Microglia use multiple mechanisms to mediate interactions with vitronectin; non-essential roles for the highly-expressed αvβ3 and αvβ5 integrins
© Welser-Alves et al; licensee BioMed Central Ltd. 2011
Received: 5 August 2011
Accepted: 10 November 2011
Published: 10 November 2011
As the primary resident immune cells, microglia play a central role in regulating inflammatory processes in the CNS. The extracellular matrix (ECM) protein vitronectin promotes microglial activation, switching microglia into an activated phenotype. We have shown previously that microglia express two vitronectin receptors, αvβ3 and αvβ5 integrins. As these integrins have well-defined roles in activation and phagocytic processes in other cell types, the purpose of the current study was to investigate the contribution of these two integrins in microglial activation.
Microglial cells were prepared from wild-type, β3 integrin knockout (KO), β5 integrin KO or β3/β5 integrin DKO mice, and their interactions and activation responses to vitronectin examined in a battery of assays, including adhesion, expression of activation markers, MMP-9 expression, and phagocytosis. Expression of other αv integrins was examined by flow cytometry and immunoprecipitation.
Surprisingly, when cultured on vitronectin, microglia from the different knockout strains showed no obvious defects in adhesion, activation marker expression, MMP-9 induction, or phagocytosis of vitronectin-coated beads. To investigate the reason for this lack of effect, we examined the expression of other αv integrins. Flow cytometry showed that β3/β5 integrin DKO microglia expressed residual αv integrin at the cell surface, and immunoprecipitation confirmed this finding by revealing the presence of low levels of the αvβ1 and αvβ8 integrins. β1 integrin blockade had no impact on adhesion of β3/β5 integrin DKO microglia to vitronectin, suggesting that in addition to αvβ1, αvβ3, and αvβ5, αvβ8 also serves as a functional vitronectin receptor on microglia.
Taken together, this demonstrates that the αvβ3 and αvβ5 integrins are not essential for mediating microglial activation responses to vitronectin, but that microglia use multiple redundant receptors to mediate interactions with this ECM protein.
Microglia are immune effector cells resident in the central nervous system (CNS), whose main role is to orchestrate immunological responses following cerebral insults [1–3]. In the resting CNS, microglia occupy a basal surveillance state, but after activation by pro-inflammatory cytokines or microorganisms, they transform into metabolically active phagocytic cells, upregulating expression of cytokines and chemokines, and migrating to the inflammatory focus. As well as playing a protective role, recent evidence suggests that in some diseases, including multiple sclerosis (MS), microglia may become inappropriately stimulated, leading to auto-immune destruction of host tissue [4–7].
To understand why microglia may become inappropriately activated in the early stages of MS, we have focused our attention on the function of certain ECM proteins present in blood at high concentrations, including fibronectin and vitronectin [8, 9]. We have demonstrated that the plasma proteins vitronectin and fibronectin promote microglial activation in vitro [10, 11]. Taken together with the observation that blood-brain barrier (BBB) breakdown is an early event in the pathogenesis of MS we proposed that leakage of these two proteins into brain parenchymal tissue pre-disposes to microglial activation and myelin damage. Results obtained with the experimental autoimmune encephalomyelitis (EAE) model demonstrated that BBB breakdown was closely associated with fibronectin and vitronectin deposits in the CNS, which closely correlated with microglial activation and expression of the matrix metalloproteinase, MMP-9 . Combined with the evidence from other groups demonstrating vitronectin and fibronectin deposition in demyelinated lesions in the brains of MS patients [12–14] and EAE mice , this supports the hypothesis that fibronectin and vitronectin promote microglial activation in vivo.
A major question yet to be fully answered is: which microglial receptors mediate the activation response to vitronectin? Our prior work has shown that microglia express the two vitronectin receptors, αvβ3 and αvβ5 integrins, and that the microglial response to vitronectin is largely mediated by αv integrins . As the αvβ3 and αvβ5 integrins have well-defined roles in activation and phagocytic processes in other cell types, the purpose of the current study was to investigate the contribution of these two integrins in this process, and thereby test our hypothesis that absence of both αvβ3 and αvβ5 integrins would render microglia unresponsive to vitronectin. To examine these events, microglial cells were prepared from wild-type, β3 integrin KO, β5 integrin KO and β3/β5 integrin DKO mice, and the behavior of these microglia evaluated in a battery of assays including cell adhesion, expression of activation markers and MMP-9, and phagocytosis of vitronectin-coated beads.
The studies described have been reviewed and approved by The Scripps Research Institute Institutional Animal Care and Use Committee. β3 integrin KO and β5 integrin KO mice (backcrossed > 10 times on the C57Bl/6 background) were maintained under pathogen-free conditions in the closed breeding colony of The Scripps Research Institute (TSRI). β3 integrin KO and β5 integrin KO mice were bred and offspring genotyped using previously described protocols [16–19] to generate homozygous β3 integrin KO (β3 -/-), homozygous β5 integrin KO (β5 -/-), and double-knockout (DKO) mice, homozygous (β3 -/-, β5 -/-). In all experiments, littermate wild-type mice were used as controls.
Pure cultures of mouse microglia were obtained as described previously , with cultures from the different strains of mice being established at the same time in parallel. Briefly, 7-10 day old mixed glial cultures were shaken for 30 minutes and the supernatant containing detached microglia was collected. Microglia were counted by hemocytometer and plated at a density of 2 × 105 cells/well in six-well plates (Nunc, Naperville, IL) previously coated for two hours at 37°C with a 10 μg/ml solution of vitronectin (Sigma). Cells were grown overnight in the mixed glial culture media, and then switched to N1 serum-fee media (DMEM supplemented with N1 (Sigma). The purity of these microglial cultures was > 99% as determined by Mac-1 positivity in flow cytometry.
Cell adhesion assays
Adhesion assays were performed as previously described . Briefly, substrates were prepared by coating the central area of 24 well plates (Nunc) with 25 μl of ECM solution (10 μg/ml of vitronectin or fibronectin, both from Sigma) for 2 hours at 37°C. Substrates were washed twice before addition of cells. Microglia were prepared as described above, centrifuged, re-suspended in N1 serum-free media, and 2000 microglia applied to the substrates in a 25 μl drop and then incubated at 37°C for 15 or 30 minutes. In the function-blocking experiments, the anti-αv monoclonal antibody (RMV-7), anti-β1 monoclonal antibody (Ha2/5) or control antibodies were included at a concentration of 5 μg/ml. The assay was stopped by adding 1 ml of DMEM and washing off any loosely attached cells. The attached cells were fixed in 4% paraformaldehyde in PBS for 20 minutes, and stored in PBS. Adhesion was quantified under phase microscopy by counting all attached cells within 5 fields of view per condition. Within each experiment each condition/time-point was performed in duplicate; the results represent the mean ± SEM of three experiments. Statistical significance was assessed by using the Student's paired t test, in which p < 0.05 was defined as statistically significant.
The following monoclonal antibodies were obtained from BD Pharmingen (La Jolla, CA): rat monoclonal antibodies reactive for MHC class I (M1/22.214.171.124), the integrin subunits α4 (MFR4.B), α5 (5H10-27 (MFR5)), αv (RMV-7), αM (M1/70), and the isotype control antibody, rat anti-KLH (A110-2), and the hamster monoclonal antibodies reactive for the β1(Ha2/5; function-blocking antibody) and β3 (2C9.G2) integrin subunits and isotype control (G235-1). Rabbit polyclonal antibodies specific for the αv integrin subunit were obtained from Chemicon (Temecula, CA). The anti-β8 integrin polyclonal antibody was a kind gift from Dr. Joseph McCarty, M.D. Anderson Cancer Center, Houston, TX.
Cell surface labeling and immunoprecipitation
Microglial cell surface molecules were labeled with biotin as previously described [10, 21]. Briefly, microglial cell cultures were incubated with NHS-LC-biotin (Pierce, Rockford, IL) for 30 minutes, washed in TRIS-containing cell wash buffer (CWB), and then removed from tissue culture plates and centrifuged. Cells were lysed in 0.5% Triton-X100 in CWB that contained a cocktail of protease inhibitors (Invitrogen, Carlsbad, CA). After 30 minutes on ice, the lysate was centrifuged to remove the insoluble fraction. The supernatants were pre-cleared for one hour with 30 μl of protein A sepharose or protein G sepharose per ml of cell lysate. Immunoprecipitations were performed overnight at 4°C on a rotating platform using the polyclonal anti-αv or anti-β8 integrin antibodies at 1:250 dilution in a tube containing 30 μl protein A sepharose. Beads were washed 5 times in immunoprecipitation wash buffer, as previously described and the integrin immune complexes were separated by boiling the beads in non-reducing sample buffer for 5 minutes before being analysed by 8% SDS-PAGE (Invitrogen) under non-reducing conditions. Proteins were electro-blotted for 1.5 hours onto nitrocellulose membranes (Invitrogen), blocked overnight in 3% BSA in TBS containing 0.1% Tween-20 (Sigma) and probed with streptavidin-HRP conjugate (Pierce) for one hour, before being extensively washed. Protein bands were visualised with the SuperSignal WestFemto ECL detection system (Pierce) according to the manufacturers' instructions.
Microglial phagocytosis of vitronectin-coated beads
Microglial cells were plated at a density of 2 × 105 cells/well in six-well plates. After one day of culture, 2.5 μl of a suspension of yellow-green fluorescent beads (Molecular Probes, Eugene, OR) previously coated in a 100 μg/ml vitronectin solution for 2 hours at 37°C, was added to the microglia, and thoroughly mixed with the tissue culture media to distribute the beads throughout the culture. After a further 24 hours, cultures were visualised for microglial uptake of beads, and microglia collected and phagocytic uptake of fluorescent beads analyzed by flow cyometry, with 10,000 events recorded for each condition. The phagocytic index of microglia was quantified and expressed as the mean fluorescent intensity of the cell population. Each experiment was repeated a minimum number of four times and the data expressed as mean ± SD. Statistical significance was assessed by using the Student's paired t test, in which p < 0.05 was defined as statistically significant.
Microglia, isolated from the four different strains of mice, were cultured in vitronectin-coated 6-well plates under serum-free conditions. After 2 days, microglia were removed from the culture plates and cell surface expression of MHC class I and the integrins α4, α5, αv or Mac-1 analyzed by flow cytometry using direct fluorescent-conjugated monoclonal antibodies, as described previously . The fluorescent intensity of the labeled cells was analyzed with a Becton Dickinson FACScan machine (San Diego, CA), 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. Each experiment was repeated a minimum number of four times and the data expressed as mean ± SD. Statistical significance was assessed by using the Student's paired t test, in which p < 0.05 was defined as statistically significant.
Gelatin zymography was used to detect MMP-9 activity as previously described [11, 22]. Microglial cells were plated at a density of 2 × 105 cells/well in six-well plates that were either left uncoated, or coated with vitronectin or fibronectin. After 2 days culture, microglial supernatants were collected and analyzed for gelatinolytic activity. Positive controls for MMP-9 and MMP-2 (obtained from R&D) were included. For quantification, gels were scanned using a Bio-Rad VersaDoc imaging system (Hercules, CA) and band intensities quantified using the NIH Image program. Each experiment was repeated a minimum number of four times and the data expressed as mean ± SD. Statistical significance was assessed by using the Student's paired t test, in which p < 0.05 was defined as statistically significant.
Absence of αvβ3, αvβ5, or both integrins does not diminish microglial adhesion to vitronectin
Microglial activation responses to vitronectin are not affected by the absence of αvβ3, αvβ5 or both these integrins
Microglia deficient in β3 and β5 integrins show low expression levels of two additional αv integrins
The microglial αvβ8 integrin acts as a functional vitronectin receptor, and αvβ3 is a functional fibronectin receptor
Microglia play a critical role in the CNS by performing immune surveillance and regulating inflammatory processes [1, 2], therefore defining the factors that control microglial activation state is of fundamental importance. Evidence suggests that ECM proteins play an important role in this process. In particular, vitronectin, present at high levels in plasma , and absent in the normal CNS, is deposited in a perivascular manner in MS tissue  and in the mouse model of MS, EAE [11, 15]. Consistent with this, we have shown that vitronectin directly promotes microglial activation in vitro [10, 11]. The next important question is to identify the microglial receptors that mediate this effect. Having shown previously that microglia express the two vitronectin receptors, αvβ3 and αvβ5 integrins [10, 23], the purpose of the current study was to investigate the contribution of these two integrins in microglial activation, and thereby test our hypothesis that absence of both αvβ3 and αvβ5 integrins would render microglia unresponsive to vitronectin. Using microglia derived from different strains of mice (wild-type, β3 integrin KO, β5 integrin KO and β3/β5 integrin DKO), different aspects of microglial activation were examined. Surprisingly, when cultured on vitronectin, microglia from the knockout strains showed no obvious defects in adhesion, activation marker or MMP-9 expression, or phagocytosis of vitronectin-coated beads. To investigate the reason for this lack of effect, we examined the expression of other αv integrins. This revealed that microglia also express low levels of the alternative vitronectin receptors, αvβ1 and αvβ8 integrins. This demonstrates that the αvβ3 and αvβ5 integrins are not essential for mediating microglial activation responses to vitronectin, but that microglia use multiple redundant receptors to mediate interactions with this ECM protein.
Non-essential roles for the αvβ3 and αvβ5 integrins in microglial activation
Evidence gathered from a variety of different cell types supports a role for the αvβ3 and αvβ5 integrins in promoting cellular activation and phagocytic responses. The αvβ3 integrin has been implicated in mediating phagocytosis in monocytes and peripheral macrophages, and the αvβ5 integrin plays a similar role in macrophages and in retinal pigment epithelial (RPE) cells [24, 25, 29]. Indeed, β5 KO mice develop accelerated age-related blindness as a result of defective phagocytic clearance of old photoreceptor cells in the retina . In addition, αvβ3 plays an essential role in osteoclast function, supported by the fact that osteoclasts in β3 KO mice have defective bone resorption . With this in mind, we were surprised to find that microglia lacking αvβ3 and αvβ5 integrins showed normal adhesion and activation responses to vitronectin, and is further demonstration that the findings of pharmacological blockade studies are not always borne out by the use of genetic KO approaches. This is perhaps best illustrated by the case of the role of αvβ3 and αvβ5 integrins in promoting angiogenesis, in which antibody blockade suggested key roles for these integrins in angiogenesis [31, 32], but mice lacking these integrins display no apparent angiogenic defect , actually displaying an enhanced angiogenic response in tumor growth .
Redundancy of microglial αv integrins
Our data show that the αvβ3 and αvβ5 integrins comprise the major fraction of total αv integrins expressed by microglia. However, we have found that microglia also express two other vitronectin receptors, the αvβ1 and αvβ8 integrins, though at appreciably lower levels than αvβ3 and αvβ5. What is surprising about our data is that despite lacking the two abundant vitronectin receptors, β3/β5 DKO microglia show no obvious defects in adhesion to vitronectin, or in the subsequent activation responses. This is in stark contrast to our finding with brain endothelial cells, where absence of the αvβ3 integrin leaves the cells totally unable to attach to vitronectin . This clearly demonstrates that the αvβ3 and αvβ5 integrins are not essential for mediating microglial activation responses to vitronectin, highlighting the redundancy of microglial vitronectin receptors. In this light it is informative to compare the αv integrin expression profile of microglia with other CNS cell types. Both neural stem cells and oligodendrocyte precursor cells also express the four αv integrins expressed by microglia, αvβ1, αvβ3, αvβ5 and αvβ8 [21, 27, 35], and it is interesting to note that all three cell types have the capacity to migrate considerable distances, even in the adult CNS. In contrast, astrocytes, which are far less motile, express only αvβ5 and αvβ8 , while brain endothelial cells express just the αvβ3 integrin, and then only when actively undergoing angiogenesis [34, 36, 37].
Regulation of αv integrin heterodimer formation
Integrins comprise a family of αβ heterodimers, composed of 11 different α and 9 different β subunits. In light of the potential to form up to 99 potential different αβ heterodimers, in reality, only 24 heterodimers have been identified . We have shown that microglia express four different αvβ heterodimers, with high levels of αvβ3 and αvβ5, and much lower levels of αvβ1 and αvβ8. This begs the question: what regulates the coupling and abundance of each of the different αv heterodimers? It has been previously suggested that the αv integrin subunit is produced in excess, and its cell surface appearance is limited by the transcription level of the associated β subunits . An alternative mechanism would be that the αv subunit is produced in a fixed amount, and that loss of one or more β subunits (e.g.: β3) would automatically lead to compensatory upregulation of the other β subunits (e.g.: β8). Our data would appear to support the first mechanism, because loss of both of the major β subunits β3 and β5 in microglia did not result in any compensatory upregulation of αvβ1 or αvβ8; rather the total amount of the αv integrin subunit was massively reduced.
The aim of this study was to define the role of the αvβ3 and αvβ5 integrins im mediating microglial activation responses to vitronectin. Microglia from β3, β5, or β3/β5 knockout strains showed no defects in adhesion, activation marker expression, MMP-9 induction, or phagocytosis of vitronectin-coated beads. Flow cytometry and biochemical analysis revealed that microglia also express low levels of the alternative vitronectin receptors, αvβ1 and αvβ8 integrins. Taken together, we conclude that the αvβ3 and αvβ5 integrins are not essential for mediating microglial activation responses to vitronectin, but that microglia employ multiple receptor systems to mediate interactions with vitronectin. On embarking on these studies, we were hopeful that identification of a single vitronectin receptor would lead to potential therapeutic targets for blocking microglial activation. The outcome of the current study suggests that pinpoint targeting of single αv integrins will not be productive, but rather a broad-spectrum blockade aimed at targeting all αv integrins is more likely to be successful.
This work was supported by the National Multiple Sclerosis Society: by a Harry Weaver Neuroscience Scholar Award to RM (JF 2125A1/1), and by a Post-Doctoral Fellowship to JVW (FG 1879-A-1). This is manuscript number 21399 from The Scripps Research Institute.
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