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David H. Chang1, Cristina Angelin-Duclos2 and Kathryn Calame1,2

B lymphocyte–induced maturation protein-1 (BLIMP-1 or PRDI-BF1) is induced when bonemarrow–derived progenitors differentiate in response to macrophage–colony stimulating factor (M-CSF) and is present in peripheral blood monocytes and granulocytes. BLIMP-1 is also induced duringdifferentiation of U937 and HL-60 cells into macrophages or granulocytes. Induction of BLIMP-1mRNA during macrophage differentiation of U937 and HL-60 shows a biphasic pattern.Overexpression of BLIMP-1 is sufficient to initiate macrophage differentiation of U937 cells whereasblocking endogenous BLIMP-1 inhibits differentiation. One target of BLIMP-1–dependenttranscriptional repression in U937 cells is c-myc, providing an explanation for cessation of celldivision. Thus BLIMP-1 is a key regulator of terminal differentiation in two separate hematopoieticlineages: myeloid cells and B lymphocytes.

1Integrated Program in Cellular, Molecular and Biophysical Studies and 2Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, NY10032, USA. Correspondence should be addressed to K.C. (klc1@columbia.edu).

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The transcriptional repressor B lymphocyte–induced maturation pro-tein-1 (BLIMP-1) is a 98-kD protein containing five Krüppel-typezinc fingers, originally identified by subtractive cloning of mRNAsinduced during differentiation of a B cell lymphoma 1 line (BCL1)1.Ectopic expression of BLIMP-1 is sufficient to drive terminal differ-entiation of BCL1 lymphoma cells to a plasma cell phenotype1,2. Thus BLIMP-1 is viewed as a master regulator of B cell terminal differentiation.

The human homolog of BLIMP-1, PRDI-BF1, was cloned by itsability to bind the PRDI site in the human interferon β (IFN-β) pro-moter3. BLIMP-1, or PRDI-BF1, is a transcriptional repressor thatassociates with human Groucho4 and histone deacetylases5. (For sim-plicity, both murine and human proteins are referred as BLIMP-1from here on.) One important target of BLIMP-1 transcriptionalrepression in B cells is the c-myc gene, where BLIMP-1 binds a pre-viously identified repressor site6,7,8. Recently, the CIITA gene, encod-ing a coactivator for class II major histocompatibility complex(MHC) gene transcription, was identified as an additional target ofBLIMP-1 in B cells8 (and J.F. Piskurich et al., unpublished data).

Although the initial report suggested that BLIMP-1 mRNA expres-sion was limited to the B cell lineage1, BLIMP-1 is expressed in ahuman osteosarcoma line3. In addition, BLIMP-1–null mice die dur-ing embryogenesis (M.M. Davis, personal communication) and theXenopus homolog of BLIMP-1 is required for anterior endomesoder-mal cell fate and head induction9. These data suggested that BLIMP-1 might be expressed and functionally important during terminal dif-ferentiation of cells outside the B lymphoid lineage.

To explore this possibility we studied cells in the myeloid lineage.We investigated the expression of BLIMP-1 during differentiation ofbone marrow progenitors in response to macrophage–colony stimu-lating factor (M-CSF also called CSF-1) and in differentiated mono-cytes and granulocytes from peripheral blood. We also took advantageof two well characterized cell lines that undergo defined programs ofmyeloid terminal differentiation. U937 is a promonocytic line that can

be induced to differentiate into macrophages by treatment with phor-bol 12-myristate 13-acetate (PMA)10. HL-60 is a pluripotentialpromyelocytic cell line that differentiates into macrophages whentreated with PMA, and into granulocytes when treated with dimethylsulfoxide (DMSO)11,12,13. We show that BLIMP-1 is induced duringdifferentiation of U937 and HL-60 cells and is both required and suf-ficient to trigger differentiation of U937 cells.

ResultsBLIMP-1 mRNA induction during myeloid differentiationTo determine whether BLIMP-1 is expressed in differentiatedmyeloid cells, we investigated BLIMP-1 mRNA in primary mono-cytes and granulocytes purified from human peripheral blood.Peripheral blood monocytes are post-mitotic but undergo further dif-ferentiation to macrophages upon entry into inflammatory sites andtissues14,15,16. BLIMP-1 mRNA was present in both monocytes andgranulocytes purified from healthy donors (Fig. 1a, two are shown).We found that BLIMP-1 mRNA was not further induced when periph-eral blood monocytes were activated by in vitro culture (Fig. 1a,Donor 1, M5).

We examined whether BLIMP-1 mRNA is induced during differ-entiation of myeloid progenitors. A population enriched for progeni-tor cells was isolated16 from mouse bone marrow and cultured for 8days in M-CSF to promote macrophage differentiation17. After 8 daysof culture in M-CSF most cells were adherent and showed membraneruffling (Fig. 1d). As the cells were heterogeneous, especially at thebeginning of the culture period, immunocytochemistry was per-formed to identify CD11b+ (the α chain of Mac-1, the complementreceptor 3) cells and to follow expression of BLIMP-1 in this popula-tion. CD11b is expressed on early bone marrow progenitors18 and ondifferentiated macrophages19 but expression is lost in common lym-phoid progenitors (CLP) 20 and early pro-B cells21, although it hasbeen found on a subset of T cell progenitors22.

In the original progenitor-enriched population 66% of the cells

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expressed CD11b. This number increased to 100% after 8 days of cul-ture in M-CSF, consistent with M-CSF–driven macrophage differenti-ation. Early in the culture period the cells were not adherent and weretherefore prepared for immunocytochemistry by cytospin (which doesnot allow analysis of their morphology.) However, at the end of theculture, most viable cells became adherent with morphology of typi-cal differentiated macrophages (Fig. 1d). These cells were analyzedby immunocytochemistry in situ. At day 0, 9% of CD11b+ cellsexpressed BLIMP-1 in their nuclei (5% of all cells) (Fig. 1b). After 3days of M-CFS treatment the number of CD11b+BLIMP-1+ cellsincreased to 51% (44% of all cells) (Fig.1c). By day 8, 100% ofCD11b+ cells expressed BLIMP-1 (Fig. 1d). The specificity of ourantiserum for BLIMP-1 was established by showing that addition ofrecombinant BLIMP-1 ablated the signal (data not shown). These datashow clearly that BLIMP-1 is induced during differentiation of bonemarrow CD11b+ progenitors to macrophages in response to M-CSF.

BLIMP-1 induction during U937 and HL-60 differentiationU937 and HL60 cells were used for further analysis of BLIMP-1induction and function during myeloid differentiation. When U937and HL-60 were grown in the presence of PMA for 3 days almost100% of cells underwent growth arrest, changed from suspension toadherent growth (Fig. 2a,b) and expressed macrophage-specific sur-face proteins CD11b and CD11c (the integrin α chain of gp150, 95)(see Fig. 4a,b)23,24. When HL-60 cells were treated with DMSO theyceased to proliferate and, after 6 days of treatment, differentiated intogranulocytes as illustrated by smaller cell size, altered morphologyand positive staining by the Nitro-blue tetrazolium–reduction assay(data not shown)25.

To investigate whether BLIMP-1 was expressed in differentiatedU937 cells, we performed immunocytochemical staining for nuclearBLIMP-1 and surface CD11b on U937 cells that became adherentafter 3 days of PMA treatment. Although the adherent cells displayeddifferent degrees of cytoplasmic extension and membrane ruffling,

virtually all of them expressed BLIMP-1 and CD11b (Fig. 2c,d).The kinetics of BLIMP-1 mRNA induction were monitored in

PMA- or DMSO-treated U937 and HL-60 cells. BLIMP-1 mRNAwas low or undetectable before treatment of either U937 or HL-60cells. In U937 cells, BLIMP-1 mRNA (determined by ribonucleaseprotection assay) was induced after 4 h of PMA treatment, subse-quently decreased, then peaked again at the end of the treatment (Fig. 2e). Similarly in HL-60 cells BLIMP-1 mRNA, determined bynorthern blotting, was rapidly induced and peaked in the second hourof PMA treatment, decreased, then peaked again late in the treatmentperiod (Fig. 2f). Three isoforms of BLIMP-1 mRNA observed bynorthern blotting differ in their 3′ untranslated sequences (C. Tunyaplin and K. Calame, unpublished data). When HL-60 cellswere treated with DMSO (Fig. 2g), BLIMP-1 mRNA stayed low dur-ing the first 3 days of treatment, then increased and stayed high after4 days of DMSO treatment. Thus BLIMP-1 mRNA is induced upondifferentiation of U937 and HL-60 cell lines into macrophages inresponse to PMA, and upon differentiation of HL-60 cells into gran-ulocytes in response to DMSO. The presence of BLIMP-1 protein(Fig. 2c,d) correlates with the expression of BLIMP-1 mRNA inPMA-treated U937 cells (Fig. 2e).

Based on its activity in B cells7 we speculated that BLIMP-1 mightrepress transcription of the c-myc gene during monocyte and granulo-cyte differentiation. Consistent with this idea, c-Myc steady-statemRNA was initially high but decreased following the first inductionof BLIMP-1 mRNA during differentiation of U937 and HL-60 cellsto macrophages after PMA treatment (Fig. 2e,f). During differentia-tion of HL-60 cells into granulocytes, c-Myc mRNA levels fell beforeinduction of BLIMP-1 mRNA, then increased slightly and decreasedagain after the later induction of BLIMP-1 mRNA (Fig. 2g).

Requirement for BLIMP-1 in U937 differentiationTo determine the functional importance of BLIMP-1 induction duringmacrophage differentiation we used a truncated form of BLIMP-1,

Figure 1. BLIMP-1 mRNA is expressed in human peripheral blood mono-cytes and granulocytes, and induced during differentiation of primarymurine macrophages. (a) The expression of BLIMP-1 mRNA in human peripher-al blood monocytes and granulocytes. Human peripheral blood leukocytes from twohealthy donors were purified using Histopaque density gradient and adherence sep-aration. Monocytes were 70–80% pure whereas granulocytes were 99.9% pure.TotalRNA was analyzed by ribonuclease protection assay with probes protecting BLIMP-1 mRNA and GAPDH mRNA. (M, purified monocytes; G, purified granulocytes; M5,monocytes after 5 days in vitro culture.) (b–d) Immunocytochemical staining forBLIMP-1 (nuclear red staining) and CD11b (cytoplasmic and cell surface blue stain-ing) in bone marrow–derived progenitors and macrophages. Bone marrow cells wereenriched from progenitors and cultured in M-CSF. (b) Day 0, starting population (66%CD11b+, 5% CD11b+ BLIMP-1+) arrows indicate CD11b+BLIMP-1+ cells. (c) Day 3(86% CD11b+, 44% CD11b+ BLIMP-1+.) (d) Day 8 (100% CD11b+, 100% CD11b+

BLIMP-1+).At days 0 and 3 staining was performed on cells prepared by cytospin. Day8 staining was done in situ on cells that adhered to coverslips. Two different fieldsfrom each day are shown.

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T-BLIMP, to interfere with endogenous BLIMP-1. T-BLIMP (Fig. 3a)retains the zinc-finger domain that confers DNA binding26 but lacks theproline-rich domain required for transcriptional repression4,5. A similar-ly truncated form of BLIMP-1 was used in the inhibition of endogenousBLIMP-1 function in B cell lines2 and to inhibit BLIMP-1 function inXenopus embryos9. U937 cells were stably transfected with a T-BLIMPexpression vector or a vector control. Five T-BLIMP–expressing trans-fectants (TB-1–TB-5) and three mock transfectants (Mock 1–3) wereused for subsequent studies. TB-3 and TB-5 have relatively high levelsof T-BLIMP mRNA, whereas TB-1, TB-2 and TB-4 have medium orlow levels of T-BLIMP mRNA (Fig. 3b).

After 3 days of PMA treatment almost 100% of U937 and the threemock transfectants became adherent and displayed macrophage-likemorphology. For the five T-BLIMP transfectants, however, weobserved variable morphological changes. For TB-3 and TB-5 (high T-BLIMP), after 3 days of PMA treatment cell growth was evident andless than one-third of the cells became adherent. For TB-1, TB-2 andTB-4 cells (low T-BLIMP), about one-third of cells were still in sus-pension while two-thirds became adherent (data not shown). About80% of cells from U937 and the mock transfectants were positive formacrophage-specific esterase (α-naphthyl butyrate esterase) whereasonly 25–40% of the five T-BLIMP transfectants were positive foresterase expression after 3 days of PMA treatment (data not shown).

In U937 parental cells and mock transfectants, CD11b and CD11cwere expressed on 80–90% of the cells after PMA treatment (Fig.4a,b—only Mock-1, TB-1, TB-3 and TB-5 are shown). For TB-3 andTB-5 (high T-BLIMP), 20–30% of cells expressed CD11b and <30%of cells expressed CD11c. For TB-1, TB-2 and TB-4 (low T-BLIMP),about 50% of cells expressed CD11b and 60% expressed CD11c.When CD11b and CD11c were monitored at intervals during PMAtreatment of TB-1 (low T-BLIMP), expression of both surface proteinswas delayed. However, CD11c (and CD11b partially) eventuallyachieved normal levels (Fig. 4c,d—only Mock-1, TB-1 and Tb-3 areshown). For TB-3 (high T-BLIMP), both CD11b and CD11c remained

at low concentrations throughout treatment. T-BLIMP also impairedthe ability of U937 to phagocytose opsonized bacteria. TB-1, TB-2and TB-4 (low T-BLIMP) had partial phagocytotic activity whereasTB-3 and TB-5 (high T-BLIMP) had little phagocytotic activity (Fig. 4e—only Mock-1, TB-1, 3 and 5 are shown).

Therefore, blocking endogenous BLIMP-1 with T-BLIMP delaysor inhibits macrophage differentiation of U937. Higher T-BLIMPexpression, which is likely to compete more fully with endogenousBLIMP-1, inhibited PMA-induced adherent growth, expression ofmacrophage surface antigens, and phagocytosis. Lower T-BLIMPexpression partially inhibited or delayed macrophage differentiation.

Figure 2. BLIMP-1 expression is induced in U937 and HL-60 cells treated with PMA, and in HL-60 cells treated with DMSO.Wright-Giemsa staining of U937cells (a) before and (b) after 3-day PMA treatment. Untreated cellls were prepared by cytospin. PMA-treated cells were allowed to adhere to cover slips before staining.Immunocytochemical staining for expression of BLIMP-1 (red) and CD11b (blue) of (c) untreated and (d) PMA-treated U937 cells. (e) BLIMP-1, c-Myc and GAPDH mRNAsin U937 cells, at times indicated following PMA treatment, were determined by ribonuclease protection assay. (f) BLIMP-1, c-Myc and GAPDH mRNAs in HL-60 cells, at timesindicated following PMA treatment, were determined by northern blotting. (g) BLIMP-1, c-Myc and GAPDH mRNAs in HL-60 cells, at times indicated following DMSO treat-ment, were determined by northern blotting.

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BLIMP-1 overexpression drives U937 differentiationKnowing that BLIMP-1 is required for U937 differentiation, wewanted to determine whether expression of BLIMP-1 might be suffi-cient to drive macrophage differentiation in this system. A retroviruscontaining BLIMP-1 cDNA, an internal ribosome entry site (IRES)and cDNA encoding green fluorescent protein (GFP) was engineered.Expression of both BLIMP-1 and GFP from the bicistronic vectorensured that cells expressing BLIMP-1 could be identified by virtueof GFP expression. Three days after infection of U937 cells with theBLIMP-1-GFP virus, about 6% of the cells were GFP+; 50% of theGFP+ cells (3.1% of the total) expressed CD11b and 73% (4.1% of thetotal) expressed CD11c (Fig. 5a–c). Some GFP– cells from theBLIMP-1-GFP virus infection also expressed CD11b (5%) andCD11c (15%) after infection. As GFP expression by the IRES-GFPvirus is lower than that of the control MFG-GFP virus, these may rep-resent infected cells that express insufficient GFP to be detected byfluorescence-activated cell sorting (FACS) but still express sufficientBLIMP-1 to induce CD11b and CD11c expression. Consistent withthis interpretation, cells infected with control virus encoding onlyGFP (MFG-GFP) had minimal expression of CD11b (0.7%) orCD11c (3.7%) demonstrating that virus infection did not induceexpression of these markers. By comparison, when U937 cells weretreated for three days with PMA, 87% expressed CD11b and 98%expressed CD11c (data not shown).

In addition to induction of surface proteins, overexpression ofBLIMP-1 was sufficient to cause morphological changes and adher-ent growth of U937 cells (Fig. 5d,e). Three days after infection 10%of BLIMP-1-GFP–infected cells became adherent, compared to con-

trol virus–infected cells where less than 0.1% were adherent.Expression of BLIMP-1 and CD11b was detected in all the adherentcells from BLIMP-1-GFP virus infection (Fig. 5f,g). This is consis-tent with the possibility raised by data in Fig. 5a–c that some BLIMP-1-GFP–infected cells differentiated even though they did not expressenough GFP to be detected by FACS. However, the BLIMP-1-GFPvirus–infected cells were generally smaller and had less cytoplasmand membrane ruffling compared to cells differentiated in response toPMA (see Fig. 2a–d). Thus, overexpression of BLIMP-1 in U937 issufficient to induce macrophage differentiation as shown by expres-sion of CD11b and CD11c on the surface, cell adherence and alteredmorphology.

BLIMP-1 targets c-myc for repressionThe correlation between BLIMP-1 mRNA induction and decreased c-Myc mRNA during U937 and HL-60 differentiation suggested thatBLIMP-1 may repress c-myc during macrophage differentiation. Totest this possibility directly we determined c-Myc, T-BLIMP andBLIMP-1 mRNA levels in U937 cells expressing T-BLIMP after treat-ment with PMA using riboprobe analysis (Fig. 6a–d). EndogenousBLIMP-1 mRNA was induced in a biphasic manner in controls aswell as T-BLIMP clones; the levels of T-BLIMP mRNA were in thesame range as those of endogenous BLIMP-1 mRNA, consistent withinhibition of BLIMP-1 by T-BLIMP. In Mock-1, c-Myc mRNAdecreased normally after 1 day of PMA treatment (Fig. 6a–d).However, after 3 days of PMA treatment c-Myc mRNA remained highin all T-BLIMP transfectants. After 5 days of PMA treatment, c-MycmRNA in TB-1 (low T-BLIMP) decreased while c-Myc mRNA in

Figure 4.T-BLIMP delays or inhibits the expression macrophage-specific surfaceproteins CD11b and CD11c, and impairs the phagocytotic ability of PMA-treat-ed U937 cells. FACS analysis of Mock-1 and T-BLIMP transfectants for (a) CD11b and (b)CD11c expression was performed. Cells were either untreated (broken line) or treatedwith PMA for three days (solid line) before FACS analysis. (Percentage of cells in the pop-ulation of PMA-treated cells indicated by the horizontal bar is shown.) Kinetics of induc-tion of surface (c) CD11b and (d) CD11c in Mock-1,TB-1 and TB-3 after PMA treatmentwas also determined. Cells were analyzed before treatment (thin broken line), after 1 day(thick broken line), 3 days (thin solid line) and 5 days (thick solid line) in PMA. (e) Phagocyticactivity of PMA-treated mock transfectant, and three T-BLIMP expressing clones. Cellsuntreated (thin solid line) and treated with PMA for 3 days (thick solid line) were incubat-ed with FITC-labeled opsonized S. aureus before FACS analysis.

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TB-3 and TB-5 (high T-BLIMP) remained high. Concomitant withdecreased c-Myc mRNA, U937 and mock transfectants underwentgrowth arrest following 3 days of PMA treatment but T-BLIMP trans-fectants continued to divide (Fig. 6e). After 5 days of PMA treatment,TB-1 (low T-BLIMP) became growth-arrested while 10% of TB-3(high T-BLIMP) cells were still in S phase (data not shown). Thus,these data show that BLIMP-1 is required for growth arrest duringU937 differentiation, and one target of BLIMP-1 repression is the c-myc gene.

DiscussionBLIMP-1 expression not limited to B cell lineageThe original report described BLIMP-1 as a B cell–specific proteinwith expression restricted to cell lines representing late stages of B

cell development, particularly plasmacytomas1. We have shown herethat BLIMP-1 mRNA is expressed in peripheral blood monocytes andgranulocytes and is induced during M-CSF–dependent differentiationof bone marrow–derived macrophages, U937 promonocytes treatedwith PMA, and HL-60 promyelocytes treated with either PMA orDMSO. Thus, BLIMP-1 is clearly induced during differentiation ofpromyelocytic cells into macrophages or granulocytes.

We also performed northern blot analyses of RNA from adult murinetissues and detected low levels of BLIMP-1 mRNA in many tissuesincluding brain, lung, heart, kidney and testis (D. H. Chang and K.Calame, unpublished data). Thus it is clear that expression of BLIMP-1 is not limited to the B cell lineage. Our data are consistent with theexpression of BLIMP-1 mRNA in a human osteosarcoma cell line3,with the embryonic lethal phenotype of BLIMP-1–/– mice

Figure 5. BLIMP-1 expression is sufficient to drive macrophage differenti-ation of U937 cells. U937 cells were either (a) untreated, (b) infected with con-trol virus MFG-GFP or (c) with BLIMP-1-GFP virus for 3 days. Surface expression ofCD11b and CD11c was determined by FACS analysis (7-AAD was used to excludedead cells).The numbers in each box correspond to the percent of total cells in eachpopulation.Wright-Giemsa staining of U937 cells 3 days after infection with (d) con-trol virus (prepared by cytospin) or (e) BLIMP-1-GFP virus (cells which adhered toa cover slip). (f,g) Immunocytochemical staining of cells prepared as described in dand e, respectively, for CD11b (blue) and BLIMP-1 (red).

Figure 6. c-myc is a target of BLIMP-1 repression during U937 differentiation. Ribonuclease protection assay for c-Myc, endogenous BLIMP-1 (human homolog)and ectopic T-BLIMP (mouse homolog) mRNAs in (a) Mock-1 (b) TB-1 (c) TB-3 and (d) TB-5 clones treated with PMA at the days indicated. (e) Cell cycle status of U937,mock and T-BLIMP clones. Cells in S phase were quantified using FACS, gating on cells with high BrdU incorporation and propidium iodide content greater than 2n.

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(M.M. Davis, personal communication) and with the recent demonstra-tion that XBLIMP-1 is necessary for Xenopus embryo development9.

BLIMP-1 represses c-myc transcription in U937 cellsGiven that BLIMP-1 is induced during myeloid differentiation, it wasimportant to identify functionally relevant target genes of BLIMP-1–dependent repression. The delayed or absent down-regulation of c-Myc mRNA observed in U937 transfectants expressing T-BLIMPprovides direct evidence that c-myc is a target of BLIMP-1 repressionduring U937 differentiation. This is consistent with the previous iden-tification of a region on the c-myc promoter, encompassing theBLIMP-1 binding site (positioned 290 bp before the P1 site), whichis required for repression of c-myc transcription in U937 cells27.

The importance of c-myc repression in myeloid differentiation hasbeen established by many previous studies. Decreased c-Myc corre-lates with U937 differentiation27,28, and ectopic expression of v-Mycblocks U937 differentiation29,30. As c-Myc promotes cell proliferationit makes sense that it must be repressed when cells cease division andundergo terminal differentiation. Thus, c-myc is a critically importanttarget of BLIMP-1–dependent transcriptional repression in U937cells. Furthermore, repression of c-myc by BLIMP-1 in bothmacrophages and B cells7 provides a mechanistic link between termi-nal differentiation in these two cell lineages.

However, BLIMP-1–dependent repression of other potential targetgenes may be required for full macrophage differentiation. This issuggested by the biphasic expression pattern of BLIMP-1 mRNAobserved during U937 and HL-60 differentiation in response to PMA.For plasma cell differentiation, we have recently shown that repres-sion of c-myc is necessary, but not sufficient, to drive differentiation(K.-I. Lin and K. Calame, unpublished data) and have identified theCIITA gene as an additional target of BLIMP-1 repression (J.F.Piskurich et al., unpublished data). It will be important to try to iden-tify other BLIMP-1 target genes in myeloid cells.

In contrast to macrophage differentiation, during granulocytic dif-ferentiation of HL-60 cells, induction of BLIMP-1 mRNA did notcorrelate with the initial decrease in c-Myc mRNA levels.Nevertheless this is consistent with a previous study showing that twomechanisms of transcriptional control operate on c-myc duringDMSO-induced differentiation of HL-60 cells: the early phaseinvolves blockage of c-myc transcriptional elongation and the latephase involves repression of transcription initiation31. Transcriptionrepression, which correlates with the induction of BLIMP-1 mRNAwe observed, is the irreversible terminal step in granulocytic differ-entiation of HL60 cells. Therefore it is likely that c-myc is also a tar-get of BLIMP-1 during granulocyte differentiation.

BLIMP-1 required for U937 differentiation to macrophagesMultiple clones of U937 expressing T-BLIMP failed to respond nor-mally to PMA treatment. The correlation between relative T-BLIMPexpression and the degree of inhibition of U937 differentiation pro-vides additional evidence that ectopic T-BLIMP competes withendogenous BLIMP-1. As endogenous BLIMP-1 continues toincrease during PMA treatment of the T-BLIMP transfectants, itseems likely that the delayed differentiation observed in some cloneswas overridden when the level of endogenous BLIMP-1 became suf-ficiently high to counter T-BLIMP’s inhibitory effect.

All aspects of macrophage differentiation we measured were affect-ed by T-BLIMP. T-BLIMP–expressing U937 cells failed to down-reg-ulate c-Myc mRNA, continued to cycle, and did not become adherentfollowing PMA treatment. CD11b, CD11c and macrophage-specific

esterases were reduced and the cells’ ability to phagocytose wasimpaired. These observations establish the importance of BLIMP-1 infull macrophage differentiation of U937 cells. Coupled with demon-strated induction of BLIMP-1 mRNA during differentiation of bonemarrow–derived macrophages, the data strongly suggest a role forBLIMP-1 in normal monocyte and macrophage differentiation.Furthermore, induction of BLIMP-1 mRNA upon differentiation ofHL-60 cells into granulocytes, and the presence of BLIMP-1 mRNAin normal peripheral blood granulocytes, suggest that BLIMP-1 mayalso be required for granulocyte development.

Overexpression of BLIMP-1 in U937 cells was sufficient to triggerdifferentiation as shown by induction of CD11b and CD11c on thesurface, cell adherence, and acquisition of partial macrophage mor-phology. However, neither the percentage of cells expressing CD11bor CD11c nor the acquisition of a macrophage morphology was ascomplete a response to overexpressed BLIMP-1 as that observed inresponse to PMA. This may simply reflect the suboptimal levels ofBLIMP-1 achieved by retroviral transduction. Alternatively BLIMP-1may drive some, but not all, aspects of U937 differentiation.

Transcription factors AP-1, PU.1, NF-κB and IRF-1 are requiredfor U937 differentiation32,33,34 and gene-targeting studies show thatPU.135, C/EBP36 ICSBP37,38 are necessary for normal macrophage dif-ferentiation. To our knowledge, however, BLIMP-1 is the only tran-scription factor capable of triggering macrophage differentiation inU937 cells. The ability of BLIMP-1 to direct macrophage terminaldifferentiation is strikingly similar to its ability to drive B cell termi-nal differentiation1. Taken together, the data suggest BLIMP-1 mayplay a critical role in terminal differentiation of many cell lineages.

MethodsCell culture and isolation. U937 (CRL-1593.2) and HL-60 (CCL-240) were purchasedfrom American Type Culture Collection (ATCC, Manassas, VA) and maintained in growthmedium (RPMI 1640, Sigma; 10% heat-inactivated fetal bovine serum (FBS), Gemini,Calabasas, CA; and 20 µg/ml of Gentamicin, Gemini). PMA (Sigma) was dissolved inDMSO (Sigma) in a 1 mg/ml stock solution and used at 10 ng/ml for all experiments. Toinduce granulocytic differentiation, DMSO (at a final concentration of 1.25%) was addeddirectly to growth media. Ecotropic Phoenix cells were a gift from G.P. Nolan at StanfordUniversity and were cultured following Nolan’s Phoenix retroviral producer line protocol(http://www.stanford.edu/group/nolan/NL-phnxr.html).

Peripheral blood monocytes and granulocytes were isolated from healthy donors (NewYork Blood Center). Mononuclear cells were isolated by density gradient usingHistopaque-1077 (Sigma), according to manufacturer’s procedure. Polynuclear cells(granulocytes) were isolated using Histopaque-1119. Purity was determined by FACSanalysis of CD45 (leukocyte common antigen), CD11b (Mac-1) and CD14 (monocyte-specific antigen), and Wright-Giemsa staining. Monocytes were further purified frommononuclear cell pools by adherence separation. The mononuclear cells were resuspend-ed in growth medium (RPMI 1640 + 30% heat-inactivated human ultraserum fromGemini, 100 U/ml penicillin G and 100 µg/ml streptomycin sulfate purchased fromSigma). Cells in growth medium were placed in 75-cm2 tissue culture flasks and incubat-ed for 2 h at 37 ˚C with 5% CO2. Nonadherent cells were removed by aspiration and theremaining adherent cells (monocytes) were rinsed twice in RPMI, scraped off by celllifter (Costar Scientific, Cambridge, MA) and centrifuged at 1,800 rpm for 5 min. The cellpellets were washed twice in RPMI then collected and tested for purity by FACS.

Murine bone marrow progenitors were purified based on an established protocol16.Bone marrow cells taken from murine femurs and tibia were flushed out and washed byEMEM (MEM with Earle salt from Gibco-BRL, Gaithersburg, MD, 100 U/ml penicillinG and 100 µg/ml streptomycin sulfate). Mononuclear bone marrow cells were purified bydensity gradient (Histopaque-1077), and B and T lymphocytes consecutively depletedwith anti-B220- and anti-Thy1.2-magnetic beads (Dynal, Oslo) following manufacturer’sprotocols. The purified mononuclear cells were cultured in EMEM-10 (EMEM with 10%heat-inactivated FBS) with 20 ng/ml of M-CSF (Sigma).

Plasmids and primers. BLIMP-1 cDNA, a gift of M.M. Davis at Stanford University,was cloned into pBluescript (pSK) (Stratagene, La Jolla, CA). PRDI-BF1 (humanBLIMP-1 homolog) cDNA, a gift of T. Maniatis at Harvard University, and human MyccDNA (exons 2 and 3) were cloned into pBluescript (pKS). The pKS-PRDI-BF1, pKS-hMYC and pTRI-human glyceraldehyde-3-phosphate dehydrogenase (pTRI-hGAPDH) (Ambion, Austin) were used for making probes for northern blottingand ribonuclease protection assay.

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The retroviral construct, pMSCV-IRES-GFP and pMFG-GFP were a gift from G.P.Nolan at Stanford University39. The VSV-G plasmid was a gift of S. Goff at ColumbiaUniversity. Ψ−ECO (packaging DNA) was a gift of O. Witte from UCLA. HA-tagged T-BLIMP, a truncated form of (mouse homolog) BLIMP-1 from bp 1612–2788 of theopen reading frame, was cloned into pSK and pBABE40,41. pBABE-HA-T-BLIMP andpBABE were linearized before using for stable transfection. The pSK-HA-T-BLIMP wasused for ribonuclease protection assay to detect T-BLIMP.

Transfection and infection. Stable transfection was performed by pulsing 0.3 ml of cellssuspended in growth medium (5–10 × 106 cells/ml), at 240 V, 960 µF (Bio-RadElectroporator, Richmond, CA). After electroporation cells were resuspended in 10 ml ofgrowth medium, transferred to 96-well plates and allowed to recover for 2 days beforeadding the selection drug, puromycin, at a final concentration of 1 µg/ml. After about 3–4weeks of drug selection, individual clones were expanded from 96-well plates. Limiteddilution was performed in case when more than 20 individual clones were drug-resistantduring 96-well plates of drug selection.

Retrovirus was prepared according to Nolan’s protocol (web address as before) withthe following modifications. Three DNA vectors (retroviral construct, VSV-G and Ψ−ECO) were used for calcium phosphate transfection into Phoenix cells. After trans-fection for 2 days the supernatant contained infectious viruses that were concentrated, asdescribed previously43, and used for infecting cells of interest. Cells were analyzed afterthree days of infection.

RNA analysis. Total RNA was prepared by the guanidium thiocyanate procedure asdescribed43. Ribonuclease protection assay was performed based on the established pro-tocol44. Antisense cRNA probes were generated using T3 or T7 RNA polymerase(Promega, Madison, WI) with α[32P] Uridine and cDNA templates (prepared by restric-tion digestion and gel purification). Probes (1 × 106 c.p.m.) were added to 10 µg of totalRNA in hybridization buffer—80% formamide, 40 mM PIPES buffer (pH 6.4), 400 mMNaCl and 1 mM EDTA—denatured at 85 ˚C for 3 min, hybridized for 12 h at 45 ˚C. Afterhybridization the reaction was treated with 1:500 dilution of RNAse cocktail (Ambion)for 1 h at 30 ˚C, treated with 0.125 mg of Proteinase K and 0.5% SDS at 37 ˚C for 15 min,extracted with phenol and precipitated in ethanol with yeast tRNA as carrier. After pre-cipitation the dry pellet was resuspended in 5 µl of loading buffer and separated at 5%sequencing gel, prepared as described44.

Northern blotting was performed essentially as described44. GAPDH mRNA level wasused as an internal control in all experiments. Gel imaging was done usingPhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Flow cytometric analysis. Cell cycles analysis and immunofluorescent staining wereperformed according to manufacturer’s protocols (Pharmingen, San Diego, CA) and ana-lyzed by FACScan and Cell Quest software (Becton Dickinson, San Jose). Phycoerythrin(PE)-conjugated antibodies to CD11b and CD11c, fluorescein isothiocyanate (FITC)-con-jugated antibodies to CD14 and CD45, and 7-AAD (Via-Probe for dead cell exclusion)were all purchased from Pharmingen. 5-Bromo-2′ deoxy-uridine (BrdU) and propidiumiodide (for cell cycle analysis) were purchased from Roche (Indianapolis, IN).

Cytostaining. For morphological studies 5 × 104 cells were washed in PBS buffer andspun down on microscope slides using Cytospin 2 (Shandon Inc., Pittsburgh, PA). Slideswere air-dried before cystostaining. For Wright-Giemsa staining, the Diff-Quik Stain Set(Baxter Healthcare Corp., Miami, FL.) was employed according to manufacturer recom-mendation. Nonspecific esterase staining was done according to manufacturer’s proce-dure (α-naphthyl butyrate esterase kit, Sigma). The nitro-blue tetrazolium (NBT) reduc-tion assay was performed as described25.

For immunocytochemical staining, cells, either grown as adherent cells on coverslipsor prepared by cytospin on slides, were dried at room temperature for 1 h, fixed for 10min in 10% buffered formalin and 10 min in methanol. Cells were stained first with pri-mary antibody (polyclonal rabbit anti-mouse–BLIMP-1) and incubated overnight in TBSbuffer with 1% bovine serum albumin. The sections were then washed in Tris-bufferedsaline (50 mM Tris pH 7.5, 0.1% Tween-20) and counterstained with 1:200 diluted biotin-conjugated, mouse and human sera adsorbed, goat anti-rabbit (Southern BiotechnologyAssociates, Birmingham, AL). Finally, HRP-streptavidin was added and, after washing,developed with aminoethyl carbzole (Sigma). After color development, sections wereincubated at room temperature overnight with monoclonal antibody to CD11b(Pharmingen) at a 1:50 dilution. After washing, sections were incubated with AP-conju-gated anti-mouse immunoglobulin (Southern Biotechnology Associates) at a 1:200 dilu-tion. AP was developed by fast blue and Naphtol AsBi-Phosphate (Sigma) substrate,which gives a blue color. Slides were lightly counterstained with hematoxylin.

Phagocytosis assay. The phagocytosis assay was carried out essentially as described45.Heat-killed Staphylococcus aureus (ATCC) were labeled with 0.02 mg/ml of FITC iso-mer I (Sigma) for 30 min at 37 ˚C with constant mixing, washed three times with Hanksbalanced salt solution (HBSS) and resuspended in aliquots of ∼ 1 × 109 cells/ml in85%/15% HBSS/glycerin v/v before being stored at -70 ˚C.

To perform the phagocytosis assay, S. aureus were sonicated and opsonized for 30min at 37 ˚C with an equal volume of human serum. Bacteria at a final concentration of1 × 108 cells/ml were incubated with approximately 1 × 106 cells/10 ml of media (treat-ed or not treated with PMA) for 2 h at 37 ˚C. To quench the signal from externally bound

bacteria, cells were incubated with red blood cell lysis solution (Sigma) for 5 min,washed twice with PBS buffer and treated with 20 µl/0.5 ml of Trypan Blue. Cells thatinternalized the FITC-labeled bacteria were detected by FACScan.

Acknowledgments

We thank R. Dalla-Favera, S. Greenberg, C. Schindler, S. Silverstein and K.-I. Lin forreading the manuscript and for discussions, D.J. Husemann and J. Rooney for discus-sions and technical advice, G. Cattoretti for technical support.We are grateful tomembers of the Calame laboratory especially J. Liao for technical assistance and J.Yufor providing the T-BLIMP construct. Supported by National Institutes of HealthCancer Biology Training grant 2 T32 CA09503-14 (to D.H.C) and RO1-AI43576 (to K.C).

Received 24 April 2000; accepted 29 June 2000.

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