Hello,
I am currently trying to standardise qPCR primers for IL-10 expression in human macrophages i.e. checking amplification, primer efficiencies. Before the actual stimulation by bacteria, I am using control treatments to establish the system using M-CSF mediated THP-1-derived macrophages.
I am using the following treatments:
1. Monocytes only
2. Differentiated but unpolarised Macrophages
3. LPS+ IFNg
4. TGFBeta1
5. Glucocorticoids mixture (GCM)
6. As a control for some experiments, PMA-mediated THP1 derived macros.
I have tested the following samples already for the expression of IL-10 and MMP12 with two separate sets of primers.
1. Monos, 2. unpolarised macros 3. LPS + IFNg macros 4. GCM macros
Problems:
1. I cannot detect IL-10 and MMP12 in monos (Amplified very late Ct 38 or undetermined).
2. Cannot detect IL-10 and MMP12 in M2-like (GCM treated) macros (Amplified very late Ct 38 or undetermined).
3. Very weak MMP12 and almost no IL-10 in M1-like (LPS+IFNg) macros.
Suggestions:
1. Should I wait for the TGFbeta1 samples before concluding anything, is it usually expected in this treatment ?
2. Should I check in PMA-treated macros ?
3. What could be a positive control for IL-10 and MMP12 ?
I would be extremely grateful if someone could show me the way here,
Cheers,
Sudip
HME-MMP12 is almost exclusively produced by dendritic cells in vivo.
In vitro two types of cells are capable of producing large amounts of MMP12: CD34 + DCs grown in GM-CSF + TNFα, and monocytes grown in GM-CSF + II4. The secretion of MMP12 is greatly increased if IFNg and especially IL10 are added.
The amounts of MMP12 produced in vitro under optimal conditions (with IL10) can reach 1 mg / ml and become comparable to the serum levels of patients with monocytosis or langerhansian histiocytosis, which can reach 1.4 mg / ml .
And if COPD has a relatively low MMP12 level, it is the pulmonary dendritic cells that secrete the enzyme responsible for pulmonary emphysema: with amounts of MMP12 that can exceed 2mg / ml in the BAL. The secretory role of type 2 pneumocytes (DCLamp +), bronchial langerhans cells (langerin +) and alveolar DCs (MMR +) is paramount.
IFN gamma down-regulates LPS induced IL10. It might be therefore better to use LPS alone.
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