1. Loss of CCR2 Expression and Functional Response to Monocyte Chemotactic Protein (MCP-1) During the Differentiation of Human Monocytes: Role of Secreted MCP-1 in the Regulation of the Chemotactic Response
by Laura Fantuzzi, Paola Borghi, Veniero Ciolli, George Pavlakis, Filippo Belardelli, and Sandra Gessani
Blood
Volume 94(3):
August 1, 1999
©1999 by American Society of Hematology

2. Expression of chemokine receptors during monocyte differentiation.
Expression of chemokine receptors during monocyte differentiation. (A) RNA-PCR assay; 1 μg of total RNA extracted from monocytes at different stages of differentiation (day 1, 3, and 7) was retrotranscribed and amplified as described in Materials and Methods. Porphobilinogen deaminase was used as internal control. Results are representative of 4 independent experiments. (B,C) RNase protection assay; 5 μg of total RNA extracted after 1 and 7 days of monocytes culture was hybridized to the hCR5 multiprobe (P) as described in Materials and Methods. Autoradiographs were exposed for 24 hours (B) or 4 days (C) to better visualize the CCR2 mRNA isoforms. Representative results from 6 independent experiments are shown.
Laura Fantuzzi et al. Blood 1999;94:
©1999 by American Society of Hematology

3. Surface expression of CCR2, CCR5, CXCR4, and CD71 in monocytes at different stages of differentiation.
Surface expression of CCR2, CCR5, CXCR4, and CD71 in monocytes at different stages of differentiation. Monocytes (day 0, 1, and 7) were directly or indirectly stained with specific antibodies and analyzed by FACS. The immunofluorescence profile obtained for each antibody was compared with that of its corresponding control and the result is shown as an open curve. The level of expression of each surface antigen is calculated by differences between the level of staining with a specific antibody and the baseline of the control antibody. Similar profiles were obtained with 3 different donors.
Laura Fantuzzi et al. Blood 1999;94:
©1999 by American Society of Hematology

4. Specific binding of 125I-MCP-1 and125I-RANTES to monocytes/macrophages at different stages of differentiation.
Specific binding of 125I-MCP-1 and125I-RANTES to monocytes/macrophages at different stages of differentiation. Cells from 4 different donors (1 × 106) were incubated with 0.25 nmol/L 125I-MCP-1 (A) or 0.1 nmol/L 125I-RANTES (B). After incubation for 2 hours at 4°C, cell pellets were extensively washed and the radioactivity was measured in a γ counter. Specific binding was defined as the differences between total binding and nonspecific binding in the presence of a 200-fold excess of unlabeled chemokines; nonspecific binding never exceeded 20% of total binding. Each point represents the average of duplicate measurements. Statistical analysis showed that the differences in the binding of MCP-1 and RANTES observed in monocytes at different stages of differentiation were statistically significant (1-way analysis of variance P = .009 for MCP-1 and P= for RANTES; Kruskall-Wallis test P = for MCP-1 and P = for RANTES).
Laura Fantuzzi et al. Blood 1999;94:
©1999 by American Society of Hematology

5. Effect of MCP-1 on intracellular [Ca2+]i in differentiating monocytes.
Effect of MCP-1 on intracellular [Ca2+]i in differentiating monocytes. Cells (5 × 106/mL) were incubated with Indo-1 probe (2 μg/mL final) at 37°C for 30 minutes, washed, and exposed in cuvette (1 × 106/mL) to recombinant MCP-1 (10 ng/mL) or RANTES (10 ng/mL) or FMLP (1 × 10−7 mol/L) in the presence of 1 mmol/L extracellular Ca2+. One representative experiment of 3 is shown in (A). (B) Average values obtained in 3 independent experiments with monocytes from different donors.
Laura Fantuzzi et al. Blood 1999;94:
©1999 by American Society of Hematology

6. Secretion of MCP-1 by monocytes/macrophages and its role in the downmodulation of MCP-1–specific binding sites.
Secretion of MCP-1 by monocytes/macrophages and its role in the downmodulation of MCP-1–specific binding sites. (A) Secretion of MCP-1 during monocytes differentiation. MCP-1 was measured by ELISA in the culture supernatants of freshly isolated monocytes (day 1) and 7-day–cultured macrophages. Results obtained with 3 different donors are shown. (B) Effect of antibody to MCP-1 on the specific binding of125I-MCP-1 to differentiating monocytes. Cells from 4 different donors were cultured in the presence (○) or in the absence (•) of polyclonal antibody to MCP-1 (5 μg/mL) for 1 or 7 days, extensively washed to remove the unbound antibody, and processed for binding studies as described in Fig 3. The mean percentage values (±SD) versus day 0 MCP-1 specific binding are shown. Statistical analysis showed that the differences in the binding of MCP-1 in monocytes cultured in the presence of antibody to MCP-1 were statistically significant (2-way analysis of variance P = .0194) with respect to untreated control cultures.
Laura Fantuzzi et al. Blood 1999;94:
©1999 by American Society of Hematology