Macrophage-Inflammatory Protein-3β/EBI1-Ligand Chemokine/CKβ-11, a CC Chemokine, Is a Chemoattractant with a Specificity for Macrophage Progenitors Among Myeloid Progenitor Cells¹

Movement of hemopoietic stem and progenitor cells (HPC)³ is an important functional process during fetal and adult life. Some cytokines/chemokines show chemotactic activity for HPC (1, 2, 3) and, thus, have the potential to direct movement of HPC to bone marrow (BM) or mobilization from BM to the peripheral blood or focal sites of inflammation.

Chemokines are small peptide molecules of 8 to 12 kDa and have a number of functions such as leukocyte trafficking, angiogenesis, regulation of hemopoiesis, suppression of HIV infection, and antitumor effect (4). Stromal cell-derived factor-1 (SDF-1) is the first chemokine shown to have the capacity to attract CD34⁺ cells and HPC (1). SDF-1, a CXC chemokine, has chemotactic activity for CFU granulocyte macrophage (CFU-GM), burst-forming unit erythrocyte (BFU-E), and CFU granulocyte, erythrocyte, macrophage, and megakaryocyte (GEMM) (1, 3). Mice deficient in SDF-1 had defects in BM hemopoiesis (5). The CC chemokine MIP-3β/EBI1-ligand chemokine/CKβ-11 (hereafter termed MIP-3β) has recently been described as a ligand for the EBI1/BLR2/CCR7 receptor (6). Its mRNA is detected in thymus and secondary lymphoid organs (6, 7). MIP-3β is distantly related to other CC chemokines, forming a separate subfamily of this group (8). The chromosomal location of MIP-3β is different from other CC chemokines in that the gene for MIP-3β is localized on chromosome 9, while most other CC chemokine genes are found on chromosome 17. EBI1/BLR2/CCR7, the only identified receptor for MIP-3β, had been initially identified on EBV-transformed B cell lines and also on T cell lines (9, 10). MIP-3β attracts thymocytes (25) and T and B lymphocytes, but not monocytes or granulocytes (11).

In this study, we report that MIP-3β is another chemoattractant for BM and cord blood (CB) CD34⁺ cells and HPC. However, unlike SDF-1, MIP-3β shows relatively specific chemotactic activity for HPC restricted to macrophage differentiation. We also observed that mRNA expression of this CC chemokine is inducible in BM stromal cells by inflammatory cytokines and LPS. MIP-3β is the first reported chemokine with relatively restricted chemotactic activity for subtypes of BM and CB HPC.

BM and CB CD34⁺ cells were purified as previously described (3). The purity of isolated BM CD34⁺ cells was 93 to 98%, and that of CB CD34⁺ cells was 85 to 95%. For short-term storage, CD34⁺ cells were maintained in X-VIVO 20 medium (BioWhittaker, Walkersville, MD) supplemented with 20% FBS (HyClone, Logan, UT), and were used within 20 h after isolation.

BM stromal cells were derived from low density BM mononuclear cells. Low density BM cells were incubated in X-VIVO 20 medium (BioWhittaker) supplemented with 20% FBS. After 2 to 3 wk, monolayers of BM stromal cells, fibroblast-like cells, were used for RT-PCR analysis of MIP-3β mRNA expression.

MIP-3β was expressed in Chinese hamster ovary (CHO) cells (11). Expression, purification, N-terminal analysis, and matrix-assisted laser desorption ionized (MALDI) mass spectrometry for MIP-3β was conducted, as previously described (11, 12). The MIP-3β expressed from CHO cells used in this study lacked 5′ carboxyl-terminal amino acids from the expected 77-amino-acid protein. Full-length MIP-3β was also expressed in a baculovirus system. Both truncated and full-length MIP-3β were equally active in inducing calcium mobilization in RBL (rat basophilic leukemia) cells that were transfected with CCR7 (EC₅₀ range: 1–4 nM). We consistently used the mammalian cell (CHO cells)-derived MIP-3β in this study.

mAb to human CD34 (clone anti-HPCA-2), conjugated with fluorescent phycoerythrin (PE), was obtained from Becton Dickinson (San Jose, CA). SDF-1 was a kind gift from Dr. Ian Clark-Lewis (University of British Columbia, Vancouver, Canada).

Chemotaxis and chemokinesis were assayed by a two-chamber cell migration system (3). Chambers were incubated at 37°C, 5% CO₂ for 4 to 5 h. Cells completely migrating into the lower chamber were counted using a FACScan (Becton Dickinson, San Jose, CA), with appropriate gating, for 20 s at a high flow rate. For counting only CD34⁺ cells, migrated cells in staining buffer (PBS containing 1% BSA and 0.01% NaN₃) were stained with PE-conjugated anti-CD34 mAb. Isotype-matched mAbs were used to identify negative cell populations. Those cells brighter than isotype-matched Ab-stained cells were counted for 30 s at a high flow rate by FACScan as positive cells.

Methylcellulose colony assay of input or migrated CD34⁺ cells in response to chemoattractants was performed as described previously (3). BFU-E colonies were scored from plates containing recombinant human (rhu) IL-3, rhuGM-CSF, and EPO, and CFU-GM and CFU-GEMM colonies were scored from plates containing rhu IL-3, GM-CSF, EPO, and steel factor (SLF). rhuEPO was purchased from Amgen (Thousand Oaks, CA) and rhu GM-CSF, IL-3, and SLF were kind gifts of Immunex (Seattle, WA).

For some experiments (Fig. 2), rhuM-CSF (final concentration of 1000 U/ml) and rhuG-CSF (100 U/ml), gifts respectively of Chiron (Emeryville, CA) and Immunex, were added to the culture systems in addition to the cytokines described above. For agarose colony assay, washed migrated cells were plated in 35-mm plastic tissue culture dishes (Costar, Cambridge, MA) containing 100 U/ml rhuG-CSF, or 1000 U/ml rhuM-CSF in 0.4% agarose (low melting temperature seaplaque agarose; FMC, Rockland, ME) culture medium containing 10% FBS (13). All results from HPC colony assays were reproduced 3 to 10 times. Cultures were incubated for 14 days at 37°C in a 100% humidified atmosphere of 5% CO₂ at lowered (5%) O₂. For general identification of colony types in granulomonocytic lineages, colonies grown in methylcellulose media were transferred to glass plates and stained with Wright-Giemsa staining (Leukostat; Fisher Scientific, Pittsburgh, PA). For specific identification of macrophage colonies, the transferred cells on glass plates were stained by α-naphthyl acetate esterase and naphthol AS-d-chloroacetate esterase staining kits (Sigma, St. Louis, MO).

Confluent BM stromal cells grown in 10-cm culture dishes were treated with LPS (10 μg/ml; Sigma), rhuIFN-γ (500 U/ml; R&D Systems, Minneapolis, MN), LPS plus IFN-γ, rhuTNF-α (50 ng/ml; R&D Systems), G-CSF (50 ng/ml), or SLF (50 ng/ml) for 14 h at 37°C and 5% CO₂. Total RNAs were isolated from the treated cells with Trizol solution (Life Technologies, Grand Island, NY), according to manufacturer’s instructions. Single-strand cDNA was made from the total RNA with SuperScript Preamplification System for First Strand Synthesis (Life Technologies). Primers used to detect MIP-3β mRNA were 5′-ATG GCC CTG CTA CTG GCC CTC AGC CTG-3′ for a forward primer, and 5′-TTA ACT GCT GCG GCG CTT CAT CTT GGC-3′ for a reverse primer, which give PCR products of about 300 bp. PCR reactions were performed for 40 cycles (94°C, 1 min; 60°C, 1 min; 72°C, 1 min). To verify that the PCR product indeed coded for MIP-3β, the PCR products were gel purified, and DNA sequence was determined by automated fluorescent dideoxy sequencing. Northern blot analysis of MIP-3β expression was performed as previously described (14). For detection of CCR7 mRNA expression, two primers, 5′-GTC ATC ATC CGC ACC CTG CT-3′ (forward primer) and 5′-GTC TCG GCC TCC ACA CTC ATG-3′ (reverse primer), were used. PCR reactions were performed in the presence of 0.1 μl of [³²P]dCTP (Amersham, Arlington Heights, IL; 800 mCi/mM/reaction) for 30 cycles (94°C, 1 min; 58°C, 1 min; 72°C, 1 min). The PCR products were resolved on a 5% nondenaturing polyacrylamide gel. After drying, the filters were analyzed by Molecular Imager (Bio-Rad, Hercules, CA), and exposed on x-ray films.

Student’s t test was used to analyze data for significance. In each experiment, three plates per point were scored per colony assay. p values less than 0.05 were regarded as significant differences.

We examined a number of new CC and CXC chemokines identified from the EST data base of Human Genome Sciences for their chemotactic activities on CD34⁺ cells. Some of them, now known, were CKβ-1/HCC-1 (15), Exodus-1/CKβ-4/MIP-3α/liver and activation-dependent chemokine (LARC) (7, 14, 16, 17), CKβ-6/MPIF-2/Eotaxin-2 (18, 19), CKβ-7/MIP-4 (20), CKβ-8/MPIF-1 (18), CKβ-10/MCP-4 (21), MIP-3β/EBI1-ligand chemokine/CKβ-11 (6, 7), and CKβ-12/IL-10-inducible chemokine (GenBank accession numbers: U91746 and AB007454). MIP-3β was the only chemokine that had significant chemotactic activity for CD34⁺ cells (data not shown). We examined chemotactic activity of MIP-3β for BM CD34⁺ cells (Fig. 1,A). It was observed that BM CD34⁺ cells were attracted to MIP-3β in the lower chamber of the chemotaxis assay system in a dose-dependent fashion. To exclude the possibility that migrated cells were not CD34⁺, but were contaminating CD34⁻ cells, we specifically monitored the migration of CD34⁺ cells by staining the migrated CD34⁺ cells with anti-CD34 mAb and analyzing them by flow cytometry. Maximum migration was observed at MIP-3β concentrations between 200 and 2000 ng/ml (Fig. 1,A). Most of the time, 10 to 15% of input BM CD34⁺ cells were attracted to MIP-3β at 200 ng/ml (Fig. 1,B). SDF-1, at an optimum concentration (200 ng/ml, as determined previously (3)), attracted approximately 50% of input CD34⁺ cells (Fig. 1,B). MIP-3β in a negative gradient showed inhibitory effects on CD34⁺ cell migration induced by a positive gradient of this chemokine (Table I). At 20 ng/ml concentration in the upper chamber, it greatly decreased CD34⁺ cell migration induced by 200 ng/ml MIP-3β in the lower chamber (Table I). Like SDF-1, it showed no significant chemokinetic activity for CD34⁺ cells, which is defined as random movement observed under zero concentration gradient with the same amount of chemoattractants in both chambers (3). Most chemokines show transient actin polymerization in cells. Regulation of actin polymerization by chemoattractants is believed to be important in many biologic processes, including cell movement. We treated BM CD34⁺ cells with MIP-3β and observed that it induced transient actin polymerization in these cells (data not shown).

To examine CCR7 mRNA expression in human CD34⁺HPC, we performed RT-PCR analysis (Fig. 1,C) since it is difficult to obtain enough CD34⁺ cells for more quantitative Northern blot analysis. CCR7 mRNA was detected in CD34⁺ cells responding to MIP-3β as well as in the input CB CD34⁺ cells used for chemotaxis (Fig. 1 C).

We performed colony-forming assays to examine chemotactic activity of MIP-3β on HPC, because not all CD34⁺ cells are HPC and able to form colonies in semisolid culture medium containing appropriate growth and differentiation factors. Another reason for analyzing colony formation was to determine whether MIP-3β had specificity for certain types of HPC. We assayed the migrated CD34⁺ cells in methylcellulose media containing growth factors (GM-CSF, SLF, IL-3, and EPO for assaying CFU-GEMM; GM-CSF, IL-3, and EPO for CFU-GM and BFU-E). Surprisingly, MIP-3β attracted mainly CFU-GM in BM CD34⁺ cells. The chemotactic activity for other BM progenitors such as BFU-E and the more immature CFU-GEMM was weak and not significant (Table II). At high concentrations of MIP-3β (500–2000 ng/ml), a significant chemoattraction of CB BFU-E and CFU-GEMM by MIP-3β was also observed in addition to that seen for CFU-GM (Table II). However, attraction of CB CFU-GM to MIP-3β was greater than that for CB CFU-GEMM and BFU-E. Maximum chemoattraction of BM CFU-GM was observed at MIP-3β concentrations between 200 and 2000 ng/ml. This was in good agreement with the results shown in Figure 1 A for CD34⁺ cells.

SDF-1 attracted BFU-E, CFU-GM, and CFU-GEMM in BM (Fig. 2,A) and CB (Fig. 2,B). In contrast, colonies formed from the BM and CB CD34⁺ cells attracted to MIP-3β were quite homogeneous in morphology, and the size of mature cells in the colonies was quite large (Fig. 2, C and D). To more precisely identify the types of colonies, we used cytochemical staining techniques including Wright-Giemsa or esterase (α-naphthyl acetate esterase and naphthol AS-d-chloroacetate esterase) staining. Most mature cells in the GM-type colonies formed from the CD34⁺ cells migrating in response to MIP-3β were large macrophages with developed cytoplasmic structures, which were positive for α-naphthyl acetate esterase and negative for naphthol AS-d-chloroacetate esterase (Fig. 2, E, F, G, and H). There were no apparent differences in the morphology of mature cells from the migrated CB and BM CD34⁺ cells in response to MIP-3β. Other types of mature cells such as neutrophils were frequently found (e.g., 58% CB granulo/monocytic colonies) in colonies formed from the input CD34⁺ cells, but not found from the CD34⁺ cells attracted to MIP-3β in a greater than background level. This demonstrated specificity of this chemokine for progenitors of the macrophage lineage among HPC of granulo/monocytic lineages.

To further verify the fact that MIP-3β attracted macrophage, but not granulocyte progenitors, we assayed the migrated BM CD34⁺ cells in agarose colony culture assay in the presence of only M-CSF to specifically detect M-CSF-responsive CFU-M, or in the presence of G-CSF to detect G-CSF-responsive CFU-G progenitors (13). M-CSF has been shown to exert differentiation and proliferation effects on cells of the mononuclear phagocyte lineages, and in the presence of M-CSF alone, all colonies formed in agarose/agar media containing 10% heat-inactivated FBS are reported to be α-naphthyl acetate esterase-positive monocyte progenitors (CFU-M) (13, 22). On the other hand, G-CSF alone in agarose/agar medium containing heat-inactivated FCS (10%) stimulates predominantly formation of pure neutrophil granulocyte colonies, which are mostly positive for naphthol AS-d-chloroacetate esterase (13, 23, 24). The BM CD34⁺ cells migrating in response to MIP-3β formed colonies in the presence of M-CSF (Fig. 3). However, in the presence of G-CSF, few colonies formed from the migrated CD34⁺ cells (Fig. 3). In contrast, CD34⁺ cells attracted to SDF-1 formed colonies in both G-CSF-containing and M-CSF-containing agarose culture media. These data support the finding that MIP-3β attracted mainly macrophage, but not granulocyte progenitors, while SDF-1 attracted both types of progenitors.

Northern blot analysis revealed that MIP-3β mRNA is expressed in several lymphoid organs, including thymus, intestines, and lymph nodes, but not BM (6, 7). Activated macrophages have also been reported to express mRNA for MIP-3β (7). We examined the possibility that MIP-3β mRNA might be expressed in the BM environment upon induction. Primary BM stromal cells were derived from BM aspirates. Confluent BM stromal cells, containing mainly fibroblast-like cells, were treated with LPS, IFN-γ, LPS plus IFN-γ, or TNF-α, and analyzed for expression of MIP-3β message. Consistent with previous Northern blot analysis data (6, 7), no MIP-3β mRNA was detected in resting BM stromal cells (Fig. 4). However, either LPS, IFN-γ, or TNF-α alone induced expression of MIP-3β mRNA and IFN-γ increased the LPS-dependent mRNA expression, demonstrating that the BM environment can be induced to express MIP-3β. In contrast, G-CSF and SLF did not induce expression of MIP-3β mRNA. We performed Northern blot analysis to examine the induction of MIP-3β mRNA in a more quantitative way from control cells, cells stimulated with 1, 5, and 25 μg/ml LPS; 5, 20, and 100 ng/ml TNF-α; or 20, 200, and 1000 U/ml IFN-γ. In two of three experiments, no mRNA expression was detected for MIP-3β, even though expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) demonstrated adequate loading of RNA in each of the lanes. In only one of the three experiments did we detect any MIP-3β mRNA expression, and this was only with cells stimulated with 25 μg/ml LPS. Thus, the induction of MIP-3β mRNA by LPS, TNF-α, or IFN-γ was not reproducibly detected by Northern blot analysis, suggesting that the induction of MIP-3β mRNA is low (data not shown).

The results herein report the first evidence that there is a chemokine specific for attraction of subtypes of HPC. This contrasts with SDF-1, a broad range chemoattractant for most types of HPC tested. MIP-3β is also the only CC chemokine to date demonstrating chemotactic activity for CD34⁺ cells. In addition to a number of newly identified CC chemokines, including CKβ-1/HCC-1 (15), Exodus-1/CKβ-4/MIP-3α/LARC (7, 14, 16, 17), CKβ-6/MPIF-2/Eotaxin-2 (18, 19), CKβ-7/MIP-4 (20), CKβ-8/MPIF-1 (18), CKβ-10/MCP-4 (21), and CKβ-12/IL-10-inducible chemokine (GenBank accession numbers: U91746 and AB007454) that lacked chemotactic activity for CD34⁺ cells, it has been reported that MIP-1α, MIP-1β, IL-8, MCP-1, RANTES, and eotaxin were also devoid in chemotactic activity for CD34⁺ cells (1). Thus, MIP-3β has a unique and rare chemotactic activity among CC chemokines. Chemoattraction by MIP-3β is highly specific to subtypes of myeloid progenitors in BM and CB CD34⁺ cells. The mechanisms underlying differences in chemotactic specificity between SDF-1 and MIP-3β are not known. The broad chemotactic spectrum of SDF-1 for not only CFU-GEMM, BFU-E, and CFU-GM, but also CFU-G and CFU-M, suggests the ubiquitous expression of CXCR4, the receptor for SDF-1, on these HPC. We speculate that distribution of EBI1/BLR2/CCR7, the receptor for MIP-3β, might be more restricted to subsets of HPC such as CFU-M. Unfortunately, until one can phenotypically define subsets of myeloid progenitors such as CFU-GEMM, BFU-E, CFU-GM, CFU-G, and CFU-M by a means that clearly distinguishes one progenitor type from another, it will be difficult to determine selective receptor distribution on these different progenitor cell types.

MIP-3β is believed to be either present at a very low level or not present under normal conditions in BM, because no MIP-3β mRNA is detected by RT-PCR and Northern blot analysis in BM. We have now shown that LPS, IFN-γ, or TNF-α induces mRNA expression of MIP-3β in BM stromal cells. Thus, it is reasonable to speculate that MIP-3β may influence trafficking of CFU-M to/from BM under inflammatory conditions.

We thank Drs. Robert Hromas and David Leibowitz (Indiana University) for their help in obtaining bone marrow samples. Thanks are also given to the following from SmithKline Beecham: Edward Appelbaum, Kyoung Johanson, and Donna Cusimono for vector construction, Joyce Mao and Stephanie Van Horn for oligonucleotide synthesis and deoxyribonucleic acid sequencing, Laura Grayson for Chinese hamster ovary cell transfection and growth, Gil Scott for purification of macrophage-inflammatory protein-3β, and Dean McNulty for analytic characterization of macrophage-inflammatory protein-3β. The assistance of Rebecca Miller and Cindy Booth (Indiana University) in preparation of this manuscript is appreciated.