Claude C.A. Bernardd, Mauro Martins Teixeirab, Juliana Carvalho-Tavaresa,T b
a Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Department of Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil c Department of Pathology, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil d La Trobe University, Victoria, Australia
Received 13 December 2004; received in revised form 31 January 2005; accepted 31 January 2005
Abstract Experimental autoimmune encephalomyelitis (EAE) models multiple sclerosis (MS) and is characterized by marked mononuclear cell influx in the brain. Several studies have demonstrated a role for chemokines during EAE. It remains to be determined whether these mediators modulate EAE primarily by mediating leukocyte influx into the CNS or by modifying lymphocyte activation and/or trafficking into lymphoid organs. After induction of EAE with MOG35–55, leukocyte recruitment peaked on day 14 and correlated with symptom onset, TNFa production and production of CCL2 and CCL5. Levels of CXCL-10 and CCL3 were not different from control animals. Using intravital microscopy, we demonstrated that leukocyte rolling and adhesion also peaked at day 14. Treatment with anti-CCL2 or anti-CCL5 antibodies just prior to the intravital microscopy prevented leukocyte adhesion, but not rolling. Our data suggest that induction of leukocyte adhesion to the brain microvasculature is an important mechanism by which CCL2 and CCL5 participate in the pathophysiology of EAE. D 2005 Elsevier B.V. All rights reserved. Keywords: Leukocyte adhesion; Brain; Chemokines; Intravital microscopy
1. Introduction The central nervous system is considered an immunologically privileged site due to the presence of the endothelial blood–brain barrier (BBB), which, under physiological conditions, protects against leukocyte traffic into the CNS. However, during inflammatory conditions of the CNS, such as Multiple Sclerosis (MS), large numbers of mononuclear cells gain access to the CNS (Ransohoff et al., 2003). Indeed, previous studies have shown that, whereas resting cells do not breach the BBB, activated T lymphocytes can rapidly cross this lining regardless of their Ag specificity (Piccio et al., 2002). However, only T cells that
T Corresponding author. Tel.: +55 31 3499 2943; fax: +55 31 3499 2924. E-mail address:
[email protected] (J. Carvalho-Tavares). 0165-5728/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2005.01.020
recognize the CNS Ag persist and are able to recruit other inflammatory cells (Piccio et al., 2002). MS is the most common non-traumatic, disabling neurological human inflammatory demyelinating disease of the CNS, affecting most commonly young adults (Noseworthy et al., 2000). Experimental autoimmune encephalomyelitis (EAE) tries to model human MS and is characterized by a CD4+ Th1 cell-mediated autoimmune disease of the CNS (Bradl and Hohlfeld, 2003). To migrate into sites of inflammation, leukocytes must first tether and roll along the vessel before they firmly adhere and emigrate out of the vasculature (McCafferty et al., 2000; Kerfoot and Kubes, 2002). Firm adhesion is mediated by the expression of adhesion molecules and their ligands on the surface of leukocytes and endothelium cells but is triggered by the action of chemoattractant molecules, such as chemokines, mediated by specific
A.C. dos Santos et al. / Journal of Neuroimmunology 162 (2005) 122–129
receptors on the leukocyte surface (Fife et al., 2000). In the context of MS, chemokines have been detected within the CNS and within active plaques lesions of MS patients, suggesting that these molecules contribute to demyelination by attracting targeted populations of leukocytes into the CNS (Glass et al., 2004). Moreover, several groups have now shown selective and differential expression and functional role of chemokines in different models of EAE (Fife et al., 2001; Juedes et al., 2000; Denkinger et al., 2003; Ambrosini et al., 2003; Glass et al., 2004). Whereas it is clear that chemokines are important for the development of CNS inflammation in experimental models of EAE, it remains to be determined whether these mediators modulate EAE primarily by mediating leukocyte influx into the CNS or by modifying lymphocyte activation and/ or trafficking into lymphoid organs. Previous studies have shown that leukocytes roll and adhere to the microcirculation of the CNS after injection of cytokines and after EAE induction (Piccio et al., 2002; Carvalho-Tavares et al., 2000). The present study was carried out to examine the possible involvement of chemokines in the trafficking of leukocytes into the microcirculation of the CNS in a model of MOG35–55induced EAE in mice. To this end, the concentrations of CCL2, CCL3, CCL5 and CXCL-10 were evaluated at different times after EAE induction and correlated to the inflammatory infiltrate, leukocyte rolling and adhesion and TNF-a production. These chemokines shown to be involved in EAE induction (Juedes et al., 2000; Tran et al., 2000; Rottman et al., 2000; Mahad and Ransohoff, 2003; Klein et al., 2004), as the expression of CCL2 and CCL5 was enhanced after EAE induction in our model, the role of these proteins for leukocyte rolling and adhesion was evaluated using specific antibodies.
2. Materials and methods 2.1. Animals Female C57BL/6 mice were obtained from Animal Care Facilities of Universidade Federal de Minas Gerais (UFMGBrazil), between 9 and 10 weeks of age. The Animal Ethics Committee of UFMG approved all experimental procedures used. 2.2. Reagents and antibody MOG peptide, sequence 35–55 (MEVGWYRSPFSRVVHLYRNGK; Auspep) was a gift from Dr. Claude Bernard (La Trobe University, Australia). Methylated bovine serum albumin (MBSA), o-phenylenediamine (OPD), and Freund’s complete adjuvant (CFA) were purchased from Sigma Chemical Co. Specific polyclonal antibodies, anti-CCL2 and antiCCL5 were a kind gift of Dr. Steve Kunkel (University of
123
Michigan, Ann Arbor). These antibodies are effect blockers of the function of their respective proteins and have been tested in several in vivo systems (Lukacs et al., 1995; Chensue et al., 1999; Coelho et al., 2002). 2.3. EAE induction EAE was induced by s.c. immunization (base of tail) with an emulsion containing 100Ag MOG35–55 peptide and CFA supplemented with 4 mg/ml Mycobacterium tuberculosis H37 RA (Difco Laboratories). Pertussis toxin, 300 ng/animal (Sigma) was injected i.p. on the day of immunization and again 48 h later. Animals were monitored daily and neurological impairment was quantified on an arbitrary clinical scale. 2.4. Clinical assessment of EAE The mice were weight before and on 7, 14 and 21 days post-immunization. The clinical status of animals was monitored daily as previously described (Kerfoot and Kubes, 2002). Scores were: 0=no clinical signs, 1=tail paralysis (or loss of tail tone), 2=tail paralysis and hind-limb weakness, 3=hind-limb paralysis, 4=complete hind-limb paralysis and front limb weakness. 2.5. Intravital microscopy in mouse brain Intravital microscopy of the mouse cerebromicrovasculature was performed as previously described (CarvalhoTavares et al., 2000). Briefly, the mice were anesthetized by intraperitoneal injection of a mixture of 150 mg/kg Ketamine and 10 mg/kg Xylazine and the tail vein was cannulated for administration of fluorescent dyes. A craniotomy was performed using a high-speed drill (Dremel, USA) and the dura matter was removed to expose the underlying pial vasculature. Throughout the experiment, the mouse was maintained at 37 8C with a heating pad (Fine Science Tools Inc., Canada) and the exposed brain was continuously superfused with artificial cerebrospinal fluid buffer, an ionic composition in mmol/L: NaCL 132, KCL 2.95, CaCL2 1.71, MgCL2 0.64, NaHCO3 24.6, dextrose 3.71 and urea 6.7, pH 7.4, at 37 8C. To observe leukocyte/endothelium interactions, leukocytes were fluorescently labeled by i.v. administration of rhodamine 6G (0.5 mg/kg body weight) and observed using a microscope (Olympus B201, X20 objective lens, corresponding a 100 Am of area) outfitted with a fluorescent light source (epi-illumination at 510–560 nm, using a 590-nm emission filter). A silicon-intensified camera (Optronics Engineering DEI-470) mounted on the microscope projected the image onto a monitor (Olympus). Rolling leukocytes were defined as white cells moving at a velocity less than that of erythrocytes cells. Leukocytes were considered adherent to the venular endothelium if they remained stationary for 30 s or longer.
A.C. dos Santos et al. / Journal of Neuroimmunology 162 (2005) 122–129
To determine whether leukocyte recruitment in MOG3555-induced EAE was mediated by chemokines, animals were treated with an injection i.p. (300 Al/animal) of rabbit anti-murine MCP-1/CCL2 Ab or anti-murine RANTES/ CCL5 Ab (Coelho et al., 2002), 2 h before craniotomy. Control animals received non-immune rabbit serum. 2.7. Histology Brains were quickly removed after intravital microscopy and preserved in 10% formalin. The sections were stained with hematoxylin and eosin (H&E) and analyzed for CNS inflammation. To exclude the possibility that neutrophils were migrating into the CNS after EAE induction, neutrophil accumulation in the CNS was assessed by measuring myeloperoxidase (MPO) activity. 2.8. ELISA of proteins in the CNS Brain tissue extracts were obtained from control and experimental mice that were sacrificed at 7, 14 and 21 days after immunization. Brains were removed after intravital microscopy, and left and right hemispheres were stored on ice. Thereafter, each hemisphere was homogenized in extraction solution (100 mg of tissue per 1 ml), containing: 0.4 M NaCL, 0.05% Tween 20, 0.5% BSA, 0.1 mM Fenil metil sulfonil fluoride, 0.1 mM benzetonio chloride, 10 mM EDTA and 20 KI aprotinin, using Ultra-Turrax. Brain homogenate was spun at 10.000 rpm for 10 min at 4 8C and supernatants were collected and stored at 70 8C. The concentration of TNF-a, MCP-1, RANTES, IP-10 and MIP1a was determined using ELISA. The supernatants of brain extraction, at a 1:3 dilution in 1% BSA in PBS, were assayed in an ELISA set-up using commercially available antibodies and the concentrations according to the procedures supplied by the manufacturer (R&D Systems, Minneapolis, MN and Pharmingen, San Diego, CA). 2.9. Statistical analysis Data is shown as meanFSEM. ANOVA parametric test with Bonferroni correction was used for multiple comparisons. Statistical significant was set at Pb0.05.
3. Results
there was no worsening of symptoms till day 21 (Fig. 1A). Tail paralysis and hind limb weakness were the major clinical feature noted. Paralleling the clinical symptoms above, there was significant weight loss which peaked at day 14 after disease induction (Fig. 1B). In H&E stained brain sections, there was a marked inflammation in perivascular area and the major infiltrating leukocytes were mononuclear cells around and within the pial venules of EAE mice. The leukocyte influx was absent in control mice. Neutrophils, the major migrating leukocyte in intravital microscopy experiments (Cara et al., 2001), were not observed in brain sections. In agreement with the latter observation, evaluation of neutrophil number by MPO analysis revealed no significant neutrophil influx in EAE mice (data not shown). 3.2. Kinetics of leukocyte recruitment in the pial microvasculature of EAE mice The leukocyte/endothelium interactions in the pial microcirculation of EAE mice were evaluated using intravital microscopy. Initial experiments evaluated leukocyteendothelial cell interactions at days 7, 14 and 21 after EAE induction. Leukocyte rolling was already apparent at day 7 but peaked on day 14 after induction (33.3F3.1 leukocyte rolling/min) (Fig. 2A). At day 21, the number of leukocyte
A 4
Clinical score
2.6. Specific chemokine neutralization
3 2 1 0 7
14
B 25 20 15 10 5 0 0
3.1. Kinetics of inflammatory changes in EAE mice The severity of MOG35–55-induced EAE was assessed daily using a previously validated scale (Kerfoot and Kubes, 2002). Clinical evidence of disease was first noticed at day 10 and peaked at day 14 after immunization. Thereafter,
21
Days post-immunization
weight baseline
124
7
14
21
Days post-immunization Fig. 1. Mice (n=15) were monitored about clinical signs (A) and body weights loss (B). This kinetics study includes the time points 7, 14 and 21 days post-induction of EAE with MOG 35-55. Data is according to maximal clinical score achieved at any time during experimental design and is based on scale previous described.
A.C. dos Santos et al. / Journal of Neuroimmunology 162 (2005) 122–129
A
3.4. EAE-induced leukocyte-endothelium interaction is dependent on CCL2 and CCL5 chemokines
leukocyte rolling/min
40
**
30
*
*
20 10 0 sham
7d
14d
21d
days post-immunization
B 10.0
leukocyte adherence/min
125
**
7.5 5.0
As leukocyte rolling and adhesion and production of CCL2 and CCL5 peaked at day 14, this time point was used in further experiments to evaluate the role of the two chemokines to leukocyte-endothelial cell interactions. The role of CCL2 was evaluated by injecting specific anti-CCL2 antisera 2 h before the intravital microscopy experiments. Blockade of MCP-1 resulted in a significant reduction of leukocyte adherence to the pia-mater vasculature of EAE mice (Fig. 4B). In contrast, neutralization of CCL2 was not able to modulate the rolling process observed at day 14 on pia-mater vessels of EAE mice (Fig. 4A). Inhibition of adhesion but not rolling was maintained even after 30 min of craniotomy (data not shown). The role of CCL5 was studied by injecting specific anti-CCL5 antisera 2 h before the intravital microscopy experiments. Visualization of brain microvasculature revealed a significant reduction by the antibody of leukocyte adherence, but not rolling, in relation to control (Fig. 4C,D).
2.5
4. Discussion 0.0 sham
7d
14d
21d
days post-immunization Fig. 2. Visualization of leukocyte-endothelium interaction during different time of disease progress. Intravital microscopy was used to assess the rolling (A) and firm arrest (B) of leukocytes on brain microvasculature. The protocol included sham group and EAE mice on days 7, 14 and 21 postimmunization. The recruitment was peaked on day 14 coinciding with time when clinical signs were more significant. Data indicate a mean (SEM of cells per minutes, and one-way ANOVA confirmed **Pb0.001.
rolling had diminished when compared to day 14. The kinetics of adhesive events following EAE induction was remarkably similar to the kinetics of leukocyte rolling and the number of events was maximal at day 14 (Fig. 2B). 3.3. Kinetics of cytokine and chemokine protein production in the CNS of EAE mice In addition to evaluating leukocyte-endothelial cell interactions, we evaluated the levels of TNF-a and chemokines in brain extracts of EAE mice at days 7, 14 and 21 after disease induction. In agreement with the clinical signs of disease from day 14 onwards, the concentration of TNF-a increased at day 14 and remained elevated till day 21 (Fig. 3A). The concentration of CCL2 and CCL5 followed a similar pattern to the number of leukocyte/endothelium interactions. Both chemokines were detected at similar levels to control mice at day 7, however, there was a marked elevation at day 14 and return to near background levels by day 21 (Fig. 3B,C). In contrast, the concentration of CCL3 and CXCL10 were not different in control or EAE mice (Fig. 3D,E).
A pathological hallmark of MS is the infiltration of immune cells across the endothelium of the blood brain barrier (BBB) and their subsequent entry into the CNS (Adams et al., 1989; Prineas and Wright, 1978). In the current study we used intravital microscopy to evaluate the functional contribution of chemokines for leukocyte rolling and adhesion in the brain microvasculature of EAE mice. Differential patterns of chemokine levels have been noted in patients with MS. Previous studies have shown the presence of CXCL10, CCL5, CCL3 and CCL2 in brain lesions and cerebrospinal fluid of MS patients (Balashov et al., 1999; Sorensen et al., 1999; Trebst and Ransohoff, 2001; Mahad and Ransohoff, 2003; Glass et al., 2004). Visualization of leukocyte-endothelial cell interaction in vivo in inflamed vessels has revealed that leukocytes must first tether and roll along the venular wall before they can attach firmly and emigrate out of the vasculature (Kerfoot and Kubes, 2002). Leukocyte rolling and adhesion in the cerebral microvasculature also occurs after cytokine treatment or EAE induction in mice (Carvalho-Tavares et al., 2000; Piccio et al., 2002). We observed a significant increase of the number of leukocyte rolling and adhesion on days 7, 14 and 21 after disease induction as compared to control mice. However, leukocyte rolling and adhesion peaked on day 14, correlating with the peak of the clinical symptoms and CNS inflammation. These results are consistent with the idea that trafficking of immune cells within the CNS may play an important role in the establishment and progression of disease. Successful leukocyte recruitment into the brain microcirculation in different inflammatory conditions depends of
126
A.C. dos Santos et al. / Journal of Neuroimmunology 162 (2005) 122–129
A
B CCL2 (pg/100mg brain tissue)
TNFα (pg/100mg brain tissue)
150 100 75 50 25 0
sham
7d
14d
* 100
50
0
21d
sham
C
14d
21d
D 300
CCL3 (pg/100mg brain tissue)
CCL5 (pg/100mg brain tissue)
7d
Days post-immunization
Days post-immunization
*** 200
* 100
0
sham
7d
14d
21d
750
500
250
0
sham
Days post-immunization
7d
14d
21d
Days post-immunization
E CXCL10 (pg/100mg brain tissue)
8000 6000 4000 2000 0
sham
7d
14d
21d
Days post-immunization Fig. 3. Kinetics of cytokine and chemokine protein production in the CNS of EAE mice. Protein concentrations were measured in brain extracts by ELISA on days 7, 14 and 21 after immunization with MOG 35-55. Assay was performed in EAE mice and sham group for TNF-((A), CCL2 (B), CCL5 (C), CCL3 (D) and CXCL10 (E). Results were expressed as the mean (SEM from five animals per group. Statistically significant differences were indicated by *Pb0.05 and **Pb0.01.
the expression of adhesion molecules (selectins, integrins) on leukocytes and their ligands on endothelial cells (VCAM-1, ICAM-1) (Tang et al., 1996; Kerfoot and Kubes, 2002; James et al., 2003). Although the mechanisms regulating leukocyte entry into the CNS are not well understood, previous evidences point to an important role for chemokines in up-regulating adhesion molecules expres-
sion on lymphocytes and facilitating the migration process (Babcock and Owens, 2003; Ransohoff, 2002; Tanaka et al., 1993). The observation that EAE mice showed leukocyte recruitment in vivo led us to assess the level of chemoattractants in brain extracts at distinct times after disease induction. These studies revealed a significant increase of CCL2 and CCL5, but not CCL3 and CXCL10, at day 14
A.C. dos Santos et al. / Journal of Neuroimmunology 162 (2005) 122–129
A leukocyte rolling/min
40 30
20 10
0 sham
EAE
anti-CCL2
B leukocyte adherence/min
20
***
10
*** 0 sham
EAE
anti-CCL2
C leukocyte rolling/min
40 30 20 10 0 sham
EAE
anti-CCL5
D leukocyte adherence/min
20
***
10
***
0 sham
EAE
anti-CCL5
Fig. 4. Effect of anti-CCL2 and anti-CCL5 antibody treatment on the recruitment of leukocyte in pial microvasculature. On day 14 postimmunization, EAE mice (n=7) were treated with specific mAb, 2 h before starting the intravital microscopy visualization. The blockade of MCP-1 (A,B) and RANTES (C,D) activity provoked a significant decrease on leukocyte adherence, and the rolling was not abrogate. Data was expressed as mean (SEM, indicating ***Pb0.001).
127
(peak of disease), suggesting these two chemokines could be playing an important role in mediating leukocyte rolling and adhesion during EAE. In order to evaluate the direct role of CCL2 and CCL5 in the rolling and adhesion observed in the EAE mice, we administrated neutralizing antisera just prior to the observation of leukocyte/endothelium interactions by intravital microscopy. The choice of this administration schedule was to allow the adequate development of the lesions in the brain and to evaluate the contribution of the chemokines solely to the rolling and adhesion processes. The findings presented clearly demonstrate that the neutralization of CCL2 was able to decrease adherence to endothelial cells without altering the rolling of cells. Similarly, the anti-CCL5 antibody resulted in a dramatic inhibition of leukocyte adhesion to the cerebral microvasculature. Altogether, our studies clearly demonstrate that both CCL2 and CCL5 are essential for the ability of leukocytes to adhere to brain microvessels. As adherence is an essential step for leukocyte migration into tissue, our results suggest that these two chemokines may play an important role in EAE by affecting the ability of leukocytes to migrate into the CNS. It is unclear why either of the two antibodies prevented adhesion almost completely. We do not have an explanation for the latter finding. However, one possibility to explain the need for both chemokines stems from our previous studies demonstrating the ability of different chemoattractants to cooperate in the induction of eosinophil migration in vivo (Klein et al., 2001). Based on the latter study, we hypothesize that relatively small amounts of both CCL2 and CCL5 are produced after EAE and synergize to induce a full-blown leukocyte recruitment. Such small quantities would be insufficient to induce leukocyte recruitment per se, but would induce so in the presence of the other chemoattractant. In such way, inhibition of one of the two chemokines would be sufficient for prevention of leukocyte influx. Further studies are necessary to confirm this hypothesis. We found that the concentration of CXCL10 in brain extracts of EAE mice was not different from the control group, suggesting that this chemokines may not play a major role in the induction of EAE in mice. Corroborating our data, Klein and cols. showed that genetic deletion of CXCL10 did not ameliorate the severity or histopathology of EAE (Klein et al., 2004). A few studies have suggested that CCL3 is important for EAE induction in mice (Karpus et al., 1995; Sorensen et al., 1999; Trebst and Ransohoff, 2001). In contrast, we did not find enhanced CCL3 expression in our experiments. As CCL3 is also important for the induction of Th1 immune responses (Manczak et al., 2002; Babcock and Owens, 2003), our results suggest that the role of CCL3 in EAE may be derived from the latter effect rather than an effect on leukocyte recruitment to the brain. Altogether, our results suggest an involvement of CCL2 and CCL5 during EAE. More specifically, our data suggest that these two chemokines play an important role in the adhesion of leukocytes to the brain microcirculation in EAE.
128
A.C. dos Santos et al. / Journal of Neuroimmunology 162 (2005) 122–129
As adhesion is essential for further migration, these two chemokines may participate in the pathophysiology of EAE by modulating the migration of leukocytes into the brain. Limiting the migration of immune cells into the CNS using CCL2 and/or CCL5-based strategies appear to be interesting targets for development for the treatment of MS.
Acknowledgments This work was supported by grants from CNPq and FAPEMIG.
References Adams, C.W., Poston, R.N., Buk, S.J., 1989. Pathology, biochemistry and immunocytochemistry of lesions in acute multiple sclerosis. J. Nat. Sci. 92, 291. Ambrosini, E., Columba-Cabezas, S., Serafini, B., Muscella, A., Aloisi, F., 2003. Astrocytes are the major intracerebral source of macrophage inflammatory protein-3a/CCL20 in relapsing experimental autoimmune encephalomyelitis and in vitro. Glia 41, 290 – 300. Babcock, A., Owens, T., 2003. Chemokines in experimental autoimmune encephalomyelitis and multiple sclerosis. Adv. Exp. Med. Biol. 520, 120. Balashov, K.E., Rottman, J.B., Weiner, H.L., Hancock, W.W., 1999. CCR5(+) and CXCR3(+) T cells are increased in multiple sclerosis and their ligants MIP-1 alpha and IP-10 are expressed in demyelinating brain lesions. Proc. Natl. Acad. Sci. 96, 6873 – 6878. Bradl, M., Hohlfeld, R., 2003. Molecular pathogenesis of neuroinflammation. J. Neurol. Neurosurg. Psychiatry 74, 1364 – 1370. Cara, D.C., Kaur, J., Forster, M., McCafferty, D., Kubes, P., 2001. Role of p38 mitogen-activated protein kinase in chemokine-induced emigration and chemotaxis in vivo. J. Immunol. 167, 6552 – 6558. Carvalho-Tavares, J., Hickey, M.J., Hutchison, J., Michaud, J., Sutcliffe, I.T., Kubes, P., 2000. A role for platelets and endothelial selectins in tumor necrosis factor-(-induced leukocyte recruitment in the brain microvasculature. Circ. Res. 87, 1141 – 1148. Chensue, S.W., Warmington, K.S., Allenspach, E.J., Lu, B., Gerard, G., Kunkel, S.L., Lukacs, N.W., 1999. Differential expression and cross-regulatory function of RANTES during mycobacterial (type 1) and schistosomal (type 2) antigen-elicited granulomatous inflammation. J. Immunol. 163, 165 – 173. Coelho, P.S., Klein, A., Talvani, A., Coutinho, S.F., Takeuchi, O., Akira, S., Silva, J.S., Canizzaro, H., Gazzinelli, R.T., Teixeira, M.M., 2002. Glycosylphosphatidylnositol-anchored mucin-like glycoproteins isolated from Trypanosoma cruzi trypamastigotes induce in vivo leukocyte recruitment dependent on MCP-1 production by IFN-g-primed-macrophages. J. Leukoc. Biol. 71, 834 – 844. Denkinger, C.M., Dekinger, M., Kort, J.J., Metz, C., Forsthuber, T.G., 2003. In vivo blockade of macrophage migration inhibitory factor ameliorates acute experimental autoimmune encephalomielytis by impairing the homing of encephalitogenic T cells to the central nervous system. J. Immunol. 170, 1274 – 1282. Fife, B.T., Huffinagle, G.B., Kuziel, W.A., Karpus, W.J., 2000. CC Chemokine receptor 2 is critical for induction of experimental autoimmune encephalomyelitis. J. Exp. Med. 192, 899 – 905. Fife, B.T., Paniagua, M.C., Lukacs, N.M., Kunkel, S.L., Karpus, W.J., 2001. Selective CC chemokine receptor expression by central nervous system-infiltration encephalitogenic T cells during experimental autoimmune encephalomyelitis. J. Neurosci. Res. 66 (4), 705 – 714. Glass, W.G., Hickey, M.J., Hardison, J.L., Liu, M.T., Manning, J.E., Lane, T.E., 2004. Antibody targeting of the CC chemokine ligant 5
results in diminished leukocyte infiltration into the central nervous system and reduced neurologic disease in a viral model of multiple sclerosis. J. Immunol. 172 (7), 4018 – 4025. James, W.G., Bullard, D.C., Hickey, M.J., 2003. Critical role of the (4 integrin/VCAM-1 pathway in cerebral leukocyte trafficking in lupusprone MRL/faz IPR mice. J. Immunol. 170, 520 – 527. Juedes, A.E., Hjelmstrfm, P., Bergman, C.M., Neild, A.L., Ruddle, N.H., 2000. Kinetics and cellular origin of cytokines in the central nervous system: insight into mechanisms of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. J. Immunol. 164, 419 – 426. Karpus, W.J., LuKacs, N.W., McRae, B.L., Strieter, R.M., Kumkel, S.L., Miller, S.D., 1995. An important role for the chemokine macrophage inflammatory protein-1a in the pathogenesis of the T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis. J. Immunol. 155, 5003 – 5010. Kerfoot, S.M., Kubes, P., 2002. Overlapping roles of P-selectin and alpha 4integrin to recruit leukocytes to the central nervous system in experimental autoimmune encephalomyelitis. J. Immunol. 169 (2), 1000 – 1006. Klein, A., Talvani, A., Silva, P.M.R., Martins, M.A., Wells, T.N.C., Proudfoot, A., Luckacs, N.W., Teixeira, M.M., 2001. Stem Cell FactorInduced Leukotriene B4 Production Cooperates with Eotaxin to Mediate the Recruitment of Eosinophils During Allergic Pleurisy in Mice1. J. Immunol. 167, 524 – 531. Klein, R.S., Izikson, L., Means, T., Gibson, H.D., Lin, E., Sobel, R.A., Weiner, H.L., Luster, A.D., 2004. IFN-inducible protein-10/CXC chemokine ligant 10-independent induction of experimental autoimmune encephalomyelitis. J. Immunol. 172, 550 – 559. Lukacs, N.W., Strieter, R.M., Shaklee, C.L., Chensue, S.W., Kunkel, S.L., 1995. Macrophage inflammatory protein-1 alpha influences eosinophil recruitment in antigen-specific airway inflammation. Eur. J. Immunol. 25, 245 – 251. Mahad, D.J., Ransohoff, R.M., 2003. The role of MCP-1 (CCL2) and CCR2 in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Semin. Immunol. 15, 23 – 32. Manczak, M., Jiang, S., Orzechowska, B., Adamus, G., 2002. Crucial role of CCL3/MIP-1a in the recurrence of autoimmune anterior uveitis induced with myelin basic protein in Lewis rats. J. Autoimmun. 18, 259 – 270. McCafferty, D.M., Kanwar, S., Granger, N., Kubes, P., 2000. E/P-selectindeficient mice: an optimal mutation for abrogating antigen but not tumor necrosis factor-a-induced immune responses. Eur. J. Immunol. 30, 2362 – 2371. Noseworthy, J.H., Lucchinetti, C., Rodriguez, M., Weinshenker, B.G., 2000. Multiple sclerosis. N. Engl. J. Med. 343 (13), 938 – 952. Piccio, L., Rossi, B., Scarpini, E., Laudanna, C., Giagulli, C., Issekutz, A.C., Vestweber, D., Butcher, E.C., Constantin, G., 2002. Molecular mechanisms involved in lymphocyte recruitment in inflamed brain microvessels: critical roles for P-selectin glycoprotein ligant-1 and heterotrimeric Gi-linked receptors. J. Immunol. 168, 1940 – 1949. Prineas, J.W., Wright, R.G., 1978. Macrophages, lymphocytes and plasma cells in the perivascular compartment in chronic multiple sclerosis. Lab. Invest. 38, 409. Ransohoff, R.M., 2002. The chemokine system in neuroinflammation: an update. J. Infect. Dis. 186, S152. Ransohoff, R.M., Kivisakk, P., Kidd, G., 2003. Three or more routes for leukocyte migration into the central nervous system. Nat. Rev. 3, 569 – 582. Rottman, J.B., Slavin, A.J., Silva, R., Weiner, H.L., Gerard, C.G., Hancock, W.W., 2000. Leukocyte recruitment during onset of experimental allergic encephalomyelitis is CCR1 dependent. Eur. J. Immunol. 30, 2372 – 2377. Sorensen, T.L., Tani, M., Jensen, J., Pierce, V., Luchinetti, C., Folcik, V.A., Qin, S., Rottman, J., Sellebjerg, F., Strieter, R.M., 1999. Expression of specific chemokines and chemokine receptors in the central nervous system of multiple sclerosis patients. J. Clin. Invest. 103, 807 – 815.
A.C. dos Santos et al. / Journal of Neuroimmunology 162 (2005) 122–129 Tanaka, Y., Adams, D.H., Hubscher, S., Hirano, H., Siebenlist, U., Shaw, S., 1993. T-cell adhesion induced by proteoglycan-immobilized cytokine MIP-1h. Nature 361, 79 – 82. Tang, T., Frenette, P.S., Hynes, R.O., Wagner, D.D., Mayadas, T.N., 1996. Cytokine-induced meningites is dramatically attenuated in mice deficient in endothelial selectins. J. Clin. Invest. 97, 2485 – 2490.
129
Tran, E.H., Kuziel, W.A., Owens, T., 2000. Induction of experimental autoimmune encephalomyelitis in C57BL/6 mice deficient in either the chemokine macrophage inflammatory protein-1a or its CCR5 receptor. Eur. J. Immunol. 30, 1410 – 1415. Trebst, C., Ransohoff, R.M., 2001. Investigating chemokines and chemokine receptors in patients with multiple sclerosis: opportunities and challenges. Arch. Neurol. 58, 1975 – 1980.
