Monocyte-derived CXCL7 peptides in the marrow microenvironment

Abstract

The marrow microenvironment consists of several different interacting cell types, including hematopoietic-derived monocyte/macrophages and nonhematopoietic-derived stromal cells. Gene-expression profiles of stromal cells and monocytes cultured together differ from those of each population alone. Here, we report that CXCL7 gene expression, previously described as limited to the megakaryocyte lineage, is expressed by monocytes cocultured with stromal cells. CXCL7 gene expression was confirmed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and secretion of protein was detected by enzyme-linked immunosorbent assay (ELISA) and Western blot. At least 2 stromal-derived activities, one yet to be identified, were required for optimal expression of CXCL7 by monocytes. NAP-2, the shortest form of CXCL7 detected in the coculture media, was confirmed to decrease the size and number of CFU-Meg colonies. The propeptide LDGF, previously reported to be mitogenic for fibroblasts, was not secreted by stimulated monocytes. The recombinant form of LDGF produced in a prokaryotic expression system did not have biologic activity in our hands. The monocytic source of CXCL7 was also detected by immunohistochemistry in normal bone marrow biopsies, indicating an in vivo function. We conclude that stromal-stimulated monocytes can serve as an additional source for CXCL7 peptides in the microenvironment and may contribute to the local regulation of megakaryocytopoiesis.

Figure 1.

Gene expression of CXCL7 in response to stromal signals. (A) CD14⁺ cells from 8 different healthy donors were cultured alone or cocultured with HS27a cells for 3 days. Nonadherent cells were harvested, and CXCL7 gene expression was determined by Syber Green real-time PCR and normalized to G3PDH gene expression. Data represent mean ± SE; P = .015 by Student t test. (B) CXCL7 gene expression in CD14⁺ cells from 10 healthy donors at day 0, day 5 in control media (RPMI with 10% FCS), and day 5 in culture in media conditioned by HS5 cells (HS5 CM). Data represent mean ± SE; P < .001 by Student t test comparing HS5 CM to either day 5 control or day 0.

Figure 2.

CXCL7 gene expression and protein secretion in CD14⁺ cells. (A) cDNA was prepared from CD14⁺ cells after 5 days of culture in control media (RPMI with 10% FCS), media conditioned by HS5 cells, media conditioned by primary long-term cultures (LTC CMs), or media conditioned by human foreskin fibroblasts (HFFs). CXCL7 gene expression was measured by Syber Green real-time PCR as in “Materials and methods”. Data represent mean ± SE of 2 experiments. Both HS5 CM and LTC CM differ from control and HFF CM by P = .004 and .003, respectively, using Student t test. (B) To measure the time course of CXCL7 up-regulation, cDNA was prepared from CD14⁺ cells cultured in HS5 CM for up to 8 days. CXCL7 gene expression, measured by real-time PCR, is shown on the y-axis on the right. The media isolated from the same cultures were assayed for CXCL7 protein content using ELISA, shown on the y-axis to the left. (C) Conditioned media obtained from HS5 cultures was fractionated by filtration with a 10-kDa cutoff. CD14⁺ cells were cultured for 3 days in control media, HS5 CM, the smaller than 10-kDa fraction, the larger than 10-kDa fraction, and a combination of both fractions. Results are shown as mean of at least 2 samples ± SE.

Figure 3.

Western blot of CXCL7 peptides. (A) Western analyses of culture media from CD14⁺ and HS27a cells. Culture media were collected, and Western analyses were performed for CXCL7 peptides. Lanes 1 and 2 represent positive controls NAP-2 and PBP, respectively. Culture conditions for the other lanes are as follows: CD14⁺ cells cocultured with HS27a cells for 3 days (lane 3), CD14⁺ cells cocultured with HS27a cells for 6 days (lane 4), CD14⁺ cells cultured alone for 6 days (lane 5), and HS27a cells cultured alone for 6 days (lane 6). At day 3, the CXCL7 peptides secreted are of comparable size to PBP, whereas by day 6, two distinct peptides are detected, PBP and NAP-2. (B) Western analyses of CD14⁺ coculture with HS5 cells. As in panel 3A, lanes 1 and 2 show NAP-2 and PBP. Culture conditions for the rest of the lanes are as follows: CD14⁺ cells cocultured with HS5 cells for 3 days (lane 3); CD14⁺ cells cocultured with HS5 cells for 6 days (lane 4); CD14⁺ cells cultured alone for 6 days (lane 5); and HS5 cells cultured alone for 6 days (lane 6). Here, only the PBP peptide is detected at days 3 and 6 of coculture. (C) CXCL7 peptides secreted by PBMCs in response to LPS stimulation. PBMCs were stimulated by 1 μg/mL LPS for 24 hours. Positive controls are β-TG, recombinant NAP-2, and recombinant PBP (lanes 1, 2, and 7). Conditions for the rest of the lanes are as follows: media from PBMCs cultured with LPS (lane 3), cell extract from PBMCs with LPS (lane 4), media and cell extract from PBMCs cultured without LPS (lanes 5 and 6). PBP is detectable in the culture media stimulated by LPS; both PBP and β-TG are detectable in the cellular extract. No CXCL7 peptides are seen when no LPS is added.

Figure 4.

Immune histochemical analysis of normal bone marrow for CD. Serial sections from B5-fixed bone marrow biopsies from healthy donors were subjected to antigen retrieval and incubated with a monoclonal antibody against CD68 (A). The arrow shows macrophages with long cellular processes staining positive for CD68, whereas the arrowhead shows a smaller positively stained mononuclear cell with no cellular processes (similar to the cell shown in panel F). Panel B shows staining with rabbit polyclonal antibody against CXCL7. The arrow points to a positively stained megakaryocyte, also identifiable by large size and multiple nuclei. The arrowhead shows a smaller mononuclear cell positive for CXCL7. The bound antibodies were detected with the HRP-conjugated Envison Plus system (DakoCytomation). Sections stained with appropriate isotype control antibodies are shown in panel C (monoclonal mouse IgG3 antibody) and panel D (polyclonal rabbit antibody). Panels E and F show double immune histochemical staining for CXCL7 and CD68 in bone marrow biopsies from healthy donors. CD68 (blue) is visualized with the Vectastain ABC-AP kit and Vector Blue, and CXCL7 (red) by EnVision Plus/HRP and NovaRED substrate. As shown in panel E, distinct patterns of staining for both CD68 and CXCL7 are observed as in panels A and B, but a small proportion of mononuclear cells without extensive cellular processes also stain for both of the antigens, suggesting that a subset of bone marrow monocytes also express CXCL7. No counterstain was used for panels E and F. All images were acquired using a Nikon Eclipse E800 microscope (Nikon Instruments, Kanagawa, Japan), Coolsnap CF Color Camera (Photometrics, Tucson, AZ), and Metamorph software (Molecular Devices, Sunnyvale, CA). For panels A-D and F, a 100 ×/1.3 NA objective with oil immersion was used; for panel E, a 60 ×/1.4 NA objective with oil immersion was used. Images were adjusted for brightness and contrast with Adobe Photoshop 7.0 (Adobe, San Jose, CA). Panel F was magnified 3 times after acquisition for better cellular detail.

Figure 5.

Recombinant CXCL7 peptides and their mitogenic properties on fibroblasts. (A) Recombinant CXCL7 peptides LDGF and PBP were prepared and purified as described in “Materials and methods.” Western analysis of recombinant LDGF and PBP along with positive controls β-TG and NAP-2 are shown. (B) Proliferative response of primary marrow-derived fibroblasts to CXCL7 peptides (LDGF, PBP, β-TG, and NAP-2) as well as PDGF was measured using the MTT assay. Results are shown as a percentage of control cultures with no added peptides. Although there was increased proliferation evident with increasing concentration of PDGF, none of the CXCL7 peptides tested stimulated proliferation. Results are shown as mean of 3 samples ± SE.

Figure 6.

CXCL7 peptides and megakaryocyte colony growth. (A) G-CSF–mobilized CD34⁺ cells were cultured in serum-free culture media using the Megac-ult-C kit (Stem Cell Technologies), fixed, stained after 10 to 14 days with anti-CD41 antibodies, and scored for colony number and size (small colonies had 3-20 cells, medium 21-50, and large more than 50 cells), all per the manufacturer's instructions. Results are shown as a percentage of control colonies. Colony numbers were significantly smaller than control colony numbers when NAP-2 was added at concentrations of either 50 ng/mL (P = .010) or 100 ng/mL (P = .018). Colony numbers were not affected by LDGF or β-TG. (B) Percentage of CFU-Meg colonies larger than 20 cells in the same assays as in panel A. NAP-2 significantly decreases the size of the colonies at concentrations of 50 ng/mL and 100 ng/mL (P values of .001 and .006, respectively). For the CFU-Meg assays, G-CSF–mobilized CD34⁺ cells from 4 different donors were plated in duplicate sets. Results shown are mean values ± SE. P values were calculated using Student t test.