Guanosine-rich oligodeoxynucleotides induce proliferation of macrophage progenitors in cultures of...

0014-2980/99/1111-3496$17.50+.50/0 © WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999

Guanosine-rich oligodeoxynucleotides induceproliferation of macrophage progenitors in culturesof murine bone marrow cells

Roland Lang1, Lothar Hültner2, Grayson B. Lipford1, Hermann Wagner1 and KlausHeeg3

1 Institute of Medical Microbiology, Immunology and Hygiene, Technical University of Munich,Munich, Germany

2 Institute of Experimental Hematology, GSF-National Research Center for Environment andHealth, Munich, Germany

3 Institute of Medical Microbiology and Hygiene, Philipps-University of Marburg, Marburg,Germany

Widely used to specifically inhibit gene expression, synthetic oligodeoxynucleotides (ODN)can exert a plethora of non-antisense effects. Immunostimulation by CpG-ODN hasattracted particular attention. ODN rich in the nucleotide guanosine (G-rich ODN) constituteanother type of sequences displaying non-antisense-mediated effects. We have examinedthe effects of CpG- and G-rich ODN on primary mouse bone marrow cells (BMC) in vitro.CpG-ODN induced rapid proliferation of B cells and production of IL-6 and IL-12p40. How-ever, when tested in agar colony assays, CpG-ODN failed to promote the formation of colo-nies. In marked contrast, G-rich non-CpG-ODN led to sustained proliferation ofmacrophage-like cells without inducing cytokines or hemopoietic growth factors. UnlikeCpG-ODN, G-rich ODN effectively induced the formation of macrophage colonies in agarassays, indicating a direct action on progenitor cells. Electrophoretic mobility shift assaysrevealed specific binding of G-rich ODN to a non-nuclear protein. The ability of a panel ofODN to compete for binding correlated with their potential to induce proliferation ofmacrophage-like cells from primary mouse BMC. As such, these data reveal a so far unrec-ognized potential of G-rich ODN to signal directly outgrowth of macrophage progenitorsfrom BMC.

Key words: Guanosine-rich oligodeoxynucleotides / Proliferation / Macrophage / Bone marrowcell

Received 20/5/99Revised 2/8/99Accepted 2/8/99

[I 19823]

Abbreviations: ODN: Oligodeoxynucleotide BMC: Bonemarrow cells EMSA: Electrophoretic mobility shift assayNSE: Nonspecific esterase

1 Introduction

Synthetic oligodeoxynucleotides (ODN) are widely usedto specifically inhibit gene expression by antisenseapproaches [1]. However, ODN not only act via an anti-sense mechanism, but exert a variety of additionaleffects in vitro and in vivo which are both sequence spe-cific and nonspecific [2]. Intriguing non-antisense effectsare exhibited by immunostimulatory CpG-ODN with thesequence motif (5’Pu-Pu-C-G-Py-Py3’). CpG-ODN aremitogenic for B cells [3] and directly activate APC such

as macrophages [4, 5] and dendritic cells [6, 7] and actas Th1-type adjuvants for B and T cell responses [8–12].Since the dinucleotide CpG is suppressed in the genomeof eukaryotic cells [13], but occurs at the expected sta-tistical frequency in bacteria, CpG-ODN may be recog-nized by pattern recognition receptors signaling in-fectious danger to cells of the innate immune system [14,15].

Due to their polyanionic properties ODN interact in asequence-nonspecific manner with the integrin Mac-1[16]. Furthermore, the macrophage scavenger receptorhas been reported to bind homooligomers of guanosine[17, 18]. These interactions of ODN with cell surface pro-teins not only increase uptake of ODN, but may havefunctional consequences. For example, in the case ofMac-1 an enhanced oxidative burst can be observed [16]and binding of polyG to the scavenger receptor can trig-

3496 R. Lang et al. Eur. J. Immunol. 1999. 29: 3496–3506

Figure 1. Proliferation of splenocytes and BMC in response to different ODN. Either 4 × 104 BMC (A–C) or splenocytes (D–F)were incubated with the indicated concentrations of ODN 1668 (A, D), ODN SP1 (B, E) or ODN GR1 (C, F). At days 2, 4 and 6 ofthe culture [3H] thymidine was added for 6 h. Data presented are mean values + SD of quadruplicate samples.

ger increased DNA synthesis [19]. Another emergingclass of non-antisense effects is mediated by ODN con-taining stretches of guanosine residues (G-rich ODN).Designed to inhibit cell proliferation by Watson-Crickbase pairing to specific mRNA, the effectiveness of syn-thetic ODN targeted to c-myc [20], c-myb [21] and rasGAP [22] appears to be independent of antisense mech-anisms but dependent on the presence of guanosineblocks in the ODN sequence. Stein and coworkers [23]have also shown sequence-selective down-regulation ofthe transcription factor RelA by G-rich ODN.

Progenitors for all immune cell lineages are generated inthe bone marrow. Descending from pluripotent stemcells, progenitors proliferate and differentiate under theinfluence of hemopoietic growth factors and cytokines[24]. Agents that interfere with the cell cycle or affect lev-els of hemopoietic cytokines thus may perturb thehomeostasis of cells generated in the bone marrow [25,26]. For example, treatment of mice with CpG-ODNinduces a wave of cytokines in the serum [27, 28], whichis followed by extramedullary hematopoiesis [29]. To fur-ther elucidate direct effects of ODN on bone marrowcells, we analyzed the response of primary bone marrowcells (BMC) to ODN in vitro. In these analyses wefocused on CpG-ODN and G-rich ODN. We report herethat CpG-ODN induced the proliferation of B lympho-cytes in cultures of primary PBM, while G-rich ODN ledto the proliferation of macrophage-like cells. G-rich ODNpromoted colony formation in soft agar assays, whileCpG-ODN did not. Using ODN as probes in electropho-retic mobility shift assays (EMSA) we show sequence-specific binding of G-rich ODN, but not CpG-ODN, to anon-nuclear protein. Competition analyses demonstrate

a correlation between the ability of G-rich ODN to inducethe selective outgrowth of macrophage-like cells fromprimary mouse BMC and binding to this protein.

2 Results

2.1 Sequence-specific response of splenocytesand BMC to different classes of ODN

In a first set of experiments, we tested the proliferativeresponse of mouce splenocytes or primary BMC to theCpG-ODN 1668, the G-rich ODN GR1 and the ODN SP1which contains CpG dinucleotides as well as stretches ofguanosines (Table 1). ODN 1668 induced proliferation ofsplenocytes peaking at day 2 (Fig. 1 D), which was alsoseen in the cultures of BMC (Fig. 1 A). Two-color flowcytometry showed expression of CD71 (transferrinreceptor) on B220+ cells in the BMC culture stimulated

Table 1. Sequences of ODN used

ODN Sequence Classifi-cation

1668 T CC A T GA CG T T CC T GA T GC T CpG

1720 T CC A T GAGC T T CC T GA T GC T controlfor CpG

GR1 T T GGAGGGGG T GG T GGGG G-rich

GR1comp

CCCC A CC A CCCCC T CC A A controlfor CpG

SP1 T CGA T CGGGGCGGGGCGAGC CpG andG-rich

Eur. J. Immunol. 1999. 29: 3496–3506 G-rich ODN promote proliferation of bone marrow cells 3497

Figure 2. Restimulation of BMC after 1 week of culture withdifferent ODN. Primary BMC were cultured for 7 days in thepresence of 1 ? M of either ODN 1668 (A), ODN SP1 (B) orODN GR1 (C). After harvesting and counting, 2 × 104/well ofthe outgrowing cells were stimulated in 96-well plates with1 ? M ODN as indicated on the y-axis. (D) GR1-induced cellswere incubated with 20 ng/ml M-CSF, 20 ng/ml GM-CSF or10 ng/ml G-CSF. At day 5 of the second culture, proliferationwas determined by a [3H] thymidine incorporation assay.Data presented are mean values + SD.

Figure 3. Morphology of GR1-induced cells stained withGiemsa and NSE content. BMC cultured in either ODN GR1or GM-CSF were harvested at day 6 of the culture and cyto-spun onto cover slides. (A) shows Giemsa staining of cellsgrown in GM-CSF, (B) GR1-induced cells. (C) shows NSEcontent of GR1-induced cells.

with ODN 1668 (data not shown), which is consistent withthe known B cell mitogenicity of ODN 1668 [3]. ODN SP1caused a much weaker proliferation of the spleno-cytes at day 2 (Fig. 1E) and ODN GR1 did not induce sple-nocyte proliferation (Fig. 1F). In contrast, both ODN with Gblocks induced sustained proliferation of primary BMC(Fig. 1B and C). At day 2 the magnitude of proliferation waslower than that caused by the CpG-ODN 1668 butincreased continuously during the 6 days of culture. Non-CpG ODN 1720 and non-G-rich ODN GR1comp did notlead to proliferation of splenocytes or BMC, indicating thatthe observed effect was not due to the polyanionic nature orthe phosphorothioate modification of the ODN backbone.

2.2 Restimulation with ODN and hemopoieticgrowth factors

Next we asked whether the cells from the primary bonemarrow culture would continue to grow after reseedingat day 7. When cells harvested after 7 days of primaryculture with either ODN 1668, SP1 and GR1 were trans-ferred to medium alone, no further proliferation wasdetected (Fig. 2). Cells from the primary culture withODN 1668 responded only weakly to addition of eitherstimuli (Fig. 2 A). In contrast, cells induced in primary cul-

tures with ODN SP1 and GR1 proliferated in secondaryculture upon restimulation with either ODN SP1 or GR1(Fig. 2 B and C). GR1-induced BMC were also tested fortheir responsiveness to the growth factors M-CSF, GM-CSF and G-CSF (Fig. 2 D). While M-CSF and GM-CSFeffectively led to further proliferation, G-CSF did not sup-port the growth of GR1-induced BMC.

2.3 GR1-induced BMC have a macrophage-likephenotype

The morphology and cytochemistry characteristics ofthe cells driven by ODN GR1 to grow out from bone mar-row were evaluated in cytospin preparations after 7 daysof culture. While BMC grown in GM-CSF displayed aheterogeneous morphology (Fig. 3 A), ODN GR1 induced


Figure 4. GR1-induced cells phagocytose fluorescent latexbeads. Either primary BMC or BMC cultured for 6 days in thepresence of ODN GR1, ODN 1668 or M-CSF or J774 cells(serving as a positive control) were tested for the ability tophagocytose beads by mixing 106 cells with 108 fluorescentlatex beads and incubating the mixture for 1 h at 37 °C. Theharvested cells were analyzed on a flow cytometer for fluo-rescence at 520 nm. Plotted is the percentage of positivecells. In addition, GR1-induced cells were treated for addi-tional 48 h with either medium, 1 ? g/ml LPS, or 10 ng/mlTNF- § before performing the phagocytosis assay.

Figure 5. Production of IL-12p40 and TNF- § by LPS-triggered GR1-induced BMC. Either primary BMC, GR1-induced BMC or J774 cells were plated at 106 cells/well in1 ml Click’s RPMI. The cells were either left with mediumalone or stimulated with 10 ? g/ml LPS or a combination of20 ng/ml IFN- + plus 10 ? g/ml LPS. Supernatants wereremoved after 8 h for detection of TNF- § (upper panel) andafter 16 h for IL-12p40 (lower panel). Data shown are meanvalues of duplicate ELISA measurements. Similar resultswere obtained in a control experiment.

a homogeneous population of mononuclear cells(Fig. 3 B) which were judged as macrophage like. Thepresence of nonspecific esterase (NSE) in the majority ofthe cells further supported this classification (Fig. 3 C).Phagocytosis is considered to be characteristic of mac-rophages. While only 12 % of BMC ex vivo took up fluo-rescent latex beads, about 30 % of the NSE-positivemacrophage-like cells from day 6 cultures with ODNGR1 displayed phagocytic activity (Fig. 4). Stimulation ofprimary BMC with M-CSF induced a similar percentageof phagocytosing cells (Fig. 4). Withdrawal of ODN GR1or treatment of the GR1-induced cells with LPS or TNF- §for 48 h further increased the number of phagocytic cellsto approximately 80 %, approaching the values ob-served with cells of the mouse macrophage cell lineJ774 (Fig. 4). We also asked whether the phagocytosingmacrophage-like cells obtained from BMC cultures withODN GR1 produce cytokines upon stimulation with LPS.As shown in Fig. 5, the amounts of TNF- § and IL-12p40secreted by GR1-induced cells in response to LPS or acombination of IFN- + and LPS were comparable to thosefrom J774 cells. In contrast, LPS treatment of BMCinduced only poor production of cytokines (Fig. 5).

2.4 Cytokine release from primary BMC inducedby CpG or G-rich ODN

The possibility that G-rich ODN exert the proliferativeeffect on primary BMC via induction of cytokines or

hemopoietic growth factors was tested and compared tothe effects of CpG-ODN 1668. BMC secreted IL-12p40and IL-6 when stimulated with ODN 1668 (Fig. 6 A andB). ODN 1668 was also found to strongly induce theexpression of IL-6 and weakly increased mRNA forM-CSF (Fig. 6 C). In contrast, the proliferation inducedby ODN GR1 was not accompanied by the induction ofdetectable amounts of cytokines or hemopoietic growthfactors (Fig. 6). Thus, the potential of G-rich ODN toinduce proliferation of BMC appeared not to be medi-ated by induction of these hemopoietic growth factorsand cytokines.

2.5 Induction of colony formation in agar assaysby ODN GR1

Next we tested the potential of G-rich ODN GR1 andCpG-ODN 1668 to induce colony formation from BMC inagar colony assays. While ODN 1668 failed to inducecolonies, ODN GR1 effectively promoted colony forma-tion (Fig. 7 A). In fact, at a concentration of 2.5 ? M ODNGR1 alone induced about 40 % of the colonies inducedby a combination of IL-3, Kit ligand and GM-CSF usedas a positive control (Fig. 7 A). Addition of ODN GR1 tosuboptimal concentrations of the growth factor cocktailresulted in an additive effect (Fig. 7 A). Finally, the depen-


Figure 6. Induction of cytokines in primary BMC by ODN1668, but not by ODN GR1. Supernatants of primary cul-tures of BMC (2 × 106/well) treated as indicated wereremoved at day 1 and day 3 of the culture and analyzed forthe presence of the cytokines IL-12p40 (A) and IL-6 (B) (nd:not detectable). In a separate experiment, total RNA wasprepared from 5 × 106 primary BMC treated for 24 h and72 h with ODN GR1, ODN 1668, M-CSF or medium only.Transcripts for hematopoietic growth factors were detectedas described in Sect. 4.4 (C). The marker lane contains theundigested in vitro transcripts of the indicated growth factortemplates. Due to flanking vector sequences, the productsof the in vitro transcription appear in a different position thanthe corresponding protected RNA species. Arrows indicatethe position of IL-6 and M-CSF. M: marker lane; lane 1:medium only for 24 h; lane 2: ODN GR1 for 24 h; lane 3:ODN 1668 for 24 h; lane 4: M-CSF for 24 h; lane 5: ODNGR1 for 72 h; lane 6: ODN 1668 for 72 h; lane 7: M-CSF for72 h.

Figure 7. ODN GR1 supports formation of colonies in softagar culture. (A) BMC (105) were seeded in the presence ofthe indicated concentrations of ODN 1668 (triangles) or ODNGR1 (circles) together with (closed symbols) or without(open symbols) a suboptimal amount of KL + IL-3 + GM-CSF. As positive control a saturating concentration of the KL+ IL-3 + GM-CSF combination was used (closed square).Open square: media control. Data presented are mean + SDfrom triplicates. (B) In a separate experiment, titrated num-bers of BMC were seeded together with 5 ? M ODN GR1without cytokine addition in triplicates and the number ofcolonies was determined on day 7. Shown are mean values± SD and the linear regression curve.

dence of the colony numbers induced by ODN GR1 with-out addition of cytokines on the number of cells seededin the soft agar assay was tested (Fig. 7 B). The linearrelationship [30] between the number of cells culturedand the number of colonies obtained indicated a directeffect of ODN GR1 on the responsive progenitor cells.

2.6 Sequence-specific binding of G-rich ODNto a non-nuclear protein correlates withinduction of BMC proliferation

To approach the mechanisms by which ODN GR1 stimu-lates the proliferation of macrophage progenitors, wemade use of EMSA to search for proteins binding to ODNGR1. In the non-nuclear fraction of J774 extracts astrong binding activity was found (Fig. 8), which was


Figure 8. Sequence-specific binding of ODN GR1 to proteinextract of J774 cells in EMSA. 33P-labeled ODN GR1 (2 ng)was incubated with 5 ? g of the non-nuclear fraction of aJ774 protein extract for 30 min at room temperature asdescribed in section 4.7. Were indicated, cold competitorswere included at a 50-fold molar excess. Lane 1: probe only;lanes 2–8: probe plus 5 ? g J774-protein; lane 3: plus coldGR1; lane 4: plus cold SP1; lane 5: plus cold 1668; lane 6:plus cold GR1comp; lane 7: plus cold SP1; lane 8: plus pro-teinase K.

Table 2. Sequence variants of ODN GR1: competition with GR1-binding in EMSA and induction of BMC proliferation

ODN Sequence % proliferation of BMCa) IC50 for competition in EMSAb)

GR1 T T G G A G G G G G T G G T G G G G 100 X 5pG18 G G G G G G G G G G G G G G G G G G 95 20GR1A T T G G A G G G G G T G G T G T T G 25 n.d.c)

GR1B T T G G A G T T G G T G G T G G G G 78 n.d.GR1C T T T T A G G G G G T G G T G G G G 46 n.d.GR1AB T T G G A G T T G G T G G T G T T G 5 n.d.GR1BC T T T T A G T T G G T G G T G G G G 75 n.d.GR1AA T T G G A G G G G G T G G T T T T T 23 80GR1BB T T G G A G T T T T T G G T G G G G 90 80GR1ABC T T T T A G T T G G T G G T G T T G X 5 G 320GR1AABB T T G G A G T T T T T G G T T T T T X 5 G 320

a) Induction of BMC proliferation by GR1 sequence variants was tested by stimulating 4 × 104 BMC/well with the indicated ODN(at concentrations of 1 ? M, 3 ? M and 10 ? M) in quadruplicates. At day 5 a [3H] thymidine incorporation assay was performed.Data are pooled from two experiments and are expressed relative to the proliferation induced by ODN GR1.

b) Competition EMSA was performed as described in legend to Fig. 8. GR1 sequence variants were used as competitors in a 5-,20-, 80- and 320-fold molar excess. Using ImageQuantR software, the density of the specific band shift was quantified foreach competitor concentration and divided by the density obtained in the absence of specific competitor. Given are the foldmolar excess values corresponding to a 50 % reduction in band intensity.

c) Not done.

sensitive to proteinase K digestion. Binding to ODN GR1was sequence specific because a 50-fold molar excessof cold ODN 1668 did not abolish the band shift (Fig. 8,lane 5) while a 50-fold excess of cold ODN GR1 waseffective (Fig. 8, lane 3). Addition of an excess of coldODN GR1comp to the binding reaction resulted in theformation of double-stranded ODN GR1 and stronglyreduced the mobility, indicating preferential binding ofthis protein to the single-stranded form of ODN GR1(Fig. 8, lane 6). ODN SP1, which induced BMCproliferation similar to ODN GR1 (Fig. 1), competed onlyweakly at a 50-fold molar excess but effectively did so athigher concentrations (Fig. 8, lane 4, and data notshown). Next we used a panel of GR1 sequence variantsto anlyze the sequence requirements for binding incompetition EMSA. These GR1 sequence variants werealso tested for their ability to induce BMC proliferation todetermine whether a correlation between these twoeffects would be observed. Starting with ODN GR1, thenumber of guanosine residues was diminished byexchanging the guanosines by thymidine, therebyreducing the total number of G and destroying the blocksof G (Table 2). In addition, a homooligomer of guanosine(pG18) was tested. GR1 variants induced proliferation ifthe sequences contained at least one or two blocks offour guanosines (ODN GR1A, GR1B, GR1AA, GR1BB inTable 2). The homooligomer of guanosine (ODN pG18)was also effective in inducing proliferation (Table 1).Sequences lacking G blocks (ODN GR1AB, GR1ABC


and GR1AABB) did not induce significant proliferation ofBMC (Table 2). In the competition EMSA, basically thesame sequence dependence was found for competitionof GR1 binding to the J774 non-nuclear protein extract:ODN GR1 efficiently competed with a 50 % reduction inband intensity at a less than fivefold molar excess, fol-lowed by ODN pG18, GR1BB and GR1AA with IC50

between 20 and 80-fold molar excess, while no competi-tion was observed with ODN GR1ABC and GR1AABB upto a 320-fold molar excess (Table 2). Taken together,these data show a correlation between binding of GR1sequence variants to a protein in the non-nuclear frac-tion of J774 cell extracts with the ability to stimulate pro-liferation of macrophage-like cells from primary BMC.While a sequence consisting entirely of guanosines(ODN pG18) is sufficient to be effective in both assays,the clustering of guanosine residues within a ODNsequence seems to be more important than the totalcontent of guanosines.

3 Discussion

In this study we analyzed the effects of different classesof phosphorothioate ODN on BMC in vitro. Both CpGand G-rich ODN induced proliferation of BMC but dif-fered substantially in the kinetics of proliferation and thephenotype of outgrowing cells. In accordance with thepreviously reported B cell mitogenicity of CpG-ODN [3],ODN 1668 led to polyclonal short-term proliferation ofB220+ B cells within BMC and splenocytes (Fig. 1 anddata not shown). Furthermore, CpG-ODN 1668 inducedproduction of cytokines like IL-12p40 and IL-6 (Fig. 6), asdescribed for macrophages [28, 4, 5]. In contrast, G-richODN induced a sustained, continuously increasing pro-liferation of BMC but not of splenocytes (Fig. 1).Although the phenotype of the GR1-responsive targetcell has not been addressed here, the differential respon-siveness of spleen and bone marrow as well as thecapacity of ODN GR1 to induce colony formation in agarassays suggest that ODN GR1 acts on progenitorcells. The outgrowing cells appeared morphologicallyand functionally macrophage-like. Furthermore, GR1-induced cells are responsive to M-CSF (Fig. 2). Thus,stimulation with G-rich ODN increased the frequency ofmacrophage-like cells in BMC cultures.

The growth-promoting effect of G-rich ODN on BMC issurprising in view of the published data on inhibition ofcellular proliferation by ODN containing G quartets [31,20, 21, 22]. We confirmed the growth inhibitory effect ofODN GR1 using the Jurkat T cell line (data not shown).However, this growth suppression was observed only atconcentrations G 5 ? M. Of note, the dose-responsecurve of GR1-induced BMC proliferation described here

shows a peak at a concentration of 1–3 ? M with declin-ing incorporation rates at higher concentrations (data notshown). We did not analyze whether the high-dose sup-pressive effect of G-rich ODN on BMC proliferation issequence dependent or due to a nonspecific toxicity. Forexample, release of toxic amounts of deoxynucleotidemonophosphates by hydrolysis from ODN in the culturemedium inhibited cellular proliferation sequence nonspe-cifically [32]. In the light of the data presented here, stud-ies on macrophage differentiation and proliferationemploying antisense ODN with blocks of guanosinesshould be interpreted with particular caution.

Unlike CpG-ODN, which mimic the structural character-istics of bacterial DNA [33], G-rich ODN have noaccepted naturally occurring counterpart. However, thetelomeres of vertebrate chromosomes share features ofG-rich ODN: the holoenzyme telomerase adds repeats ofthe sequence TTAGGG to the ends of the chromosomecreating a single-stranded overhang of G-rich DNA [34,35]. Of note, a synthetic 18-mer phosphorothioate ODNconstructed of three repeats of a telomeric hexamersequence (TTAGGG)3 induced BMC proliferation likeODN GR1 although at slightly higher concentrations(unpublished observation). Release of G-rich telomericDNA in the context of cell death may thus trigger prolifer-ation of responsive macrophage progenitors to augmentoverall phagocytotic potential.

The mechanisms by which G-rich ODN induce out-growth of macrophage-like cells from BMC are at pre-sent unclear. While CpG-ODN induced cytokine produc-tion and transcription of hemopietic growth factors inBMC cultures, G-rich ODN failed to do so (Fig. 6). Wethus consider an indirect action of G-rich ODN via induc-tion of growth factors unlikely. The effectiveness of ODNGR1 in the colony formation agar assay in the absence ofadded cytokines (Fig. 7 A), especially the linear relation-ship between the cell number seeded and the number ofcolonies obtained (Fig. 7 B), is taken as evidence thatG-rich ODN directly cause and maintain proliferation ofsensitive progenitor cells [30].

The use of GR1 sequence variants revealed that BMCproliferation could be induced by ODN containing atleast one block of four contiguous G (Table 1). Since anantisense mode of action is very unlikely for G-rich ODNof such divergent sequences, the question ariseswhether binding proteins for G-rich ODN are involved intriggering cell proliferation. The macrophage scavengerreceptor binds polyG [17, 18] and ligands for this recep-tor have been reported to stimulate DNA synthesis inmacrophages [19, 36]. When tested on BMC, the scav-enger receptor ligands fucoidan and dextran sulfate nei-ther led to proliferation by themselves nor did they spe-


cifically inhibit the effect of G-rich ODN (data not shown).Although the use of these competitors may not beunequivocal [18], these results do not support a role forthe macrophage scavenger receptor. The integrin sur-face molecule Mac-1 (CD11b/CD18) binds ODN in asequence-nonspecific manner [16]. Since around 30 %of the ex vivo BMC and the vast majority of GR1-inducedcells are Mac-1+, we depleted Mac-1+ cells from BMCand observed that ODN GR1-induced proliferativeresponses were not abolished (unpublished results). Inview of these data we employed the EMSA technique toidentify proteins binding to G-rich ODN in extracts of themouse macrophage cell line J774. Indeed, we observedthe presence of a non-nuclear binding protein displayingsequence selectivity for G-rich ODN (Fig. 8 and Table 2).This binding protein may be involved in mediating thegrowth-inducing effects of G-rich ODN, since binding ofGR1 sequence variants correlated with their potential toinduce proliferation of progenitor cells from mouse bonemarrow.

In conclusion, our studies describe for the first time adirect growth-promoting effect of G-rich ODN on macro-phage precursors within BMC. Identification of the bind-ing protein described here may provide unique informa-tion on the signaling pathways involved. Finally, there is aneed to determine if the effect of G-rich ODN on macro-phage development and proliferation can be replicatedin vivo.

4 Materials and methods

4.1 Reagents

Synthetic ODN were synthetized by MWG (Ebersberg, Ger-many) and TIB MOLBIOL (Berlin, Germany) in single-stranded form and are given in Tables 1 and 2. Thesequence of ODN GR1 is identical to the sequence reportedby Tam et al. [37] as inhibiting up-regulation of CD28 expres-sion in T cells. Except for the EMSA studies, all ODN weresynthetized with a fully phosphorothioate-modified back-bone to render them nuclease resistant. Recombinantmurine GM-CSF and antibodies for flow cytometry (PE-conjugated B220-PE, FITC-conjugated, FITC-conjugatedCD71) were purchased from Pharmingen (Hamburg, Ger-many). Recombinant murine M-CSF and G-CSF was fromR&D Systems (Wiesbaden, Germany). LPS was from Sigma(Deisenhofen, Germany).

4.2 Preparation and culture of cells; proliferation assay

From female C57/BL6 mice (8–12 weeks old) femur andtibia were isolated by dissection. Using a 25-gauge needle,ice-cold PBS was forced through the bone cavity. Cells were

drawn several times through the syringe to break up clumps.Splenocyte suspensions were prepared by teasing spleensthrough a steel mesh. In both instances, erythrocytes werelysed by incubation with NH4Cl for 5 min at room tempera-ture. After two washing steps in Click’s RPMI with 5 % FCS(Seromed, Berlin, Germany), cells were counted. Click’sRPMI supplemented with 50 ? M 2-ME, 2 mM L-glutamine,100 U/ml penicillin G, 100 ? g/ml streptomycin, 10 mMHepes and 5 % FCS was also the culture medium used in allexperiments. For proliferation assays, cells were seeded at adensity of 4 × 104/well in 96-well round-bottom plates in atotal volume of 200 ? l. Cultures were pulsed with 1 ? Ci[3H] thymidine for 6–12 h, cells were harvested onto filterpapers and radioactivity incorporated into DNA was mea-sured. For other experiments, BMC were cultured in 24-wellplates at a density of 2 × 106/well in a volume of 2 ml.

The mouse macrophage cell line J774 was cultured in RPMI1640 (Seromed, Berlin, Germany) supplemented with 8 %endotoxin-free FCS.

4.3 Agar colony assay

The proliferative and differentiation-inducing effects ofhemopoietic cytokines or ODN GR1 on murine bonemarrow-derived hemopoietic progenitor cells (colony form-ing cells) were analyzed in a soft agar colony assay asdescribed previously [38]. Cellular aggregates containing atleast 50 cells were scored as colonies. Individual colonieswere picked from culture plates and typed morphologically.

4.4 Cytokine determination with ELISA and RNaseprotection assay

For measurement of TNF- § , IL-12p40 and IL-6, antibodypairs and recombinant proteins for standard curve genera-tion were obtained from Pharmingen (Hamburg, Germany).Streptavidin-coupled peroxidase was bought from Sigma.The assays were performed according to the manufacturer’sinstructions, using optimized concentrations of reagents. Toanalyze the expression of hematopoietic growth factors, acommercially available RNase protection assay kit (mCK4from Pharmingen, Hamburg, Germany) was performed fol-lowing strictly the manufacturer’s protocol. RNA was pre-pared from 5 × 106 cultured BMC by a guanidinium-basedmethod (Tri-Reagent, Sigma). Overnight exposure on aphosphoimager screen (Molecular Dynamics) was used fordetection of radioactive signals on the dried gel.

4.5 Giemsa staining and cytochemistry of BMC

For cytology, cultured cells were harvested by trypsin diges-tion, counted and cytospun onto glass slides. After fixationwith methanol, the slides were incubated in Giemsa stainingsolution for 15 min, rinsed with water and dried. Cytochemi-


cal analysis of NSE was performed after immersing theslides in formaldehyde-acetone fixative by incubating theslides in a solution containing § -naphthylacetate (Sigma)according to a standard protocol [39].

4.6 Phagocytosis of fluorescent latex beads

Cultured cells were harvested and reseeded at 106/well in a24-well plate in Click’s RPMI with 5 % FCS. Fluorescentlatex beads of 1 ? m diameter (Molecular Probes) wereadded at a 100:1 ratio (beads:cells). The mixture was incu-bated for 1 h at 37 °C. After harvesting, cells were washedwith PBS and analyzed with a flow cytometer (Coulter EPICSXL). Cells which had taken up fluorescent beads gave a sig-nal which was detected on the FITC channel and corre-sponded in its intensity to the number of beads taken up. Bysetting appropriate gates, the percentage of FITC-positivecells was determined.

4.7 EMSA

Since the phosphorothioate modification inhibits T4 polynu-cleotide kinase [40], phosphodiester ODN were used forEMSA studies. ODN GR1 (250 ng) was labeled at the 5’ endusing T4 polynucleotide kinase (Promega, Madison, WI) and[ + -33P] ATP. The labeled probe was purified on a push-column (Stratagene, Heidelberg, Germany). Protein extractsof the mouse macrophage cell line J774 cells were preparedfrom approximately 108 cells, according to a standardmethod [41]. Briefly, after washing with PBS, cells were incu-bated in buffer A (10 mM Hepes, 1.5 mM MgCl2, 10 mM KCl,0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride) for10 min on ice, pelleted and resuspended in buffer A contain-ing 0.05 % NP40 and lysed with 20 strokes of a douncehomogenizes. The nuclei were pelleted by 10 min centrifu-gation at 250 × g, the supernatant harvested and clearedfrom debris by centrifugation at 30 000 × g for 30 min. Afterthis step, the supernatant containing the non-nuclear cellu-lar proteins was saved. Protein concentration was deter-mined using the Bio-Rad Protein Assay (Bio-Rad, Munich,Germany). The binding reaction for the EMSA was carriedout at room temperature for 30 min in a total volume of 12 ? l.Five to ten micrograms protein were used per reaction, bind-ing buffer was 100 mM Tris-Cl (pH 8.0), 50 mM NaCl, 1 mMDTT with 15 % glycerol, and 2 ng of labeled ODN wereadded to each binding reaction. As a nonspecific competitor1 ? g random sequence ODN was included. For competitionanalysis, cold phosphodiester ODN were added to the bind-ing reaction at the indicated concentrations before the addi-tion of labeled probe. Complexes were separated on a 5 %non-denaturing polyacrylamide gel at 100 V for 3.5 h. Afterdrying, the gel was read by a phosphoimager (MolecularDynamics).

Acknowledgments: This work was supported by grantsfrom BMBF and CpG – Immunopharmaceuticals GmbH.

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Correspondence: Roland Lang, Institute of Medical Micro-biology, Immunology and Hygiene, Technical University ofMunich, Trogerstr. 32, D-81675 München, GermanyFax: +49-89-41404942e-mail: immuno.rdi — lrz.tu-muenchen.de


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