Hematopoietic Placental Protein 14 - Europe PMC

American Journal of Pathology, Vol. 145, No. 6, December 1994 Copyright C) American Society for Investigative Pathologv

Hematopoietic Placental Protein 14 An Immunosuppressive Factor in Cells of the Megakaryocytic Lineage

Dwight M. Morrow,* Na Xiong,* Robert R. Getty,* Mariusz Z. Ratajczak,t Doris Morgan,* Markku Seppala,§ Leena Riittinen,§ Alan M. Gewirtz,t and Mark L. Tykocinski* From the Institute of Pathology,* Case Western Reserve University, Cleveland, Ohio; Departments of Pathology and

Internal Medicine,t University of Pennsylvania, Philadelphia, Pennsylvania; Department of Hematology,* Hahnemann University, Philadelphia, Pennsylvania; and First Department of Obstetrics and Gynecology,5 Helsinki University Central Hospital, Helsinki, Finland

Placentalprotein 14 (PP14), an immunosuppressive molecule previously known to be expressed in the female and male reproductive tracts only, was shown to be expressed by hematopoietic ceUs of the megakaryocytic lineage. Northern blot analysis confirmed the induction specificity of PP14 mRNA in phorbol ester-treated K562 ceUs. Potent immunosuppressive activity in conditioned medium from phorbol ester-treated K562 cells was attributed to hematopoietic PP14 by anti-PP14 antibody blocking. Immunoprecipitation with anti-PP14 antibodies from conditioned medium revealed two distinct PP14 protein isoforms, designated PP14.1 and PP14.2. Polymerase chain reaction cloning and analysis demonstrated the presence of distinct mRNA counterparts to PP14.1 and PP142 that had not been resolved by Northern blot analyses. Hematopoietic PP14.1 mRNA corresponds in size to endometrial PP14 mRNA, whereas the smaUer hematopoietic PP14.2 mRNA displays an internal in-frame 66-nucleotide deletion that can be explained by alternative splicing and predicts a 22amino-acid deletion in the encoded gene product. Both PP14 mRNA isoforms were additionaly detected by reverse transcriptase polymerase chain reaction analyses in two human megakaryocytic ceU lines and in normal human

megakaryocytes and platelets. PP14 mRNA was not detected by reverse transcriptasepolymerase chain reaction in a panel of nonhematopoietic, nonendometrial tissues examined. Thefinding of hematopoietic PP14 within the megakaryocytic lineage provides an additional regulatory link between the coagulation and immune systems in normal and pathological settings. (Am J Pathol 1994, 145:1485-1495) Human placental protein 14 (PP14) is a glycoprotein expressed by endometrial decidua during the first and second trimesters of pregnancy. During this period, it constitutes from 5 to 10% of the total secreted decidual protein.1' 2 PP14 is present in high concentrations in the amniotic fluid during the first half of pregnancy and accumulates to significant levels in serum from pregnant women as well.1 In addition, PP14 is found at high concentrations in male seminal fluid (19 to 515mg/1).3,4 To date, no systematic search for the expression of PP14 in tissues outside of the reproductive tracts of women and men has been reported. Whereas the precise physiological roles of PP14 remain uncertain, the most intriguing possibilities stem from its documented immunoregulatory function. PP14 has been shown to inhibit lymphocyte proliferation.5 In these experiments, extracts of human decidual tissue were added to mixed lymphocyte culSupported in part by grants from the NIH (MLT and AG), the Sigrid Juselius Foundation (MS), the Cancer Society of Finland (MS), the University of Helsinki (MS), and the Finnish Social Insurance Institution (MS and LR). Accepted for publication August 16, 1994. Address reprint requests to Dr. Mark L. Tykocinski, Institute of Pathology, Case Western Reserve University, Biomedical Research Building, Room 925, 10900 Euclid Avenue, Cleveland, OH 441064943. Current address of Dr. Morrow is: Department of Molecular Biology and Genetics, The Johns Hopkins University, Baltimore, MD 21205.

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tures (MLCs), and a linear relationship was observed between the quantity of PP14 present and the inhibition of lymphocyte proliferation observed. Moreover, anti-PP14 antibodies added to the MLCs inhibited the anti-proliferative effect, verifying the functional link between PP1 4 and anti-proliferative activity. Subsequent studies have noted an inhibitory effect of PP14 on the synthesis of interleukins 1 and 2 and soluble interleukin 2 receptors by peripheral blood mononuclear cells, suggesting potential explanations for the anti-proliferative effect of PP14 on lymphoid cells.6-8 It has been further shown that PP14 can suppress natural killer activity.9 Although it is tempting to speculate that there may be a link between PP14's immunosuppressive capacity and survival of the developing fetal semi-allograft (expressing both paternal and maternal antigens), as well as the absence of significant immune reactions to paternal antigens on spermatozoa and in seminal fluid, there is as yet no definitive data to substantiate such physiological roles. In this study, we describe the intriguing finding of PP14 mRNA and protein species in hematopoietic cells. This first observation of PP14 outside of the female and male reproductive tracts has ensued from experiments with the human myelogenous leukemia cell line K562. This leukemic line has bipotential differentiative capacities, as it can be chemically induced to differentiate along erythroid10 and megakaryocytic11 lineages. In the course of cloning cDNAs that are differentially expressed after induction along the megakaryocytic lineage, we identified PP14 cDNAs. We now report the characterization of the two mRNA and protein isofQrms that comprise hematopoietic PP14. It is further shown that mRNA encoding this potent immunoregulatory cytokine is present in normal megakaryocytes and platelets, providing a potentially significant connection between the coagulation and immune systems.

Materials and Methods Cell Culture The human myelogenous leukemic cell line K562 (ATCC 243) was maintained in RPMI medium (Whittaker Bioproducts, Walkersville, MD), supplemented with 10% heat-inactivated fetal calf serum, 10 mmol/L HEPES (pH 7.2), 40 pg/ml gentamicin, and 2 mmol/L glutamine. MBO-2 and HU-3 megakaryocytic cell lines12 were grown in RPMI 1640 containing 10% hu-

man serum and granulocyte-macrophage colonystimulating factor supplement. Cells were grown at 37

C with 5% CO2. The phorbol ester used was phorbol 12-myristate 13-acetate (PMA; Sigma, St. Louis, MO) which was diluted in dimethyl sulfoxide and stored at -20 C at 1 to 3 mmol/L until use. Final concentrations of PMA used ranged between 100 and 10 nmol/L. To determine the effect of PMA on K562 cell differentiation, K562 cells were cultured at 2 x 105 cells/ml in tissue culture flasks. PMA was added to the cell cultures for 24, 48, and 72 hours before cells or cell supernatants were removed for analysis. For K562 cells, differentiation induction by PMA and hemin was monitored by using platelet glycoprotein IIIA (gpllla) protein (PMA), granulocytemacrophage colony-stimulating factor RNA (PMA), erythrocyte-potentiating activity RNA (PMA), and glycophorin protein (hemin) as differentiation markers, respectively. For the other leukemic lines, differentiation markers included CD14 protein (HL-60/PMA); PLB-985/PMA) and the capacity to reduce nitroblue tetrazolium (HL-60/DMSO; U937/PMA).

Isolation of Normal Human Megakaryocytes Human megakaryocytes were enriched from bone marrow aspirated from normal consenting, remunerated donors. Aspirated marrow (-15 ml) was suspended in megakaryocyte collection medium13 and then subjected to counterflow centrifugal elutriation (Beckman J2-21M; standard elutriation rotor; Beckman Instruments, Mountain View, CA) as we have previously reported.14,15 Megakaryocytes enriched approximately 200-fold by elutriation were further enriched to >99% purity by isolating morphologically recognizable mature megakaryocytes with a micromanipulator (Narishige, Japan). Individual megakaryocytes were aspirated into a microcapillary tube pipet and transferred into a 1.5-ml microcentrifuge tube containing Iscove's modification of Dulbecco's minimal essential medium (GIBCO BRL, Gaithersburg, MD) supplemented with 10% bovine calf serum (Hyclone Laboratories, Logan, UT). RNA was extracted from the cells immediately after collection of approximately 1000 megakaryocytes.

Isolation of Platelets Citrated blood was centrifuged at 140 x g for 15 minutes at room temperature. The platelet-rich plasma supernatant was isolated and centrifuged at 2500 x

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g for 15 minutes at room temperature. Platelet pellets were stored at -70 C.

cDNA Library Assembly and Differential Screening Total RNA was extracted from cells by the guanidinium isothiocyanate/CsCI method, and poly(A)+ RNA was isolated by using oligo(dT) cellulose chromatography as described.16 The cDNA library was constructed with a pool of poly(A) RNA isolated from K562 cells treated with PMA (100 nmol/L) for 24, 48, and 72 hours (1.5 pg per time point; 4.5 pg poly(A)+ RNA total). The library was constructed with the Uni-ZAP XR cloning kit according to manufacturer's recommendations (Stratagene, La Jolla, CA). After synthesis of double-stranded cDNA, the cDNA was ligated into the Uni-ZAP XR vector and packaged into Gigapack 11 Gold packaging extracts (Stratagene) according to the manufacturer's protocol. To differentially screen the induced K562 cDNA library, 60,000 individual primary clones were used to infect PLK-F' bacteria (Stratagene). Duplicate membrane lifts were taken using Stratagene Duralon UV membranes. The membranes were denatured, neutralized, and UV crosslinked according to manufacturer's recommendations. Membranes for the primary screens were hybridized with single-stranded cDNA probe from untreated K562 cells and compared to duplicate lifts hybridized with subtracted, single-stranded cDNA probe from PMA-treated K562 cells. Secondary and tertiary screens were hybridized with single-stranded cDNA probe from untreated K562 cells and compared to duplicate lifts hybridized with nonsubtracted, singlestranded cDNA probe from PMA-treated K562 cells. To generate the cDNA probe, 1 pg of poly(A)+ RNA was primed with oligo(dT)12-18 and reverse transcribed using 10 U Superscript (GIBCO) according to manufacturer's recommendations. To generate the subtracted, induced cDNA probe, poly(A)+ RNA (1 pg) from K562 cells treated with PMA was reverse transcribed as described. After RNA hydrolysis and cDNA precipitation, the singlestranded cDNA was annealed with a 30-fold excess of photobiotinylated poly(A)+ RNA from untreated K562 cells and subtracted according to manufacturer's suggestions (Invitrogen, San Diego, CA). The Duralon UV membranes were prehybridized, hybridized, and washed according to manufacturer's protocol.

Northern Blot and Polymerase Chain Reaction (PCR) RNA Analyses Total RNA (10 pg) was isolated as described above, heated to 65 C for 15 minutes in 50% formamide, 6% formaldehyde, 1X EPPS (N-(2-hydroxyethyl)-piperazine-N'-3-propanesulfonic acid) buffer (1X EPPS buffer contains 20 mmol/L EPPS (pH 8.2), 10 mmol/L sodium acetate (pH 5.2), and 2 mmol/L EDTA) and separated on 1.2% agarose gels containing 1X EPPS buffer and 6% formaldehyde. The RNA was passively transferred to Duralon UV membranes and UV crosslinked. Membranes were prehybridized as described above. Probe was generated by random priming 20 ng of purified PP14 DNA. The DNA was denatured by heating to 100 C for 10 minutes, then incubating for 30 minutes at 37 C in 50 pCi a-[32P]dCTP (Amersham, Arlington Heights, IL), 0.2 mmol/L each dGTP, dTTP, and dATP, and 10 U Klenow enzyme in a buffer containing random hexanucleotides (Boehringer Mannheim, Indianapolis, IN). Probe was hybridized overnight at 42 C. Membranes were washed and exposed as described above. For the reverse transcriptase (RT)-PCR analysis shown in Figure 6, total RNA was reverse transcribed with primer 1 (5'-GGATCCCATGCTCCAAGGGTTTATTAATAACCTCTGC-3') which includes a BamHI site. The resulting single-stranded DNA product was PCR amplified by using primer 2 for the 5' primer (5'-

GGTACCGCTCCAGAGCTCAGAGCCACCCACAG-3') which includes a Kpnl site, and primer 3 as the 3' primer (5'-GTGCAGAACGATCTCCAGGTTG3'). For the RT-PCR analysis shown in Figure 7, RNA was isolated from the megakaryocytic cell lines and platelets with the Ultraspec RNA isolation system (Biotecx Laboratories, Houston, TX). Total RNA was reverse transcribed using primer 4 (5'-ATGCTCCAAGGGTTTATTAATAACCTC-TGC-3') lacking the BamHI site of primer 1. Resulting product was PCR amplified with primer 5 (5'-CCCCAGACCAAGCAGGACCTGGAGC-3') and primer 6 (5'-CTTCTTTGGAT-

TCCCAGTCTTCTC-3'). For the RT-PCR analysis shown in Figure 8, normal megakaryocyte mRNA was isolated with a QuickPrep Micro mRNA purification kit (Pharmacia Biotech, Piscataway, NJ). To carry out RT-PCR, megakaryocyte mRNA was heated to 65 C for 10 minutes and then cooled on ice for 3 minutes. MMuLV-RT (100 U; GIBCO), 50 ng of random primers (Boehringer Mannheim), 40 U of RNAzin (Promega, Madison, WI) and dNTPs (50 pM) were added to the mRNA in the buffer previously described. This was incubated for 1 hour

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at 37 C. This cDNA was used for the nested PCR amplification of PP14. The primers used for the PP14 amplification were primer 7 as 5' primer (5'-GCTCAGAGCCACCCACAGCCGCAG-3') lacking the Kpnl site of primer 2 and primer 6 as 3' primer. A total of 30 ng of primers was added to the reaction tube along with 2.5 U Taq polymerase (Promega). PCR was performed for 30 cycles of 1 minute at 95 C and 1.5 minutes at 72 C. Two microliters of this product was used as a template for the second, nested PCR. Run conditions were identical and used 30 ng of primer 8 (which overlaps primer 5) as 5' primer (5'-CCCCAGACCAAGCAGGACCTGGAG-3') and primer 9 (which overlaps primer 3) as 3' primer (5'-GTGCA-

GAACGATCTCCAGGTTGTC-3'). For the RT-PCR analysis shown in Figure 9, total RNA was reverse transcribed with primer 1 and then amplified with primer 2 and primer 1. In this experiment, the tissue RNAs analyzed were from human brain, liver, small intestine, meninges, and kidney (kind gifts from Dr. Todd Golde and Linda Younkin, Case Western Reserve University, Cleveland, OH). RNA obtained from these tissues was shown to be intact by actin hybridization (data not shown). Other RNA samples were obtained from fibroblasts (Dr. Rick Hershberger, Case Western Reserve University), stromal cells (Dr. Matt Weber, Case Western Reserve University), and placenta. The PCR reactions were carried out in a buffer containing 25 mmol/L TAPS-HCI (pH 9.3), 50 mmol/L KCI, 2 mmol/L MgCI2, 1 mmol/L DTT, 200 pmol/L each dATP, dCTP, dGTP, and dTTP, and 2.5 U Taq polymerase (Perkin Elmer, Norwalk, CT). Amplification was performed on a PTC-100 thermal cycler (MJ Research, Watertown, MA) for 25 cycles of 1 minute at 94 C, 45 seconds at 65 C, and 1 minute at 72 C.

Immunoprecipitation and SDS-PAGE Analysis K562 cells were cultured at 5 x 106 cells/ml and treated with PMA as described above. Cells were labeled with 0.5 mCi [35S]cysteine and [35S]methionine (ICN Biomedicals, Inc., Irvine, CA) in cysteine- and methionine-free media supplemented with 10% fetal calf serum that had been extensively dialyzed in phosphate-buffered saline. The next morning cells were collected by centrifugation and supernatants were saved. Cells were lysed in 1% Triton X-100, 1% bovine serum albumin and 1 mmol/L phenylmethylsulfonylfluoride (Sigma). Supernatants were used directly. After preclearing cell lysates or cell supernatants with protein-G Sepharose (Pharmacia), 100 pl of

a 50% slurry of protein-G Sepharose was added with 2 pl of mouse monoclonal anti-PP14 antibody (1 05DH 1 Fl ).17 Alternatively, the preclearing step was carried out with protein-A Sepharose (Pharmacia), and immunoprecipitation was performed with 100 pl of a 50% slurry of protein-A Sepharose and 2 p1 (0.125 mg/ml) of affinity-purified-polyclonal anti-PP14 antibodies. The latter polyclonal antibodies were prepared by immunizing rabbits with purified human PP1 417 and affinity purifying polyclonal anti-PP14 antibodies from rabbit sera with PP14 Sepharose. After incubating overnight at 4 C with gentle rotation the beads were pelleted by centrifugation and washed as follows: five times in 0.1% Triton X-100 and 0.1% bovine serum albumin; one time in 0.01 mol/L Tris-HCI (pH 8.0), 0.14 mol/L NaCI, and 0.025% NaN3; and finally one time in 0.01 mol/L Tris-HCI (pH 7.5). Bound protein was eluted by boiling and electrophoresed on a 3% stacking, 12% resolving SDS-PAGE gel. The gel was dried and exposed as above.

MLC To assess the effect of supernatants from K562 cells on peripheral blood lymphocyte proliferation, MLCs were set up as follows. Blood from two unrelated donors was collected in heparin (10 U/ml blood). Blood was diluted two times in 1 x phosphate-buffered saline, and 0.3 volumes Ficoll-Paque (Pharmacia, Uppsala, Sweden) was under-layered and then centrifuged for 30 minutes (1000 x g). The leukocyte layer was removed, washed three times with 1 x phosphate-buffered saline and lymphocytes were counted in a hemocytometer. Cells were cultured in 96-well plates at 2 x 105 cells per well in triplicate. Supernatants, when used, were added to 50% v/v in cell culture wells. Antibody was added directly at a 1:100 dilution. Cells were cultured for 5 to 7 days as described above. Twelve to fifteen hours before harvesting 0.5 pCi [3H]thymidine (New England Nuclear, Boston, MA) was added to each well. Cells were harvested with a MiniMash 11 cell harvester, filters were dried, and counted in a 1-ml scintillation cocktail with a Beckman (Fullerton, CA) LS3801 beta counter.

PCR Cloning and DNA Sequencing PCR cloning was performed by annealing a PP14 3'-specific primer (5'-CATGCTCCAAGGGTTTATTAATAACCTCTGC-3') and reverse transcribing as described above. The resulting product was used directly in a PCR (described above) by using the

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3'-specific primer and a 5'-specific primer (5'-AGCTCAGAGCCACCCACAGCCGCAG-3'). The PCR product was gel purified and cloned into a T vector. DNA sequencing was performed with the Sanger sequencing method and the Sequenase sequencing kit according to the manufacturer's recommendations (US Biochemical, Cleveland, OH).

Results K562 leukemia cells can be induced to differentiate along the megakaryocytic lineage by PMA.11 Megakaryocytic markers known to be expressed by such cells include gplla, platelet-derived growth factor a and 1 chains, and transforming growth factor f3 (TGF-f3).18 To study changes in the mRNA expression profile that accompany K562 differentiation along this lineage, we set out to clone mRNAs that are differentially expressed after PMA induction of this cell line. A phage cDNA library was constructed from a pool of three groups of K562 cells treated with PMA for 24, 48, and 72 hours, respectively. Such a pool was chosen to maximize chances of finding different genes activated throughout the stochastic K562 differentiation program. Duplicate lifts of the induced K562 cDNA library were differentially screened with subtracted (induced minus uninduced K562) and nonsubtracted (uninduced K562) single-stranded probes, as detailed in Materials and Methods. Approximately 60,000 cDNA clones were analyzed, and 127 putative positives were selected after the first round of screening. After analysis and sequencing of all the clones in a random fashion and subsequent sequence comparisons with the Genbank sequence database,19 2 of the 127 cDNA clones were identified as partial PP14 cDNAs. Verification of PP14 expression in the K562 line and confirmation of its inducibility by PMA was accomplished by Northern blot analyses (Figures 1 and 2). Total cellular RNA from untreated K562 cells or from 0

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Figure 1. Kinetics qf PP14 mnRNA inducibility in PMA-induced K562 cells. Total RNA from K562 cells induced with PMA (10 nmol/L) for 0, 0.5, 1, 2, 3, 6, 12, or 24 houts uwas isolatced atnd hybridized to a [32PI-labeled PP14 cDNA probe. Actin RNA hybridization controls wvere performed to verzify RNA quality and comparable RNA loading

(not shoun). The approximately 800-bp band corresponding to PP14 mRNA is indicated by an arrouhead. The size estimate was on the basis of rRNA markers.

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Figure 9. Search for PP14 mRNA outside of the reproductive and hematopoietic systems. Total RNA (2.5 lig) uas reverse transcribed uisitng a 3'end-specific primer/or PP14. The resulting product u'as utsed directly itn a PCR reaction with a 5-end- specific primerfor PP14. Lane 1. untinduced K562 cells; lane-s 2 and 3, PMA-induiced K562 cells at 24 anid 48 houirs, respectiv'ell; lane 4, brain; lane 5, spleen; lane 6, small intestine, lanle 7, liver; lane 8, kidnievy latne 9, braini meninges; lane 10, KI I-102 bone marroufibroblastoid stromal cell/, lane I1, placenta at 18 uveeks; lane 12, placenta at term; latne 13, skin fibroblasts; lane 14, primers alone; lane 1imiolecular ueight markers.

tissues examined are all negative for PP14 transcripts. These tissues include brain (lane 4), spleen (lane 5), small intestine (lane 6), liver (lane 7), kidney (lane 8), and meninges (lane 9). Similarly, two fibroblastic cell lines, KM-102 (human bone marrow stromal cells; lane 10) and skin fibroblasts (lane 13) were also negative. The smaller PCR products seen in these lanes are PCR artifacts. Hence, PP14 mRNA expression seems to be restricted to the reproductive and hematopoietic systems.

Discussion PP14, originally named after the placental tissue it later shown to originate from associated endometrial tissue.2 Subsequent studies indicated that PP14 was present not only in the endometrial decidua and serum of pregnant women, but also in the seminal fluid of men.3 We now report that PP14 is produced in cells outside of the reproductive tract as well. PP14 mRNA and protein has been identified in a human leukemic cell line induced to differentiate along the megakaryocytic lineage, as well as in megakaryocytic cell lines, normal megakaryocytes, and platelets. PP14 mRNA analyses have further established that whereas endometrial PP14 mRNA is composed of a single dominant species, 28 hematopoietic PP14 mRNA is composed of two molecular species. To simplify considerations of these two mRNA species and the encoded proteins, we have proposed referring to the larger hematopoietic isoform comigrating with the dominant endometrial PP14 species as PP14.1 and to the smaller hematopoietic isoform as PP14.2. The hematopoietic PP14.2 mRNA isoform contains an internal sequence gap, relative to PP14.1 mRNA, that is predicted to yield a 22-amino-acid deletion in the encoded protein. The correspondence of the PP1 4.2-associated deletion to the 5' end of exon 2 of the PP14 gene, as well as the presence of a consensus splice acceptor site at the deletion boundary, are consistent with the likelihood that the two PP14 variants arise through alternative splicing. Interestingly, the PP1 4.2 mRNA species corresponds to one of several minor variant endometrial a2-globulin mRNAs

was thought to derive from,27 was

previously reported-28 Endometrial a2-globulin cDNA sequence is almost identical to that of PP14, and hence the two are presumed to be the same, with isolated sequence differences reflecting polymorphisms. In that study, the endometrial a2-globulin 22amino-acid deletion variant represented only 1 of 34 endometrial a2-globulin mRNAs cloned from first trimester endometrial tissue. Moreover, the investigators of that study reported difficulty in demonstrating the existence of polypeptides corresponding to this and the other mRNA variants, suggesting to them that either translation is poor or the proteins are unstable. In contrast, in the case of hematopoietic PP14, the shorter mRNA variant and its associated protein are readily detectable. The rarity of the 22-amino-acid deletion variant clone in endometrium is consistent with the possibility that the single clone of the previous study originated from hematopoietic cells contaminating the endometrial tissue used as the source of RNA. If so, PP14.2 would indeed be hematopoietic cell specific. Hematopoietic PP14.1 comigrates with the dominant endometrial a2-globulin mRNA that lacks internal deletions in the coding sequence. However, Garde et al28 demonstrated three other minor variants with the same coding sequence and differing only by short stretches in 3'-untranslated sequence. To date, we have characterized a single hematopoietic PP14.1 clone that actually corresponds to one of these endometrial minor variants, displaying a 29-bp insertion in the 3'-untranslated region (Y. Feng, D. Morrow, and M. Tykocinski, unpublished observations). Such a sequence variation could affect mRNA translatability and/or stability. However, additional RTPCR cloning and analysis will be required to determine how representative this clone is for the molecular species comigrating in the hematopoietic PP14.1 band. The amino acid sequence of hematopoietic PP1 4. 1 absent from hematopoietic PP14.2 is slightly hydrophobic. Of interest, one of the three potential N-glycosylation sites of hematopoietic PP14.1 resides within this stretch. It remains to be determined whether loss of a short hydrophobic stretch and/or

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loss of a potential N-glycosylation site in hematopoietic PP14.2 has functional consequences. Moreover, inasmuch as the functional form of endometrial PP14 has been reported to be a homodimer,29 it will be of interest to determine whether PP14.2 is dimeric as well. The anti-PP14 monoclonal antibody used in this study co-immunoprecipitated both PP14.1 and PP14.2, a finding consistent with heterodimer formation and/or antibody cross-reactivity. PP14.2 isoformspecific antibodies and co-transfection analyses will be required to definitively resolve this issue. Inasmuch as endometrial PP14 homodimers are relatively unstable, a PP1 4.2-containing dimer could potentially turn out to be more useful as an immunoregulatory protein for T cell and/or natural killer cell inhibition in treating acute inflammatory disorders. The survey of human leukemic cell lines reported in this study indicates that PP14 is not promiscuously expressed in leukemic lines. In fact, even in K562 cells, it is only expressed after chemical induction with one particular chemical inducer. Nonetheless, it is tempting to speculate that under in vivo conditions, leukemic cells that share the differentiative phenotype of K562 may be triggered into a more differentiated state in which they can express PP14. This could occur spontaneously or in response to therapeutic agents. If this were the case, PP14, by virtue of its potent immunosuppressive function, could play a pathogenic role in blocking effective anti-tumor immune responses. This could certainly be the case for genuine megakaryocytic leukemias. Such a role has been suggested for tumor-derived TGF-P,30'31 another cytokine with potent anti-proliferative activity directed at T cells.32 However, unlike PP1 4, TGF-,3 must be activated by proteolysis in order to exert its immunosuppressive effects.33 As was shown here for K562 leukemic cells, the secreted PP14, but not necessarily secreted TGF-1, is already in the biologically active form. It has been shown that at least one tumor, a glioblastoma, which produces significant amounts of TGF-13 and whose conditioned medium is immunosuppressive, can be inhibited by protease inhibitors.31 It remains to be determined whether PP14 expression is a property of leukemic cells in patients in whom it could play a role in the initiation and/or maintenance of leukemia. The present finding of hematopoietic PP1 4 mRNAs in the end cell of the megakaryocytic lineage, the platelet, may be clinically significant. Potentially, PP14 might provide a critical link between the coagulation and immune systems. For instance, it is tempting to speculate that platelet-derived PP1 4 could play a role in the resolution of inflammatory processes at wound healing sites. Moreover, it also seems possible

that PP14 release could contribute to immunosuppression in the context of coagulopathies. Ongoing studies should resolve some of these issues, shedding light on the physiological and pathophysiological roles of hematopoietic PP14.

Acknowledgments We thank Dr. R. Yomtovian for providing us with human platelets and Dr. J. Ilan for endometrial decidual RNA. We also thank Drs. R. Redline, S. Emancipator, and C. Kaetzel for their valuable advice. We appreciate Dr. M. Lamm for his support throughout this project.

References 1. Julkunen M, Rutanen E-M, Koskimies Al, Ranta T, Bohn H, Seppala M: Distribution of placental protein PP14 in tissues and body fluids during pregnancy. Br J Obstet Gynaecol 1985, 92:1145-1151 2. Julkunen M, Koistinen R, Sjoberg J, Rutanen E-M, Wahlstrom T, Seppala M: Secretory endometrium synthesizes placental protein 14. Endocrinology 1986, 118:1782-1786 3. Julkunen M, Wahlstrom T, Seppala M, Koistinen R, Koskimies Al, Stenman U-H, Bohn H: Detection and localization of placental protein 14-like protein in human seminal plasma and in the male genital tract. Arch Androl 1984, 12:59-67 4. Pockley AG, Barratt CLR, Bolton AE: Placental protein 14 (PP14) content and immunosuppressive activity of human cervical mucus. Symp Soc Exp Biol 1989, 43: 317-323 5. Bolton AE, Pockley AG, Clough KJ, Mowles EA, Stoker RJ, Westwood OMR, Chapman MG: Identification of placental protein 14 as an immunosuppressive factor in human reproduction. Lancet 1987, 1:593-595 6. Pockley AG, Bolton AE: Effect of decidual placental protein 14 on interleukin-2 lymphocyte interactions. Biochem Soc Trans 1988, 16:794 7. Pockley AG, Bolton AE: Placental protein 14 (PP14) inhibits the synthesis of interleukin-2 and the release of soluble interleukin-2 receptors from phytohaemagglutinin-stimulated lymphocytes. Clin Exp Immunol 1989, 77:252-256 8. Pockley AG, Bolton AE: The effect of human placental protein 14 (PP14) on the production of interleukin-1 from mitogenically stimulated mononuclear cell cultures. Immunology 1990, 69:277-281 9. Okamoto N, Uchida A, Takakura K, Kariya Y, Kanzaki H, Riittinen L, Koistinen R, Seppala M, Mori T: Suppression by human placental protein 14 of natural killer cell activity. Am J Reprod Biol 1991, 26:137-142 10. Andersson LC, Jokinen M, Gahmberg CG: Induction of erythroid differentiation in the human leukaemia cell line K562. Nature 1979, 278:364-365

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11. Gewirtz A, Berger D, Rado TA, Benz EJ, Hoffman R: Constitutive expression of platelet glycoproteins by the human leukemia cell line K562. Blood 1982, 60: 785-789 12. Morgan DA, Gumucio DL, Brodsky I: Granulocytemacrophage colony-stimulating factor-dependent growth and erthropoietin-induced differentiation of a human cell line MB-02. Blood 1991, 78:2860-2871 13. Gewirtz A, Boghosian-Sell L, Catani L, Ratajczak M, Shen Y-M, Schreiber AD. Expression of FCyRII CD4 receptors by normal human megakaryocytes. Exp Hematol 1992, 20:512-516 14. Gewirtz A, Keefer M, Doshi K, Annamali A, Chiu HC, Colman RW: Biology of human megakaryocyte factor V. Blood 1986, 67:1639-1648 15. Gewirtz, A.: Recent methodologic advances in the study of human megakaryocyte development and function. Pharmacologic Methods for the Investigation of Coagulation and Platelets. Edited by RW Colmun and BJ Smith. New York, Alan R. Liss, 1987, pp 1-17 16. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Current Protocols in Molecular Biology. Boston, Greene Publishing Associates and Wiley-lnterscience, 1994, pp 4.2.1-4.5.3 17. Riittinen L, Narvanen 0, Viranen I, Seppala M: Monoclonal antibodies against endometrial protein PP14 and their use for purification and radioimmunoassay of PP14. J Immunol Methods 1991, 136:85-90 18. Alitalo R: Induced differentiation of K562 leukemia cells: a model for studies of gene expression in early megakaryoblasts. Leuk Res 1990, 14:501-514 19. Pearson WR, Lipman DJ: Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 1988, 85:2444-2448 20. Julkunen M, Seppala M, Janne 0: Complete amino acid sequence of human placental protein 14: a progesterone-regulated uterine protein homologous to f-lactoglobulins. Proc NatI Acad Sci USA 1988, 85: 8845-8849 21. Rovera G, Santol D, Damski C: Human promyelocytic leukemia cells in culture differentiate into macrophagelike cells when treated with a phorbol diester. Proc Natl Acad Sci USA 1979, 76:2779-2783 22. Tucker KA, Lilly MB, Heck L, Rado TA: Characterization of a new human diploid myeloid leukemia cell line (PLB-985) with granulocytic and monocytic differentiating capacity. Blood 1987, 70:372-378 23. Riittinen L, Stenman U-H, Alfthan H, Suikkari A-M, Bohn H, Seppala M: Time-resolved immunofluoromet-

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