Immunofluorescence assay of Actin to check BV2 cell spread

In BV2 cells the immunofluorescence assay for actin was done 4 and 6 hrs after treated with or without LPS (1 µg/ml). Without LPS, that is control condition, the cell showed round shape cell body sign of having process like short proximal process and long distal ramification and cell membrane looks even in both 4 and 6 hrs (Fig.3). In the other hand, actin expression in the LPS-treated group showed changes in the cell shape and sign of having more than two pole, possible retraction of process, their uneven membrane (Fig.3).

Fig. 3. Immunofluorescence assay of BV2 cell morphology. Microglial cell line BV2 cell were treated with or without LPS (1 μg/ml) for 4hrs and 6hrs and labeled with antibody for actin. Treatment of LPS increased the number of cells containing uneven membrane, thick proximal process with short or no distal ramification, which indicated the spread BV2 cells. Blue arrow shows the round cell, green arrow shows the ramified cells and red arrow shows the spread cells. Immunefluorescence photomicrograph of the corresponding fields is X400.

18 C. Role of cytoskeleton in cell shape change

Cytoskeleton is one of the main factors that are responsible for the maintenance of the cell shape. Therefore, I performed immunoassay for the stress fibers, components of cytoskeleton, in BV2 cells. After treatment with LPS (1 μg/ml) for 4hrs and 6hrs, the cells were fixed and labeled with antibody against a focal adhesion molecule vinculin and phalloidin specific for F-actin. It showed that LPS increased the focal adhesion area and changed the arrangement of stress fibers (Fig.4A). In LPS-treated group, the numbers of spread cells increased while the numbers of round cells decreased than the control group, which was same as previous data in phase contrast picture. In cytoskeleton-stained pictures, the cell shape change is suggested as due to rearrangement of the actin filaments.

Fig. 4. Immunofluorescence assay cytoskeleton of BV2 cell. Microglial cell line BV2 cell were treated with or without LPS (1 μg/ml) for 4hrs and 6hrs and labeled with antibody for vinculin (red) stress fiber (green). LPS increased the focal adhesion area of the cells and changed the rearrangement of F-actin (A). Blue arrow shows the round cell, green arrow shows the ramified cells and red arrow shows the spread cells. According to the cell shape the cell numbers also counted into three different groups as previously mentioned (B). Immunefluorescence photomicrograph of the corresponding fields is X200.

D. LPS-induced cell adhesion molecule mRNA expression in BV2 cells

Cell adhesion molecules expressed by LPS (1 ug/ml) in BV2 cells were evaluated by RT-PCR. The BV2 cells were treated with or without LPS and incubated for 1, 10 and 30 mins, 1hr, 2hr and 4 hrs. The mRNA for ICAM-1, LFA-1 and subunits of different integrin (β1 & β2) were done. Results showed that there was no change of ICAM-1 mRNA expression in the control group in any time course, whereas LPS induced ICAM-1 mRNA expression as early as 1 hr and maintained in 2 and 4 hrs (Fig. 5), which coincided with the time of BV2 cell spread. However, the expression of LFA-1 and integrin subunits (β1 & β2) mRNA was not much changed in time or by LPS treatment.

E. ICAM-1 protein expression in BV2 cells

LPS induced ICAM-1 mRNA expression in BV2 cell as early as 1 hr. The ICAM-1 protein expression was observed at 2, 4 and 6 hrs with western blotting. There was no change in ICAM-1 protein expression in the control group in any time course (Fig.

6), while LPS began to induce ICAM-1 protein expression at 2hrs and increased the expression significantly at 4 and 6 hrs (Fig. 6).

Fig. 5. Cell adhesion molecule express in BV2 cells The cells were treated with or without LPS for 0, 10 and 30 min, 1hr, 2 hrs and 4hrs to check mRNA expression of different CAMs. The expression of ICAM-1 mRNA increased in BV2 cells treated with LPS at 1 hr and continued in 2 hrs and 4 hrs, which also correlated with change of morphology. In case of CAMs from integrin family there were no significant changes in their mRNA expression by LPS.

Fig. 6. ICAM-1 protein expression with or without LPS. Western blotting was done to measure ICAM-1 protein expression in BV2 cells treated with or without LPS for 2, 4 and 6 hrs. It showed that the ICAM-1 protein expression increased at 2hr and more increased at 4 and 6hrs. This result coincided with the ICAM-1 mRNA expression and the increase in number of spread cells.

24 F. Cytokine expression By the BV2 cell

After seeding BV2 cells, the expression of cytokines IL-β and TNF-α was observed at the time of seeding, 30 min, 1hr, 2hr, 4hr and 6hr (Fig. 7). Both of the cytokines gradually increased until 1 hr, and gradually decreased after 2 hrs and later at 6hr become hardly traceable. The treatment of LPS (1ug/ml) increased the expression of both cytokines as early as 30 mins and the expression remained same until even 4hrs.

Therefore, the LPS-treated cells started spread after 2 hrs but the cytokines expressed far earlier than the cell spread (Fig. 8). As cytokines expressed ICAM1 within 1-4 hrs, they may have some roles in cell spread in the earlier spread of BV2 cell.

Fig. 7. Cytokines Expression by BV2 cells without treatment. Cytokines IL-1β and TNF-α mRNA expression was measured in the BV2 cells without LPS treatment. Both of the cytokine expression increased as early as 30 min but gradually decreased after 2 hrs and become hardly traceable in 6 hrs. This suggested that the cytokines had little roles in BV2 cell morphology without LPS treatment.

Fig. 8. Cytokines by LPS-treated BV2 cells. BV2 cells were treated with or without LPS for 30 min, 1 hr, 2hrs and 4hrs to check the effect of LPS on the expression of cytokines IL-1β and TNF-α mRNA. LPS increased the expression of both cytokines at 30 min and remained same until 4 hrs (A). In case of control group the expression of mRNA showed as previously mentioned. Both cytokines increased the ICAM1 expression at 2 hrs but the expression was not much significant in the earlier time (B).

G. Spread inhibition by monoclonal neutralizing ICAM1antibody in BV2 cells

As BV2 cells showed spreading at 4 and 6 hrs, the cellular shape changes were observed at those time course after treatment with or without LPS and a monoclonal

neutralizing ICAM1antibody YN1/1.7.4 (2.5 ). In Fig. 9A phase contrast pictures showed that the treatment of neutralizing antibody to ICAM1 attenuated the number of spread of BV2 cells and although the numbers of ramified cells was reduced, there was no significant change (Fig. 9). However, the portion of spread cells showed a significant reduction in LPS and YN1/1.7.4 treated group compared with the only LPS-treated group (Fig. 9A and 9B). This result indicates that ICAM-1 plays roles in LPS –induced morphologic changes of BV2 cells.

Fig. 9. Inhibition of ICAM1 by monoclonal neutralizing antibody. BV2 cell were treated with or without LPS (1ug/ml) and ICAM1 monoclonal antibody YN1/1.7.4 (2.5uM/ml) for 2, 4 and 6hrs. In (A). Phase contrast picture shows that monoclonal neutralizing antibody to ICAM1 reduce the number of spread of BV2 cell. Pai chart shows that Neutralizing antibody for ICAM-1 reduce the number of spread cells in LPS treated groups in both 4 and 6 hrs (B). But there is no significant difference in other type of cells. Blue arrow shows the round cell, green arrow shows the ramified cells and red arrow shows the spread cells. Phase contrast photo micrograph of the corresponding fields X100.

H. Immunoflruorecense Assay to check the effect of monoclonal antibody to ICAM1.

To confirm the effect of the YN 1/1.7.4 the monoclonal antibody to ICAM1 on the morphologic changes by LPS in BV2 cells, I examined the effect of ICAM1 mAb on actin remodeling, LPS-induced spread, which was evident in the phase contrast. The actin remodeling was observed 4 and 6hrs after LPS treatment. After fixation with 4%

paraformaldehyde the BV2 cells were stained with Phalloidin. LPS treatment increased the actin rearrangements and those were attenuated significantly by treatment of mAb to ICAM1 (Fig.10). It gives that idea that monoclonal antibody to ICAM1 blocks the effect of LPS-induced ICAM1 which causes the actin rearrangement.

Fig. 10. Expression of actin in LPS and monoclonal antibody treated BV2 cell LPS (1μg/ml) and ICAM1 monoclonal antibody YN1/1.7.4 (2.5 μg/ml) were treated in BV2 cell for 4 and 6hrs. The cells were fixed with 4% PFA and later was stained with phalloidin. It showed that mAb for ICAM1 attenuated the spread of BV2 cells by reducing the rearrangement of actin fiber. Blue arrow shows the round cell, green arrow shows the ramified cell and red arrow shows the spread cell. Immunefluorescence photomicrograph of the corresponding fields is X200.

I. Inhibition of ICAM1 downstream molecule Rho/ROCK.

Rho/ROCK, serine-threonine kinases are the downstream molecule of ICAM1 signaling pathway. The effect of Rho/ROCK kinase inhibitor Y27632 (10 μM/ml) on BV2 cells with or without LPS was observed. It showed that Y27632 developed long process in most cells which resembled the ramified cells (Fig.10). When the cells were treated with LPS for 4 and 6 hrs, there was little changing in the cell shape or process.

However, treatment of LPS without Y27632 showed the similar result as previously shown.

Fig. 11. Inhibition of ICAM1 downstream pathway by Rho/ROK inhibitor. BV2 cells were first incubated with or without Rho/ROK inhibitor Y27632 (10 μM/ml) and later with or without LPS (1μg/ml). The time course was chosen as 4 and 6hrs. Then the pictures was taken in the contrast phase. Blue arrow shows the round cell, green arrow shows the ramified cells and red arrow shows the spread cells. Phase contrast photomicrograph of the corresponding fields is X100.

IV. DISCUSSION

In this study it is characterized that BV2 microglial cells change in morphology, which is time dependent, and the expression of ICAM-1 is parallel for the time course change of microglial shape especially for spreading. When the BV2 cells was pre-incubated with ICAM-1 neutralizing antibody YN1/1.7.4 prior to LPS, the number of spreading cells was significantly reduced.

Microglial cells, the resident macrophages of the CNS, are exquisitely sensitive to brain injury and disease, altering their morphology and phenotype to adopt a so-called activated state in response to pathophysiological brain insults. Morphologically activated microglial cells like other tissue macrophages, exist as many different phenotypes, depending on the nature of tissue injury (Perry et al, 2010). Lipopolysaccharides (LPS), also known as lipoglycans, are large molecules consisting of a lipid and a polysaccharide joined by a covalent bond; they are found in the outer membrane of Gram-negative bacteria, act as endotoxins and elicit strong immune responses in animals. LPS treatment has been used extensively in inflammatory studies (Carter et al, 2003; Maekawa et al, 2002). It has also been shown that LPS activates microglia and exerts neurocytotoxic effects in both in vitro and in vivo systems (Hughes et al, 2004; Nakamura et al, 1999). Microglia shows diverse morphological changes which also indicate activation after treated with LPS. Morphologically, this activation was characterized by the swelling of the microglial cell body, a thickening of the proximal processes, and a reduction in distal ramification (Christian et al, 2000). In this study the types of BV cells were classified into three groups round, ramified and spread.

The classification was done according to their cell size, condition of the membrane,

proximal process and distal ramification. Round cells with smooth cell surface membrane resembles as either myelomonocytic cells or activated BV2 cell (amoeboid activated cells), ramified are bipolar with small cell body and have distal ramification and proximal process, lastly the spread cells tri- or multipolar with short or no distal ramification, thickened proximal process and uneven cell surface membrane. In primary microglial cells, treatment of LPS has a more extended morphology than untreated cells on glass (Summers et al, 2009). In this study BV2 microglial cells treated with LPS (1 µg/ml) showed different time duration but the result supported the previous study. In present study BV2 cells started to take ramified morphology after 2 hours in both LPS treated and untreated group. However, LPS increased the number of spread cells at 4 and 6 hrs in Fig. 1 and 2, which also could be manifested as pro-amoeboid cells (Fig. 2). I also confirmed the changes of the cell shape with immunofluorescence assay, by using antibody binds to actin, in the control and LPS-treated group at 4 and 6 hours. It showed that BV2 cells increased in size and got spread shape in LPS-treated group in both 4 and 6 hrs. Thus, it is certain that LPS induce the spreading of BV2 cells and it is time dependent.

Cell adhesion molecules (CAMs) are located on the cell surface, which are involved with the binding with other cells or with the extracellular matrix (ECM) in the process called cell adhesion. Microglial cells express a number of CAMs like ICAM-1 (CD54), lymphocyte function associated antigen-1 (LFA-1) (CD11a) and α5β1 integrin in neurodegenerative diseases (Milner et al, 2003). Previous study reveals that cell adhesion molecules like ICAM-1, LFA-1 has a strong role in change of size in human dermal microvessel endothelial cells (HDMEC), human monocyte cell line THP-1, T-cell and other cells (Ronald et al, 2001; Mueller et al, 2004). In present study, cell adhesion

molecule expression with or without LPS (1 µg/ml) in BV2 cells was measured. The most of the CAMs or their subunits expressed by microglia such as LFA-1, β2 and β1 subunits of MAC-1 and α5β1 were present after seeding in the plastic (Milner et al, 2003; Färber et al, 2008). After seeding, mRNA of these integrins expressed at earlier time and there are no significant changes of their expression in the course of time and also irrespective of the control and the LPS-treated group (Fig. 5). In contrast, the expression of ICAM-1, a cell adhesion molecule related to Immunoglobulin super family, increased significantly after 1 hr and it continued even until 4 hrs (Fig. 5). In morphology, BV2 cells showed spreading after 2 hrs and at 4 hrs and it was suggested that the increase of ICAM-1 mRNA expression might have somewhat relationship with the spreading. Further the expression of ICAM-1 protein in BV2 cells was measured at 2, 4 and 6hrs. LPS increased ICAM-1 protein expression at 2 hrs, more significantly increased at 4 hrs and continued at 6hrs (Fig. 6). ICAM-1 protein expression fully coincided with the BV2 cell spreading, which was observed previously.

LPS induces proinflammatory cytokines including Tumor necrosis factor (TNF)-α and Interleukin beta (IL- β) in microglial cells (Ye et al, 2001; Astrid et al, 2010). The release of these factors was time-dependent (Wang et al, 2005). In the present study, the expression of TNF-α and IL- β in microglial cells increased after seeding cells on the plastic in 1^st hr but after 2^nd hr gradually decreased and the mRNA expression of both cytokines in 6 hr diminished. When LPS was treated, the expression of both cytokines increased as early as 30 mins after seeding and remained same until 4 hrs in Fig. 4. Also, the treatment of TNF-α and IL- 1β did not significantly enhance ICAM-1 mRNA levels in microglial cells, and change little in the morphology. Therefore, cytokines such as TNF-α and IL- 1β had little role in spreading.

Later to ensure the role of ICAM-1 in cell spreading, ICAM-1 was blocked by YN1/1.7.4 a neutralizing antibody specific for ICAM-1. The YN1/1.7.4 monoclonal antibody efficiently blocks the binding of ICAM-1 to LFA-1 and Mac-1 and inhibits ICAM-1-mediated functions including cell-cell adhesion, antigen presentation to T-cells and leukocyte migration to inflammatory tissues (Johnson LA, 2006; Kooyk YV, 1994).

The YN1/1.7.4 monoclonal antibody was examined whether the ICAM-1 is responsible for LPS-induced cell spreading in BV2 cells. When the BV2 cells were pre-incubated with YN1/1.7.4 for 30 mins, the number of spreading cell in LPS-treated BV2 cell was reduced. It was also observed when the cells were stained with phalloidin. Blocking of ICAM-1 attenuated the LPS-induced rearrangement of the actin filament, however there was no significant change in the number of ramified cells, which indicated that the ICAM-1 exerted on the spreading rather than ramification. However, when Rho kinase ICAM1 downstream molecule was inhibited with Y27632, BV2 cells became ramified with long process in the control group while LPS-induced spreading was not affected by Rho kinase inhibitor. It suggested that LPS-induced Rho activation is closely related with retraction of the process.

V. CONCLUSION

ICAM-1 is a well known cell adhesion molecule from the Immunoglobulin super family, and associates with receptors of the integrin (LFA-1) family, thereby mediating cell-cell interactions and allowing for signal transduction. In this study LPS significantly increased a high level of ICAM1 mRNA and protein expression using RT-PCR and western blotting and concomitantly induced the morphologic changes, which increased spreading as early as 4 and 6 hrs with actin rearrangement. The monoclonal neutralizing antibody for ICAM-1 (YN1/1.7.4) attenuated the number of the spreading cells. The LPS-induced spreading was related with retraction of ramification, which might be closely involved with ICAM-1 downstream signaling ROCK/Rho activation. Thus ICAM-1 plays an important role in the cell shape or morphological change.

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