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Carboxypeptidase-M is regulated by lipids and CSFs in macrophages and dendritic cells and expressed selectively in tissue granulomas and foam cells

Granulomatous inflammations, characterized by the presence of activated macrophages (MAs) forming epithelioid cell (EPC) clusters, are usually easy to recognize. However, in ambiguous cases the use of a MA marker that expresses selectively in EPCs may be needed. Here, we report that carboxypeptidase-M (CPM), a MA-differentiation marker, is preferentially induced in EPCs of all granuloma types studied, but not in resting MAs. As CPM is not expressed constitutively in MAs, this allows utilization of CPM-immunohistochemistry in diagnostics of minute granuloma detection when dense non-granulomatous MAs are also present. Despite this rule, hardly any detectable CPM was found in advanced/active tubercle caseous disease, albeit in early tuberculosis granuloma, MAs still expressed CPM. Indeed, in vitro both the CPM-protein and -mRNA became downregulated when MAs were infected with live mycobacteria. In vitro, MA-CPM transcript is neither induced remarkably by interferon-γ, known to cause classical MA activation, nor by IL-4, an alternative MA activator. Instead, CPM is selectively expressed in lipid-laden MAs, including the foam cells of atherosclerotic plaques, xanthomatous lesions and lipid pneumonias. By using serum, rich in lipids, and low-density lipoprotein (LDL) or VLDL, CPM upregulation could be reproduced in vitro in monocyte-derived MAs both at transcriptional and protein levels, and the increase is repressed under lipid-depleted conditions. The microarray analyses support the notion that CPM induction correlates with a robust progressive increase in CPM gene expression during monocyte to MA maturation and dendritic cell (DC) differentiation mediated by granulocyte–MA-colony-stimulating factor+IL-4. M-CSF alone also induced CPM. These results collectively indicate that CPM upregulation in MAs is preferentially associated with increased lipid uptake, and exposure to CSF, features of EPCs, also. Therefore, CPM-immunohistochemistry is useful for granuloma and foam MA detections in tissue sections. Furthermore, the present data offer CPM for the first time to be a novel marker and cellular player in lipid uptake and/or metabolism of MAs by promoting foam cell formation.


Immunohistochemistry (IHC)
To check the specificity, positive and negative controls were included for each immunohistochemistry run. Negative controls for CPM and the reference macrophage markers consisted of slides with isotype-matched control mAB IgG (Dako-Cytomation) in place of the primary antibody 11 . For all immune-peroxidase stainings, the nuclear counterstainings were with methyl-green or hematoxylin. Normal placenta served as a positive control for CPM-immunostainings where trophoblasts show membrane positivity ( Figure S1-A). The immune-stained specimens for macrophage labeling were judged semi-quantitatively as no staining (0); weak staining (1+); moderate staining (2+); intense staining (3+).

Supplementary information for monocyte separation
Cell viability was at least 98% as determined by trypan-blue exclusion, and monocyte purity was varied between 95%-98% as judged by morphology and flow cytometry analysis for CD14 expression. Separated cells were resuspended (1.5 x 10 6 /ml) in RPMI1640 supplemented with 10% heat inactivated fetal bovine serum (FBS) (Invitrogen), 500 U/ml penicillin-streptomycin and 2mM L-glutamine (Gibco). For cell culturing 25cm 3 tissue culture flasks (TPP; Trasadingen, Switzerland) were used. All cultures were maintained in a humidified 37°C incubator venting 5% CO 2 . [28][29]. The cells were grown on coverslip for in situ immunofluorescence combined with nile-red lipid staining.

Supplementary information for macrophage-infection with mycobacteria
Mycobacterium bovis-BCG tuberculosis bacilli (TB) was purchased in lyophilized form (Statens Serum Institut, Copenhagen, Denmark) and dissolved in culture medium right before infection, and clumps were removed by a 10 minutes centrifugation at 20 g. The density of bacteria in the cultures was adjusted to a dose of 1 bacterium/cell assuming that an OD 600 of 0.2 equals a concentration of 10 7 bacteria/ml. At the indicated time points, the cells were collected with centrifugation, and the pellets were then subjected to formalin fixation, paraffin embedding and sectioning for immuno-fluorescence staining as earlier described 29 .

Statistical analysis
In real-time quantitative RT-PCR experiments, the mean and SD were calculated for both the normalized and the normalizer values. To incorporate the random errors of the measurements, we used the propagation of errors to determine the SD of the normalized values. We made at least three biologic replicates for all experiments. In real-time quantitative PCR experiments, we performed an unpaired, two-tailed, t test. p < 0.03 was considered statistically significant.

The sensitivity and specificity of MA-CPM IHC on routinely processed tissues
The IHC findings for carboxypeptidase-M expression in macrophages are summarized in supplementary Table S1. By comparing the CPM expression patterns with CD68 and CD163 on tissue sections, it is obvious that CPM detects only small subpopulation(s) of macrophages (i.e., epithelioid and lipid-laden cells, only), as opposed to reference markers which decorate virtually any types of macrophages ( Figure 1B Therefore, the sensitivity of CPM for macrophage detection is low when used as a universal (pan-)macrophage IHC-marker on paraffin tissue sections. However, the marker CPM appears to be highly specific and sensitive for the detection of EPCs and lipid-laden macrophages, respectively. To allow specificity, no reactivity was seen in normal condition in adipose tissues, fatty changed hepatocytes, non-activated Kuppfer cells of the liver (not shown), as well as in resting macrophages, "regular" non-specific inflammatory cells and alveolar macrophages e.g., associated with congestive hear failure (supplementary Figure S1), persistent chronic inflammation with no significant cell damages. Also, within normal lymph nodes, basically no CPM + "resting" sinus histiocytes are observed unless lipid uptake or cytokine-effects induced foamy or antigenpresenting dendritic cells, e.g., due to the draining components of tumor cells to the lymph node (Fig. 8C-D).

Technical notes for CPM-IHC in paraffin sections versus cryosections
3 Due to lipid-association of CPM-binding to cell membranes [6][7][13][14] and the use of organic solvents (acetone, xylene, ethanol) during paraffin tissue processing (which partially removes the lipids along with the majority of the coupling proteins), the CPM immune-staining intensities may decrease in paraffin-sections. Nevertheless, the positive staining can still clearly be recognized on commercially processed paraffin tissues ( Figure 7A-C), but cryosections proved to be more effective in some cases (not shown).