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The Journal of Immunology, 2006, 176: 6225-6234.
Copyright © 2006 by The American Association of Immunologists

Reevaluation of P-Selectin and {alpha}4 Integrin as Targets for the Treatment of Experimental Autoimmune Encephalomyelitis1

Steven M. Kerfoot2, M. Ursula Norman, Benoit M. Lapointe, Claudine S. Bonder3, Lori Zbytnuik and Paul Kubes

Immunology Research Group, Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
There has been a great deal of interest in adhesion molecules as targets for the treatment of multiple sclerosis and other inflammatory diseases. In this study, we systematically evaluate {alpha}4 integrin and P-selectin as targets for therapy in murine models of multiple sclerosis–for the first time directly measuring the ability of their blockade to inhibit recruitment and relate this to clinical efficacy. Experimental autoimmune encephalomyelitis was induced in C57BL/6 or SJL/J mice and intravital microscopy was used to quantify leukocyte interactions within the CNS microvasculature. In both strains, pretreatment with blocking Abs to either {alpha}4 integrin or P-selectin reduced firm adhesion to a similar extent, but did not block it completely. The combination of the Abs was more effective than either Ab alone, although the degree of improvement was more evident in SJL/J mice. Similarly, dual blockade was much more effective at preventing the subsequent accumulation of fluorescently labeled leukocytes in the tissue in both strains. Despite evidence of blockade of leukocyte recruitment mechanisms, no clinical benefit was observed with anti-adhesion molecule treatments or genetic deletion of P-selectin in the C57BL/6 model, or in a pertussis toxin-modified model in SJL/J mice. In contrast, Abs to {alpha}4 integrin resulted in a significant delay in the onset of clinical signs of disease in the standard SJL/J model. Despite evidence of a similar ability to block firm adhesion, Abs to P-selectin had no effect. Importantly, combined blockade of both adhesion molecules resulted in significantly better clinical outcome than anti-{alpha}4 integrin alone.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The recruitment of inflammatory cells from the circulation is a critical element of any inflammatory event, including pathological inflammatory diseases. It follows that the mechanisms mediating recruitment have often been proposed as potential therapeutic targets for the treatment of inflammatory disorders, including ischemia/reperfusion injuries, sepsis, and chronic inflammatory diseases such as multiple sclerosis (MS).4 Leukocyte recruitment is mediated by adhesion molecules expressed on circulating leukocytes as well as the endothelial cells lining blood vessels. These molecules mediate a cascade of events that result in leukocyte entry into the tissue parenchyma. First, leukocytes tether to and then roll along the vascular wall. If they encounter appropriate signals, they firmly adhere and migrate into the tissue (1, 2, 3, 4). Attempts to therapeutically block leukocyte recruitment have largely targeted these adhesion molecules.

Unfortunately, despite initial promising results in animal models, human clinical trials of therapies targeting adhesion molecules have mostly been disappointing (see Ref. 5 for review of this topic). Typically, a single adhesion molecule has been targeted in these studies, and failure is presumably due to the inability to sufficiently block recruitment into the tissue. Investigations by us and others have demonstrated that, during inflammation, multiple adhesion molecules contribute to recruitment. Therefore, a careful evaluation of recruitment mechanisms to specific tissues under specific inflammatory conditions will be required to design appropriate therapies that will be effective in human patients (5).

One apparent exception to the list of failures of therapies targeting adhesion molecules is the use of anti-{alpha}4 integrin for the treatment of MS. The efficacy of {alpha}4 integrin blockade to reduce both neurological signs of disease as well as inflammatory markers in the CNS was initially demonstrated in numerous animal studies (6, 7, 8, 9, 10, 11, 12, 13, 14). Similar effectiveness was subsequently observed in human clinical trials (15, 16). However, recent reports of a serious complication associated with anti-{alpha}4 integrin have halted the use of this once promising drug (reviewed in Ref. 17).

The therapeutic, and now pathogenic, mechanisms of {alpha}4 integrin blockade have widely been presumed to be through the blockade of steps of the recruitment cascade, i.e., leukocyte interactions with the endothelium of the cerebrovasculature. However, to date this contention has not been systematically assessed. Although a few animal studies reported some reduction in the number of inflammatory cells found within the CNS of treated mice (8, 9, 18), this may reflect an overall inhibition of the inflammatory response rather than a block of recruitment per se. Direct observation of leukocyte/endothelial interactions within the CNS vasculature revealed that blockade of {alpha}4 integrin limited some T cell adhesion in uninflamed CNS vasculature (19) or cytokine-activated vasculature (20, 21). Our own recent study was the first to extend this to a model of MS, experimental autoimmune encephalomyelitis (EAE) (22). In that study (22), we also demonstrated that, in addition to {alpha}4 integrin, endothelial P-selectin is very important to mediate leukocyte/endothelial interactions. It is not yet known how blockade of one or both of these molecules impacts leukocyte recruitment into the CNS parenchyma during disease and how this translates to disease progression.

In this study, we use our knowledge of the specific mechanisms mediating leukocyte/endothelial interactions in the CNS vasculature during EAE (22) to design optimal adhesion molecule targeting therapy to block recruitment. We then evaluated the efficacy of this treatment to prevent disease progression. Based on our previous observations (22), our hypothesis was that combined blockade of both {alpha}4 integrin and P-selectin would be more effective than the current therapy of blocking {alpha}4 integrin alone. Indeed, we demonstrate that combined blockade is more effective at reducing leukocyte/endothelial interactions, leukocyte accumulation in the CNS, and disease development in some, but not other models of EAE. We also identify a number of complexities of adhesion molecule targeting treatments. Therapeutic efficacy was highly dependent on the disease model. In addition, there was no linear relationship between the degree of inhibition of leukocyte adhesion and the inhibition of disease. Although Abs to both P-selectin and {alpha}4 integrin blocked firm adhesion equally well, only blockade of {alpha}4 integrin had any impact on disease, raising the possibility that anti-{alpha}4 integrin may have additional disease-limiting mechanisms.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Abs and reagents

Anti-murine P-selectin (RB40.34), anti-{alpha}4 integrin (R1-2), and all isotype control Abs were purchased from BD Pharmingen. A second blocking anti-{alpha}4 integrin (PS-2) was a gift from Merck. FITC- or PE-conjugated anti-murine CD3{epsilon} (clone 145-2C11), Mac-1 (clone M1/70), B220 (RA3-6B2), and NK1.1 (PK136) for use in FACS analysis of inflammatory cells were also purchased from BD Pharmingen. Both the myelin oligodendrocyte glycoprotein (MOG)35–55 (MEVGWYRSPFSRVVHLYRNGK) peptide and the proteolipid protein (PLP)139–151 (HSLGKWLGHPDKF) peptide were generated in our own institute’s peptide synthesis laboratory. Freund’s adjuvant was purchased from Sigma-Aldrich and pertussis toxin was purchased from List Biological Laboratories.

Mice

C57BL/6 mice were purchased from Charles River Laboratories. SJL/J mice, P-selectin–/–, DO11.11 transgenic, and BALB/c mice were purchased from The Jackson Laboratory. P-selectin–/– mice were backcrossed 10 generations onto the C57BL/6 background. PSGL-1–/– mice were initially a gift from Dr. D. Bullard (University of Alabama, Birmingham, AL), but were later purchased from The Jackson Laboratory. These mice have been backcrossed onto the C57BL/6 background for five generations, and therefore are insipient congenic to C57BL/6 control mice. Animal protocols were approved by the University of Calgary Animal Care Committee and met the Canadian Guidelines for Animal Research.

Induction of MOG35–55-induced EAE in C57BL/6 mice

EAE was induced in mice as previously described (23). Briefly, 9- to 11-wk-old female C57BL/6 mice were immunized s.c. with the murine MOG35–55 peptide (50 µg/mouse) in CFA. Mice were immunized twice, 1 wk apart. Pertussis toxin (200 ng) was injected i.p. on the day of the first immunization and then again 2 days later. Mice were weighed and graded for disease severity daily. Disease scores were recorded as follows: 0, healthy mouse with no signs of disease; 1, limp tail; 2, hind limb weakness; 3, hind-limb paralysis; 4, fore-limb weakness; 5, death. Half scores were awarded for intermediate signs.

Induction of PLP139–151-induced EAE in SJL/J mice

EAE was generated in SJL/J mice through immunization with the PLP139–151 peptide as described previously (6, 24). Briefly, 9- to 11-wk-old female SJL/J mice were immunized s.c. with the PLP139–151 peptide (100 µg/mouse) in CFA. Mice were immunized once. When noted, 200 ng of pertussis toxin was injected i.p. on the day of the first immunization and then again 2 days later. Mice were weighed and graded for disease severity as described above. Relapsing/remitting disease was defined as mice who, after recovering from the initial attack (as measured by a reduction in disease score by at least a full point and maintained for a minimum of 2 days), suffered a subsequent increase of at least a full disease score maintained for a minimum of 2 days.

Intravital microscopy of the cerebromicrovasculature

Intravital microscopy of the mouse cerebromicrovasculature was performed as previously described (22, 25). Mice were anesthetized with a mixture of ketamine (200 mg/kg) and xylazine (10 mg/kg). The tail vein was cannulated for the administration of additional anesthetic, fluorescent dyes, Abs, and other reagents. A craniotomy was performed using a high-speed drill (Fine Science Tools) and the dura matter was removed to expose the underlying pial vasculature. Throughout the experiment, the mouse was maintained at 37°C and the exposed brain kept moist with an artificial cerebrospinal fluid buffer.

Circulating leukocytes were fluorescently labeled by i.v. administration of rhodamine 6G (Sigma-Aldrich; 0.3 mg/kg body weight). Labeled cells were observed using a microscope (Axioskop; Carl Zeiss Canada; x10 eyepiece and x25 objective lens) outfitted with a fluorescent light source (epi-illumination at 510–560 nm using a 590-nm emission filter). A low light intensifier charge-coupled device (ICCD) camera (Stanford Photonics) mounted on the microscope was used to project the image to a monitor. Three different postcapillary venules with a diameter between 30 and 70 µm were chosen for observation. All experiments were recorded for later analysis. Rolling leukocytes were defined as those cells moving at a velocity less than that of erythrocytes within a given vessel. Rolling flux is the number of rolling cells that pass a particular point in a vessel over the course of a minute. Leukocytes were considered adherent if they remained stationary for 30 s or longer. Data from the three vessels were averaged to get results from each time point.

FACS analysis of inflammatory cells in the CNS

Infiltrating inflammatory cells in the CNS were analyzed by flow-assisted cytometry (FACS) using a protocol modified from Krakowski et al. (26), as previously described (22). Anesthetized mice were perfused through the heart with PBS to clear circulating blood from the vasculature. Brain and spinal cord tissue was dissected and then dissociated through a wire mesh. Inflammatory cells were then separated on a Percoll (Amersham Pharmacia Biotech) gradient (90, 37, and 30% isotonic Percoll). Once the cells were washed, they were then incubated with Abs to cell surface markers conjugated to fluorescent labels and analyzed with a BD Biosciences FACScan. Healthy mice were used as controls to ensure that mononuclear cell counts were not simply due to vascular leukocyte content.

In other experiments, this technique was used to recover cultured T cells (5 x 107/mouse) that had been labeled with 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (CFDA; 30 min, 5 µM; Molecular Probes) before i.v. administration to EAE mice as a measure of T cell accumulation in the tissue. Alternatively, unsorted lymph node and buffy coat leukocytes isolated from whole blood (1:1 donor to recipient ratio) were isolated from EAE mice, labeled and transferred i.v. to other EAE mice.

T cell purification and generation of OVA-specific Th1 cells

Culture of Th1 T cells was performed as previously described (27, 28). Briefly, CD4+ T cells were isolated from pooled spleens of DO11.10 mice using mouse anti-CD4 (L3T4) MACS Microbeads as per the manufacturer’s protocol (Miltenyi Biotec). Cells were then activated and driven to a Th1 phenotype through culture with irradiated congenic splenocytes in Iscove’s medium (Invitrogen Life Technologies) supplemented with 5 µg/ml OVA peptide 323–339 (New England Peptides), antibiotics, 10% FCS, 50 U/ml IL-12 (R&D Systems), and 10 µg/ml anti-IL-4 (11B11). Cells were cultured for 5–9 days before use.

Statistics

Data in graphs are shown as mean ± SEM unless indicated otherwise. ANOVA followed by a Student’s t test with Bonferroni correction was used for multiple comparisons. Statistical significance was set at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Evaluation of adhesion molecule blockade in MOG-induced EAE in C57BL/6 mice

Previously, we demonstrated that acute administration of blocking Abs to P-selectin or {alpha}4 integrin could block leukocyte/endothelial interactions within the CNS microcirculation of C57BL/6 mice with MOG35–55-induced EAE (22). The following experiments were designed to evaluate the effectiveness of more prolonged blockade of these two molecules, alone or in combination, to prevent the accumulation of leukocytes in the inflamed CNS vasculature during disease. EAE was induced in C57BL/6 mice as described (Materials and Methods). Anti-{alpha}4 integrin (R1-2, 70 µg/mouse) and/or anti-P-selectin (RB40.34, 20 µg/mouse) were administered i.v. to mice in the acute phase of disease (2–5 days postonset of neurological signs). Intravital microscopy of the cerebromicrovasculature was performed 24 h later to quantify leukocyte/endothelial interactions. Anti-{alpha}4 integrin alone had little impact on the number of rolling cells observed in CNS blood vessels of EAE mice (Fig. 1A). In contrast, leukocyte firm adhesion was reduced by nearly 70% (Fig. 1B). Anti-P-selectin alone completely blocked all leukocyte rolling (Fig. 1A). Interestingly, the prolonged blockade of P-selectin also reduced firm adhesion to the same extent as anti-{alpha}4 integrin (71%, Fig. 1B), presumably due to the elimination of upstream rolling events. Clearly, some cells adhere directly without prior rolling; a relatively rare yet measurable event. Combined application of both anti-{alpha}4 integrin and anti-P-selectin eliminated all leukocyte rolling (Fig. 1A) and reduced firm adhesion by 82% (Fig. 1B).


Figure 1
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  		FIGURE 1. Combination therapy with anti-{alpha}4 integrin and anti-P-selectin blocks leukocyte-endothelial interactions within the cerebromicrovasculature in the MOG35–55-induced model of EAE in C57BL/6 mice. Mice in the acute phase of EAE were given the blocking anti-adhesion molecule Abs (R1-2 and/or RB40.34) alone or in combination. Intravital microscopy of the brain microvasculature was performed 24 h later to quantify leukocyte rolling (A) and firm adhesion (B). Results are shown as mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs untreated. n = 8 for untreated, n = 4 for anti-{alpha}4 integrin, n = 5 for anti-P-selectin and combined treatment.

 
Next, we asked whether Th1 cell interactions within the pial vasculature were also blocked by Abs to P-selectin and/or {alpha}4 integrin. CD4+ T cells were isolated, cultured, and driven to a Th1 phenotype. They were then labeled with CFSE and transferred i.v. to EAE mice in the presymptomatic or early acute phase of EAE. We have previously demonstrated that these cells are recruited to tissues (e.g., liver) using similar mechanisms to endogenous Th1 T cells (28). Blockade of anti-{alpha}4 integrin (R1-2, 20 µg/mouse) had no effect on Th1 cell rolling (Fig. 2A), while subsequent administration of anti-P-selectin (RB40.34, 20 µg/mouse) (Fig. 2A), or anti-P-selectin alone (Fig. 2B), completely eliminated rolling. Pretreatment of Th1 cells with anti-{alpha}4 integrin (R1-2, 70 µg) blocked firm adhesion by 65% (Fig. 2C). Therefore, similar to our observations of the whole circulating population of leukocytes (Fig. 1), both P-selectin and {alpha}4 integrin contribute to Th1 T cell interactions with the cerebrovascular endothelium.


Figure 2
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  		FIGURE 2. Anti-adhesion molecule therapy prevents the accumulation of adoptively transferred Th1 T cells in the CNS of C57BL/6 EAE mice. Initial experiments were performed to identify the mechanisms mediating Th1 T cell/endothelial interactions in the CNS vasculature during EAE. A, Fluorescently labeled Th1 T cells were administered i.v. and leukocyte rolling was quantified. Rolling was then quantified after the administration of a blocking anti-{alpha}4 integrin Ab (R1-2) and again after the administration of a blocking anti-P-selectin Ab (RB40.34). Control mice received the appropriate isotype control Abs. Results normalized to isotype control ± SEM. *, p < 0.05 vs control; n = 5. In other experiments, anti-P-selectin alone or isotype control was administered alone (B). Results normalized to isotype control ± SEM. *, p < 0.05 vs control; n = 3. To determine the contribution of {alpha}4 integrin to Th1 T cell firm adhesion, cells were pretreated with anti-{alpha}4 integrin or isotype control before transfer (C). Results are shown as mean ± SEM. *, p < 0.05; n = 6 for isotype control; n = 4 for anti-{alpha}4 integrin. The ability of anti-adhesion molecule Abs to prevent the ultimate accumulation of transferred Th1 T cells in the CNS of EAE mice was then investigated (D). Fluorescently labeled Th1 T cells were administered i.v. into mice in the presymptomatic or early acute phase of EAE (5 x 107/mouse). Isotype control Abs (iso), blocking anti-{alpha}4 integrin Ab (PS/2), or a combination of anti-{alpha}4 integrin and anti-P-selectin (RB40.34) were administered at the same time as the cells. Cells were isolated from the CNS of these mice 18 h later for analysis by FACS. Inflammatory cells were gated on based on forward- and side-scatter profiles. Fluorescent cells as percent of total inflammatory cells are shown. Results are shown as mean ± SEM. *, p < 0.05; n = 5.

 
Next, we went on to determine whether adhesion molecule blockade would prevent the ultimate accumulation of Th1 cells in the CNS of EAE mice. Anti-{alpha}4 integrin (PS/2, 200 µg/mouse) alone or in combination with anti-P-selectin (RB40.34, 20 µg/mouse) was administered at the time of Th1 cell transfer. Control mice received isotype control Abs. PS/2 is another {alpha}4 integrin-blocking Ab and was used in these and all subsequent experiments for reasons of availability and because it has a long history of use in murine EAE (9, 12, 13). Preliminary experiments did not detect any difference between PS/2 and R1-2 in our model. We previously showed that a dose of 200 µg/mouse was required to fully block {alpha}4 integrin-mediated interactions in a liver model of inflammation in vivo (29) and therefore this dose was used. CFSE-labeled T cells could be easily detected by FACS analysis of inflammatory cells recovered from the CNS of isotype control-treated mice 24 h posttransfer (Fig. 2D). Although {alpha}4 integrin Ab reduced T cell accumulation by ~55% compared with isotype-treated mice, only the administration of both anti-{alpha}4 integrin and anti-P-selectin together resulted in a significant reduction in the number of Th1 cells in the CNS (~80% reduction). Therefore, anti-adhesion molecule therapy designed to interfere with leukocyte/endothelial interactions does indeed prevent the recruitment and accumulation of new Th1 cells to the CNS in EAE, and combination therapy may be more effective than anti-{alpha}4 integrin alone.

Targeting P-selectin alone is not effective in EAE

Unlike {alpha}4 integrin, P-selectin has not been well-characterized as a therapeutic target for EAE. We and others have reported a role for P-selectin in mediating rolling within the inflamed CNS vasculature (20, 21, 22, 30, 31). In addition, our intravital microscopy experiments clearly demonstrate that blockade of P-selectin is as effective in reducing firm adhesion in the CNS vasculature as blockade of {alpha}4 integrin (Fig. 1). EAE was induced in either wild-type C57BL/6 mice, or mice genetically deficient in P-selectin (P-selectin–/–) on the C57BL/6 background (ensuring a complete elimination of P-selectin-mediated recruitment). No difference was observed in the time of onset or disease severity between the two groups (Fig. 3A). Although in the example shown the onset of neurological signs appears to be slightly delayed in P-selectin–/– mice, this delay was not observed in other experiments. Our preliminary studies of mice deficient in the primary ligand for P-selectin, P-selectin glycoprotein ligand-1 (PSGL-1) (Fig. 3B, consistent with recent observations by others (32, 33)), or mice deficient in both P-selectin and E-selectin (data not shown) confirm that no benefit is observed with interference of P-selectin-mediated recruitment alone.


Figure 3
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  		FIGURE 3. Targeting the P-selectin/PSGL-1 pathway alone is not effective for the treatment of MOG35–55-induced EAE in C57BL/6 mice. EAE was induced in C57BL/6 mice, P-selectin–/– (A) or PSGL-1–/– mice (B). Mice were evaluated daily for clinical disease. Mean scores are shown; n = 5 for each group. One example of five experiments in shown for A. Intravital microscopy was performed on wild-type or P-selectin–/– mice in presymptomatic or acute phase of EAE. Leukocyte rolling (C) and firm adhesion (D) was quantified. Results are shown as mean ± SEM. **, p < 0.01, ***, p < 0.001 vs control; n = 4 for control and 3 days postsymptom mice in both strains, n = 4 for presymptomatic mice in both strains.

 
Intravital microscopy of the cerebrovasculature was performed on wild-type and P-selectin–/– EAE mice to confirm that the absence of P-selectin did indeed result in a defect in leukocyte/endothelial interactions. As expected, leukocyte rolling was completely eliminated in P-selectin–/– mice during both the developing and acute phases of disease (Fig. 3C). Firm adhesion was significantly reduced by at least 50% (Fig. 3D) compared with wild-type EAE mice. These results are comparable to our observations with P-selectin, or {alpha}4 integrin, blocking Ab (Fig. 1). Despite this, FACS analysis of inflammatory cells recovered from the CNS of wild-type and P-selectin–/– EAE mice revealed no differences in the numbers or profiles of inflammatory cells between the two groups (data not shown). Therefore, the reduction in leukocyte adhesion resulting from the elimination of the P-selectin recruitment pathway alone was not sufficient to have any impact on the overall accumulation of inflammatory cells within the diseased CNS or the ultimate development of disease.

Evaluation of combined adhesion molecule blockade as therapy for MOG35–55-induced EAE

Based on the above studies of the mechanisms of leukocyte recruitment to the CNS in MOG35–55-induced EAE, our interest was to determine whether blockade of both {alpha}4 integrin and P-selectin together would translate into a more effective therapy than the current protocol blocking {alpha}4 integrin alone. EAE was induced in three groups of C57BL/6 mice: one group of mice received either isotype control Abs or vehicle (saline), the second group received anti-{alpha}4-integin alone (PS/2, 200 µg/mouse/treatment), while the third received both anti-{alpha}4-integin and anti-P-selectin (RB40.34, 20 µg/mouse/treatment). Abs were administered i.p., three times a week for up to 3 wk beginning as early as 3 days following immunization. Mice were observed daily for clinical signs of disease. Despite the clear demonstration that the Abs block leukocyte/endothelial interactions within the cerebromicrovasculature (Fig. 1) and subsequent accumulation of proinflammatory Th1 T cells within the CNS (Fig. 2D), neither the blockade of {alpha}4 integrin alone nor the combined blockade of {alpha}4 integrin and P-selectin had any impact on disease development or severity (Fig. 4).


Figure 4
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  		FIGURE 4. Evaluation of anti-adhesion molecule therapy in MOG35–55-induced EAE in C57BL/6 mice. Starting as early as 3 days following immunization with MOG, EAE mice were treated with either isotype control Ab or saline (control), anti-{alpha}4 integrin (PS/2), or a combination of anti-{alpha}4 integrin and anti-P-selectin (RB40.34). Mice were evaluated for disease severity (A) and weight loss (B) daily. Mean disease scores and relative weight loss is shown; n = 15 for each group (a combination of three separate experiments with five mice per group).

 
Evaluation of adhesion molecule blocking therapy in PLP-induced EAE in SJL/J mice

In the literature, most studies of adhesion molecule blockade in murine EAE have made use of models in SJL/J mice (6, 7, 9, 14). Therefore, we repeated the above experiments in the PLP139–151-induced model of EAE in this strain. Control mice developed neurological signs ~14 days postimmunization, which coincided with a rapid loss of weight (Fig. 5, A and B, respectively). Disease progressed very rapidly over the next 2–3 days, after which some improvement was observed. Half of these mice went on to develop a relapsing/remitting disease course (Table I), while the rest maintained a chronic disease course.


Figure 5
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  		FIGURE 5. Evaluation of anti-adhesion molecule therapy in PLP139–151-induced EAE in SJL/J mice. Starting 5 days following immunization with PLP, EAE mice were treated with either isotype control Ab or saline (control), anti-{alpha}4 integrin (PS/2), anti-P-selectin (RB40.34), or a combination of anti-{alpha}4 integrin and anti-P-selectin. A total of four treatments were administered over 7 days. Mice were evaluated for clinical signs of disease (A) and weight loss (B) daily. Mean disease scores and relative weight loss is shown. *, p < 0.05 vs control; n = 10 for each group (a combination of two separate experiments with five mice per group).

 

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  		Table I. PLP-induced EAE in SJL/J mice

 
Consistent with our observations of P-selectin–/– mice in the MOG35–55-induced model of EAE, Abs to P-selectin alone (RB40.34, 20 µg/mouse/treatment) had no impact on any aspect of disease (Fig. 5 and Table I). In contrast, administration of anti-{alpha}4 integrin alone (PS/2, 200 µg/mouse/treatment) resulted in a significant delay in the onset of clinical signs (Fig. 5A and Table I). However, from day 20 on, mice treated with anti-{alpha}4 integrin alone were just as sick as control mice (Fig. 5A). In addition, the cumulative disease score over the course of the experiment was no different from control (Table I). Therefore, disease processes were merely delayed rather than inhibited with anti-{alpha}4 integrin alone.

Importantly, and consistent with our original hypothesis, combined treatment with both anti-{alpha}4 integrin and anti-P-selectin resulted in significantly better clinical outcome than treatment with anti-{alpha}4 integrin alone. The onset of clinical signs of disease was delayed compared with control, similar to mice treated with anti-{alpha}4 integrin alone (Fig. 5A and Table I). Weight loss of groups treated with anti-{alpha}4 integrin alone or both Abs were also similarly delayed (Fig. 5B). However, in contrast to treatment with anti-{alpha}4 integrin alone, no rebound in disease severity was observed with combined treatment as, at the end of the experiment, disease severity in the group treated with both Abs was only approximately one-third that of the control group (Fig. 5A). In addition, the cumulative clinical score was significantly lower (approximately one-half) than the cumulative score of the mice receiving anti-{alpha}4 integrin alone (Table I).

Intravital microscopy was performed to characterize the effectiveness of anti-{alpha}4 integrin and anti-P-selectin to block leukocyte/endothelial interactions in the PLP139–151-induced model of EAE in SJL/J mice (Fig. 6A). As observed in MOG-induced EAE in C57BL/6 mice, a large number of both rolling (Fig. 6B) and adherent (Fig. 6C) cells were observed in the SJL/J mice (isotype control Abs or untreated). Again, administration of anti-{alpha}4 integrin (PS/2, 200 µg/mouse) had no impact on the number of rolling cells (Fig. 6B). Although firm adhesion was reduced by 47% in this model (Fig. 6C), this inhibition was less than that observed in MOG-induced EAE (70%; Fig. 1B). Blockade of P-selectin (RB40.34, 20 µg/mouse) alone almost completely eliminated leukocyte rolling (Fig. 6B) and reduced firm adhesion to a similar degree as anti-{alpha}4 integrin (34%, Fig. 6C), but again not to the degree as observed in MOG-induced EAE (71%; Fig. 1B). Combined blockade of both {alpha}4 integrin and P-selectin was clearly much more effective than targeting either molecule alone in this model, as both rolling and firm adhesion were almost entirely eliminated (Fig. 6, B and C).


Figure 6
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  		FIGURE 6. Evaluation of adhesion molecule blockade to block leukocyte recruitment in PLP139–151-induced EAE in SJL/J mice. Mice in the developing phase of EAE (days 6–8, to coincide with the time frame of Ab treatments in Fig. 5) were given the blocking anti-adhesion molecule Abs (PS/2 and/or RB40.34) alone or in combination. Control animals were either left untreated or given isotype control Abs. Intravital microscopy of the brain microvasculature was performed 24 h later (A). Vessels have been outlined, and arrows point to adherent leukocytes. Rolling leukocytes are unmarked. Leukocyte rolling (B) and firm adhesion (C) was subsequently quantified. Results are shown as mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs control; n = 4 for control; n = 3 for all other groups.

 
Similar results were observed with the accumulation of fluorescently labeled Th1 cells in the CNS. Although anti-{alpha}4 integrin alone did not reduce Th1 cell entry into the CNS, anti-P-selectin alone or combined blockade of both molecules effectively prevented accumulation (Fig. 7A). In separate experiments, unsorted inflammatory cells (isolated from lymph nodes and whole blood of EAE mice) were labeled and transferred into new EAE mice instead of Th1 cells. Interestingly, blockade of anti-{alpha}4 integrin was effective in preventing the accumulation of this unsorted population in the CNS (Fig. 7B), suggesting that {alpha}4 integrin alone may be able to block the recruitment of some leukocyte population other than Th1 T cells.


Figure 7
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  		FIGURE 7. The ability of anti-adhesion molecule Abs to prevent the ultimate accumulation of transferred Th1 T cells in the CNS of EAE mice was then investigated (A). Fluorescently labeled Th1 T cells were administered i.v. into mice on day 7 following immunization (5 x 107/mouse). Isotype control Abs or adhesion molecule blocking Abs (PS/2 and/or RB40.34) were administered at the same time as the cells. As controls, separate mice with EAE did not receive any fluorescently labeled cells ("No cells"). Inflammatory cells were then isolated from the CNS of these mice 18 h later for analysis by FACS and identification of fluorescently labeled, transferred cells. Inflammatory cells were gated on based on forward- and side-scatter profiles. B, The above experiment was repeated using unsorted inflammatory cells isolated from lymph nodes and whole blood leukocytes (LN/Inflammatory cells) from EAE mice. These cells were pooled and fluorescently labeled before i.v. administration, as described above (1:1 ratio between donors and recipients). One representative experiment of three is shown.

 
Therapeutic efficacy is dependent on the disease model

Our studies clearly demonstrate therapeutic efficacy of adhesion molecule blockade in PLP-induced EAE in SJL/J mice (Fig. 5), but not in MOG-induced EAE in C57BL/6 mice (Fig. 4). These disease models reflect different disease courses, in that the former is a model of relapsing-remitting disease, while the latter models a chronic disease course. EAE in SJL/J mice can be modified to be more chronic by the administration of pertussis toxin at the time of immunization (34, 35). These mice developed neurological signs of disease 2 wk following immunization with PLP and administration of pertussis toxin (Fig. 8, A and B). Signs rapidly progressed over the next 4–5 days, after which some recovery was observed. As in MOG35–55-induced EAE in C57BL/6 mice, blockade of {alpha}4 integrin, P-selectin, or both molecules combined did not have any measurable impact on disease development (Fig. 8, A and B). Intravital microscopy was performed to determine the efficacy of blockade of leukocyte/endothelial interactions in this model of EAE. The number of rolling and adherent cells in the cerebrovasculature was fewer in this model compared with either of the others (Fig. 8, C and D, compared with Fig. 1, and Fig. 6, B and C, respectively). Neither anti-{alpha}4 integrin alone, nor in combination with anti-P-selectin, was effective at reducing the small number of adherent cells (Fig. 8D). Finally, FACS analysis of inflammatory cells recovered from the CNS of EAE mice did not reveal any differences in the numbers or profiles of cells from control or either groups of Ab-treated mice (data not shown).


Figure 8
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  		FIGURE 8. Therapeutic efficacy of adhesion molecule blockade is dependent on the disease type. PLP139–151-induced EAE in SJL/J mice was driven to a more chronic disease course through the administration of pertussis toxin at the time of immunization and again 2 days after. Mice were treated with blocking Abs (PS/2 and/or RB40.34) starting on day 7. Control mice either received isotype control Abs or vehicle (saline). A, Mice were evaluated for clinical signs of disease daily. B, Relative weight loss is shown; n = 5 for anti-P-selectin; n = 10 for all other groups. Intravital microscopy was used to test the ability of the Abs to block leukocyte/endothelial interactions in this model of EAE. Mice in the acute phase of EAE were administered anti-{alpha}4 integrin, or a combination of anti-{alpha}4 integrin and anti-P-selectin. Control mice were either untreated or administered isotype control Abs. Intravital microscopy of the brain microvasculature was performed 24 h later to quantify leukocyte rolling (C) and firm adhesion (D). Results are shown as mean ± SEM. *, p < 0.05 vs control; n = 4 for control; n = 3 for anti-{alpha}4 integrin and combined treatment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
When we began this study, there was a great deal of excitement surrounding therapy targeting {alpha}4 integrin (Natalizumab) for the treatment of MS. Results from human trials were very promising and there were high expectations that this new drug would represent a significant improvement over current therapies (36, 37, 38). However, very recent reports of complications surrounding the drug (see below) call for a closer evaluation of adhesion molecule targeting therapy. In this study, we systematically assess the efficacy of adhesion molecule blockade to interfere with the mechanisms of leukocyte recruitment and, ultimately, to limit disease progression. Our findings 1) reveal a previously unreported role for P-selectin in disease development, 2) demonstrate differential efficacy of treatment across disease models, and 3) further elucidate the therapeutic mechanism of the current treatment targeting {alpha}4 integrin–namely that efficacy is not related to the degree that adhesion is blocked, suggesting that it may target recruitment events downstream of leukocyte firm adhesion or that it may act on some nonrecruitment-related event.

Our study, for the first time, demonstrates a role for P-selectin in the development of EAE. The role of P-selectin in CNS autoimmunity has been controversial. A number of studies making direct observations in vivo have demonstrated a central role for P-selectin in mediating leukocyte rolling in the CNS vasculature in a number of inflammatory models (20, 21, 25, 31), including EAE (22). Despite this, other studies have reported that neither anti-P-selectin Abs (39) nor, more recently, genetic deficiency in the leukocyte ligand for P-selectin (PSGL-1) (32, 33) had any impact on the incidence, severity, or development of EAE. The inference was made that because P-selectin blockade (unlike blockade of {alpha}4 integrin) did not have any impact on disease, it has no role in leukocyte recruitment. We stress that our own observations are in complete agreement with these previous studies; interference with the P-selectin recruitment pathway alone has absolutely no impact on the development of EAE. This is presumably because leukocyte recruitment to the tissue was not sufficiently impaired. Indeed, consistent with our hypothesis, only by combining the blockade of P-selectin with blockade of {alpha}4 integrin could recruitment be sufficiently reduced to impact disease and reveal the role of P-selectin in disease development. This is evidenced by the almost complete elimination of both leukocyte rolling and firm adhesion with dual treatment as well as the prevention of both Th1 T cell and unsorted inflammatory cell entry to the CNS. Dual treatment also translated into a significant inhibition of disease development as demonstrated by not only a delay in the onset of clinical signs of disease, but also the significant recovery following the initial attack. No such recovery was observed with anti-{alpha}4 integrin alone, clearly demonstrating that dual blockade is more effective to limit disease progression. Interestingly, similar observations of improved efficacy with dual blockade of P-selectin and {alpha}4 integrin compared with {alpha}4 integrin alone were made in a murine model of colitis (40), further supporting the use of this combination to limit inflammatory disease.

The second important finding of our study is that adhesion molecule targeting therapy had differential effectiveness in different models of EAE. Indeed, the discussion in the previous paragraph applies only to PLP-induced EAE in SJL/J mice. Neither blockade of {alpha}4 integrin alone nor dual blockade of {alpha}4 integrin with P-selectin were at all beneficial in either MOG induced EAE in C57BL/6 mice or in the pertussis toxin-modified PLP-induced model in SJL/J mice. It is possible that this is because these different models represent different stages of the same disease, or possibly different pathological mechanisms under the umbrella of MS. Indeed, while many rolling and adhering leukocytes were observed in the two other EAE models, only very few interactions were observed in the pertussis-toxin modified model in SJL/J mice, and anti-adhesion molecule Abs were unable to reduce adhesion any further. In contrast, there was clear evidence that anti-adhesion molecule Abs or genetic deletion of P-selectin significantly reduced leukocyte access to the CNS in the MOG-induced EAE model in C57BL/6 mice. Despite this, no impact on disease development was observed. Clearly, in both the MOG-induced model and the PTX-modified PLP-induced model, but not the unmodified PLP-induced model, the very low residual level of leukocyte firm adhesion following adhesion molecule blockade (1–2 cells/100 µm of vessel in each) is fully capable of supporting inflammatory cell entry into the tissue and, ultimately, disease development.

Third, our study challenges some of our assumptions regarding the therapeutic mechanism of adhesion molecule-targeting therapy for the treatment of EAE and, by association, human MS. First, we clearly demonstrate that there is no linear relationship between the blockade of leukocyte adhesion and clinical efficacy. As already discussed above, adhesion molecule blockade had very different clinical outcomes in the PLP-induced model in SJL/J mice compared with MOG-induced disease in C57BL/6 mice, despite its similar effectiveness in reducing adhesion in both models. Indeed, anti-{alpha}4 integrin treatment resulted in the greatest inhibition of leukocyte firm adhesion in MOG-induced EAE, yet this did not translate into therapeutic efficacy. In addition, anti-P-selectin alone was just as effective at reducing firm adhesion as anti-{alpha}4 integrin alone, yet only anti-{alpha}4 integrin resulted in a delay in disease onset in PLP-induced EAE. Therefore, {alpha}4 integrin may contribute to recruitment mechanisms downstream of firm adhesion. In support of this, anti-{alpha}4 integrin reduced the accumulation of unsorted inflammatory cells in the CNS of EAE mice, while anti-P-selectin did not. Integrins may be involved in the transmigration of leukocytes across the endothelium, and therefore blockade of {alpha}4 integrin may limit this process. However, this process is usually attributed to other adhesion molecules. Indeed, studies of T cell migration across monolayers of brain endothelium have implicated {alpha}4 integrins in firm adhesion but not transmigration, which was instead mediated by LFA-1 (41, 42). Alternatively, {alpha}4 integrin may be involved in the retention of inflammatory cells that have infiltrated the CNS parenchyma (43).

Our study also challenges the idea that the primary therapeutic mechanism of {alpha}4 integrin blockade is to limit the entry of Th1 cell entry to the CNS. This contention is primarily based on the observations that {alpha}4 integrin blockade largely prevented the accumulation of activated T cells in the uninflamed CNS (19). However, our studies clearly demonstrate that under inflammatory conditions this is not the case. It is possible that our in vitro-derived Th1 T cells do not reflect the behavior of endogenous T cells in vivo. However, we have used these in vitro-derived Th1 cells to study recruitment mechanisms to other tissues and have demonstrated that they use similar mechanisms to endogenous cells (44, 45). Recent evidence that IL-17-producing T cells distinct from Th1 T cells are the primary pathogenic T cells in EAE must also be considered (46, 47). It is possible, therefore, that anti-{alpha}4 integrin blocks the recruitment of these cells. Indeed, blockade of {alpha}4 integrin alone did prevent the accumulation of unsorted inflammatory cells in the CNS, suggesting that some other inflammatory cell type or types are targeted.

A final possibility must also be considered, namely that the primary therapeutic mechanism of {alpha}4 integrin blockade is through some other mechanism entirely separate from its role in leukocyte recruitment. As already discussed above, clinical efficacy of {alpha}4 integrin blockade had no relationship to its ability to inhibit mechanisms of recruitment. Other potentially pathogenic roles for {alpha}4 integrin have been described including a role in T cell activation (13, 48, 49) and retention within the CNS, possibly due to a role in target recognition (43). Indeed, we demonstrated that anti-{alpha}4 integrin blocked effector mechanisms but not recruitment in a model of cardiac injury (50).

To conclude, our study has important implications to the recent reports of incidences of PML in MS and Crohn’s patients receiving anti-{alpha}4 integrin (Natalizumab), most often in combination with other drugs (17, 37, 38, 51). PML is a severe and often fatal demyelinating disorder of the CNS caused by a lytic infection of oligodendrocytes by the JC virus (17, 52). JC virus infections are very common and normally latent in immunocompetent individuals. However, in rare cases of severe immunodeficiency, such as with HIV infection, the infection is not controlled, enters the CNS and causes disease (52). Why Abs to {alpha}4 integrin would facilitate PML in the absence of other indications of immunodeficiency is not known. Although it is not yet clear whether our findings in EAE translate to human patients, they do not support the idea that blockade of {alpha}4 integrin alone could result in severe immunosuppression through the blockade of leukocyte recruitment, at least under the inflamed conditions found in EAE and MS. Indeed, over the course of our investigations of organ-specific mechanisms of recruitment (5, 53), we have yet to identify a tissue in which blockade of {alpha}4 integrin alone could have such a profound impact, implying that alternate mechanisms may be responsible. Indeed, as discussed above, {alpha}4 integrin has previously been demonstrated to be involved in T cell activation (48, 49) and proliferation to Ag (13). Any of these mechanisms could interfere with the ability of the immune system to control JC virus. It is possible that, through careful reevaluation of all of the biological mechanisms of {alpha}4 integrin blockade, we may be able to separate its beneficial and pathogenic activities and rescue this once promising therapeutic target. Indeed, if it is possible to prevent the occurrence of PML by using a lower dose or shorter half-life {alpha}4 integrin-blocking reagent, our study suggests that the loss in therapeutic efficacy could be made up for through the inclusion of anti-P-selectin in the treatment regime.


   Acknowledgments
 
We thank Dr. Stephen Miller for his constructive comments and suggestions regarding this study.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was funded by grants and fellowships from the Multiple Sclerosis Society of Canada, the Canadian Institutes of Health Research, and the Alberta Heritage Foundation for Medical Research. Back

2 Address correspondence and reprint requests to Dr. Steven M. Kerfoot at the current address: Section of Allergy and Clinical Immunology, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar Street P.O. Box 208013, New Haven, CT 06520-8013. E-mail address: steven.kerfoot{at}yale.edu Back

3 Current address: Vascular Biology Laboratory, Human Immunology, Institute for Medical and Veterinary Science, Adelaide, Australia. Back

4 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; PLP, proteolipid protein; PSGL, P-selectin glycoprotein ligand; PML, progressive multifocal leukoencephalopathy. Back

Received for publication November 29, 2005. Accepted for publication March 6, 2006.


   References
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 Introduction
 Materials and Methods
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M. Bauer, C. Brakebusch, C. Coisne, M. Sixt, H. Wekerle, B. Engelhardt, and R. Fassler
{beta}1 integrins differentially control extravasation of inflammatory cell subsets into the CNS during autoimmunity
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D. P. Stirling, S. Liu, P. Kubes, and V. W. Yong
Depletion of Ly6G/Gr-1 Leukocytes after Spinal Cord Injury in Mice Alters Wound Healing and Worsens Neurological Outcome
J. Neurosci., January 21, 2009; 29(3): 753 - 764.
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 JEMHome page
B. McDonald, E. F. McAvoy, F. Lam, V. Gill, C. de la Motte, R. C. Savani, and P. Kubes
Interaction of CD44 and hyaluronan is the dominant mechanism for neutrophil sequestration in inflamed liver sinusoids
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S. S. Smith and S. R. Barnum
Differential expression of {beta}2-integrins and cytokine production between {gamma}{delta} and {alpha}{beta} T cells in experimental autoimmune encephalomyelitis
J. Leukoc. Biol., January 1, 2008; 83(1): 71 - 79.
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 J. Immunol.Home page
A. Doring, M. Wild, D. Vestweber, U. Deutsch, and B. Engelhardt
E- and P-Selectin Are Not Required for the Development of Experimental Autoimmune Encephalomyelitis in C57BL/6 and SJL Mice
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