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(Journal of Leukocyte Biology. 2000;68:757-764.)
© 2000 by Society for Leukocyte Biology

Selective stimulation of G-CSF gene expression in macrophages by a stimulatory monoclonal antibody as detected by a luciferase reporter gene assay

Yoshiko Aoki, Shiken Sha, Hidehito Mukai and Yoshisuke Nishi

Laboratory of Life Science & Biomolecular Engineering, Japan Tobacco Inc., Yokohama, Kanagawa, Japan

Correspondence: Yoshisuke Nishi, Ph.D., Director, Laboratory of Life Science & Biomolecular Engineering, Japan Tobacco, Inc., 6-2 Umegaoka, Aoba-ku, Yokohama, Kanagawa 227-8512, Japan. E-mail: yoshisuke.nishi{at}ims.jti.co.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have identified a stimulatory monoclonal antibody (mAb) from autoimmune mice that selectively stimulates granulocyte colony-stimulating factor (G-CSF) gene expression in a mouse macrophage cell line. The induction was observed not only in the cell line, but also in normal peritoneal macrophages. This mAb bound to the monocyte/macrophage cell lines and pre-B leukemia cell lines, but also in normal peritoneal macrophages, whereas it did not bind to normal T and B cells in the spleen or fibroblastic cell lines. It could even bind to a human promyelocytic leukemia cell line, when they were differentiated into monocytic cells. On Western blotting, this mAb mainly recognized an approximately 30-kDa band and it was unique because there have been no reports of membrane-associated proteins with a similar molecular mass found in macrophages. These results suggest that there could be a specific gateway molecule to induce G-CSF in macrophages.

Key Words: RAW264.7 • peritoneal exudate macrophages


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Granulocyte colony-stimulating factor (G-CSF) is one of cytokines that stimulate and activate segmented neutrophils in bone marrow and in the periphery. G-CSF is known to be produced mainly by monocytes/macrophages, and also by T lymphocytes and endothelial and stromal cells [1 2 3 ]. In animal models of chemotherapy and radiotherapy, G-CSF accelerates the recovery of neutrophils [4 5 6 ]. Administration of recombinant human G-CSF has shown marked efficacy in recovering neutrophils in cancer patients suffering from neutrophil agranulocytosis due to chemotherapy or radiotherapy, and in aplastic anemia patients after bone marrow transplantation [3 , 7 ]. G-CSF has an advantage over other cytokines for therapeutic use because it has a selective activity to proliferate only neutrophils.

In stromal cells, fibroblasts, and endothelial cells, G-CSF is stimulated by several soluble factors including lipopolysaccharide (LPS) [8 ] and cytokines, such as interleukin (IL)-1 [9 ], IL-17 [10 ], tumor necrosis factor (TNF) [11 ], and oncostatin M [12 ], and by cellular contact with other cells [13 , 14 ]. G-CSF is induced in monocytes/macrophages by soluble factors, such as LPS [15 , 16 ], IL-1 [17 ], IL-3 [18 ], IL-4 [19 ], M-CSF [20 ], interferon-{gamma} (IFN-{gamma}) [21], and also cell-to-matrix interactions [22 ]. We previously showed that G-CSF was induced in the mouse macrophage cell line RAW264.7 by contact with extracellular matrix molecules, such as fibronectin (FN), vitronectin (VN), and laminin, and one of the RGD peptides, which are recognized by FN and VN receptors, i.e., integrins [23 ]. FN-mediated G-CSF induction was blocked by adding neutralizing antibodies (Abs) against integrin molecules to the medium. In addition, contact of this macrophage cell line with a pre-B leukemia cell line, NFS-60, induced G-CSF in a cell number-dependent fashion. These results suggested that there could be one or more cell-mediated signal pathways through which G-CSF is induced in macrophage cell lines.

During the course of our study, Fine et al. identified a small-molecular-weight compound that selectively induced G-CSF in human peripheral mononuclear cells, but it did not induce other cytokines or colony-stimulating factors (CSFs) with hemato-regulatory activity [24 ]. Their results raised the possibility of a signal pathway specific for G-CSF induction in macrophages. These results and our own results inspired us to determine whether there are specific gateway molecules that selectively transmit intracellular signals to induce G-CSF in macrophages. Most of our efforts have focused on the production of Abs that stimulate G-CSF gene expression. Some mAbs have been reported to support or inhibit hematopoiesis through interactions with CD18, VLA-4, or other unknown antigens [25 26 27 28 29 ]. In contrast, no mAbs have been reported to induce cytokine production in macrophages. In the present work, we found a cell membrane-associated gateway molecule that is uniquely recognized by a stimulatory antibody through which expression of G-CSF gene was triggered in a macrophage cell line. In this study we used a sensitive assay system using luciferase as a reporter for G-CSF and an autoimmune mouse as the source of monoclonal antibodies (mAbs).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and cells
For immunization, 8- to 12-week-old inbred female MRL/MPJ-lpr/lpr mice were purchased from Charles River (Atsugi, Japan). For preparation of normal peritoneal exudate cells from the peritoneal cavity, we used 7- to 8-week-old outbred male ICR mice and inbred male C57BL/6 mice (both from Charles River).

For reverse transcriptase-polymerase chain reaction (RT-PCR), we used outbred male ICR mice because they gave a higher yield of peritoneal exudate cells. For a cytofluorometric analysis, we used inbred male C57BL/6 mice, because this strain is a type B haplotype, and the secondary antibody we used was against IgMa, which is cross-reactive with MRL/MPJ-lpr/lpr haplotype and not cross-reactive with surface IgM on splenic B cells from C57BL/6 mice. The hypoxanthine-aminopterin-thymidine (HAT)-selective nonsecretory murine myeloma cell line PAI, which is used for making hybridomas, was obtained from the Japanese Collection of Research Bioresources (Tokyo). It was maintained in ASF104 medium (Ajinomoto, Tokyo) supplemented with 10% fetal bovine serum (FBS, Bio-Whittaker, Walkersville, MD), 100 U/mL penicillin, and 100 µg/mL streptomycin (GIBCO-BRL, Rockville, MD). The mouse macrophage cell line RAW264.7 obtained from the American Type Culture Collection (Manassas, VA) was maintained in Eagle’s minimal essential medium (EMEM; GIBCO) supplemented with 10% FBS plus nonessential amino acids (GIBCO) at 37°C under 5% CO2 in humidified air.

Reagents and treatment
Mouse FN and VN were obtained from GIBCO. LPS from Escherichia coli 026:B6 was from Sigma (St. Louis, MO). For the cytofluorometric analysis, we used phycoerythrin (PE)-anti-mouse IgMa, fluorescein isothiocyanate (FITC)-anti-CD14, FITC-anti-CD45, and FITC-anti-CD3 obtained from PharMingen (San Diego, CA) and a cell sorter EPICS ALTRA (Beckman-Coulter, Fullerton, CA). For detection of induced levels of TNF-{alpha}, IL-1{alpha}, IL-1ß, and IL-6, cytokine kits from Endogen (Cambridge, MA) were used. Treatments were done in 96-well microtiter plates (Falcon, Oxnard, CA). For treatment with extracellular matrix (ECM) proteins, substratum-coated plates were prepared by incubating the plates with ECM proteins dissolved in phosphate-buffered saline (PBS) at room temperature for 2 h followed by rinsing twice with PBS. For treatment with LPS, it was dissolved in PBS.

Immunization, preparation of hybridomas, and antibody purification
MRL/MPJ-lpr/lpr mice were injected intraperitoneally with RAW264.7 cells at 2 x 107 cells/mouse in 0.5 mL of PBS and boosted twice with an equal number of the cells at 14-day intervals. Three days after the final boost, spleens were excised and splenocytes were harvested. The splenocytes were fused with the PAI cells following the standard protocol [30 ]. Briefly, fusions were performed using 50% polyethylene glycol 1500 (w/v) in 75 mM HEPES (pH8.0) (Roche Diagnostics, Mannheim, Germany) at a spleen cell-to-myeloma cell ratio of 8:1. Hybrids were plated in 96-well microtiter plates at a concentration of 105 viable cells/well in ASF104 medium supplemented with HAT (Roche Diagnostics). The medium was replaced every 3–4 days and aminopterin was removed after 21 days. Hybridomas were then subcloned by limiting dilution in the culture medium containing hypoxanthine and thymidine. For preparation of the antibodies, the cloned hybridoma cells at 2 x 108 cells were cultured in 500 mL serum-free ASF104 medium for 5 days. The mAbs were purified from the culture supernatants by the euglobulin precipitation method [31 ]. The mAbs were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Samples with >95% purity were used for further analyses.

ELISA assays
RAW264.7 cells were used to determine antibody binding to cell surfaces. The cells were plated at 5 x 104 cells/well on flat-bottomed 96-well microtiter plates (Falcon) and cultured overnight. Supernatants from the hybridomas were added, and then incubated for 60 min at room temperature. The plates were washed twice with PBS and horseradish peroxidase (HRP)-anti-mouse Igs (Zymed Laboratories, South San Francisco, CA) were added and incubated for 30 min. Peroxidase was detected by adding 2,2’-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) and read on a spectrophotometer (M-Tmax, Molecular Dynamics, Uppsala, Sweden) at 405 nm. In some cases, the RAW264.7 cells were fixed with 0.25% glutaraldehyde for 5 min and washed thoroughly with PBS. Nonreactive sites were then blocked with 1.0% gelatin. Plates were washed and blocked with 1.0% gelatin again. In other cases, the RAW264.7 cells were washed twice with PBS, lysed with buffer containing 0.5% Igepal CA-630 [an equivalent product for Nonidet P-40, (octylphenoxy)-polyethoxyethanol, Sigma] and 0.1% EDTA at 4°C for 1 h, and centrifuged at 14,000 g. The supernatant was then used to coat flat-bottomed 96-well microtiter plates at 5 µg protein/well.

Measurements of G-CSF induction using a luciferase reporter gene
G-CSF activity was measured quantitatively using the cloned RAW264.7 cell line with the mouse G-CSF promoter gene linked to the luciferase gene as a reporter [23 ]. As a promoter of G-CSF gene, we used a 1.7-kb upstream region from the translation-initiation point of G-CSF gene [32 ]. This sequence was screened from a mouse genomic library, digested with XhoI and NcoI and inserted into a multiple cloning site of Picagene Enhancer Vecter 2 (Wako, Osaka, Japan). Then, neomycin-resistant gene was cut off from the pMC1NeoPolyA 3854 (Stratagene, La Jolla, CA) at restriction sites of XhoI and SalI and inserted into the SalI site, which is located just downstream from an SV40 enhancer of Picagene Enhancer Vector 2 with the G-CSF promoter region. RAW264.7 cells were electroporated with the plasmid DNA. One of the stable transformants that showed highest response to LPS was isolated and used for the following experiments.

Cells were cultured in growth medium containing mAbs or other stimulants as indicated. The assays were carried out using the Picagene system (Wako), and a CT9000 luminometer (Dia-Iatron, Tokyo).

RT-PCR analysis of G-CSF gene expression in peritoneal macrophages
The cells at 1.5 x 105 cells/mL were cultured for 18 h either in the presence (10–40 µg/mL) or absence of 3-4H7 mAb, and washed with PBS. Total RNA was extracted with acid guanidium thiocyanate and phenol/chloroform as described previously [33 ]. Detection of G-CSF mRNA contents was performed by the quantitative PCR method [34 ]. Briefly, 1 µg of each total RNA was allowed to form first-strand cDNA in a 33-µL reaction mixture using oligo(dT)12–15 as a primer and a first-strand cDNA synthesis kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). From the reaction mixture, for PCR of ß-actin, 1 µL of cDNA solution was used directly for PCR, whereas for PCR of G-CSF, 5 µL was used. The reaction mixture contained 10 pmol of specific primers for ß-actin or 20 pmol for G-CSF, a final concentration of 200 µM of dNTP, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 1 mg/mL gelatin, and 1 unit Taq DNA polymerase (Takara, Kyoto) in a final volume of 50 µL. PCR was performed for 30 cycles under the following conditions: 1 min at 94°C for denaturation, 1 min at 55°C for annealing, and 1 min at 72°C for extension with a Mastercycler gradient thermal cycler (Eppendorf, Hamburg, Germany). Ten µL of amplified cDNA products were electrophoresed in a 2% (w/v) agarose gel (Takara) and stained with ethidium bromide. We performed more than three independent experiments to confirm the reproducibility. Primers of G-CSF were synthesized, and the sequences of the primers were as follows: G-CSF sense, 5’-GCTGTGGCAAAGTGCACT-3’ (position 121–138); G-CSF antisense, 5’-ATCTGCTGCCAGATGGTG-3’ (position 520–537) [35 ]. Primers for ß-actin were purchased from Clontech (Palo Alto, CA).

Flow cytometric analysis
The antigens were cytofluorometrically detected. After washing with PBS, 1 x 106 cells were treated on ice for 30 min with 50 µL of PBS, 1% FBS, 0.02% NaN3 (PBS/FBS/NaN3 buffer) containing 40 µg/mL of 3-4H7 mAb or 40 µg/mL of purified mouse IgM as a negative control. The cells were then treated for 30 min with 50 µL of PBS/FBS/NaN3 buffer containing 20 µg/mL of affinity-purified PE mouse anti-mouse IgMa (PharMingen). After washing with PBS/FBS/NaN3 buffer at 4°C, cells were fixed with 3.7% formaldehyde in PBS. For analysis of primary cells, cells were stained with both 20 µg/mL of FITC anti-lineage marker (CD3, CD14, and B220) mAb and with 40 µg/mL of 3-4H7 mAb, or with both FITC anti-lineage marker mAbs and the negative isotype control mAb for 30 min on ice. These cells were then stained with 20 µg/mL of PE-anti-mouse IgMa for 20 min. After washing with PBS/FBS/NaN3 buffer at 4°C, the stained cells were fixed with 100 µL of 3.7% formaldehyde in PBS, and washed again with PBS/FBS/NaN3 buffer.

Using an EPICS ALTRA flow cytometer and EXPO 2 software (Beckman-Coulter), flow cytometric analysis was performed. Based on the forward and side scatter profiles, a gate range was set around the whole cell population, and 20,000 events in this gate range were acquired for each sample. For the cell lines, the antigens were detected by the cell-surface PE anti-mouse IgMa. For the primary cells, a two-color analysis was performed. Positive cell populations detected by FITC anti-lineage marker mAbs were further analyzed by the cell-surface PE anti-mouse IgMa. Macrophages were identified by their positive staining for FITC-conjugated CD14 monitored in FL1 (log scale), B cells were identified by B220+ and T cells were identified by CD3+. Compensation was applied to correct for FITC emissions entering the FL2 channel and for PE emissions entering the FL1 channel.

Immunoblotting
For Western blotting, total cell lysates or plasma membrane fractions of the RAW264.7 cells (5 µg protein/lane) were subjected to 10% PAGE in the presence of SDS. After electrophoresis, the proteins were transferred to a polyvinylidene difluoride (PVDF, ProBlottTM, PE Biosystems, Foster City, CA) sheet. The PVDF sheet was then pretreated with BlockAce (SnowBrand, Tokyo) for 1 h, washed by PBS containing 0.1% Tween 20 for 20 min, and reacted with 20 µg/mL of the purified 3-4H7 mAb or control IgM at room temperature. The reaction was detected by an alkaline phosphatase pNPP kit (BIO-RAD Laboratories, Hercules, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of mAbs that stimulate G-CSF gene expression
We produced mAbs against the target cell surface and then tested their ability to stimulate G-CSF gene expression. The macrophage cell line we are interested in and the mouse strain conventionally used for immunization are isogenic, so using the normal BALB/c mouse strain would not raise an immune response against the antigens. Therefore, we used the autoimmune mouse (MRL/MPJ-lpr/lpr) and the normal rat (WKY) for hybridoma preparations. Immunization with whole cells from the macrophage cell line RAW264.7 resulted in increases in the titers against both strains, but it failed to boost the titer against the normal BALB/c mice, as expected (data not shown). As a result, we were able to obtain hybridomas from both MRL/MPJ-lpr/lpr mice and WKY rats. More than 6000 wells were screened for 7 MRL/MPJ-lpr/lpr mice and more than 5000 wells were screened for 4 WKY rats (Table 1 ). Of the approximately 12,000 wells examined, a total of more than 400 wells reacted positively to the RAW264.7 cells by ELISA. We cloned monoclonal hybridomas from 58 wells that showed strong responses. mAbs were purified from these hybridomas and their ability to stimulate G-CSF gene expression in RAW264.7 cells was examined with the reporter gene assay using luciferase. Seven mAbs from the MRL/MPJ-lpr/lpr mice (three IgG and four IgM) were found to increase the expression of G-CSF gene by factors of 6 to 40, but none from the WKY rats induced G-CSF gene in the RAW264.7 cells. The mAb that showed the highest stimulatory activity, 3-4H7, was chosen for further detailed examination.


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  		Table 1. Summary of the Hybridoma Screenings

 
Dose-dependency and time-course of mAb-induced G-CSF gene expression
Expression of G-CSF gene in RAW264.7 by the purified 3-4H7 mAb was examined at 18 h after addition of the mAb to the culture medium. Dose-dependent stimulation of G-CSF gene expression was observed at mAb concentrations ranging from 2.0 to 60 µg/mL. Thirty- to sixty-fold increases in expression occurred at concentrations in the range 30–60 µg/mL (Fig. 1A ). Under identical conditions, LPS, a potent stimulatory factor for cytokines, at 100 ng/mL resulted in a 200- to 300-fold increase in expression (Fig. 1B) . The time-course of induction was examined using 3-4H7 mAb at a concentration of 40 µg/mL. G-CSF gene expression by 3-4H7 mAb was initially observed at 2 h after the addition of mAb, rapidly increased up to 8 h, and then decreased in the following 16-h period (Fig. 2 ). Under identical conditions, LPS at 100 ng/mL was more effective in inducing G-CSF gene than 3-4H7 mAb, while its maximum stimulation was observed at 12 h, some hours after the maximum stimulation of 3-4H7 mAb.



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  		Figure 1. Effect of various concentrations of 3-4H7 mAb or LPS on induction of G-CSF gene expression in RAW264.7 cells. RAW264.7 cl.27-3 cells were cultured with varying concentrations of 3-4H7 mAb (A) or LPS (B) for 18 h. After the treatments, the cells were lysed and assayed for luciferase activity. Data are shown as a mean of four experiments ± SE.

 


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  		Figure 2. Time-course of G-CSF gene expression by 3-4H7 mAb or LPS. RAW264.7 cl.27-3 cells were cultured for 0, 2, 4, 8, 12, and 24 h with 40 µg/mL of 3-4H7 mAb or 100 ng/mL of LPS. Luciferase activity was measured as described in Materials and Methods. Luciferase activity was expressed as described in Figure 1 . Data are expressed as described in Figure 1 and shown as a mean of three experiments ± SE.

 
Selective stimulation of G-CSF gene expression by mAb
The ability of the 3-4H7 mAb to stimulate G-CSF gene and other cytokines in RAW264.7 cells is shown in Figure 3A . The mAb stimulated G-CSF at least 10 times more than it stimulated four other cytokines (IL-1{alpha}, IL-1ß, IL-6, and TNF-{alpha}?. In contrast, LPS (Fig. 3B) was much more effective than 3-4H7 in stimulating the four cytokines. LPS has its greatest stimulatory effect on IL-6, and decreasing stimulatory effects in the order TNF-{alpha}, G-CSF, IL-1{alpha}, and IL-1ß. The adhesion molecule FN (Fig. 3C) stimulated G-CSF gene expression about twice as much as the 3-4H7 mAb, but it also stimulated TNF-{alpha} and IL-6 about as much as one-half to one-third of that of G-CSF. The adhesion molecule VN (Fig. 3D) was about as effective as FN in stimulating G-CSF, but it also stimulated TNF-{alpha} and IL-6 much more than did FN. Direct contact between RAW264.7 cells and cells of the pre-B leukemia cell line NFS-60 enhanced G-CSF gene expression (Fig. 3E) , but the NFS-60 cells stimulated the expression of IL-6 as well, although to a lesser extent. These figures thus demonstrate that the specific stimulation of G-CSF gene expression by the 3-4H7 mAb was unique compared with the stimulations by the other stimulants.



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  		Figure 3. Stimulation of G-CSF gene expression and other inflammatory cytokines by 3-4H7 mAb and other stimulants. Cells (5 x 104) were cultured for 18 h at 37°C in wells of 96-well microtiter plates after stimulation with 40 µg/mL of 3-4H7 mAb (A), 100 ng/mL of LPS (B), or the cells were cultured for 18 h in wells previously coated with FN (C) at 3 µg/cm2 or VN (D) at 1 µg/cm2, respectively. The cells were co-cultured with 3 x 105 NFS-60 cells in a well (E). After the treatments, the cells were lysed and assayed for luciferase activity. The concentrations of TNF-{alpha}, IL-1{alpha}, IL-1ß, and IL-6 in the medium were determined by ELISA. Data are shown as a mean of three experiments ± SE for 3-4H7 mAb and LPS, and shown as a mean of three wells from a single experiment for FN, VN, and NFS-60 cells.

 
mAb 3-4H7 stimulates expression of G-CSF mRNA in normal peritoneal macrophages
The mAb clearly triggered G-CSF mRNA expression in peritoneal macrophages from normal mice (Fig. 4 ). In addition, this effect was concentration-dependent for mAb concentrations in the range 10–40 µg/mL, which is consistent with the G-CSF gene expression observed using the luciferase reporter assay. These results show that the specificity of the mAb for G-CSF induction was not due to the choice of cell lines.



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  		Figure 4. RT-PCR analysis of G-CSF gene expression in primary peritoneal macrophages. Quantitative RT-PCR was performed as described in Materials and Methods. The size of the G-CSF transcripts measured was approximately 416 bp.

 
Flow cytometric detection of antigen(s) of the 3-4H7 mAb
A flow cytometric analysis was performed to determine whether the antigen(s) recognized by the 3-4H7 mAb is also unique to the RAW264.7 macrophage cell line or a property of all normal monocytes/macrophages, or found in other types of cells. We tested several murine cell lines, including monocytic/macrophage-like cell lines, myelocytic leukemia cell lines, and fibroblastic cell lines. In addition, we tested five human cell lines to determine whether they show cross-reactivity with this mAb. We also tested normal mouse peritoneal macrophages, splenocytic T and B cells, and bone marrow B cells. The results are summarized in Table 2 and Fig. 5A B C D E . These results demonstrated that the antigen(s) was found on the cell surface of not only the RAW264.7 cells but also other cell lines, although at weaker intensities. Positive results were obtained from the monocytes/macrophage-like cells and some undifferentiated promyelocytic leukemia cell lines, although none of the fibroblastic cell lines were positive for the antigen(s) recognized by this mAb. It is interesting to note that human promyelocytic leukemia (HL-60) cells were negative to this mAb when they were in an undifferentiated state or when they differentiated into granulocytic cells by N6,2'-o-dibutryryladenosine 3':5'-cyclic monophosphate (dbcAMP), but they were positive when they differentiated into monocytic cells by phorbol 12-myristate 13-acetate (PMA). Among the normal hematopoietic cells obtained from normal mice, only peritoneal macrophages reacted positively with the 3-4H7 mAb, whereas bone marrow B, spleen T, and spleen B cells were completely negative (Fig. 5A 5B 5C 5D 5E) .


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  		Table 2. Flow Cytometric Analysis of the Distribution of Antigen(s) Recognized by the 3-4H7 mAb Among Various Cell Types

 


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  		Figure 5. FACS analysis of RAW264.7 cells and normal hematopoietic cells stained with 3-4H7 mAb. RAW 264.7 cells (A), and the macrophages from the peritoneal exudate (B), bone marrow B cells (C), splenic T cells (D), and splenic B cells (E) freshly isolated from the C57BL/6 mice were stained with 40 µg/mL of the 3-4H7 mAb and PE-conjugated anti-mouse IgMa and analyzed on an EPICS ALTRA flow cytometer (solid lines). Controls were stained with anti-TNP IgM and PE-conjugated anti-mouse IgMa (dashed lines). Histograms show the intensity of the fluorescence on a logarithmic scale (x-axis) and the relative cell number (y-axis), respectively.

 
Detection of antigen(s) by Western blotting
The above evidence strongly suggested that the antigen(s) recognized by this mAb reside on the cell membrane. To confirm this, we performed Western blotting against the whole-cell lysates of the RAW264.7 cells. A single band was identified at about 30 kDa in the Western blot with the 3-4H7 mAb, whereas no signals were found in the blot with the control mAb (Fig. 6 ). This band must correspond to an antigen that specifically bound to the mAb 3-4H7.



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  		Figure 6. Western blotting of antigen(s) on the RAW264.7 cells recognized by the 3-4H7 mAb. Lane 1, the gel was stained with Coomassie brilliant blue; lane 2, Western blotting with the 3-4H7 mAb; lane 3, Western blotting with the control IgM. Molecular weight standards are shown on the left.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrate that a stimulatory mAb (3-4H7) selectively potentiates G-CSF gene expression in a mouse macrophage cell line, i.e., it does not measurably induce other inflammatory cytokines, such as IL-1{alpha}, IL-1ß, IL-6, and TNF-{alpha}. Our success in obtaining these mAbs was due to two factors. One was to set up a reliable reporter gene assay system for quantitatively detecting G-CSF gene expression [23 ]. For this we used a cell line cloned from a macrophage cell line in which expression of G-CSF gene is especially sensitive to various stimuli, such as LPS. There have been no practical methods like ELISA to detect murine G-CSF because antibodies against murine G-CSF suitable for use in ELISA are not yet available. Therefore, as an alternative assay, a reporter gene assay was developed by introducing a fusion gene into three macrophage cell lines. The fusion gene has a promoter region from a mouse G-CSF gene juxtaposed to a luciferase gene at a 3’ downstream of the promoter [23 ]. Among the macrophage cell lines, the cell line RAW264.7 was found to be the best because it showed the lowest background and the highest signal of induced levels of G-CSF gene by several stimulants [23 ]. The other factor was the use of an autoimmune mouse strain for immunizations. We first immunized the commonly used BALB/c mice with this cell line, but the immunization failed to boost the titer in these mice. As a second choice, we used a rat strain (WKY) for immunization, hoping that a different species would show a greater immune reaction. Immunization of the rats with the cells successfully boosted the titer, but unfortunately, no stimulatory antibodies were recovered. As another alternative, we tried using an autoimmune mouse strain. We chose the MRL/MPJ-lpr/lpr strain only because we had previously succeeded in isolating catalytic antibodies from this strain in much greater quantities than we did with the conventionally used strain [36 ]. As we found in this study, this strain was also a good one for obtaining stimulatory antibodies.

In this study, we carefully checked, and ruled out, the possibility that trace contaminants in the medium were responsible for the expression of G-CSF gene. First of all, in order to minimize the possibility that trace elements in the serum were responsible for the expression of G-CSF gene, all antibody samples used for the assays were carefully isolated from the hybridomas by cultivating them in medium completely free of bovine serum. In our previous experiments, we found that LPS was a potent inducer of G-CSF gene expression at the nanogram per milliliter level [23 ], so contaminants like endotoxins may affect the results even at a trace level. Soluble factors such as cytokines also may trigger G-CSF gene expression even at a trace level. Trace levels of the cytokines IL-4, M-CSF, and oncostatin M might be important because low levels of these cytokines were found to induce G-CSF in monocytes/macrophages [13 , 20 , 21 , 32 ]. To evaluate these possibilities, we examined how the expression of G-CSF gene depends on the concentration of the mAb, and determined whether there is difference in the time-course patterns among the stimulatory factors. The induction was dependent on the concentration of the mAb. The kinetics of induction by this mAb were different from the kinetics of induction by LPS in that the time of the maximum induction was earlier for the mAb than for LPS. In addition, we found that induction of G-CSF gene expression by LPS was not suppressed by the addition of the 3-4H7 mAb (data not shown). We also found that M-CSF was not responsible for the induction (data not shown), although we have not yet tested other stimulants. The present data strongly suggested that the stimulatory effect was due to the mAb, and not to residual amounts of other contaminants. The 3-4H7 mAb did not stimulate the luciferase activity to the RAW264.7 cells, which were transfected with just a Picagene Enhancer Vector 2, but without the promoter region of G-CSF. In general, antibodies do not penetrate into the cells. Taken together, it was quite unlikely that the 3-4H7 mAb nonspecifically activated the luciferase. In addition, we have directly tested the trace amount of LPS in the 3-4H7 mAb samples through the use of an LPS-detecting kit (Seikagaku Kogyo, Tokyo). It was 7.23 pg/µg of the 3-4H7 mAb protein in the testing sample. So, the 3-4H7 mAb sample of 60 µg/mL should contain 432 pg/mL. As shown in Figure 1 , the induction of luciferase at 1 ng/mL of LPS over the control was 6.26 ± 5.91. So the luciferase induction by LPS could be about a half of 6.26, whereas at this concentration induction of the luciferase was 57.8 ± 17.1. Therefore, contribution of LPS in the sample for luciferase induction could be around 5% at most. We thought that this level of contamination affected our results little, if at all.

An important finding of this study was that this mAb was selective in the induction of the cytokines. It induced G-CSF gene, but not other inflammatory cytokines, such as IL-1{alpha}, IL-1ß, IL-6, and TNF-{alpha}. This result was quite different from the results obtained by ECM proteins that are known to induce G-CSF gene expression [22 ]. None of them selectively induced G-CSF gene expression, but they did show rather pleiotropic potency toward the induction of inflammatory cytokines. Based on these observations, we speculated that the signal pathway stimulated by the mAb was selective and not shared by the pathways stimulated by these other stimulants.

We then attempted to determine whether this phenomenon was specific to RAW264.7 cells or a relatively common property of macrophages. None of the methods for detecting G-CSF gene expression is better than the reporter gene assay, and only quantitative RT-PCR permitted us to measure G-CSF gene expression in normal tissues. We found that the mAb was also effective in stimulating G-CSF gene expression in the primary macrophages of the peritoneal exudate cells from the normal mice. This result suggested that a stimulatory signal for G-CSF gene expression via the mAb to the monocytic/macrophage lineage was more common than we expected. The signal or signals mediated by the mAb were blocked by Herbimycin A (data not shown). So, the tyrosine kinase cascade seems to be involved in induction of G-CSF gene expression. Whether these are novel molecules or molecules that have been previously described was of particular interest to us. Therefore, we tried to identify the molecule(s) through the use of flow-cytometric analysis. The results of this analysis demonstrated that this mAb was bound to the surface of RAW264.7 cells. Antigens were also found to be distributed in monocytic/macrophage cell lines, and some lymphoblastic and/or myeloblastic cell lines as well. An important finding was that the molecule(s) was distributed in the normal peritoneal macrophages, but it was not distributed in the normal T and B lymphocytes from the spleen or in the normal B lymphocytes from the bone marrow. In the case of the human promyelocytic leukemia cell line HL-60, the mAb cross-reacted with the molecule(s). It is interesting that a cross reaction was observed in the cells upon their differentiation into macrophage-like cells by PMA. The Western blotting indicated that the size of the molecule mainly recognized by this stimulatory mAb was about 30 kDa. We have recently cloned a candidate cDNA by expression cloning and now we are trying to verify the details. To the best of our knowledge, membrane-associated molecules involved in signaling with a molecular mass around this range have not yet been reported in monocytic/macrophage cells. These results, taken together, strongly suggested that there are some signal gateways on the macrophage cell surface that lead to the induction of G-CSF gene expression. Among them, there is a specific gateway on the cell membrane, through which the extracellular stimulatory signal is transmitted to the intracellular signal molecule(s) for induction of G-CSF gene expression.

G-CSF is a widely used therapeutic cytokine for boosting neutrophils in bone marrow transplant patients and in patients with neutropenia after treatment with anticancer agents [2 , 3 ]. However, due to the high cost of these therapies, the requirement for subcutaneous or intravenous administration, and the potential side effects, such as medullary bone pain, a small-molecular-weight compound that could be delivered with a minimal cost and low side effects would be of clinical value. Fine et al. found a small synthetic compound that selectively induced G-CSF [24 ]. The target of this compound could be one or more intracellular signal molecules. The present results obtained with the mouse cells could easily be extended to search for human counterparts of the molecule(s) recognized by this mAb. Such an effort will facilitate the development of biologically active molecules, such as peptides or antibodies as well as small molecular weight molecules that specifically induce G-CSF in peripheral monocyte/macrophage cells.


   ACKNOWLEDGEMENTS
 
This work was supported in part by the New Energy and Industrial Technology Development Organization as the R & D Project of Industrial Science and Technology Frontier Program. We thank Dr. J. Ihle at St. Jude Children’s Hospital for providing us with the NFS-60 cell line, Dr. Y. Yamasaki for technical suggestions in the cytometric analysis, Ms. K. Inoue and Ms. F. Saito for excellent technical assistance, and Drs. N. Takahashi, M. Nakamura, and D. Hirano for valuable discussions and comments.

Received March 19, 2000; revised May 16, 2000; accepted June 23, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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