Monocyte differentiation and macrophage priming are regulated differentially by pentraxins and their ligands

All protocols were approved by the local ethical committees and performed in accordance with national guidelines and regulations. Human peripheral blood was collected from healthy adult volunteers who gave written consent and with specific approval from the Texas A&M University human subjects Institutional Review Board. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood using Ficoll-Paque Plus (GE Healthcare Biosciences, Piscataway, NJ), as described previously [9]. PBMC were cultured at 37 °C in a humidified incubator with 5% (vol/vol) CO₂ in either 8-well glass slides (Falcon-Corning, Tewksbury, MA or EMD-Millipore, Billerica, MA) or 96 well μ-plates (ibidi, Madison, WI) with 200 μl/well at 5 x 10⁵ cells per ml in RPMI-1640 (Lonza, Walkersville, MD) containing 100 U/ml penicillin, 100 μg/ml streptomycin (Lonza), and 10% fetal calf serum (FCS; Seradigm, Radnor, PA) [9, 11, 25]. Although both human AB serum and FCS can be used for human monocyte/macrophage cultures, we used FCS as it contains low levels of pentraxins and their ligands [26‐31].

For monocyte differentiation, PBMC were incubated for 6 days in the presence or absence of the indicated concentrations of SAP (EMD Millipore, Billerica, MA), CRP (#30-ac05AF, Fitzgerald Industries, Acton, MA), or PTX3 (R&D Systems, Minneapolis, MN) [9, 25]. As commercial SAP preparations contain 0.1% azide, we buffer-exchange the SAP into 20 mM sodium phosphate, pH 7.4, as described previously [9, 32]. CRP and PTX3 preparations were purchased free of azide. For macrophage priming, PBMC were incubated in the presence or absence of 25 ng/ml M-CSF or GM-CSF (BioLegend, San Diego, CA) for 6 days [4, 33, 34]. The medium was then removed and fresh medium containing M-CSF or GM-CSF was then added, containing the indicated concentrations of pentraxins in the presence or absence of 30 μg/ml C1q (Fitzgerald Industries), 100 μg/ml factor H (Fitzgerald), or 2 μg/ml MBL (NovoProtein, Summit, NJ) for an additional 2 days. To polarize macrophages, PBMC were incubated for 6 days in the presence of 25 ng/ml M-CSF or GM-CSF and then polarized into M1 macrophages with 10 ng/ml IFN-γ (BioLegend) and LPS (Sigma, St. Louis, MO) or M2 macrophages with 10 ng/ml IL-4 (BioLegend) [33, 35, 36]. All reagents were isolated from human material and/or tested for LPS/endotoxin and found to be <0.01 EU/ml or <1.0 EU per μg of protein (apart from the LPS), as determined by the manufacturer. After 6-day monocyte differentiation experiments or 8-day macrophage priming experiments, supernatants were collected and stored at either 4 °C (for less than 2 days) or at −20 °C, and the plates were air-dried as described previously [9]. Supernatants were analyzed by ELISA using kits for IL-4 (Peprotech, Rocky Hill, NJ), IL-10 (BioLegend), IL-12 (BioLegend), and IFN-γ (Peprotech) following the vendor’s protocol.

For 8-well slides and 96 well plates, cells were fixed with acetone for 15 min, air dried for 15 min and then non-specific binding was blocked by incubation in PBS containing 4% BSA (PBS-BSA) for 60 min. Slides were then incubated with 5 μg/ml primary antibodies (Table 1) in PBS-BSA for 60 min as described previously [9]. Isotype-matched irrelevant mouse and rat monoclonal antibodies (BioLegend), or irrelevant rabbit or goat polyclonal antibodies (R&D Systems, Minneapolis, MN), at 5 μg/ml in PBS-BSA were used as controls. Primary antibodies were detected with either biotinylated donkey F(ab’)₂ anti-mouse IgG, biotinylated mouse F(ab’)₂ anti-rat IgG, or biotinylated donkey F(ab’)₂ anti-rabbit IgG (all cross-adsorbed against human Ig; Jackson ImmunoResearch, West Grove, PA). All secondary antibodies were used at 1 μg/ml in PBS-BSA for 30 min. Biotinylated antibodies were detected by a 1/500 dilution of ExtrAvidin alkaline phosphatase (Vector Laboratories, Burlingame, CA) in PBS-BSA. Staining was developed with the Vector Red Alkaline Phosphatase Kit (Vector Laboratories) for 5–7 min, and then counterstained with Gill’s hematoxylin #3 (Sigma-Aldrich, St. Louis, MO) following the manufacturer’s directions. Macrophages were identified as 15–40 μm diameter cells with a large nucleus and pronounced cytoplasm.

There is no definitive set of markers to identify polarized macrophages [4, 5]. Therefore, we first assessed the effect of pentraxins on macrophage polarization using markers previously identified as being expressed on M1 macrophages including IFN-γ receptor (CD119), MHC class II, CCR7, and IRF5; M2a macrophages including Dectin-1, resistin-like molecule-β (RELMβ; FIZZ2), IRF4, and fibronectin (Fn); and M2c macrophages including sphingosine kinase-1 (SPK), TNFRSF14 (CD270) and SIRPα (CD172a) [4, 35‐42]. As discussed by Murray et al. [4], all macrophages regulate the immune response, therefore we will use the term M2c, as defined by Mantovani [3], to define the IL-10 producing regulatory/resolving macrophage phenotype. We specifically did not isolate monocytes away from the other cell types found in PBMC, so that we could determine the effect of pentraxins and their ligands on monocyte differentiation and macrophage polarization in the presence of other immune cells. PBMC were cultured for 6 days in the presence of GM-CSF to differentiate the monocytes into M1-like macrophages, or M-CSF to differentiate the monocytes into M2-like macrophages [4, 33, 43, 44], and then pentraxins were added for additional 2 days to prime the macrophages. Compared to no pentraxin, there was no significant effect of SAP, CRP, or PTX3 on the percent of morphologically-identifiable macrophages expressing the markers listed above (Fig. 1). Antibodies against many of the markers only stained a subset of macrophages, indicating a heterogeneity of macrophages in these standard culture conditions. To confirm that we could identify polarized macrophages, PBMC were cultured for 6 days in the presence of M-CSF or GM-CSF and then polarized into M1 or M2 macrophages with either IFN-γ and LPS or IL-4. We found that the M1 marker IRF5 was upregulated in cultures containing IFN-γ and LPS, and the M2 marker IRF4 was upregulated in cultures containing IL-4 [33, 35, 36] (Fig. 2). These results suggest that pentraxins do not appear to affect the percent of macrophages expressing CD119, MHC class II, CCR7, IRF5, Dectin-1, RELMβ, IRF4, fibronectin, SPK, CD270, or CD172a.

Testing other markers, we then found that the hemoglobin-haptoglobin complex receptor CD163, the sialoadhesin CD169, and the C-type lectin CD206 were differentially expressed by human macrophages when cultured in the presence or absence of pentraxins (Fig. 3). CD163 has been used as both a tumor-associated and M2 macrophage marker, CD169 has been used as a marker of subsets of macrophages in lymph nodes, lung, and GI tract independent of the M1/M2 classification system, and CD206 has been used as a general M2 marker [4, 37, 45, 46]. However, the association of these three receptors with the broad M2 classification is difficult to interpret, as the M2 subset of macrophages contains pro-fibrotic M2a macrophages, M2c anti-inflammatory macrophages, and tumor-associated macrophages [4]. Compared to macrophages cultured with M-CSF in the absence of pentraxins, SAP increased the percentage of macrophages expressing CD169 and CD206, CRP increased the percentage expressing CD169, and PTX3 increased the percentage expressing CD169 and CD206 (Fig. 3a-c). Compared to macrophages cultured with GM-CSF in the absence of pentraxins, SAP increased the percentage expressing CD206, CRP increased the percentage expressing CD163, CD169, and CD206, and PTX3 increased the percentage expressing CD169 (Fig. 3d-f). We occasionally observed (by morphology) dendritic cells in cultures with GM-CSF (Fig. 3g insert). These results suggest that in cultures with M-CSF or GM-CSF, pentraxins affect the percentage of macrophages expressing detectable levels of CD163, CD169, and CD206.

In healthy humans the plasma levels of CRP and PTX3 are low (<2 μg/ml and < 25 ng/ml respectively) and SAP is approximately 30 μg/ml, whereas during inflammation CRP and PTX3 levels may rise to 50–500 μg/ml and 200–800 ng/ml respectively, but SAP levels remain constant [7]. Pentraxins bind to several plasma proteins. SAP, CRP, and PTX3 all bind the complement component C1q [20‐22], CRP and PTX3 bind Factor H, while SAP does not [7, 23], and SAP and PTX3, but not CRP, bind mannose-binding lectin (MBL) [24]. The plasma concentrations of C1q (50–200 μg/ml), Factor H (200–600 μg/ml), and MBL (1–3 μg/ml) are relatively constant and are not significantly altered during inflammation [47‐51]. To determine if the above factors affect the response of macrophages to pentraxins, we cultured human PBMC with either M-CSF or GM-CSF for 6 days and then added increasing concentrations of pentraxins in the presence or absence of a single concentration of each pentraxin-binding ligand, and cultured the cells for an additional 2 days. For the cells cultured with M-CSF, neither the pentraxins nor the ligands had any significant effect on the percentage of macrophages expressing CD163 (Fig. 4a-c). 3 to 30 μg/ml SAP, 1 to 300 μg/ml CRP, and 20 to 200 ng/ml PTX3 increased the percentage of cells expressing CD169 (Fig. 4d-f). At 1 and 60 μg/ml SAP, all three ligands increased the percentage of macrophages expressing CD169. In the presence of CRP, the ligands had no significant effect, and in the presence of 20 to 200 ng/ml PTX3, C1q significantly reduced the percentage of macrophages expressing CD169. 10 μg/ml SAP, 30–600 μg/ml CRP (higher concentrations than used for the data in Fig. 3), and 20 to 800 ng/ml PTX3 increased the percentage of cells expressing CD206 (Fig. 4f-i). In the presence of 20 ng/ml PTX3, MBL reduced the percentage of macrophages expressing CD206 (Fig. 4i). These results suggest that for macrophages cultured with M-CSF, pentraxins and the ligands C1q and MBL can modulate the expression of CD169 and CD206.

For the cells cultured with GM-CSF, we found that neither the pentraxins nor the ligands had any significant effect on the percentage of macrophages expressing CD163, apart from CRP at 10 μg/ml (Fig. 5a-c). 1 to 300 μg/ml CRP and 20 to 400 ng/ml PTX3 increased the percentage of cells expressing CD169 (Fig. 5d-f). At 3 to 30 μg/ml SAP, MBL and C1q increased the percentage of macrophages expressing CD169, in the presence of CRP the ligands had no effect, and at 50 to 200 ng/ml PTX3, C1q significantly reduced the percentage of macrophages expressing CD169. 3 and 10 μg/ml SAP and 10 μg/ml CRP increased the percentage of macrophages expressing CD206 (Fig. 5g). These results suggest that for macrophages cultured with GM-CSF, pentraxins and the ligands C1q and MBL can modulate the expression of CD169 and CD206.

Besides cell surface receptors, M1 and M2 primed macrophages also secrete different cytokines, M1 macrophages secrete elevated levels of IL-12, M2a fibrotic macrophages secrete IL-4, and M2c macrophages secrete IL-10 [4, 6]. We collected supernatants from cells cultured in the presence of M-CSF and GM-CSF, and then primed with pentraxins in the presence or absence of ligands, and assayed for IL-4, IL-10, IL-12, and IFN-γ. We only found detectable levels of IL-10 in our culture conditions. As previously described [8], in the presence of M-CSF or GM-CSF, SAP increased IL-10 accumulation (Fig. 6a and d). In M-CSF, but not GM-CSF, 30 to 300 μg/ml CRP increased IL-10 accumulation (Fig. 6b). PTX3 had no significant effect on IL-10 accumulation. With M-CSF and no pentraxins, MBL and C1q increased IL-10 accumulation (Fig. 6a-c). With M-CSF and 100 and 300 μg/ml CRP, Factor H and MBL decreased IL-10 accumulation (Fig. 6b). These results suggest that CRP can affect IL-10 production and that this is reduced by the presence of GM-CSF, Factor H, or MBL.

To determine if the lack of signal for IL-12 was due to the pentraxins not stimulating the cells to secrete IL-12, or a technical issue with ELISA sensitivity, we tested the supernatants from M1- and M2-primed macrophages. When cells were cultured with M-CSF or GM-CSF and then polarized with LPS+ IFN-γ, we could detect high levels of both IL-10 and IL-12 (Additional file 1: Figure S1). These results suggest that the inability to detect IL-12 in cultures of PBMC with pentraxins and/or ligands indicates that these molecules do not generate a signal that induces IL-12 production.

Depending on the health of an individual, monocytes will be exposed to different concentrations of pentraxins in the blood and as they differentiate into macrophages in the tissues. To model these conditions, we cultured PBMC for 6 days to induce monocyte differentiation into macrophages in the presence or absence of pentraxins and ligands. None of the pentraxins had a significant effect on the percentage of macrophages expressing CD163 (Fig. 7a-c). 1 to 30 μg/ml CRP and 20 to 50 ng/ml PTX3 increased the percentage of macrophages expressing CD169 (Fig. 7e-f). 0.3 to 60 μg/ml SAP, 0.1 to 100 μg/ml CRP, and 10 to 400 pg/ml PTX3 increased the percentage of macrophages expressing CD206 (Fig. 7g-i). The ligands did not significantly alter the number of macrophages expressing CD163 (Fig. 7a-c). At 0.3 to 10 μg/ml SAP, MBL and C1q increased the percentage of macrophages expressing CD169 (Fig. 7d). At 20 to 50 ng/ml PTX3, MBL and C1q decreased the percentage of macrophages expressing CD169 (Fig. 7 f), and at 20 to 100 ng/ml PTX3, C1q also decreased the percentage of macrophages expressing CD206 (Fig. 7i). These results suggest that for monocytes differentiating into macrophages, pentraxins and the ligands C1q and MBL can modulate the expression of CD169 and CD206.

To determine if pentraxins and/or their ligands also regulate extracellular cytokine accumulation as monocytes differentiate into macrophages, we collected supernatants from cells cultured in the presence of pentraxins in the presence or absence of ligands, and assayed for IL-4, IL-10, IL-12, and IFN-γ. We only found detectable levels of IL-10 in our culture conditions. As described previously [8], SAP modestly increased IL-10 accumulation (Fig. 8). CRP strongly potentiated IL-10 accumulation, and PTX3 had no significant effect. The addition of ligands did not significantly affect IL-10 accumulation (Fig. 8). These results suggest that for monocytes differentiating into macrophages, CRP can also affect IL-10 production and this is independent of ligands.