Dear colleagues,
Does anyone of you have experience with the differentiation between A and B synoviocytes in cell culture and could give me an advice which antibody is best / most specific? I've read aboout UDPGD for B synoviocytes, is this truly specific? Would love to hear about your experiences.
Thanks a lot! Stephanie
Dear Stephanie,
We abolished A and B synoviocytes in about 1980 but you will still see people suing these terms in the literature. What you are referring to are synovial intimal macrophages and intimal fibroblasts. If you disaggregate tissue and put it in culture you will not be able to know whether the macrophages and fibroblasts in the culture are intimal or subintimal so you will have to call them just synovial macrophages and synovial fibroblasts. The exception is if you look at the cells absolutely fresh after gentle collagenase digestion when if you are lucky you can see the intimal cells still in mixed clumps. I think there were some of these in the original paper on UDPGD and CD68 labelling we did with Andy Pitsillides and Linda Wilkinson.
Once in culture you can pick out the macrophages easily with CD68 or a lot of other myeloid related markers. Intimal macrophages are different in expressing CD16 and CR3 but in culture other macrophages will catch up on that after a few days. In tissue only the intimal fibroblasts have special features like UDPGD, VCAM-1 and DAF expression. In culture they tend to lose these quickly but you can still tell they are fibroblasts as they will have an elongated morphology and be CD68 negative. They are likely to keep standard fibroblast markers (I think we once used procollagen III).
Rheumatoid tissue will contain subintimal fibroblasts that also express VCAM-1 and DAF in association with lymphocyte aggregates but these do not have UDPGD even in tissue.
We spent ten years sorting all this out in the 1990s and I would be happy to give further details. The most useful answers are likely to depend on exactly why you want to identify these cells. Do remember that you will not be able to identify intimal cells specifically after a day or two, because of convergent changes.
Another simple point is that the UDPGD assay is almost impossible for normal human beings to perform. Andy P had intensive training in the cytochemical methods needed over a period of years at the Kennedy Institute. I think I only ever got it to work once. You have to get the lemon sorbet absolutely right before you put it on the cells!
Dear Jonathan,
Thank you very much for your very useful reply and the detailed explanation!
I was thinking about CD68 for picking out the macrophage population in cell culture, and the additional markers you mentioned seem to be useful as well. I am however wondering if I might have problems with contamination by fibroblasts from other locations or chondroblasts / chondrocytes and how to distinguish them from FLS. We are trying to culture mouse FLS, so I wouldn't exclude other cells to be present as well.
I will have a look into your publications you cited and talk with my colleages about your suggestions. If you don't mind I will then contact you again and would very much appreciate getting further details, especially on the UDPGD Assay.
Thanks again!
Best wishes, Stephanie
Fair enough. Stephanie. Remember, however, that 'FLS' do not exist in culture - it is a description that can be applied to intact tissue but has been erroneously applied to cultures under the false assumption that fibroblasts from synovium in culture come from intima. In the 1990s I wrote a lot of reviews to try to sort this out - in book chapters and other places, but the confusion tends to persist.
The key question is why do you think you are interested in 'FLS" rather than just stromal cells from synovial subintima for instance? The cells that associate with lymphoid cells are subintimal cells, not 'type B cells' or strictly 'FLS'. My conclusion after our embryological work was that all these cells have the same potential for gene expression - probably in common with bone marrow stromal cells and may be a class of 'blastemal core fibroblasts' in terms of long term differentiation. That ebing the case it does not really matter where your fibroblasts come from if they come from skeletal tissue I suspect.
It all depends on what idea you are trying to test - over the years it was my experience that people working on synovial cells tended not to have clearly thought out what their idea was!
the 'A' and 'B' cells are actually intimal macrophages and intimal fibroblasts. apart from UDPGD, the intimal fibroblasts show CD55 expression.
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