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The Journal of Immunology, 2007, 178, 8158 -8167
Copyright © 2007 by The American Association of Immunologists, Inc.
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Fibronectin- and Vitronectin-Induced Microglial Activation and Matrix Metalloproteinase-9 Expression Is Mediated by Integrins {alpha}5beta1 and {alpha}vbeta51

Richard Milner2,*, Stephen J. Crocker{dagger}, Stephanie Hung*, Xiaoyun Wang*, Ricardo F. Frausto{dagger} and Gregory J. del Zoppo*

* Department of Molecular and Experimental Medicine and {dagger} Molecular and Integrative Neurosciences Department, The Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Early in the pathogenesis of multiple sclerosis, the blood-brain barrier is compromised, which leads to deposition of the plasma proteins fibronectin and vitronectin in cerebral parenchyma. In light of our previous finding that microglial activation in vitro is strongly promoted by fibronectin and vitronectin, we set out to examine the possibility that modulation of microglial activation by fibronectin or vitronectin is an important regulatory mechanism in vivo. In an experimental autoimmune encephalomyelitis mouse model of demyelination, total brain levels of fibronectin and vitronectin were strongly increased and there was a close relationship between fibronectin and vitronectin deposition, microglial activation, and microglial expression of matrix metalloproteinase-9. In murine cell culture, flow cytometry for MHC class I and gelatin zymography revealed that microglial activation and expression of pro-matrix metalloproteinase-9 were significantly increased by fibronectin and vitronectin. Function-blocking studies showed that the influence of fibronectin and vitronectin was mediated by the {alpha}5beta1 and {alpha}vbeta5 integrins, respectively. Taken together, this work suggests that fibronectin and vitronectin deposition during demyelinating disease is an important influence on microglial activation state. Furthermore, it provides the first evidence that the {alpha}5beta1 and {alpha}vbeta5 integrins are important mediators of microglial activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Microglia are important immune effector cells resident within the CNS and, as such, they act as a liaison between the immune system and the CNS (1, 2, 3). In addition to playing a protective role in host defense, evidence suggests that microglia may also be actively involved in the initiation and maintenance of the chronic inflammatory response in a number of CNS disorders, including the demyelinating state that characterizes multiple sclerosis (MS)3 (4, 5, 6, 7, 8). Following stimulation, the normally quiescent microglia are activated into highly aggressive cells that proliferate and migrate to sites of injury. Like peripheral macrophages (9, 10), microglia can secrete a plethora of factors including cytokines, chemokines, and proteases, which can initiate and promote tissue damage (1, 2, 3).

The matrix metalloproteinases (MMPs) are a large family of zinc-dependent endopeptidases that are responsible for dynamic remodeling of the extracellular matrix (ECM) (11). Within the MMP family, MMP-9 has been implicated in demyelinating disease. MMP-9 levels are strongly up-regulated during demyelination (12, 13, 14) and MMP-9 knockout mice are resistant to experimental autoimmune encephalomyelitis (EAE), at least early in life (15). Known functions of MMP-9 within the CNS include degradation of vascular basal lamina proteins such as laminin and collagen IV (11, 16), degradation of myelin-specific proteins such as myelin basic protein (MBP) (17, 18), and cytotoxic effects on neurons following cerebral ischemia (19, 20).

Inflammatory disorders of the CNS are associated with changes in both the interendothelial junctions and the basal lamina ECM that together comprise the blood-brain barrier (BBB) (21, 22, 23). Early in the course of MS (24, 25) and focal cerebral ischemia (26), the BBB appears compromised which allows deposition of the plasma proteins fibronectin and vitronectin into the CNS (27, 28, 29). In light of this, we have focused on the potential role of these proteins and their cell surface receptors, integrins, in the promotion of microglial activation and of myelin destruction. Integrins are a large family of ECM receptors that play essential roles during development, inflammation, and neoplasia (30, 31, 32, 33). They are expressed at the cell surface as noncovalently linked {alpha}beta heterodimers. Integrins regulate cell behavior by forming a physical transmembrane link between the ECM and the cytoskeleton and by transducing signals from the ECM (34, 35, 36, 37).

Previous studies have shown that microglial activation in vitro is strongly promoted by fibronectin and vitronectin, but not by laminin (38). In the current study, we tested the hypothesis that fibronectin and vitronectin deposition during demyelinating disease directly promotes microglial activation and expression of MMP-9. By using an EAE mouse model of demyelination, we quantified fibronectin and vitronectin levels in the brains of mice with demyelinating disease and examined the relationship between fibronectin and vitronectin deposition, microglial activation, and expression of the protease MMP-9. To determine whether this was a causal relationship, we extended these findings to investigate microglial activation and MMP-9 expression in response to fibronectin and vitronectin in pure cultures of microglia in vitro and then identified the specific microglial integrins that mediate these effects.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

The studies described have been reviewed and approved by The Scripps Research Institute (TSRI) Institutional Animal Care and Use Committee. All animals were maintained under pathogen-free conditions in the closed breeding colony of TSRI.

The EAE model of demyelination

For the EAE studies, C57BL/6 mice, between 8 and 10 wk of age, were immunized s.c. with a 1:1 emulsion of 100 µl of myelin oligodendrocyte glycoprotein peptide (MOG35–55, TSRI Peptide Synthesis Core Facility) in 100 µl of CFA (Sigma-Aldrich) containing Mycobacterium tuberculosis (200 ng/ml; Difco), as described previously (39). In addition, each mouse received an i.p. injection of 500 ng of pertussis toxin (List Laboratories) on days 0 and 2. Control animals received CFA without the MOG peptide (CFA controls). This protocol leads to robust induction of clinical EAE on days 13–14 following immunization. Animals were monitored daily for clinical signs and scored as follows: 0, no physical signs; 0.5, partial loss of tail tonus; 1, complete tail atony; 2, hindlimb paraparesis; 3, full bilateral hindlimb paralysis; 4, moribund; and 5, death due to EAE. Mice were euthanized at different time points defined by their clinical status: CFA controls (clinical score = 0), acute symptomatic (clinical score = 2–4, day 21) and chronic symptomatic (clinical score = 0–1, day 40).

Immunohistochemistry and Abs

Immunohistochemistry was performed as described previously (40) on frozen sections of perfused brains. The following mAbs were obtained from BD Pharmingen: rat mAbs reactive for the integrin subunits {alpha}4 (R1-2 and MFR4.B), {alpha}5 (5H10-27 (MFR5)), {alpha}v (RMV-7), {alpha}M (M1/70), and hamster mAbs reactive for the integrin subunits beta1 (Ha2/5), {alpha}5 (HM{alpha}5-1), and the isotype control Abs, rat anti-keyhole limpet hemocyanin (A110-2), and hamster anti-trinitrophenol-keyhole limpet hemocyanin (G235-1). Other monoclonals included anti-MMP-9 (MAB909; R&D Systems), F4/80 (Serotec), and anti-glial fibrillary acidic protein (GFAP; DakoCytomation). The following rabbit polyclonal Abs were used: fibronectin (Sigma-Aldrich), vitronectin (Molecular Innovation), GFAP (Sigma-Aldrich), MBP (Chemicon International), and beta-actin (Neo-marker). Goat anti-mouse albumin was obtained from Bethyl Laboratories. Texas Red-conjugated anti-rabbit and Texas Red-conjugated anti-rat secondary Abs were obtained from Jackson Immunologicals. The DGR and RGD peptides were obtained from Sigma-Aldrich. Quantification of the numbers of activated microglia was performed using dual immunofluorescence (IF) by selecting regions of interest (ROI; area = 1.5 mm2) displaying abnormal perivascular deposits of fibronectin or vitronectin and then counting the number of Mac-1- or F4/80-positive cells within five ROI per brain section of four different sections per animal. Each experiment was performed three separate times with different animals and the results expressed as the mean ± SD of the number of Mac-1- or F4/80-positive cells per field of view. Quantification of the proportion of microglia expressing MMP-9 was performed in a similar manner. Statistical significance was assessed by using the Student t test in which p < 0.05 was defined as statistically significant.

Western blotting

Brains were removed from perfused mice, separated into forebrain and hindbrain, and then homogenized in lysis buffer, consisting of PBS containing 1% Nonidet P-40 (Sigma-Aldrich) and a mixture of protease inhibitors (Invitrogen Life Technologies). After 30 min on ice, the homogenate was centrifuged to remove the insoluble fraction and the protein concentration of the brain lysate quantified (Bio-Rad). Ten-microgram amounts of protein were then mixed with nonreducing sample buffer, boiled for 5 min, and analyzed on 8% resolving gels (Invitrogen Life Technologies) under nonreducing conditions. Proteins were electroblotted for 3 h onto nitrocellulose membranes (Invitrogen Life Technologies), blocked for 1 h in 5% nonfat milk in PBS containing 0.1% Tween 20 (Sigma-Aldrich) and membranes were probed for 1 h, washed, then incubated with anti-rabbit HRP conjugate (Sigma-Aldrich) for 1 h, before being extensively washed. Finally, protein bands were visualized with the ECL detection system (Amersham Biosciences) according to the manufacturer’s instructions. For protein quantification, gels were scanned using a Bio-Rad VersaDoc imaging system and band intensities were quantified using the NIH Image program. Within each brain sample, levels of fibronectin and vitronectin were first normalized to the level of beta-actin and then expressed as the fold-increase over the level present within the brain of CFA control animals.

Cell culture

Pure cultures of mouse microglia were obtained as described previously (41). Microglia were counted by hemocytometer and plated at a density of 2 x 105 cells/well in 6-well plates (Nunc) previously coated for 2 h with a 10 µg/ml solution of fibronectin, laminin, or vitronectin (all from Sigma-Aldrich). Cells were grown in N1 serum-fee medium (DMEM supplemented with N1 (Sigma-Aldrich). The purity of these microglial cultures was >99% as determined by Mac-1 positivity in flow cytometry.

Flow cytometry

After 2 days in culture, microglia were removed from the culture plates and cell surface expression of MHC class I and the integrins {alpha}4, {alpha}5, and Mac-1 was analyzed by flow cytometry using direct fluorescent-conjugated mAbs, as described previously (41). The fluorescent intensity of the labeled cells was analyzed with a BD Biosciences FACScan machine, with 10,000 events recorded for each condition. For each experimental condition, the mean fluorescent intensity was compared with the control state and expressed as the percentage change relative to the control condition.

Integrin inhibition studies

For the investigation of integrin functions, microglia were first plated down for 2 h before function-blocking reagents were added to the culture medium (5 µg/ml for the anti-integrin Abs and 0.1 mg/ml for the DGR/RGD peptides), and maintained for the duration of the experiment (2 days). In these experiments, the control condition was an isotype control Ab. Each experiment was repeated a minimum number of four times and the data were expressed as mean ± SD. Statistical significance was assessed by using the Student paired t test, in which p < 0.05 was defined as statistically significant.

Gel zymography

Gelatin zymography to detect MMP activity was performed as previously described (42). Microglial cells were plated at a density of 2 x 105 cells/well in 6-well plates. After 2 days of culture, microglial supernatants were collected and analyzed for gelatinolytic activity. Positive controls for MMP-9 and MMP-2 (obtained from R&D Systems) were always included. For quantification, gels were scanned using a Bio-Rad VersaDoc imaging system and band intensities were quantified using the NIH Image program. Each experiment was repeated a minimum number of four times and the data were expressed as mean ± SD. Statistical significance was assessed by using the Student paired t test, in which p < 0.05 was defined as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Fibronectin and vitronectin levels are increased in the CNS of mice undergoing demyelination in EAE

To test the notion that fibronectin and vitronectin promote microglial activation during demyelinating disease, we examined these events in EAE, a demyelinating animal model of MS. As described in Materials and Methods, C57BL/6 mice were immunized s.c. with the MOG35–55 peptide emulsified in CFA and assessed clinically on a daily basis for the development of EAE. Control mice were injected with CFA without the MOG peptide. Mice began developing EAE signs at 14 days post-MOG immunization. The mean score of clinical disease peaked between days 21 and 28 (acute phase), and then the animals made a partial recovery to stabilize after day 42 (chronic phase), though consistent with previous studies performed in C57BL/6 mice (43, 44), some residual clinical impairment was apparent and was maintained for the duration of the experiment (52 days).

In the first set of experiments, we examined the amounts of fibronectin and vitronectin present in the brain of EAE mice by performing Western blot on brain lysates taken from three different stages of EAE: CFA control, acute, and chronic. Brain lysates made from the hindbrain of individual animals were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with polyclonal Abs against fibronectin or vitronectin. This showed that fibronectin and vitronectin were barely detectable in the brain of CFA control animals, but were dramatically up-regulated in the acute phase before falling back to a lower level during the chronic phase of disease (Fig. 1). Compared with the level in the CFA control animals, the amount of fibronectin protein was increased 11.39- ± 1.78-fold in the brain of acute-phase animals (p < 0.02) and 4.37- ± 0.52-fold in the brain of chronic-phase animals (p < 0.05). Likewise, compared with the level in the CFA control animals, the amount of vitronectin protein was increased 9.2- ± 0.80-fold in the brain of acute-phase animals (p < 0.005) and 1.55- ± 0.41-fold in the brain of chronic-phase animals (p < 0.01). Thus, there was a clear association between the severity of EAE and the total amount of fibronectin and vitronectin protein within the CNS. This work confirms the findings of a previous study, which showed by an immunohistochemical approach that fibronectin and vitronectin levels in the CNS are increased during EAE (45).


Figure 1
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  		FIGURE 1. Western blot analysis of fibronectin and vitronectin protein levels in the brain of EAE mice. A, Western blots. Protein lysates obtained from the hindbrain of CFA control, acute, and chronic EAE animals were resolved by SDS-PAGE on 8% nonreducing gels, transferred to nitrocellulose membranes, and amounts of fibronectin and vitronectin protein were detected as described in Materials and Methods. B, Graphical representation of Western blots. Levels of fibronectin or vitronectin are expressed as the fold-increase over the CFA control condition; all points represent the mean ± SD of three separate brain samples. Note that fibronectin (Fibro) and vitronectin (Vitro) were barely detectable in the brain of CFA control animals, but were dramatically up-regulated in the brain of acute EAE animals, before falling back to a lower level in the brain of chronic EAE animals.

 
Fibronectin and vitronectin deposition in EAE is associated with microglial activation

Having established that fibronectin and vitronectin levels are strongly up-regulated in the CNS during EAE, we next investigated whether fibronectin or vitronectin deposition in the CNS of EAE animals is associated with microglial activation. In this EAE model, the areas of brain most affected were the cerebellum, pons, and medulla. These areas displayed the highest level of inflammatory infiltrates and also showed the most abundant deposition of fibronectin and vitronectin. Therefore, we focused on these areas of the brain and used dual-color IF to examine the association between fibronectin or vitronectin distribution and the presence of activated microglia, as defined by the activation integrin Mac-1. As shown in Fig. 2, in the brain of CFA control animals, no extravascular deposits of fibronectin or vitronectin were detected and this correlated with very small numbers of Mac-1-positive cells. In contrast, in the brain of acute-phase EAE animals, many extravascular deposits of fibronectin and vitronectin were present and these were always surrounded by Mac-1-positive cells. To characterize this association, ROI (1.5 mm2) were selected on the basis of the appearance of perivascular deposits of fibronectin or vitronectin, and within these ROI, numbers of Mac-1-positive cells were quantified. This revealed a significant increase in Mac-1-positive cells during the acute phase of EAE, as compared with the CFA controls (for the fibronectin/Mac-1 dual IF, 40.6 ± 13.2 Mac-1-positive cells in the acute phase vs 1.2 ± 1.1 Mac-1-positive cells in the CFA controls, p < 0.05, and for the vitronectin/Mac-1 dual IF, 35.8 ± 9.5 Mac-1-positive cells in the acute phase vs 1.73 ± 1.0 cells in the CFA controls, p < 0.05). Parallel studies using the F4/80 Ag as a marker for activated microglia gave very similar results (45.6 ± 10.7 F4/80-positive cells in the acute phase vs 1.6 ± 0.9 F4/80-positive cells in the CFA controls, p < 0.05 (Fig. 2C)). In the ROI, dual-color IF showed that deposition of fibronectin and vitronectin was also associated with GFAP-positive astrocyte fibers (Fig. 2D). This is consistent with the geographical distribution of astrocyte foot processes running along cerebral blood vessels and the leakage of fibronectin and vitronectin across the vessels. To address whether deposition of fibronectin is associated with increased microvascular permeability, dual-color IF examining fibronectin and albumin distribution was performed. In the brain of CFA control mice, no fibronectin or albumin was detected. In contrast, in the brain of acute EAE animals, a strong association between fibronectin deposition and albumin leakage into the CNS parenchyma was observed (Fig. 2E), indicating a correlation between reduced BBB integrity and fibronectin deposition.


Figure 2
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  		FIGURE 2. Association between fibronectin/vitronectin deposition and activated microglia, astrocytes, and albumin in the CNS of EAE mice. Dual-color IF histochemistry was performed on frozen sections of brain stem taken from CFA control or acute symptomatic EAE mice. A, Fibronectin-Texas Red (Fibro), Mac-1-FITC. B, Vitronectin-Texas Red (Vitro), Mac-1-FITC. C, Fibronectin-Texas Red (Fibro), F4/80-FITC. D, Fibronectin/Vitronectin-Texas Red (Fibro/Vitro), GFAP-FITC. E, Fibronectin-Texas Red (Fibro), Albumin-FITC. Scale bar, 50 µm. Numbers of activated microglia were quantified by selecting ROI showing perivascular deposits of fibronectin or vitronectin and counting numbers of Mac-1- or F4/80-positive cells within five ROI of four slides. All points represent the mean ± SD of three separate animals. Note that in the CFA control brain, no deposits of fibronectin or vitronectin were present and this correlated with very few Mac-1- or F4/80-positive cells (A–C). In contrast, in the acute symptomatic phase of EAE, many perivascular deposits of fibronectin and vitronectin were present and these deposits were always surrounded by aggregates of Mac-1- or F4/80-positive cells. In addition, fibronectin and vitronectin deposits were associated with GFAP-positive astrocyte processes (D) and albumin leakage into the cerebral parenchyma (E).

 
Activated microglia in EAE lesions express MMP-9

In the pathogenesis of MS, MMP-9 has attracted a lot of interest because it is strongly induced during demyelination (12, 13, 14), and this has been substantiated by the finding that MMP-9 knockout mice show resistance to EAE (15). In light of the observation that activated microglial cells express MMP-9 (46, 47), we next examined whether activated microglia in an EAE model also express MMP-9. As shown in Fig. 3A, very few Mac-1-positive cells and no MMP-9-positive cells were detected in the brain of CFA control mice. In contrast, in ROI showing perivascular fibronectin deposits in the brain of acute-phase mice, we observed many Mac-1-positive cells that also stained positive for MMP-9. Because previous reports have also described MMP-9 expression in astrocytes (48, 49) and oligodendrocytes (50, 51, 52), we also examined whether these cell types express MMP-9 in the brains of acute EAE mice. While GFAP-positive cells were often closely associated with regions containing MMP-9-positive microglia, GFAP-positive cells did not express MMP-9 themselves (Fig. 3B). Furthermore, MBP-positive oligodendrocytes within myelinated tracts did not express MMP-9 either. Hence, MMP-9 is expressed specifically by Mac-1-positive microglial cells within the brains of EAE mice. However, not all Mac-1-positive cells expressed detectable levels of MMP-9 (Fig. 3A). To estimate the proportion of microglia that expressed MMP-9, we performed dual-color IF combined with the nuclear marker Hoechst. This revealed that within these ROI, 30.5 ± 8.4% of Mac-1-positive cells expressed detectable amounts of MMP-9.


Figure 3
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  		FIGURE 3. MMP-9 is expressed in activated microglia, but not astrocytes or oligodendrocytes. A, Dual-color immunofluorescent histochemistry was performed on frozen sections of brain stem taken from CFA control or acute symptomatic EAE mice, to detect activated microglia (Mac-1-FITC) and MMP-9 (Cy3 conjugate). Scale bar, 50 µm. Note that in the CFA control, no activated microglia or MMP-9-positive cells were present. In contrast, in the acute symptomatic EAE mice, many Mac-1-positive activated microglia were present, and many of these cells expressed MMP-9. B, Dual-color IF was performed on frozen sections of brain stem taken from acute symptomatic EAE mice, to examine whether astrocytes (GFAP-FITC) or oligodendrocytes (MBP-FITC) express MMP-9 (Cy3). Scale bar, 100 µm. Note that astrocytes and oligodendrocytes do not express MMP-9.

 
Fibronectin and vitronectin promote microglial activation and pro-MMP-9 expression in vitro

In an EAE model of demyelinating disease, we have shown that in the acute phase of the disease, deposition of perivascular fibronectin and vitronectin is associated with microglial activation, and that these activated microglia express MMP-9. Although this association suggests that in EAE, fibronectin and vitronectin stimulate microglial activation and MMP-9 expression, it is not possible to conclude this in vivo because of the complex nature of the multitude of mechanisms that act during neuroinflammation. Thus, to determine whether there is a causal relationship between fibronectin or vitronectin deposition and microglial activation/MMP-9 expression, we next examined microglial responses in vitro.

Pure cultures of mouse microglia derived from postnatal mixed glial cultures were grown in serum-free medium on different substrates including uncoated plastic (control), laminin, fibronectin, and vitronectin, as previously described (41). We used serum-free culture conditions throughout the experiments as serum contains many factors known to modulate the microglial activation state including LPS (53) and some of the ECM proteins this study was designed to investigate (54, 55). In the initial set of studies, we examined the influence of ECM substrate on microglial activation, as assessed by MHC class I expression, and on microglial production of MMP-9, as measured by gelatin zymography. After 2 days in serum-free culture on the different substrates, microglial MHC class I expression was determined by flow cytometry. Relative to the uncoated plastic control substrate, microglial MHC class I expression was increased by fibronectin (by 89 ± 19.6%, p < 0.005) and vitronectin (by 101 ± 14.8%, p < 0.001), but decreased by laminin (by 29.4 ± 8.2%, p < 0.01) (Fig. 4A). These results confirm our previously published observation that fibronectin and vitronectin directly stimulate microglial activation (38). To quantify microglial expression of MMP-9, gelatin zymography was performed on supernatants taken from microglia cultured on the different ECM substrates. Gelatin zymography reveals both the active and inactive (proform) forms of MMP-9, as a result of the SDS-denaturation and -renaturation steps of this technique (56, 57). Gelatin zymography revealed that microglia in culture produce the higher molecular mass (92 kDa) pro-MMP-9 form, but negligible amounts of the lower molecular mass (82 kDa) activated form. Interestingly, microglial production of pro-MMP-9 was increased by fibronectin (by 170 ± 42%, p < 0.005) and vitronectin (by 260 ± 54%, p < 0.005), but not by laminin, relative to the uncoated plastic (Fig. 4, B and C). Microglial MMP-2 activity was not altered by the different ECM substrates (see Fig. 4). Taken together, this data shows that fibronectin and vitronectin directly promote microglial activation and expression of pro-MMP-9.


Figure 4
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  		FIGURE 4. The influence of ECM proteins on microglial expression of MHC class I and MMP-9. Microglia were purified from mixed glial cultures, as described in Materials and Methods, and then cultured in serum-free medium on uncoated plastic, laminin (Lam), fibronectin (Fibro), or vitronectin (Vitro). After 2 days in culture, microglial expression of MHC class I was quantified by flow cytometry (A) and microglial supernatants were analyzed for MMP-9 activity by gel zymography. B, A representative gel zymogram; C, the mean ± SD of four independent experiments. Note that fibronectin and vitronectin increased microglial expression of MHC class I, whereas laminin reduced expression. In addition, fibronectin and vitronectin, but not laminin, significantly promoted microglial production of pro-MMP-9, relative to the uncoated plastic control substrate.

 
Fibronectin and vitronectin promote microglial activation via the {alpha}5beta1 and {alpha}vbeta5 integrins, respectively

To investigate which integrins mediate the microglial activation response to fibronectin and vitronectin, microglial cells were cultured on fibronectin or vitronectin in the presence of function-blocking reagents specific for the different integrins. In a previous study, we demonstrated that microglia express the fibronectin integrin receptors, {alpha}4beta1 and {alpha}5beta1, and the vitronectin integrin receptor, {alpha}vbeta5 (38). Therefore, to target these integrins, we used the following blocking reagents: isotype control Ab, the control DGR peptide, the integrin function-blocking peptide RGD, or function-blocking Abs against the beta1, {alpha}v, {alpha}4, or {alpha}5 integrin subunits. After 2 days of incubation with these reagents, the microglial activation state was determined in flow cytometry by measuring cell surface expression of MHC class I and three different integrins ({alpha}4beta1, {alpha}5beta1, and Mac-1), previously shown to be increased during microglial activation (38, 58). As shown in Fig. 5, A and B, expression of MHC class I by microglia grown on fibronectin was reduced by RGD peptides (by 25.5 ± 2.3%, p < 0.005) and by Abs against the beta1 subunit (by 28.2 ± 1.6%, p < 0.002) and the {alpha}5 subunit (by 27.4 ± 2.3%, p < 0.005), but was not affected by the control DGR peptides or Abs against the {alpha}v or {alpha}4 integrin subunits. In a similar manner, on fibronectin, RGD peptides reduced microglial expression of the integrins {alpha}4 (by 39.7 ± 8.8%, p < 0.02), {alpha}5 (by 23.1 ± 3.3%, p < 0.01), and Mac-1 (by 24.4 ± 2.3%, p < 0.01), the anti-beta1 integrin Ab reduced microglial expression of the integrins {alpha}4 (by 47.5 ± 11.1%, p < 0.02), {alpha}5 (by 38.7 ± 6.9%, p < 0.02), and Mac-1 (by 27.1 ± 2.6%, p < 0.005), and the anti-{alpha}5 integrin Ab reduced microglial expression of the integrins {alpha}4 (by 44.3 ± 8.3%, p < 0.02), {alpha}5 (by 34.6 ± 6.0%, p < 0.01), and Mac-1 (by 26.7 ± 4.9%, p < 0.02), relative to the isotype control Ab (Fig. 5C).


Figure 5
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  		FIGURE 5. Examination of the role of specific integrins in mediating microglial activation. Microglia were purified from mixed glial cultures, as described in Materials and Methods, and then cultured in serum-free medium on fibronectin (A–C) or vitronectin (D–F) in the presence of function-blocking reagents specific for the different integrin subunits (DGR control peptides, RGD peptides, and mAbs reactive for the beta1, {alpha}4, {alpha}5, and {alpha}v integrin subunits). After 2 days in culture, microglial expression of MHC class I (A, B, D, and E), or expression of the "activation integrins" {alpha}4, {alpha}5, and Mac-1 (C and F), was quantified by flow cytometry and expressed as the percentage change relative to control conditions (isotype control Ab). All points represent the mean ± SD of four separate experiments. Note that microglial activation on fibronectin was inhibited by RGD peptides and the beta1 and {alpha}5 Abs, while microglial activation on vitronectin was inhibited by RGD peptides and the {alpha}v Ab.

 
Expression of MHC class I by microglia grown on vitronectin was reduced by RGD peptides (by 37.7 ± 9.1%, p < 0.02) and by Abs against the {alpha}v integrin subunit (by 32.7 ± 3.6%, p < 0.01), but was not affected by the control DGR peptides or the Ab against the beta1 integrin subunit (Fig. 5, D and E). In a similar manner, on vitronectin, RGD peptides reduced microglial expression of the integrins {alpha}4 (by 35.3 ± 6.8%, p < 0.02), {alpha}5 (by 27.1 ± 3.8%, p < 0.01), and Mac-1 (by 29.6 ± 6.5%, p < 0.02), and the anti-{alpha}v integrin Ab reduced microglial expression of the integrins {alpha}4 (by 40.0 ± 6.3%, p < 0.01), {alpha}5 (by 36.7 ± 7.8%, p < 0.02), and Mac-1 (by 27.9 ± 6.8%, p < 0.02), relative to the isotype control Ab (Fig. 5F). In summary, this flow cytometric analysis showed that microglial activation by fibronectin is mediated via the RGD-dependent {alpha}5beta1 integrin, while microglial activation by vitronectin is mediated via the RGD-dependent {alpha}vbeta5 integrin.

Integrin blockade reverses microglial activation on fibronectin and vitronectin

Microglial activation is associated with a morphological switch from a resting ramified form to an activated amoeboid form (2, 3, 58). Consistent with our previous observations (38, 41), microglia cultured on fibronectin or vitronectin showed the amoeboid morphology typical of activated microglia (Fig. 6). In these experiments, the presence of the beta1 integrin blocking Ab reverted microglial morphology on fibronectin from amoeboid back to the resting ramified form. In contrast, microglia cultured on vitronectin only showed this morphological reversion in the presence of the {alpha}v integrin blocking Ab (Fig. 6). Taken together, this data implies that microglial activation on fibronectin is mediated by the integrin {alpha}5beta1 and microglial activation on vitronectin is mediated by the integrin {alpha}vbeta5.


Figure 6
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  		FIGURE 6. The role of beta1 and {alpha}v integrins in mediating microglial activation state. Microglia were purified from mixed glial cultures, as described in Materials and Methods, and then cultured in serum-free medium on fibronectin (Fibro, top panels) or vitronectin (Vitro, lower panels) in the presence of isotype control Ab, or function-blocking Abs against the beta1 or {alpha}v integrin subunits. Photomicrographs were taken 2 days after culture. Scale bar, 50 µm. Note that on fibronectin, only the anti-beta1 integrin Ab reverted the microglial morphology from the classical activated amoeboid form to the resting ramified form. In contrast, on vitronectin, only the anti-{alpha}v integrin Ab promoted this reversion in phenotype.

 
Fibronectin and vitronectin promote pro-MMP-9 expression via the {alpha}5beta1 and {alpha}vbeta5 integrins, respectively

Because we found that fibronectin and vitronectin directly stimulated microglial production of pro-MMP-9, we also conducted parallel experiments to examine which integrins are important in mediating this influence. In a similar manner to the evaluation of integrin roles in promoting microglial activation, microglial cells were cultured on fibronectin or vitronectin for 2 days, in the presence of the same integrin-specific function-blocking reagents. After this time, microglial supernatants were collected and analyzed for gelatinolytic activity by gelatin zymography. This showed that the fibronectin induction of microglial pro-MMP-9 was reduced by the RGD peptides (by 56.7 ± 6.9%, p < 0.005), anti-beta1 integrin Ab (by 52.2 ± 5.0%, p < 0.005) and the anti-{alpha}5 integrin Ab (by 52.5 ± 8.0%, p < 0.01), but was not affected by the DGR control peptides or Abs against the {alpha}4 or {alpha}v integrin subunits (Fig. 7, A and B). In contrast, the microglial pro-MMP-9 response to vitronectin was inhibited by the RGD peptides (by 63.1 ± 13.5%, p < 0.02), and the anti-{alpha}v integrin Ab (by 56 ± 14%, p < 0.05), but not affected by the DGR control peptides or Ab against the beta1 integrin subunit. (Fig. 7, C and D). Interestingly, microglial expression of MMP-2 on fibronectin or vitronectin was not altered by any of the integrin function-blocking reagents (data not shown). Taken together, these in vitro studies show that microglial activation and pro-MMP-9 expression are directly promoted by fibronectin and vitronectin, via the {alpha}5beta1 and {alpha}vbeta5 integrins, respectively. This provides the first evidence that the {alpha}5beta1 and {alpha}vbeta5 integrins are important mediators of microglial activation.


Figure 7
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  		FIGURE 7. Examination of the role of specific integrins in mediating microglial expression of pro-MMP-9. Microglia were purified from mixed glial cultures, as described in Materials and Methods, and then cultured in serum-free medium on fibronectin (A and B) or vitronectin (C and D) in the presence of function-blocking reagents specific for the different integrin subunits (DGR control peptides, RGD peptides, and mAbs reactive for the beta1, {alpha}4, {alpha}5, and {alpha}v integrin subunits). After 2 days in culture, levels of pro-MMP-9 in the microglial supernatants were examined by gel zymography. A and C, Representative gel zymograms; B and D, represent the mean ± SD of four separate experiments, in which each point is expressed as the percentage change relative to control conditions (isotype control Ab). Note that fibronectin induction of microglial pro-MMP-9 was blocked by RGD peptides and the beta1 and {alpha}5 integrin Abs, whereas the vitronectin influence was inhibited by RGD peptides and the {alpha}v Ab.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Early in the pathogenesis of a number of CNS disorders, the BBB is compromised (21, 24, 25, 26, 59). This increased permeability leads to deposition of a number of plasma proteins, including fibrinogen, fibronectin, and vitronectin within the cerebral parenchyma (27, 28, 29, 45, 60). Based on the previous observation that microglial activation can be induced by fibronectin and vitronectin in vitro (38), we set out to test the hypothesis that fibronectin and vitronectin deposition during demyelinating disease directly promotes microglial activation and expression of MMP-9 in that setting. In the EAE-affected brain, fibronectin and vitronectin levels were strongly increased and there was a close spatial relationship between fibronectin and vitronectin deposition and microglial activation/MMP-9 expression. Cell culture studies demonstrated that microglial activation and pro-MMP-9 expression were directly promoted by fibronectin and vitronectin in a coincident manner, and that these effects were mediated by the {alpha}5beta1 and {alpha}vbeta5 integrins, respectively. Taken together, this work suggests that fibronectin and vitronectin deposition during demyelinating disease is an important influence on microglial activation state.

Regulation of microglial activation by the ECM

In addition to playing a protective role, in which microglia destroy invading microorganisms, accumulating evidence suggests that in the pathological conditions of MS, focal ischemia, Alzheimer’s disease, and infection-related dementias, microglia appear stimulated and contribute to host tissue destruction (1, 2, 3, 4, 5, 6, 7, 61). Following stimulation, resting microglia rapidly transform into migratory aggressive cells that secrete a variety of soluble factors, many of which promote and exacerbate the inflammatory response (1, 2, 3). Microglial activation is promoted by cytokines, chemokines (2, 3, 38), bacterial cell components like LPS (58) and beta-amyloid protein (62). Because an early event in the pathogenesis of MS is breakdown of the BBB (24, 25), we became interested in the concept that proteins present within blood might pass through the leaky BBB and influence microglial activation state. In this study, we observed a close correlation between albumin and fibronectin deposition in the cerebral parenchyma of EAE-affected mice, supporting the notion that fibronectin leaks into brain tissue at sites of reduced BBB integrity. In a prior study, we found that two ECM proteins, fibronectin and vitronectin, present at high concentrations in blood (both ~0.3 g/L) (54, 55), stimulate microglial activation relative to uncoated plastic, while laminin inhibits microglial activation (38). In this regard, ECM-dependent regulation of microglial activation is entirely consistent with the location of these proteins in MS. Fibronectin and vitronectin are present at only very low levels in the normal adult CNS (40, 63, 64); therefore, microglia are not activated. However, as previously suggested (38), any event that leads to increased levels of fibronectin or vitronectin within the CNS parenchyma could promote microglial activation. This includes the deposition of plasma-derived fibronectin and vitronectin, as a result of BBB breakdown, not only in MS, but also during inflammation, ischemia, trauma, and tumor invasion (28, 64). It also includes the de novo synthesis of fibronectin and vitronectin by CNS-resident cells, as described in epilepsy (65, 66, 67) and tumor formation (68, 69).

Potential functions of microglial MMPs within the demyelinating CNS

The MMPs are a large family of zinc-dependent endopeptidases that can selectively degrade many ECM proteins including laminin, collagen, and fibronectin (11, 16, 70, 71). MMP-9 and MMP-12 also degrade myelin proteins including MBP (17, 18, 72) and MMP-9 has been implicated in neuronal cell death (19, 20). Current evidence suggests that MMP-9 is an important effector molecule in demyelinating disease. In MS, MMP-9 expression is increased, both in the affected CNS tissue (12, 13, 14) and in the cerebrospinal fluid (73, 74). Pharmacological blockade of MMP-9 improves the clinical outcome of EAE (75, 76). Furthermore, mice deficient in MMP-9 are resistant to EAE, at least early in life (15). In terms of the role of MMP-9 in demyelinating disease, most studies to date have focused on the contribution of inflammatory cells entering the CNS. These studies support the concept that peripheral inflammatory cells mediate BBB breakdown, at least in part by MMP-9-mediated degradation of basal lamina ECM components (16, 71, 77, 78, 79).

In this study, we have shown that in EAE lesions, MMP-9 is expressed by microglia, but not astrocytes or oligodendrocytes, and further demonstrated in vitro, that microglial activation and production of pro-MMP-9 are directly promoted by fibronectin and vitronectin. This raises the following question: what is the contribution of microglial MMP-9 to the pathogenesis of demyelination and what are the relevant substrates? In light of the remodeling role of microglia, we postulate that microglial production of MMP-9 may be part of a normal physiological response to remove the extraneous fibronectin and vitronectin deposited in the CNS parenchyma. However, the downside of this response could be that MMP-9 degrades other substrates, including: 1) ECM proteins within the vascular basal lamina, thus causing further BBB breakdown, 2) ECM proteins associated with axons, shown to mediate survival signals for oligodendrocytes (80), and 3) myelin-specific components such as MBP (17, 18). Clearly, degradation of any of these substrates will have deleterious effects on oligodendrocytes, leading to a demyelinated state. As evidence suggests that activated microglia may mediate oligodendrocyte damage in demyelinating disease (4, 5, 6, 7, 8), it now becomes important to test the hypothesis that microglial-secreted MMP-9 is destructive for oligodendrocytes. In future studies, we will test this idea by examining whether microglial-derived MMP-9 can degrade oligodendrocyte myelin membranes and/or induce oligodendrocyte death in cell culture.

We found that pure cultures of microglial cells secrete the inactive 92-kDa form of MMP-9, but none of the active 82-kDa form. This raises the question of what activates microglial pro-MMP-9 in vivo? Several different purified proteases have been shown to activate pro-MMP-9 in vitro, including plasmin (81), trypsin (82), MMP-2 (83), and MMP-3 (stromelysin-1) (81, 82). More significantly, the work of Ramos-DeSimone et al. (84) demonstrated that MMP-3 is a potent activator of pro-MMP-9 in vivo. Interestingly, recent work has shown that MMP-3 released from apoptotic neurons directly stimulates microglial activation (85). Thus, it seems likely that MMP-3 is an important upstream molecule for stimulating microglial activity, first by directly promoting microglial activation and second, by mediating activation of microglial pro-MMP-9. Taken together with our recent finding that MMP-3 is produced specifically by astrocytes, but not microglia, stimulated by LPS or IL-1beta (86), this implies that within the inflamed CNS, astrocyte production of MMP-3 would be one mechanism leading to activation of microglial pro-MMP-9.

The role of integrins in promoting microglial activation

Another important finding of this study is that the influence of fibronectin and vitronectin on microglial activation was mediated by the integrins {alpha}5beta1 and {alpha}vbeta5, respectively. This is the first evidence that these integrins mediate microglial activation. It follows that these integrins represent potential targets for therapeutic intervention to prevent excessive microglial activation during brain injury. Previous studies have shown that select integrins can modulate microglial behavior. In a hippocampal slice culture model of excitotoxicity, antisense inhibition of the beta2 integrin LFA-1 ({alpha}Lbeta2) blocked microglial activation and migration (87). In addition, RGD peptides blocked microglial uptake of apoptotic neurons in cell culture (88) and also inhibited amyloid peptide uptake and microglial activation in hippocampal slice cultures (89). The RGD-dependent {alpha}vbeta5 integrin is particularly interesting because of its described role in phagocytosis in other cell types, including macrophages and retinal pigment epithelial cells (90, 91, 92). This has been substantiated by the recent finding that beta5 integrin null mice develop accelerated age-related blindness as a result of defective phagocytosis of photoreceptor cells in the retina (93). In light of the view that microglia may actively contribute to the pathogenesis of MS, in part by phagocytosing oligodendrocytes (8, 94, 95), in future studies it will be important to examine whether the {alpha}vbeta5 integrin is actively involved in microglial phagocytosis of oligodendrocytes.

In conclusion, we have shown that in an EAE animal model of demyelination, deposition of fibronectin and vitronectin in the cerebral parenchyma were tightly associated with microglial activation and MMP-9 expression. Furthermore, in vitro experiments revealed that both responses were directly promoted by fibronectin and vitronectin, and that this influence was mediated by the {alpha}5beta1 and {alpha}vbeta5 integrins, respectively. Taken together, this work implies that fibronectin and vitronectin deposition during demyelinating disease is an important influence on microglial activation state. Furthermore, this is also likely to be true in other CNS disorders that involve deposition of fibronectin and vitronectin into the parenchyma, including inflammation, ischemia, trauma, epilepsy, and tumor formation (64, 65, 66, 67, 68, 69). Finally, this work suggests that targeting the microglial integrins {alpha}5beta1 and {alpha}vbeta5 may represent a promising therapeutic approach to inhibit the excessive microglial activation seen in a number of pathological states, including MS.


   Acknowledgment
 
We thank Professor J. Lindsay Whitton for comments on the manuscript. This is manuscript number 18358 from The Scripps Research Institute.


   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 supported in part by a Harry Weaver Neuroscience Scholar Award from the National Multiple Sclerosis Society (to R.M.), by an Advanced Postdoctoral Fellowship from the National Multiple Sclerosis Society (to S.J.C.), and by National Institutes of Health Grants R01 NSO26945, R01 NSO38710, and R01 NSO53716. Back

2 Address correspondence and reprint requests to Dr. Richard Milner, Department of Molecular and Experimental Medicine, MEM-132, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: rmilner{at}scripps.edu Back

3 Abbreviations used in this paper: MS, multiple sclerosis; MMP, matrix metalloproteinase; ECM, extracellular matrix; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; BBB, blood-brain barrier; MOG, myelin oligodendrocyte glycoprotein; GFAP, glial fibrillary acidic protein; ROI, region of interest; IF, immunofluorescence. Back

Received for publication August 24, 2006. Accepted for publication April 3, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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