Dear Andrew,

From our work and others CCR2 is not expressed on resident microglia. However, your comment is important. I think, we will need to address whether expression of CCR2 on recruited inflammatory monocytes can be suppressed in the CNS inflamed milieu  or other neurodegenerative conditions. Thus, CCR2 may not be a stable surface/genetic marker to distinguish recruited inflammatory monocytes in the CNS. Moreover, only small portion of myeloid recruited cell express CCR2. Therefore, CCR2-RFP is not an optimal model to study at least all recruited myeloid cells.

Dear Oleg and Andrew,

Thanks for your answer and discussion about this. I just wonder someone reported that in vitro microglia can response to CCL2. Is that because in vitro the microglia lose the CNS milieu and upregulate the ccr2 ?

You, I have had a quick look at some microglia microarray data that I generated as part of my PhD work. I used primary microglia. Without going into all of the minutiae of the analysis, we determined that signal intensity of approximately 25 was our threshold, so anything expressed below this was considered background and therefore unreliable.

Ccr2 had a signal intensity of about 34.

For comparison:

GLAST, an astroglia marker had a signal intensity of 22 (as did many other markers specific to other cell types (e.g, CD4, CD8).

CD11b, a microglia/macrophage marker had a signal intensity of 1448.

So, although we would say that Ccr2 is expressed in primary cells based strictly on our background cutoff, it is at a level that that would be of little interest to anyone. If we wanted to say anything meaningful about Ccr2 we would first have to validate by qPCR and WB. By the way, in our experiment we treated primary microglia with IL6 and I did not observe a change in Ccr2 levels.

Practically, I think it is safe to say that under primary culture conditions, Ccr2 is not expressed - unless validated.

Hope this is helpful to you. Best of luck.

@Ricardo: Your answer is so convincing and I would say yes, you're right.  However, in the brain the ccl2 level is pretty high. Do you think it is expressed by astrocyte and do some effect on infiltrating immune cells?

By the way, did you do the microglia microarray on adult microglia? We are also trying to do RNA array on microglia. I sorted 1 million microglia from mice, but get little RNA. So i feel so frustrated. Do you have any clue about this by chance?

Thanks,

You LI

Dear Li,

CCL2 is expressed by astrocytes and particularly by resident microglia associated with disease phenotype.

Please contact my assistant Zain Fanek ([email protected]) to provide you the protocol to sort adult microglia and RNA isolation. You should be able to isolate around 200K of FACS-sorted FCRLS+ resident microglia from healthy (homeostatic) brain and to get enough RNA. All the best.

Dear Oleg,

Thank you so much for your help! I read your publications before. "Identification of a unique TGF-β–dependent molecular and functional signature in microglia". it is amazing. I remembered in your paper anti-FCRLS antibody was made by yourself lab. we use cd11b+ and cd45 medium to gate microglia population. Hopefully I can get enough RNA. Thanks again.

Dear You Li,

No, the arrays were performed on primary cultures of astroglia and microglia isolated from 3-day old pups. I am not aware of anyone culturing these glia from adult mice.

I would be surprised if CCL2 is expressed in normal mouse brain, I suppose the high expression is in a disease model you are working with?

If it is of interest to you here are my results for Ccl2 expression in primary cultures of astroglia and microglia (untreated or treated with IL6). I can tell you with some confidence that Ccr2 is not expressed in primary astroglia or microglia. Ccl2 is not expressed in untreated primary astroglia, but may be expressed (low level) in untreated primary microglia.

Astroglia: 25 vs. 347 (13.8 FC)

Microglia: 43 vs. 201 (4.6 FC)

These results remain to be evaluated in vivo, but at least this is what happens in primary cultures. Hope that it is of help to you.

Best of luck.

Dear Ricardo,

Thanks for your response. I just did antibody array with brain tissue samples. Compared to other chemokines, ccl2 expression is pretty high even in naive group. While other chemokines are undetectable. I also did ccl2 ELISA  with the naive brain. Ccl2 is 130pg/mg total protein. So probably in tissue level, ccl2 is well expressed.

Best,

You Li

That is interesting You Li. I'm curious to know the role of Ccl2 in the normal brain. Has ISH been performed for the mRNA to determine the cells that are expressing it?

I hope the best for your research.

In our own hands, using a CCR2-RFP reporter mouse as well as immunostaining for CCR2, we were not able to detect any CCR2 positive microglia under normal conditions. 

Ricardo, we did not yet define what cells that express ccl2. Someone reported it was astrocytes. It must be interesting to determine the function of ccl2 in normal brain.

Best,

Can anybody provide any papers that demonstrate CCR2- myeloid cells enter the brain during inflammation? 

I think this paper may provide some interesting insights into dynamic changes of temporal CCR2 expression in myeloid cells under diseased conditions.

Article CCR2 is localized in microglia and neurons, as well as infil...