The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Milner, R.
Right arrow Articles by Campbell, I. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Milner, R.
Right arrow Articles by Campbell, I. L.
The Journal of Immunology, 2003, 170: 3850-3858.
Copyright © 2003 by The American Association of Immunologists

The Extracellular Matrix and Cytokines Regulate Microglial Integrin Expression and Activation1

Richard Milner2 and Iain L. Campbell

Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Microglia are the primary immune effector cells resident within the CNS, whose activation into migratory, phagocytic cells is associated with increased expression of cell adhesion molecules of the integrin family. To determine which specific factors are important regulators of microglial activation and integrin expression, we have examined the influence of individual cytokines and extracellular matrix (ECM) substrates by quantifying cell surface expression of MHC and individual integrins by flow cytometry. We found that the proinflammatory cytokines TNF and IFN-{alpha} promoted microglial activation, as assessed by amoeboid morphology and increased expression of MHC class I, and also increased expression of the {alpha}4{beta}1 and Mac-1 integrins. In contrast, TGF-{beta}1 had the opposite effect and was dominant over the other cytokines. Furthermore, the ECM substrates fibronectin and vitronectin, but not laminin, also promoted microglial activation and increased expression of the {alpha}4{beta}1, {alpha}5{beta}1 and Mac-1 integrins, but significantly, the influence of fibronectin and vitronectin was not diminished by TGF-{beta}1. Taken together, this work suggests that, in addition to cytokines, the ECM represents an important regulatory influence on microglial activity. Specifically, it implies that increases in the local availability of fibronectin or vitronectin, as a result of blood-brain barrier breakdown or increased expression in different pathological states of the CNS, could induce microglial activation and increased expression of integrins.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Microglia constitute between 10 and 15% of cells within the CNS, and are considered the primary immune effector cells resident within this tissue (1, 2). Following injury or infection within the CNS, the normally quiescent microglia are rapidly transformed into highly activated cells, which migrate to the site of injury, proliferate, and phagocytose both microorganisms and damaged host tissue. In performing these functions, microglia play an important role in protecting the CNS against harmful pathogens, and also in removing tissue debris after CNS injury, thereby promoting tissue regeneration and remodeling. In addition to this protective role, evidence suggests that microglia may also exert a destructive influence in the CNS in several neuropathological conditions, including multiple sclerosis (MS),3 Alzheimer disease (AD), and HIV infection-associated dementia (reviewed in Ref. 3). In demyelinating MS lesions, activated microglia are present and contain lipid derived from destroyed myelin membranes. More recently, it has been shown that microglia make abnormal physical contacts with myelin membranes before the onset of demyelination, something not observed in unaffected brain (4, 5, 6), prompting the suggestion that microglial contact with oligodendrocyte membranes may be an important initiating event that triggers demyelination and inflammation (3, 6, 7).

In addition to their function as primary immune effector cells, microglia also play an important role in orchestrating the behavior of other immune cells that enter the CNS, by secreting cytokines and chemokines, key regulatory molecules that are highly up-regulated during CNS inflammation (1, 2), and members of the matrix metalloproteinase family, which degrade the extracellular matrix (ECM), thus facilitating inflammatory cell migration within the CNS (2). Microglia also up-regulate MHC molecule expression following activation (1, 2), supporting the notion that microglia act as APC within the CNS. Taken together, this evidence suggests that microglia play a critical role in contributing to inflammation within the CNS. Furthermore, it implies that adhesive interactions between microglia and CNS substrates, such as myelin, may be of fundamental importance in the pathogenesis of tissue injury.

Integrins are a major family of cell adhesion molecules expressed by all cell types (reviewed in Refs. 8 and 9). They are expressed as cell surface {alpha}{beta} heterodimers, which act as receptors for molecules of the ECM and for cell surface counterreceptors on other cells. At present, 16 different {alpha} and 8 different {beta} mammalian integrin subunits have been identified; these associate to form 22 recognized {alpha}{beta} heterodimers, grouped into three main classes: {beta}1, {beta}2, and {alpha}v. Integrins are recognized as an important class of molecules that regulate immune cell behavior (10). In particular, the {beta}2 integrins and {alpha}4{beta}1 and {alpha}6{beta}1 are expressed by most cells of the immune system and play a major role in mediating leukocyte adhesion, migration, and extravasation across the endothelium during inflammation. Microglia express several different integrins, and although it has been shown that expression of some of these is regulated in different pathological conditions and by injection of LPS (11), what remains to be determined is which specific factors exert the major influence on microglial integrin expression. To address this question, the goal of this study was to perform a comprehensive investigation to examine the influence of individual cytokines and ECM molecules on the state of microglial activation and integrin expression. Specifically, we have investigated the effect of these factors in vitro by 1) examining microglial cell morphology and 2) quantifying cell surface expression levels of MHC and integrin molecules by flow cytometry.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

Mixed glial cultures were prepared as described previously (12) using a technique modified from McCarthy and de Vellis (13). Briefly, forebrains from postnatal mice (days 0–2) were stripped of meninges, chopped into small chunks, and dissociated in papain, before being cultured for ~10 days on poly-D-lysine (Sigma-Aldrich, St. Louis, MO)-coated T75 tissue culture flasks (Falcon, Franklin Lakes, NJ) in DMEM (Sigma-Aldrich) supplemented with 10% FCS (Sigma-Aldrich). After establishment of the astrocyte monolayer, the flasks were then shaken for 1 h to obtain the loosely attached microglia. The microglia were then cultured in six-well tissue culture plates (Nunc, Naperville, IL) at a density of 2 x 105 cells/well in serum-free N1 medium (DMEM supplemented with N1 (Sigma-Aldrich)) for analysis of MHC and integrin expression by flow cytometry or immunoprecipitation. The purity of these microglial cultures was >99% as determined by Mac-1 positivity in flow cytometry.

Antibodies

The following Abs used in immunoprecipitations and FACS analysis were obtained from BD PharMingen (San Diego, CA): the mAbs specific for the integrin subunits {alpha}1 (Ha31/8), {alpha}2 (Hm{alpha}2), {alpha}4 (9C10), {alpha}5 (5H10-27), {alpha}6 (GoH3), {alpha}L (M174), {alpha}M (M1/70), {alpha}X (HL3), {beta}4 (346-11A), {beta}7 (FIB27), and the isotype control mAb (anti-KLH) and the polyclonal donkey anti-rat PE-conjugated secondary Ab. The rat mAb specific for the {alpha}v integrin subunit (14) was kindly provided by Dr. C. Streuli (University of Manchester, Manchester, U.K.). mAbs specific for the MHC class I (M1/42.3.9.8) and MHC class II (M5/114.15.2.) molecules were obtained as hybridomas from the American Type Culture Collection (Manassas, VA). The mouse mAb P1F6 specific for the rat {alpha}v{beta}5 integrin heterodimer and the anti-{alpha}v integrin antiserum were obtained from Chemicon (Temecula, CA).

Cell surface labeling and immunoprecipitation

Cell surface labeling and immunoprecipitation were performed as described previously (12). Immunoprecipitations were conducted with 250 µl of cell lysate and the anti-{alpha}v antiserum at 1/250 dilution, and the P1F6 mAb at 1/100 dilution.

FACS analysis

Microglia were cultured in six-well plates (Nunc) in serum-free N1 medium in the presence or absence of the following cytokines: IL-1{beta} (PeproTech, Rocky Hill, NJ), IL-3 (R&D Systems, Minneapolis, MN), IL-6 (R&D Systems), TNF (Genentech, San Francisco, CA), TGF-{beta}1 (R&D Systems), IFN-{alpha} (Life Technologies, Grand Island, NY), and IFN-{gamma} (R&D Systems). Preliminary experiments were performed to investigate the dose-response relationship of each cytokine to microglial activation, as assessed by MHC class I expression in flow cytometry. The lowest concentration of each cytokine that induced the greatest change in MHC class I expression was then selected and used for all subsequent experiments. These concentrations were the following: IL-1{beta} (2 ng/ml), IL-3 (1 ng/ml), IL-6 (0.2 ng/ml), TNF (40 ng/ml), TGF-{beta}1 (2 ng/ml), IFN-{alpha} (103 U/ml), and IFN-{gamma} (5 U/ml = 1.6 ng/ml). To investigate the influence of different ECM substrates on microglial expression of integrins and MHC class molecules, six-well plates were coated with a 10 µg/ml solution containing the individual ECM proteins murine laminin, bovine fibronectin, or bovine vitronectin diluted in PBS (all from Sigma-Aldrich) for 2 h at 37°C. After 2 days in culture, microglial MHC and integrin expression were analyzed by flow cytometry as described previously (15). For each condition, the mean fluorescent intensity was compared with the control state, which was absence of cytokine in the case of the cytokine experiments, and uncoated plastic in the case of the ECM substrate experiments. Within each experiment, the mean fluorescent intensity was then expressed as the percentage change relative to the control condition. Each experiment was repeated a minimum of three times, and the data were expressed as mean ± SD. Statistical significance was assessed by using Student’s paired t test in which a value of p < 0.05 was defined as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cytokines influence microglial morphology and activation state

To investigate the influence of cytokines on microglial behavior, we first examined microglial morphology in the presence of the cytokines IL-1{beta}, IL-3, IL-6, TNF, TGF-{beta}1, IFN-{alpha}, or IFN-{gamma}. As described in Materials and Methods, preliminary experiments were performed to establish the concentration of each cytokine that would induce the maximal change in microglial activation, as assessed by MHC class I expression, and this concentration was used for all subsequent experiments. After 2 days in culture, the microglia showed clear morphological differences in some of these different conditions. Under control conditions, microglia displayed two basic types of morphology: some cells showed a well-spread, amoeboid morphology, whereas the remainder exhibited an elongated processed morphology. Interestingly, microglia incubated with the proinflammatory cytokines TNF, IFN-{alpha}, or IFN-{gamma} were induced toward the amoeboid form (Fig. 1, C, D, and E, respectively), although the vast majority of microglia incubated with TGF-{beta}1 displayed the elongated processed form (Fig. 1B). To see which cytokine would exert the dominant effect, microglia were also exposed to a combination of TNF plus TGF-{beta}1, IFN-{alpha} plus TGF-{beta}1, and IFN-{gamma} plus TGF-{beta}1. Under these conditions, the vast majority of microglia displayed the elongated processed form (for example, IFN-{alpha} plus TGF-{beta}1, shown in Fig. 1F), indicating that TGF-{beta}1 exerts a dominant effect over these other cytokines, on microglial morphology. This dominant effect of TGF-{beta}1 was observed at a range of concentrations from 2.0 ng/ml to as low as 0.1 ng/ml. In these experiments, the cytokines IL-1{beta}, IL-3, and IL-6 had no obvious effect on microglial cell morphology. Although microglial morphology is thought to correlate with activation state—amoeboid representing the activated form and the elongated processed shape representing the resting state (1, 2)—we wanted to further examine the influence of individual cytokines on microglial activation by examining cell surface expression of the MHC molecules, class I and II, which are up-regulated following microglial activation in vivo (1, 2). Following 2-day incubation with the different cytokines, microglial expression of the MHC molecules was examined by flow cytometry. This showed that microglial MHC class I expression was significantly increased following incubation with the proinflammatory cytokines TNF (by 46 ± 2.5%; p < 0.001), IFN-{alpha} (by 120 ± 23.8%; p < 0.001), and IFN-{gamma} (by 104 ± 24.5%; p < 0.001), but was significantly decreased following incubation with TGF-{beta}1 (by 49 ± 5.9%; p < 0.001) (Fig. 2). Furthermore, the effect of TGF-{beta}1 dominated over the other cytokines and suppressed MHC class I expression, which correlated with the dominant effect of TGF-{beta}1 on microglial morphology. MHC class II was not expressed by microglia under control conditions, nor was it induced by any of the cytokines tested, with the exception of IFN-{gamma} (not shown).



View larger version (166K):
[in this window]
[in a new window]
  		FIGURE 1. Regulation of microglial morphology by cytokines. Microglia were purified from mixed glial cultures, as described in Materials and Methods, and then cultured in serum-free medium on uncoated plastic in the presence of different cytokines. Magnification, x200. Note that under control conditions (A), microglia showed two basic types of morphology, either well-spread amoeboid or elongated and processed. After 2 days in culture, microglia incubated with the proinflammatory cytokines TNF (C), IFN-{alpha} (D), and IFN-{gamma} (E) were induced toward the amoeboid form, whereas the vast majority incubated with TGF-{beta}1 (B) displayed the elongated processed form. The effect of TGF-{beta}1 dominated over the proinflammatory cytokine IFN-{alpha} in mixed cytokine experiments (F).

 


View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 2. A, The influence of cytokines on microglial expression of MHC class I. Microglia were purified from mixed glial cultures, as described in Materials and Methods, and then cultured in serum-free medium on uncoated plastic in the presence of different cytokines. After 2-day incubation with the different cytokines, microglial expression of MHC class I was quantified by flow cytometry, and is expressed as the percentage change relative to control conditions (no cytokine). All points represent the mean ± SD of three experiments. Note that MHC class I expression was increased by TNF, IFN-{alpha}, and IFN-{gamma}, but decreased following incubation with TGF-{beta}1. The influence of TGF-{beta}1 dominated over the other cytokines, and suppressed MHC class I expression. B, Representative flow cytometry profile showing microglial MHC class I expression in the absence of cytokine (light line) or in the presence of TGF-{beta}1 (dark line) or IFN-{alpha} (broken line). Note that TGF-{beta}1 reduced microglial MHC class I expression, whereas IFN-{alpha} had the opposite effect.

 
Microglia express integrins of the three main classes: {beta}1, {beta}2, and {alpha}v

As the principal immune effector cells resident within the CNS, microglia form adhesive interactions with other cell types and CNS substrates (1, 2, 3, 16), and we hypothesized that the regulation of these adhesive interactions may profoundly influence microglial morphology, migration, and the ability to phagocytose foreign organisms or dying cells. The integrin family of cell adhesion molecules has attracted a lot of interest because these molecules are up-regulated following microglial activation (11), and inhibition of integrin expression and function has been shown to block microglial migration and phagocytosis (17). Therefore, the main focus of this study was to examine how microglial integrin expression may be regulated by extracellular factors including cytokines and the ECM. As a first step, we analyzed expression of the three different classes of integrins by flow cytometry. This revealed that microglia express a wide repertoire of integrins, including members from each of the three main classes: {beta}1, {beta}2, and {alpha}v (Fig. 3A). Specifically, this showed that microglia express the {beta}1 integrins, {alpha}4{beta}1, {alpha}5{beta}1, and {alpha}6{beta}1, and the {beta}2 integrins, {alpha}L{beta}2 and {alpha}M{beta}2, also known as LFA-1 and Mac-1, respectively. Microglia were negative for the integrin subunits {alpha}1, {alpha}2, {alpha}X, {beta}4, and {beta}7 (not shown).



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 3. Integrin expression by microglia. A, Microglia were purified from mixed glial cultures, as described in Materials and Methods, and then cultured in serum-free medium on uncoated plastic. After 2 days in culture, microglial expression of different integrin subunits was analyzed by flow cytometry. Note that microglia express the {alpha}4, {alpha}5, and {alpha}6 {beta}1 integrins, the LFA-1 and Mac-1 {beta}2 integrins, and also {alpha}v integrins. B, Microglial expression of {alpha}v integrins was analyzed by immunoprecipitation. Mouse and rat microglia were purified from mixed glial cultures, as described in Materials and Methods, and then immunoprecipitations were performed on biotin-labeled cell surface lysates with an antiserum against the {alpha}v integrin subunit or the mAb P1F6, specific for the rat {beta}5 integrin subunit. The immunoprecipitated proteins were then separated on nonreducing SDS-PAGE gels. Note that mouse and rat microglia show an identical pattern of {alpha}v integrin expression, with the {alpha}v subunit running at 140 kDa and a single {alpha}v-associated {beta} subunit running at 90 kDa. The P1F6 Ab immunoprecipitated the same pattern, thereby identifying the microglial {alpha}v integrin as {alpha}v{beta}5.

 
FACS analysis with a mAb (C8F12) against the {alpha}v integrin subunit showed that microglia also express {alpha}v integrins. Most cells express several different {alpha}v integrin heterodimers, in which the {alpha}v subunit is associated with different {beta} subunits (12, 18, 19, 20). To define which specific {alpha}v heterodimers are expressed by microglia, we examined {alpha}v integrin expression by immunoprecipitation of biotin-labeled microglial cell surface lysates, using both the C8F12 mAb and anti-{alpha}v antiserum. When {alpha}v immunoprecipitates were separated on nonreducing SDS-PAGE gels, this showed a pattern of two bands: the {alpha}v subunit running at the expected molecular mass of ~140 kDa and a single {alpha}v-associated {beta} subunit running at ~90 kDa (Fig. 3B). Based on molecular mass, candidates for this {beta} subunit include {beta}3, {beta}5, {beta}6, and {beta}8. In trying to identify this {beta} subunit, further immunoprecipitations were conducted with polyclonal Abs against the {beta}3, {beta}6, or {beta}8 subunits, but failed to immunoprecipitate any bands. To ascertain whether this subunit was {beta}5, it was necessary to switch species to rat, because no specific reagents for the mouse {beta}5 integrin subunit are currently available. An {alpha}v immunoprecipitation on rat microglial cells showed the same pattern of bands as mouse cells, indicating one {alpha}v integrin heterodimer with a {beta} subunit running at 90 kDa. When we used the {beta}5 specific mAb P1F6 to immunoprecipitate {beta}5 integrins from rat microglial cells, we detected an identical pattern of bands as for the {alpha}v immunoprecipitate, namely the {beta}5 subunit (90 kDa) and the associated {alpha}v subunit (140 kDa). This shows that microglial cells express just one {alpha}v integrin heterodimer, {alpha}v{beta}5.

Cytokines regulate the expression of microglial integrins

In light of previous evidence that microglial expression of integrins is regulated during CNS pathology, and also modulated by LPS and some cytokines in vitro (reviewed in Ref. 11), we therefore performed a comprehensive study to examine the influence of individual cytokines on microglial integrin expression. Because the proinflammatory cytokines and TGF-{beta}1 appeared to have opposite effects on microglial cell morphology and MHC expression, we also examined the combined influence of individual proinflammatory cytokines and TGF-{beta}1 together. After 2-day incubation, microglia were analyzed by FACS for integrin expression. This showed that cytokines influenced the expression of integrins in a cytokine- and integrin subunit-specific manner (Fig. 4). First, expression of the {alpha}4 integrin subunit was significantly increased by TNF (by 37.0 ± 13.1%; p < 0.01), IFN-{alpha} (by 47.3 ± 11.3%; p < 0.005), and IFN-{gamma} (by 48.0 ± 14.1%; p < 0.01), but significantly reduced by TGF-{beta}1 (by 37.3 ± 6.4%; p < 0.005). Second, expression of Mac-1 was significantly increased by TNF (by 26.3 ± 6.4%; p < 0.01), IFN-{alpha} (by 31.3 ± 10.9%; p < 0.02), IL-1{beta} (by 43.2 ± 3.9%; p < 0.005), IL-3 (by 39.3 ± 9.0%; p < 0.01), and IL-6 (by 24.3 ± 5.8%; p < 0.01), but significantly decreased by TGF-{beta}1 (by 19.0 ± 8.0%; p < 0.05). Interestingly, in mixed cytokine experiments, the effect of TGF-{beta}1 to reduce expression of the {alpha}4 and Mac-1 integrins dominated over the proinflammatory cytokines. Third, expression of the LFA-1 integrin was significantly increased by TGF-{beta}1 (by 60.1 ± 12.8%; p < 0.001), IFN-{alpha} (by 53.3 ± 15.7%; p < 0.01), and IFN-{gamma} (by 111.3 ± 18.6%; p < 0.002). In addition, TGF-{beta}1 also significantly increased expression of the {alpha}5 subunit (by 76.0 ± 14.3%; p < 0.005) and the {alpha}v subunit (by 63.2 ± 12.1%; p < 0.005), and IFN-{alpha} also significantly increased {alpha}5 expression (by 53.7 ± 15.3%; p < 0.01) (not shown). Expression level of the {alpha}6 subunit was not significantly altered by any of the cytokines tested in this study. These results showed that expression of individual integrin subunits is regulated in a cytokine-specific manner, and points to a dominant regulatory role for TGF-{beta}1 in this process. As part of this study, we also examined the possibility that cytokines might induce microglial expression of the {alpha}1, {alpha}2, {alpha}x, {beta}4, or {beta}7 integrin subunits, but we failed to detect expression of any of these integrins by flow cytometry under any of the conditions tested (not shown). We also performed additional immunoprecipitation experiments on microglia treated with different cytokines to test the possibility that {alpha}v integrins other than {alpha}v{beta}5 might be induced by these cytokines, but this revealed that microglia express only one {alpha}v integrin, {alpha}v{beta}5, under all conditions tested (not shown).



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 4. The influence of cytokines on microglial integrin expression. Microglia were purified from mixed glial cultures, as described in Materials and Methods, and then cultured in serum-free medium on uncoated plastic in the presence of different cytokines. After 2 days in culture, microglial expression of different integrin subunits was quantified by flow cytometry, and is expressed as the percentage change relative to control conditions (no cytokine). All points represent the mean ± SD of three experiments. The lower panels show representative flow cytometry profiles for {alpha}4, Mac-1, and LFA-1 in the absence of cytokine (light line) or in the presence of TGF-{beta}1 (dark line) or IFN-{alpha} (broken line). Note that {alpha}4 expression was increased by TNF, IFN-{alpha}, and IFN-{gamma}, whereas Mac-1 expression was increased by TNF, IFN-{alpha}, IL-1{beta}, IL-3, and IL-6. TGF-{beta}1 decreased the expression of {alpha}4 and Mac-1, and this effect dominated over the other cytokines. In addition, LFA-1 expression was increased by TNF, IFN-{alpha}, IFN-{gamma}, and TGF-{beta}1.

 
Microglial activation and integrin expression is also regulated by the ECM

Our previous studies have shown that microglial adhesion and morphology vary greatly between different ECM substrates (15). In short-term adhesion assays, we found that microglia adhere strongly to plastic, fibronectin, and vitronectin, but only weakly to laminin, unless activated. In light of this strong ECM regulation of microglial behavior and previous evidence showing that integrin expression in some cell types is regulated by the ECM (i.e., ligand regulation of its own receptor) (21), we also wanted to examine the influence of individual ECM molecules on microglial integrin expression. To do this, mouse microglia were cultured on tissue culture plastic or the different ECM substrates, fibronectin, laminin, or vitronectin, in serum-free medium for 2 days. Consistent with our previous observations, we found that after 1-h incubation, microglia attached well to fibronectin, vitronectin, and the uncoated tissue culture plastic, but only weakly to laminin. Interestingly, after 2-day culture, microglia on plastic had become less attached and had changed their morphology from well-spread, predominantly amoeboid, to smaller, less spread cells, and some showed the elongated processed morphology (Fig. 5A). In contrast, microglia cultured on fibronectin or vitronectin (Fig. 5, C and D, respectively) maintained the phase-dark amoeboid phenotype. This shows that the different ECM substrates influence microglial morphology: fibronectin and vitronectin promoted the amoeboid phenotype, whereas laminin induced the nonadherent rounded-up phenotype (Fig. 5B). After 2 days in culture, microglial MHC and integrin expression was quantified by FACS analysis. Relative to the tissue culture plastic substrate, microglial MHC class I expression was significantly increased by fibronectin (by 112 ± 18%; p < 0.002) and vitronectin (by 84 ± 6%; p < 0.001) and significantly decreased by laminin (by 19 ± 6%; p < 0.02) (Fig. 6A). MHC class II was not detected on microglia cultured on plastic or on any of the ECM substrates.



View larger version (117K):
[in this window]
[in a new window]
  		FIGURE 5. Microglial morphology on different ECM substrates after 2 days in culture. Microglia were purified from mixed glial cultures, as described in Materials and Methods, and then cultured in serum-free medium on uncoated plastic (A), laminin (B), fibronectin (C), or vitronectin (D). Magnification, x200. Note that after 2 days in culture, microglia on plastic were either elongated processed cells or smaller spread cells. Microglia on fibronectin or vitronectin displayed a well-spread amoeboid phenotype, whereas, in total contrast, microglia on laminin were poorly attached, phase-bright cells.

 


View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 6. The influence of the ECM on microglial expression of MHC class I and integrins. Microglia were purified from mixed glial cultures, as described in Materials and Methods, and then cultured in serum-free medium on uncoated plastic, laminin, fibronectin, or vitronectin. After 2 days in culture, microglial expression of MHC class I (A) and different integrin subunits (B) was quantified by flow cytometry, and is expressed as the percentage change relative to control conditions (plastic substrate). All points represent the mean ± SD of three experiments. Note that the fibronectin and vitronectin substrates increased microglial expression of MHC class I, whereas laminin reduced expression. In addition, fibronectin and vitronectin, but not laminin, increased the expression of the {alpha}4, {alpha}5, and Mac-1 integrins, whereas laminin increased {alpha}v integrin expression. All three ECM substrates increased LFA-1 expression and decreased {alpha}6 expression.

 
FACS analysis revealed that integrin expression on microglia was highly regulated by the ECM in a substrate-specific manner (Fig. 6B). Microglia cultured on fibronectin or vitronectin, but not laminin, showed significantly increased levels of the {alpha}4, {alpha}5, and Mac-1 integrins relative to the control tissue culture plastic substrate. Fibronectin increased the level of {alpha}4 by 83.3 ± 18.3% (p < 0.005), {alpha}5 by 67 ± 17.5% (p < 0.005), and Mac-1 by 66 ± 17.8% (p < 0.005). Vitronectin increased the level of {alpha}4 by 63.7 ± 15.1% (p < 0.005), {alpha}5 by 137 ± 43% (p < 0.002), and Mac-1 by 62 ± 19.4% (p < 0.005). Laminin significantly increased {alpha}v integrin expression on microglia (by 53 ± 13.7%; p < 0.005), whereas fibronectin and vitronectin had no significant effect. In addition, all three ECM substrates, fibronectin, laminin, and vitronectin, significantly increased microglial expression of the LFA-1 integrin (fibronectin by 208 ± 33.5%, p < 0.001; laminin by 147 ± 46%, p < 0.005; and vitronectin by 209 ± 35.3%, p < 0.001) and down-regulation of the {alpha}6 integrin (fibronectin by 45.6 ± 7.9%, p < 0.005; laminin by 51.9 ± 7.7%, p < 0.005; and vitronectin by 50.1 ± 6.6%, p < 0.005) relative to the control tissue culture plastic substrate. These findings are particularly interesting because serum contains high levels of fibronectin and vitronectin (22), but not laminin, and therefore, breakdown of the blood-brain barrier associated with CNS inflammation or trauma will greatly increase the availability of these proteins within the CNS parenchyma, and thus influence the behavior of microglial cells. Taken together, these observations showed that fibronectin and vitronectin, but not laminin, promote microglial activation, which results in the amoeboid morphology, increased expression of MHC class I, and increased expression of the {alpha}4{beta}1, {alpha}5{beta}1, and Mac-1 integrins.

Microglial activation induced by fibronectin and vitronectin is not blocked by TGF-{beta}1

We next investigated the combined influence of cytokines and ECM molecules on microglial integrin expression. This is important for two reasons. First, we have shown that the integrins up-regulated on activated microglia ({alpha}4{beta}1 and Mac-1) are increased both by certain proinflammatory cytokines and by fibronectin and vitronectin, and furthermore, that the influence of the proinflammatory cytokines to increase {alpha}4{beta}1 and Mac-1 expression is blocked by TGF-{beta}1. Therefore, it was of interest to see whether TGF-{beta}1 also blocked the influence of fibronectin and vitronectin to increase microglial expression of the {alpha}4{beta}1 and Mac-1 integrins. Second, inflammation is associated with up-regulation both of proinflammatory cytokines (1, 2, 16) and ECM molecules like fibronectin and vitronectin (reviewed in Ref. 23), and as both types of stimuli increased {alpha}4{beta}1 and Mac-1 expression on microglia, it was highly pertinent to test whether these two different kinds of stimuli had additive effects on microglial integrin expression. Mouse microglia were cultured for 3 days on three different substrates, tissue culture plastic, fibronectin, or vitronectin, under serum-free conditions, in the presence of TNF, IFN-{alpha}, TGF-{beta}1, or combinations of TNF plus TGF-{beta}1 or IFN-{alpha} plus TGF-{beta}1, and then integrin expression was analyzed by FACS. As shown in Fig. 7, on tissue culture plastic, microglial expression of the {alpha}4 and Mac-1 integrins was increased by TNF and IFN-{alpha}, but decreased by TGF-{beta}1, and TGF-{beta}1 had the dominant effect in mixed cytokine experiments, thus confirming the findings from our earlier experiments (Fig. 4). On fibronectin or vitronectin substrates, microglia showed increased expression of the {alpha}4 and Mac-1 integrins compared with microglia grown on tissue culture plastic, and on these substrates, the cytokines had little further influence on {alpha}4 and Mac-1 expression. This experiment made two points. First, it showed that the proinflammatory cytokines do not increase {alpha}4 and Mac-1 expression over and above that increase already induced by fibronectin or vitronectin. Second, and perhaps more importantly, it showed that, in contrast to the dominant effect of TGF-{beta}1 over the proinflammatory cytokines, to suppress increased expression of {alpha}4 and Mac-1, the effect of fibronectin and vitronectin to induce increased expression of {alpha}4 and Mac-1 integrins was unaffected by TGF-{beta}1.



View larger version (69K):
[in this window]
[in a new window]
  		FIGURE 7. The combined influence of the ECM and cytokines on microglial integrin expression. Microglia were purified from mixed glial cultures, as described in Materials and Methods, and then cultured in serum-free medium on either uncoated plastic, fibronectin, or vitronectin, in the absence of cytokines or in the presence of TGF-{beta}1, TNF, IFN-{alpha}, TNF plus TGF-{beta}1, or IFN-{alpha} plus TGF-{beta}1. After 2 days in culture, microglial expression of different integrin subunits was quantified by flow cytometry and is expressed as the percentage change relative to control conditions (plastic substrate with no cytokine). All points represent the mean ± SD of three experiments. Note that, on a plastic substrate, expression of {alpha}4 and Mac-1 was increased by TNF and IFN-{alpha}, and decreased by TGF-{beta}1, with TGF-{beta}1 being dominant in mixed cytokine experiments. Fibronectin and vitronectin also increased {alpha}4 and Mac-1 expression relative to the plastic substrate. Significantly, cytokines had little influence on {alpha}4 and Mac-1 expression on these substrates, showing that TGF-{beta}1 did not suppress the influence of fibronectin and vitronectin to increase {alpha}4 and Mac-1 expression by microglia.

 
In these experiments, expression of the {alpha}5 integrin subunit was significantly increased, both by fibronectin (by 79 ± 16.4%; p < 0.005) and vitronectin (by 129 ± 21.4%; p < 0.002), and then significantly increased further by TGF-{beta}1 (to 124 ± 26.4% (p < 0.05) on fibronectin and 221 ± 38% (p < 0.01) on vitronectin), showing that the influence of cytokines and ECM had an additive effect on microglial expression of this integrin. Interestingly, in all cytokine conditions tested, vitronectin induced a significantly higher expression level of the {alpha}5 integrin than did fibronectin, the {alpha}5 integrin ligand (129 ± 21.4% vs 79 ± 16.4% (p < 0.05) with no cytokine present; 221 ± 38% vs 124 ± 26.4% (p < 0.01) with TGF-{beta}1; 161 ± 36.2% vs 64 ± 13.9% (p < 0.01) with TNF; and 175 ± 32.4% vs 91 ± 17.8% (p < 0.01) with IFN-{alpha}). Taken together, these studies showed that microglial integrin expression is influenced both by cytokines and molecules of the ECM, and indicated that, although the immunomodulatory cytokine TGF-{beta}1 can block microglial activation and increased integrin expression induced by proinflammatory cytokines, the ability of fibronectin and vitronectin to induce microglial activation and increased integrin expression is independent of TGF-{beta}1.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have used pure populations of microglial cells in culture to investigate the influence of individual cytokines and ECM substrates on microglial morphology, microglial activation, and expression of integrins. There were two main conclusions from this study. First, the proinflammatory cytokines TNF and IFN-{alpha} promoted microglial activation and increased expression of the {alpha}4{beta}1 and Mac-1 integrins, whereas TGF-{beta}1 had the opposite effect, with TGF-{beta}1 being dominant over the other cytokines. Second, the ECM substrates fibronectin and vitronectin, but not laminin, promoted microglial activation and increased expression of the {alpha}4{beta}1, {alpha}5{beta}1 and Mac-1 integrins, but significantly, the influence of fibronectin and vitronectin was not altered by TGF-{beta}1.

Regulation of microglial activation by cytokines and the ECM

Following stimulation, microglia transform from resting quiescent cells into migratory cells capable of phagocytosing foreign organisms and cellular debris. Many different factors have been shown to regulate microglial activity, including cytokines, chemokines, and components of bacterial cell walls, such as LPS (1, 2, 16). In the current study, we have shown that certain cytokines have very clear effects on microglial activation, as assessed by microglial morphology and expression of MHC molecules. In particular, we found that the proinflammatory cytokines TNF, IFN-{alpha}, and IFN-{gamma} all promoted microglial activation, resulting in a homogeneous population of microglia, displaying the classical activated amoeboid phenotype and expressing increased levels of the activation marker MHC class I. In contrast, TGF-{beta}1 influenced microglia to become elongated processed cells and down-regulated MHC class I expression, and this effect dominated in mixed cytokine experiments. In this study, we found that microglia did not express MHC class II, except after stimulation by IFN-{gamma}. These results are consistent with the findings of others, in showing that proinflammatory cytokines activate microglia whereas TGF-{beta}1 has an immunosuppressive influence (reviewed in Refs. 1 and 2). Furthermore, they support the idea that microglial activation state is determined by the overall balance between activating and suppressing factors.

Within the CNS, the ECM is altered in many different pathological states, including AD, MS, epilepsy, and tumor formation (23, 24). To shed some light on the influence of different ECM components on microglial behavior, we previously determined that microglia attach well to fibronectin and vitronectin, but only weakly to laminin and an astrocyte monolayer (15). In the current study, we found that fibronectin and vitronectin promoted microglial activation, as assessed by the amoeboid morphology and increased expression of MHC class I, whereas laminin was a relatively nonadherent substrate and decreased MHC class I expression. The significance of this observation is that serum contains high levels of fibronectin and vitronectin, but not laminin (22), and therefore breakdown of the blood-brain barrier associated with trauma or neuroinflammatory events will dramatically increase the concentration of these ECM proteins within the CNS parenchyma. Fibronectin and vitronectin expression within the CNS is also increased in epilepsy, MS, and tumor formation (reviewed in Refs. 23 and 24). Taken together, this suggests that regulation of the expression and distribution of fibronectin and vitronectin within the CNS is an important factor influencing the degree of microglial activation. In future studies, it will be important to define the nature of any ECM molecules that might be produced by microglia, because this will undoubtedly influence microglial activation and integrin expression. In light of the increased expression of fibronectin and vitronectin documented in various pathological states, it is possible that activated microglia themselves are induced to express these ECM substrates, which might act in a feed-forward fashion to further activate neighboring microglia and lead to a chronic inflammatory state within the CNS.

Regulation of integrin expression by cytokines and the ECM

Microglia are highly dynamic cells that can rapidly change their adhesive interactions with surrounding cells and substrates in response to stimulation (1, 2). One way that microglia can achieve this is by altering the expression and function of cell adhesion molecules of the integrin family, and many lines of evidence have described regulation of integrin expression with microglial activation, both in vivo and in vitro. Increased expression of different microglial integrins has been described in many pathological states of the CNS, including facial nerve lesion (25), AD (26), and MS lesions (5), and historically the Mac-1 integrin has been well established as a marker of microglial activation (1). In vitro experiments show that microglial activation is accompanied by a morphological switch, from ramified to amoeboid form, and that this is associated with increased expression of the {alpha}4{beta}1 and {beta}2 integrins, including Mac-1 (27). A recent study by Kloss et al. (11) examined the influence of LPS on microglial morphology and expression of integrins in vivo and in vitro. LPS induced a morphological switch to the amoeboid form and increased expression of the {alpha}4, {alpha}5, and Mac-1 integrins. In the current study, we took a reductionist approach to examine the influence of individual factors on pure populations of microglial cells in totally serum-free culture conditions, thus removing any indirect influences from other cell types or direct influences from trace components of growth factors or cytokines or high concentrations of ECM proteins present in serum. Our results showed that microglial activation induced by proinflammatory cytokines or the ECM substrates, fibronectin and vitronectin, promoted increased expression of the {alpha}4, {alpha}5, and Mac-1 integrins. Taken together with previous work, this suggests that, irrespective of the stimulus, microglial activation leads to a stereotypic up-regulation of specific integrins. Importantly, we found that the immunosuppressive influence of TGF-{beta}1 dominated over the proinflammatory cytokines, but did not override the influence of fibronectin or vitronectin.

Unlike the work of Kloss (11), we found that microglia express the LFA-1 integrin, although only at low levels under control conditions. However, the LFA-1 integrin showed the most dramatic up-regulation of all integrins, and was increased both by the proinflammatory cytokines TNF, IFN-{alpha}, and IFN-{gamma}, and also by TGF-{beta}1. LFA-1 expression was also up-regulated by fibronectin, vitronectin, and laminin, compared with the control plastic substrate. Why the LFA-1 integrin should be up-regulated by all these different factors remains to be determined.

Potential functions of microglial integrins

Having completed the characterization of microglial integrins and defined some of the influences that regulate microglial integrin expression, the next important aim is to define the functions of the individual integrins. Broadly speaking, integrins have two basic roles: to mediate adhesion and signaling (8, 9). In terms of adhesion, we can predict, based on experiments from other cell types, that up-regulation of the {alpha}4 and {beta}2 integrins will increase the ability of microglia to adhere to other cell types expressing the counterreceptors for these integrins, VCAM-1 and ICAM-I/II, respectively (28). These changes in adhesion will result in morphological and migratory changes in microglia, which may either increase their migratory ability, and thus speed their passage toward the attracting focus, or serve to home-in or dock the microglia directly onto their target cells. This will also serve to regulate microglial interactions with other effector cells of the immune system, e.g., invading T lymphocytes, so as to further activate these cells for immune clearance of target structures. In support of this, a recent study by Ullrich et al. (17) showed that antisense inhibition of one of the {beta}2 integrins, LFA-1, blocked microglial migration and activation in a model of CNS injury.

In this study, we have shown that microglia express the {alpha}v{beta}5 integrin. This integrin has been shown to mediate phagocytosis in several different cell types including macrophages and retinal pigment epithelial cells (29, 30). Recently, a phagocytic role for this integrin was suggested in microglia, by the observation that arginine-glycine-aspartate (RGD) peptides (which block the function of all {alpha}v integrins) inhibited microglial uptake of apoptotic neurons (31).

In addition to adhesion, integrins also play an important role in mediating bidirectional signals across the plasma membrane (8, 9, 32). Indirect support that integrins regulate microglial behavior comes from our own study, showing that different ECM substrates regulate integrin expression in a specific manner, and from previous work, showing that laminin and fibronectin exert a major influence on the synthesis and secretion of amyloid precursor protein by microglia cells (33), and by the recent observation that integrin antagonists modulate amyloid peptide uptake and microglial activation in hippocampal slice cultures (34). Also, laminin and fibronectin induce different patterns of matrix metalloproteinase expression within cultured microglial cells (our unpublished observations), which is additional evidence that the ECM, via integrin signaling, is able to regulate gene expression and subsequent behavior in microglial cells. Clearly, much work is required to elucidate the precise roles for microglial integrins. Ultimately, a better understanding of these mechanisms should prove useful in the design of therapeutic strategies aimed at reducing the devastating effects of microglial activation, and offer new hope in the treatment of CNS disorders like MS and AD.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants MH62231, MH62261, and DA12444. R.M. was the recipient of a Wellcome Trust International Prize Traveling Research Fellowship. This is manuscript no. 15406-NP from The Scripps Research Institute. Back

2 Address correspondence and reprint requests to Dr. Richard Milner at the current address: Department of Pathology, The University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, United Kingdom. E-mail address: drm27{at}hermes.cam.ac.uk Back

3 Abbreviations used in this paper: MS, multiple sclerosis; AD, Alzheimer disease; ECM, extracellular matrix. Back

Received for publication November 15, 2002. Accepted for publication February 6, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kreutzberg, G. W.. 1996. Microglia: a sensor for pathological events in the CNS. Trends Neurosci. 19:312.[Medline]
  2. Raivich, G., M. Bohatschek, C. U. Kloss, A. Werner, L. L. Jones, G. W. Kreutzberg. 1999. Neuroglial activation repertoire in the injured brain: graded response, molecular mechanisms and cues to physiological function. Brain Res. Brain Res. Rev. 30:77.[Medline]
  3. Gonzalez-Scarano, F., G. Baltuch. 1999. Microglia as mediators of inflammatory and degenerative diseases. Annu. Rev. Neurosci. 22:219.[Medline]
  4. Bo, L., S. Mork, P. A. Kong, H. Nyland, C. A. Pardo, B. D. Trapp. 1994. Detection of MHC class II antigens on macrophages and microglia, but not on astrocytes and endothelia in active multiple sclerosis lesions. J. Neuroimmunol. 51:135.[Medline]
  5. Bo, L., J. W. Peterson, S. Mork, P. Hoffman, W. M. Gallatin, R. M. Ransohoff, B. D. Trapp. 1996. Distribution of immunoglobulin superfamily members ICAM-1, -2, -3, and the {beta}2 integrin LFA-1 in multiple sclerosis lesions. J. Neuropath. Exp. Neurol. 55:1060.[Medline]
  6. Trapp, B. D., L. Bo, S. Mork, A. Chang. 1999. Pathogenesis of tissue injury in MS lesions. J. Neuroimmunol. 98:49.[Medline]
  7. Hickey, W. F.. 1999. The pathology of multiple sclerosis: a historical perspective. J. Neuroimmunol. 98:37.[Medline]
  8. Hynes, R. O.. 1992. Integrins: versatility, modulation and signaling in cell adhesion. Cell 69:11.[Medline]
  9. Hemler, M. E.. 1999. Integrins. T. Kreis, and R. Vale, eds. Guidebook to the Extracellular Matrix, Anchor and Adhesion Proteins 196. Oxford Univ. Press, New York.
  10. Springer, T. A.. 1990. Adhesion receptors of the immune system. Nature 346:425.[Medline]
  11. Kloss, C. U., M. Bohatschek, G. W. Kreutzberg, G. Raivich. 2001. Effect of lipopolysaccharide on the morphology and integrin immunoreactivity of ramified microglia in the mouse brain and in cell culture. Exp. Neurol. 168:32.[Medline]
  12. Milner, R., C. ffrench-Constant. 1994. A developmental analysis of oligodendroglial integrins in primary cells: changes in {alpha}v-associated {beta} subunits during differentiation. Development 120:3497.[Abstract]
  13. McCarthy, K. D., J. de Vellis. 1980. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Cell Biol. 85:890.[Abstract/Free Full Text]
  14. Milner, R., E. E. Frost, S. Nishimura, M. Delcommenne, C. Streuli, R. Pytela, C. ffrench-Constant. 1997. Expression of {alpha}v{beta}3 and {alpha}v{beta}8 integrins during oligodendrocyte precursor differentiation in the presence and absence of axons. Glia 21:350.[Medline]
  15. Milner, R., I. L. Campbell. 2002. Cytokines regulate microglial adhesion to laminin and astrocyte extracellular matrix via protein kinase C-dependent activation of the {alpha}6{beta}1 integrin. J. Neurosci. 22:1562.[Abstract/Free Full Text]
  16. Lee, S. J., E. N. Benveniste. 1999. Adhesion molecule expression and regulation on cells of the central nervous system. J. Neuroimmunol. 98:77.[Medline]
  17. Ullrich, O., A. Diestel, I. Y. Eyupoglu, R. Nitsch. 2001. Regulation of microglial expression of integrins by poly(ADP-ribose) polymerase-1. Nat. Cell Biol. 3:1035.[Medline]
  18. Milner, R., M. Wilby, S. Nishimura, K. Boylen, G. Edwards, J. Fawcett, C. Streuli, R. Pytela, C. ffrench-Constant. 1997. Division of labour of Schwann cell integrins on peripheral nerve extracellular matrix ligands. Dev. Biol. 185:215.[Medline]
  19. Milner, R., X. Huang, J. Wu, S. Nishimura, R. Pytela, D. Sheppard, C. ffrench-Constant. 1999. Distinct roles for astrocyte {alpha}v{beta}5 and {alpha}v{beta}8 integrins in adhesion and migration. J. Cell Sci. 112:4271.[Abstract]
  20. Delannet, M., F. Martin, B. Bossy, D. Cheresh, L. Reichardt, J. Duband. 1994. Specific roles of the {alpha}v{beta}1, {alpha}v{beta}3, and {alpha}v{beta}5 integrins in avian neural crest cell adhesion and migration on vitronectin. Development 120:2687.[Abstract/Free Full Text]
  21. Delcommenne, M., C. Streuli. 1995. Control of integrin expression by extracellular matrix. J. Biol. Chem. 270:26794.[Abstract/Free Full Text]
  22. Felding-Habermann, B., D. A. Cheresh. 1993. Vitronectin and its receptors. Curr. Opin. Cell Biol. 5:864.[Medline]
  23. Venstrom, K. A., L. F. Reichardt. 1993. Extracellular matrix 2: role of extracellular matrix molecules and their receptors in the nervous system. FASEB J. 7:996.[Abstract]
  24. Sobel, R. A.. 1998. The extracellular matrix in multiple sclerosis lesions. J. Neuropath. Exp. Neurol. 57:205.[Medline]
  25. Kloss, C. U., A. Werner, M. A. Klein, J. Shen, K. Menuz, J. C. Probst, G. W. Kreutzberg, G. Raivich. 1999. Integrin family of cell adhesion molecules in the injured brain: regulation and cellular localization in the normal and regenerating mouse facial motor nucleus. J. Comp. Neurol. 411:162.[Medline]
  26. Akiyama, H., P. L. McGeer. 1990. Brain microglia constitutively express {beta}2 integrins. J. Neuroimmunol. 30:81.[Medline]
  27. Hailer, N. P., J. D. Jarhult, R. Nitsch. 1996. Resting microglial cells in vitro: analysis of morphology and adhesion molecule expression in organotypic hippocampal slice cultures. Glia 18:319.[Medline]
  28. Dustin, M. L., T. A. Springer. 1991. Role of lymphocyte adhesion receptors in transient interactions and cell locomotions. Annu. Rev. Immunol. 9:27.[Medline]
  29. Finnemann, S. C., V. L. Bonilha, A. D. Marmorstein, E. Rodriguez-Boulan. 1997. Phagocytosis of rod outer segments by retinal pigment epithelial cells requires {alpha}v{beta}5 integrin for binding but not for internalization. Proc. Natl. Acad. Sci. USA 94:12932.[Abstract/Free Full Text]
  30. Finnemann, S. C., E. Rodriguez-Boulan. 1999. Macrophage and retinal pigment epithelium phagocytosis: apoptotic cells and photoreceptors compete for {alpha}v{beta}3 and {alpha}v{beta}5 integrins, and protein kinase C regulates {alpha}v{beta}5 binding and cytoskeletal linkage. J. Exp. Med. 190:861.[Abstract/Free Full Text]
  31. Witting, A., P. Muller, A. Herrmann, H. Kettenmann, C. Nolte. 2000. Phagocytic clearance of apoptotic neurons by microglia/brain macrophages in vitro: involvement of lectin-, integrin-, and phosphatidylserine-mediated recognition. J. Neurochem. 75:1060.[Medline]
  32. Diamond, M. S., T. A. Springer. 1994. The dynamic regulation of integrin adhesiveness. Curr. Biol. 4:506.[Medline]
  33. Monning, U., R. Sandbrink, A. Weidemann, R. B. Banati, C. L. Masters, K. Bayreuther. 1995. Extracellular matrix influences the biogenesis of amyloid precursor protein in microglial cells. J. Biol. Chem. 270:7104.[Abstract/Free Full Text]
  34. Bi, X., C. M. Gall, J. Zhou, G. Lynch. 2002. Uptake and pathogenic effects of amyloid {beta} peptide 1–42 are enhanced by integrin antagonists and blocked by NMDA receptor antagonists. Neuroscience 112:827.[Medline]



This article has been cited by other articles:


Home page
 J. Neurosci.Home page
S. Wakselman, C. Bechade, A. Roumier, D. Bernard, A. Triller, and A. Bessis
Developmental Neuronal Death in Hippocampus Requires the Microglial CD11b Integrin and DAP12 Immunoreceptor
J. Neurosci., August 6, 2008; 28(32): 8138 - 8143.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Biol.Home page
P. M. Hermann, J. J. Nicol, A. G. M. Bulloch, and W. C. Wildering
RGD-dependent mechanisms in the endoneurial phagocyte response and axonal regeneration in the nervous system of the snail Lymnaea stagnalis
J. Exp. Biol., February 15, 2008; 211(4): 491 - 501.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y.-J. Kim, S.-Y. Hwang, J.-S. Hwang, J.-W. Lee, E.-S. Oh, and I.-O. Han
C6 Glioma Cell Insoluble Matrix Components Enhance Interferon-{gamma}-stimulated Inducible Nitric-oxide Synthase/Nitric Oxide Production in BV2 Microglial Cells
J. Biol. Chem., February 1, 2008; 283(5): 2526 - 2533.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. M. Clarke, A. Lyons, F. O'Connell, B. F. Deighan, C. E. Barry, N. G. Anyakoha, A. Nicolaou, and M. A. Lynch
A Pivotal Role for Interleukin-4 in Atorvastatin-associated Neuroprotection in Rat Brain
J. Biol. Chem., January 25, 2008; 283(4): 1808 - 1817.
[Abstract] [Full Text] [PDF]

Home page
 StrokeHome page
R. Milner, S. Hung, X. Wang, G. I. Berg, M. Spatz, and G. J. del Zoppo
Responses of Endothelial Cell and Astrocyte Matrix-Integrin Receptors to Ischemia Mimic Those Observed in the Neurovascular Unit
Stroke, January 1, 2008; 39(1): 191 - 197.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
R. Milner, S. J. Crocker, S. Hung, X. Wang, R. F. Frausto, and G. J. del Zoppo
Fibronectin- and Vitronectin-Induced Microglial Activation and Matrix Metalloproteinase-9 Expression Is Mediated by Integrins {alpha}5beta1 and {alpha}vbeta5
J. Immunol., June 15, 2007; 178(12): 8158 - 8167.
[Abstract] [Full Text] [PDF]

Home page
 StrokeHome page
G. J. del Zoppo, R. Milner, T. Mabuchi, S. Hung, X. Wang, G. I. Berg, and J. A. Koziol
Microglial Activation and Matrix Protease Generation During Focal Cerebral Ischemia
Stroke, February 1, 2007; 38(2): 646 - 651.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
S. A. Austin, A. M. Floden, E. J. Murphy, and C. K. Combs
{alpha}-Synuclein Expression Modulates Microglial Activation Phenotype
J. Neurosci., October 11, 2006; 26(41): 10558 - 10563.
[Abstract] [Full Text] [PDF]

Home page
 BrainHome page
M. Schroeter, P. Zickler, D. T. Denhardt, H.-P. Hartung, and S. Jander
Increased thalamic neurodegeneration following ischaemic cortical stroke in osteopontin-deficient mice
Brain, June 1, 2006; 129(6): 1426 - 1437.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
T. Wang, W. Zhang, Z. Pei, M. Block, B. Wilson, J. M. Reece, D. S. Miller, and J.-S. Hong
Reactive microgliosis participates in MPP+-induced dopaminergic neurodegeneration: role of 67 kDa laminin receptor
FASEB J, May 1, 2006; 20(7): 906 - 915.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
W.-C. Shyu, S.-Z. Lin, M.-F. Chiang, C.-Y. Su, and H. Li
Intracerebral peripheral blood stem cell (CD34+) implantation induces neuroplasticity by enhancing beta1 integrin-mediated angiogenesis in chronic stroke rats.
J. Neurosci., March 29, 2006; 26(13): 3444 - 3453.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
I. Mazon Pelaez, S. Vogler, U. Strauss, P. Wernhoff, J. Pahnke, G. Brockmann, H. Moch, H.-J. Thiesen, A. Rolfs, and S. M. Ibrahim
Identification of quantitative trait loci controlling cortical motor evoked potentials in experimental autoimmune encephalomyelitis: correlation with incidence, onset and severity of disease
Hum. Mol. Genet., July 15, 2005; 14(14): 1977 - 1989.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
F. Giuliani, W. Hader, and V. W. Yong
Minocycline attenuates T cell and microglia activity to impair cytokine production in T cell-microglia interaction
J. Leukoc. Biol., July 1, 2005; 78(1): 135 - 143.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D. S. Gregerson, T. N. Sam, and S. W. McPherson
The Antigen-Presenting Activity of Fresh, Adult Parenchymal Microglia and Perivascular Cells from Retina
J. Immunol., June 1, 2004; 172(11): 6587 - 6597.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Milner, R.
Right arrow Articles by Campbell, I. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Milner, R.
Right arrow Articles by Campbell, I. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS