Protease distribution in J774 macrophages

Abstract

Cathepsin, matrix metalloproteinase (MMP) enzyme and tissue inhibitor of MMP (TIMP) distribution in J774 mouse macrophages has not been comprehensively studied. The distribution and vesicle regulation, trafficking and release of these is important, possibly suggesting drug targets for the therapeutic regulation of inflammatory disease and phagosomal killing of pathogenic organisms in J774 and other macrophages. Percentage

immunofluorescence and ultrastructural enzyme and inhibitor distribution, together with LysoTracker (acidity) and lysosome-associated membrane proteins (LAMPs) colocalisation (both indicating late endosome or “lysosomal” association), western blot estimates of percentage processed- and unprocessed intracellular and secreted enzyme and inhibitor, and vesicle size was used to assign enzyme and inhibitor to “classical” vesicle types. Antibodies against TIMP-1 and TIMP-2 were raised and all antibodies characterised for this purpose.

Together these were used to assign cathepsins H, S, D, B and L to possible secretory vesicles (±20 nm, non-acidic, LAMPs-negative, containing precursor enzymes) and identify at least 6 other endosome-“lysosome-like” vesicles. Cathepsin H appears to be present in classical early endosomes (±100 nm, non-acidic, LAMPs-negative) and cathepsin S in late endosomes(±50 nm, acidic, LAMPs-positive) and possibly “lysosomal” (“hybrid” or digestive organelles) (±150-200 nm, acidic, LAMPs-positive). Both cathepsins H and S, however, seem only reliable markers if used together with additional markers. Cathepsin D appearsmainly associated with “lysosomal” (“hybrid” or digestive organelles) (±150-200 nm, acidic,

LAMPs-positive), possibly consisting of further subpopulations which requires furtherinvestigation e.g. labelling for LAMP-1 and LAMP-2 and cathepsin D. Cathepsins B and Lmay occur in late endosomes and/or hybrid organelles and “secretory lysosomes” containing cathepsins B, D and L may also exist (±30-50 nm, acidic, LAMPs-positive).

The distribution of MMP-9, TIMP-1 and -2 in vesicles (non-acidic, LAMP-2-negative) thatappear novel and distinct from late endosome-“lysosome” vesicles were also demonstrated.

In LPS-stimulated cells, the identity of the large (±450 nm), possible recycling endosomes (Rab11-positive, LAMPs-negative), containing colocalised MMP-9 and TIMP-1, needs investigation i.e., requires further verification with triple labelling and EM. Possible cell membrane and recycling endosome localisation of TIMP-2 needs confirmation with labelling

of non-permeabilised cells and labelling for MT1-MMP and proMMP-2, respectively.