Microglia use multiple mechanisms to mediate interactions with vitronectin; non-essential roles for the highly-expressed αvβ3 and αvβ5 integrins

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Figure 1

Evaluating the role of αv integrins in mediating microglial adhesion to vitronectin. A. Adhesion to vitronectin of microglia derived from wild-type or β3/β5 integrin DKO mice in the presence or absence of an anti-αv blocking antibody was examined as described in Materials and Methods. Adhesion is expressed as the number of cells adherent within a given field of view after 30 minutes adhesion. All points represent the mean ± SEM of three experiments. B. Time course of adhesion to vitronectin for microglia derived from wild-type, β3 integrin KO, β5 integrin KO and β3/β5 integrin DKO mice. Adhesion is expressed as the number of cells adherent within a given field of view, after 15 and 30 minutes of cell adhesion. All points represent the mean ± SEM of three experiments. C. Phase pictures of wild-type, β3 integrin KO, β5 integrin KO and β3/β5 integrin DKO mice microglia adherent to vitronectin after 60 minutes. Scale bar = 50 μm. Note that none of the KO strains showed defects in their adhesion to vitronectin.

Figure 2

Evaluating the role of the αvβ3 and αvβ5 integrins in promoting microglial activation state in response to vitronectin. Wild-type, β3 integrin KO, β5 integrin KO and β3/β5 integrin DKO microglia were purified from mixed glial cultures as described in Materials and Methods, and then cultured in serum-free medium on vitronectin. After two days in culture, microglial expression of the activation marker MHC class I (panel A) or the α4, α5 and αM (Mac-1) integrin subunits (panel B) was analyzed by flow cytometry. All points in the graphs are expressed as the mean fluorescent index (MFI), and represent the mean ± SEM of three experiments. Note that all four strains of microglia expressed equivalent levels of the activation markers, implying that β3 integrin KO, β5 integrin KO and β3/β5 integrin DKO microglia had no defect in their activation response to vitronectin.

Figure 3

Examination of the role of the αvβ3 and αvβ5 integrins in mediating vitronectin induction of microglial activation. Wild-type, β3 integrin KO, β5 integrin KO and β3/β5 integrin DKO microglia were purified from mixed glial cultures as described in Materials and Methods, and then cultured in serum-free medium on uncoated plastic, fibronectin or vitronectin. After 2 days culture, levels of MMP-9 in the microglial supernatants were examined by gel zymography. A. Representative gel zymogram. B. Summary of zymography experiments. Each point is expressed as the percentage change in MMP-9 relative to control (wild-type microglia on uncoated plastic) and represents the mean ± SD of three separate experiments. Note that culture on fibronectin and vitronectin increased MMP-9 expression in microglia from all strains of mice, with no obvious differences detected between wild-type and integrin KO strains on any substrate. C. Examination of the role of the αvβ3 and αvβ5 integrins in mediating microglial phagocytosis. Microglia from all 4 strains were purified as described in Materials and Methods, and cultured in serum-free medium on uncoated plastic for 24 hours before 2 μl of vitronectin-coated yellow-green fluorescent 2 μm beads were added to the cultures. 24 hours later cultures were washed to remove undigested beads and the microglial uptake of fluorescent beads examined by flow cytometry. Each point is expressed as the mean fluorescent index of the microglial population, and represents the mean ± SD of three experiments. Note that none of the integrin null microglia showed defects in their ability to phagocytose vitronectin-coated beads.

Figure 4

Characterization of αv integrin expression on microglia derived from wild-type, β3 integrin KO, β5 integrin KO and β3/β5 integrin DKO mice. A. Flow cytometry analysis on microglia derived from wild-type or β3/β5 integrin DKO mice. Microglia were purified from mixed glial cultures as described in Materials and Methods, and then cultured in serum-free medium on vitronectin. After two days in culture, microglial expression of the αv integrin subunit was analyzed by flow cytometry. Note that β3/β5 integrin DKO mice microglia express the αv integrin subunit, though at much reduced levels compared to wild-type cells. B. Biochemical analysis. An αv integrin imunoprecipitation of wild-type microglia revealed a pattern of three bands: αv (140 kD), β5 (90 kD) and β3 (80 kD). As expected, αv imunoprecipitations of β3 KO microglia showed only two dominant bands: αv and β5, while that on β5 KO microglia showed only two dominant bands: αv and β3. Significantly, αv imunoprecipitations of β3/β5 DKO microglia showed that the αv subunit was still present, though at much reduced levels compared to wild-type cells, and in association with weak levels of two β subunits running at the molecular weights of 110 and 80 kD, which correspond to β1 and β8 integrin subunits, respectively. C. Confirmation that microglia express the αvβ8 integrin. Immunoprecipitations of DKO microglia with a β8 integrin polyclonal antibody detected a pattern of two bands running at 140 kD and 80 KD, that co-migrate with the αv and lower β integrin subunit detected in the αv immunoprecipitation. This confirms that the extra 80 kD band expressed by microglia is the β8 integrin subunit.

Figure 5

Examination of the role of β1 integrins in mediating microglial adhesion to vitronectin or fibronectin. A. Adhesion to vitronectin or fibronectin of microglia derived from wild-type or β3/β5 integrin DKO mice was examined in the presence of a β1 integrin function-blocking antibody. Adhesion is expressed as the number of cells adherent within a given field of view after 30 minutes of cell adhesion. All points represent the mean ± SEM of three experiments. Note that β1 integrin blockade had no impact on the adhesion of wild-type or β3/β5 integrin DKO microglia to vitronectin, but that it inhibited microglial adhesion to fibronectin by approximately 50% (wild-type) or greater than 80% (DKO). B. Adhesion to fibronectin of microglia derived from wild-type, β3 KO, β5 KO or β3/β5 integrin DKO mice was examined in the presence of a β1 integrin function-blocking antibody. Note that β1 integrin blockade of wild-type or β5 integrin KO microglia resulted in approximately 50% inhibition of adhesion to fibronectin, but in β3 integrin null or DKO microglia, the inhibition was greater than 80%.