©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Lactoferrin Down-modulates the Activity of the Granulocyte Macrophage Colony-stimulating Factor Promoter in Interleukin-1-stimulated Cells (*)

Silvana Penco , Sandra Pastorino , Giovanna Bianchi-Scarr , Cecilia Garrè (§)

From the (1) Institute of Biology and Genetics, University of Genova, 16132 Genova, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The human neutrophil lactoferrin (Lf), a cationic iron-binding glycoprotein, has an inhibitor role on granulocyte macrophage colony-stimulating factor (GM-CSF) production via interleukin-1 (IL-1). The nuclear localization of Lf suggests that it may be involved in the transcriptional regulation of GM-CSF gene expression. To explore this possibility, the effect of Lf on GM-CSF gene expression was investigated in various cell lines and in primary cultures of fibroblasts. Down-regulation of GM-CSF mRNA level was observed in Lf-transfected embryonic fibroblasts induced to produce GM-CSF by IL-1. In 5637 cell-line and in embryonic fibroblasts, co-transfection experiments, in which an Lf expression vector was used together with a vector carrying a reporter gene linked to the GM-CSF promoter, revealed that Lf reduces the activity of the GM-CSF promoter. This effect is marked in IL-1-stimulated cells. These findings suggest that Lf plays a negative role in GM-CSF expression at the transcriptional level, perhaps through the mediation of IL-1.


INTRODUCTION

Human lactoferrin (Lf)() is a strongly cationic, 80-kDa glycoprotein, secreted at a high concentration in milk from glandular epithelia, and present, at a lower concentration, in other exocrine secretions. It is present in small amounts (1-10 nM) in plasma, and is derived from neutrophils in which the secondary granules synthesize and store Lf.

Various biological functions are attributed to Lf although its exact biological role remains unclear (1) .

The expression of neutrophil Lf begins at the myelocyte stage and appears to be a useful marker of terminal myeloid differentiation. Therefore, reduced or absent Lf gene expression is also found in conditions characterized by disordered myeloid differentiation, such as acute leukemia and myelodysplastic syndromes (2) . Neutrophil Lf has been implicated in immune and inflammatory responses and has been reported to be an inhibitor of myelopoiesis, since it decreases granulocyte macrophage colony-stimulating factor (GM-CSF) production/release (3) . This latter effect is secondary to the inhibited production of interleukin-1 (IL-1), an inducer of GM-CSF in physiological and pathological conditions (4, 5) .

Regulation of GM-CSF expression involves a combination of both transcriptional and post-transcriptional controls (6) . Recently, a conserved AU-rich sequence in the 3`-untranslated region of mRNA encoding GM-CSF was found to be responsible for selective mRNA degradation (7) , and binding proteins that recognize the AU-rich motif have been described (8) . Transcriptional regulation of GM-CSF plays an important role in activated T-lymphocytes, in fibroblasts and in endothelial cells. A region of 629 base pairs upstream of the transcriptional starting site has been well characterized, as well as some transcription factors that bind to this region (6) .

The molecular mechanism by which Lf controls GM-CSF production is still unknown. It has been shown that in mice macrophages 10 nM Lf is able to decrease the GM-CSF mRNA level after stimulation with fetal calf serum (9) .

Our aim was to find out how Lf can negatively modulate GM-CSF production. A possible clue is provided by the ability of Lf to interact with DNA, thus suggesting its possible involvement in GM-CSF gene regulation as a nuclear factor. We previously demonstrated that Lf, after binding to its specific receptor, can be translocated into the nucleus of K562 cells, where it binds DNA (10, 36) . Moreover, Lf DNA-binding fragments have been found in infant urine (11) . Recently it has been shown that Lf is able to specifically bind three consensus sequences supporting the hypothesis that Lf is a nuclear factor (12) .

We tested Lf action on GM-CSF gene expression in two cellular systems: continuous cell lines that constitutively produce GM-CSF and/or IL-1 and embryonic fibroblasts induced by IL-1 to produce GM-CSF. We report here that Lf can inhibit GM-CSF mRNA in embryonic fibroblasts. In IL-1-stimulated cells (5637 and fibroblasts) Lf down-regulates GM-CSF promoter activity.


EXPERIMENTAL PROCEDURES

Cells

The following cells were used: bladder carcinoma cell line 5637, which constitutively produces GM-CSF and IL-1 (13, 14) , histiocytic lymphoma cell line U937 producing IL-1 (15) , and T-lymphocyte cell line Mo, which produces GM-CSF (16) . Primary cultures of human embryonic lung fibroblasts (PEU cells), obtained from Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia-Brescia, Italy, were used in passages 10-25 in all experiments. Cell lines were maintained in RPMI 1640 medium, and PEU cells were maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal calf serum, 2 mM glutamine, 1 mM sodium pyruvate, and 0.05 mg/ml gentamycin. All induction experiments were performed at 70% confluence for adherent cells or at a concentration of 5 10 cells/ml for nonadherent cells. Cells were incubated with different amounts of iron-saturated Lf (Sigma), ranging from 1 pM to 0.1 µM at 37 °C, for the times indicated. Cells were utilized for total RNA preparation. Cellular conditioned medium was used for GM-CSF protein assay.

Transient Transfections

5637 cell line and PEU cells were plated at 70% confluence 24 h before transfection in 10-cm diameter plastic dishes in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, and 0.05 mg/ml gentamycin. Cells were transfected by the calcium phosphate coprecipitate method (17) . Transfections were performed with 10 µg of the p91023-B expression vector (16) containing Lf cDNA() (pLf), kindly provided by Dr. O. Conneely (Baylor College of Medicine-Houston-Texas) or with an equimolar concentration of a control plasmid pBluescript (pBS). Cells, 18 h later, were reincubated for a further 18 h with or without 15 units/ml IL-1 before harvesting. In other experiments, cells were co-transfected with 10 µg of pLf and 6 µg of pPF2000 containing the promoter region (position -2010 to +26) of the GM-CSF gene cloned in pBLCAT3 vector.() Transfection experiments included 4 µg of pRSV--galactosidase, as an internal standard. In control experiments, pBS in equimolar concentration was used instead pLf. For all plasmids, at least two different preparations were used. All experiments were performed in duplicate. Harvested cells were utilized for total RNA extraction and for cytoplasmatic extract preparation. Conditioned medium of transfected-cell was utilized for GM-CSF protein assay.

RNA Analysis

Total cellular RNA was purified by the guanidium isothiocyanate/cesium chloride method (18) and, from transfected cells, according to Chomczynski and Sacchi (19) . The integrity of RNA was assayed by evaluation of the 28 S/18 S RNA ratio on ethidium bromide-stained agarose gel. Purified RNA was size-fractionated by formaldehyde/agarose gel electrophoresis and transferred onto a nylon filter (20) . The blots were hybridized with Lf cDNA (2.0-kilobase pair EcoRI-TthIII fragment in p91023-B), GM-CSF cDNA, (0.8-kilobase pair XhoI fragment in pXM), IL-1 cDNA (1.3-kilobase pair PstI fragment in pSP64) kindly provided by Dr. S. C. Clark (Genetics Institute, Cambridge, MA), and -actin cDNA (1.8-kilobase pair HindIII fragment in pEMBL8); all of the probes were labeled by random priming. -actin probe was used to compare the level of specific hybridization with the amount of RNA separated in each assay. The amount of each mRNA were quantitated by densitometric scanning of autoradiographs. The experiments, performed in duplicate, were repeated at least three times.

GM-CSF Assay

Assay of GM-CSF protein in cellular conditioned medium was performed by a solid-phase immunoenzymetric assay (EASIA, Medgenix Diagnostics, Fleurus, Belgium). This assay is based on oligoclonal system in which several monoclonal antibodies directed against distinct epitopes of GM-CSF are used. The use of several distinct monoclonal antibodies avoids hyperspecificity and allows high sensitive assay with extended standard range and short incubation time. This assay allows to detect a minimum protein concentration of 3 pg/ml and cross-reactions with M-CSF, G-CSF, IL-1, IL-1, IL-2, IL-3, IL-4, interferon-, interferon-, interferon-, tumor necrosis factor-, and tumor necrosis factor- are insignificant.

Lf Immunoblot

Transfected 5637 cells were washed twice, scraped with cold phosphate-buffered saline and then solubilized at 4 °C for 1 h using a lysis containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM aprotinin, and 1 µg/ml leupeptin. Concentrated samples (about 100 µg of proteins), suspended in gel loading buffer, were incubated for 15 min at 25 °C and subjected to SDS-polyacrylamide gel electrophoresis (21) . The resolved proteins were transferred to nitrocellulose filters using the Western blot procedure (22). Lf was immunodetected as described previously (10) , except that 5% nonfat carnation dried milk was used instead of 3% bovine serum albumin to block membranes, and 2% nonfat carnation dried milk was used during antibodies incubation.

Nuclear Run-on Assay

Evaluation of GM-CSF transcriptional rate was performed in 5637 cells. Briefly, cells untreated or treated with 0.1 µM Lf for 18 h were lysed with 10 mM Tris-HCl, pH 7.4, in the presence of 10 mM NaCl, 3 mM MgCl, 0.5% Nonidet P-40, 2.75 mM dithiothreitol, and 20 units/ml RNasin. Pelleted nuclei were resuspended in 10 mM Tris-HCl, pH 7.5, containing 5 mM MgCl, 0.5 MD-sorbitol, 2.5% Ficoll, 0.008% spermidine, 1 mM dithiothreitol, and 50% glycerol. Elongation of nascent radiolabeled RNA was performed at 27 °C for 35 min in 40 mM Tris-HCl, pH 8.3, in the presence of 200 µCi [-P]UTP, 0.63 mM ATP, 0.31 mM GTP, 0.31 mM CTP, 150 mM NHCl, 7.5 mM MgCl, and 200 units/µl RNasin. The transcription reaction was terminated by the addition of 40 units of DNase-RNase-free at 37 °C for 10 min. The extracted RNA was dissolved in 10 mM Tris-HCl, pH 8.0, containing 1 mM EDTA. Samples were normalized by radioactivity quantification in a liquid scintillation counter. Plasmids containing the GM-CSF cDNA, IL-1 cDNA, -actin cDNA, and pBS, linearized by appropriate enzymatic digestion, were denatured and immobilized on nylon filters in a slot-blot apparatus. The filters were prehybridized at 42 °C for at least 10 h; equal amount of transcribed RNAs (2.5 10 cpm/ml) were then added, and the hybridization was performed for 72 h. After appropriate washing, filters were exposed to film. -actin cDNA was used as a positive control, and pBS was used as a negative control. After radioactive counting of transcripts with reference to -actin, the reduction in GM-CSF and IL-1 transcriptional rates of Lf-treated cells compared with untreated cells was expressed as percent of reduction. This experiment was repeated 3 times.

Chloramphenicol Acetyltransferase (CAT) Assay

Transfected cells were washed twice with phosphate-buffered saline, scraped in 0.25 M Tris-HCl, pH 7.9, and lysed by three sequential cycles of freeze-thawing. Cell debris was removed by centrifugation at 15,000 g for 10 min. Supernatants were assayed for protein concentration using the Bradford reagent according the manufacturer's instructions (Bio-Rad). The amounts of cell extract used for CAT assay were normalized for -galactosidase activity, and the assay was carried out as described (23) . After separation by thin-layer chromatography (TLC), reaction products were detected by autoradiography and quantified by liquid scintillation counting of thin-layer chromatography plate areas containing [C]chloramphenicol and its acetylated derivatives. CAT activity is expressed as a percentage of chloramphenicol conversion.

RESULTS

Lf Does Not Affect Constitutive Expression of GM-CSF

The 5637, Mo, and U937 cell lines incubated with increasing concentrations of Lf from 1 pM to 0.1 µM for different times (from a few hours to some days) did not show significant modifications of IL-1 and GM-CSF mRNA levels. A representative Northern blot analysis performed from 5637 cells showed that the mRNA levels of GM-CSF and IL-1 were not significantly modified by treatment with 0.1 µM Lf for some hours (Fig. 1). As shown in , 5637 cells secreted variable amounts of GM-CSF in culture-conditioned medium. This variability was dependent on culture conditions, although the cells were used at about 70% confluence. Nevertheless, a constant but moderate reduction (about 20%) in GM-CSF protein concentration was observed after 24 h with 0.1 µM Lf treatment. Lf synthesis in 5637 cells, which do not contain endogenous Lf, was obtained by transfecting the pLf expression vector. We were able to detect Lf mRNA (Fig. 2, lane1) as well as the Lf protein by SDS-polyacrylamide gel electrophoresis (Fig. 3, lane3). From these experiments it appears that transfection with pLf was effective for expression of Lf mRNA and protein, while IL-1 and GM-CSF mRNA levels were not significantly modified by Lf (Fig. 2). In culture medium of pLf transfected-cells, the level of GM-CSF protein was 20% lower than that of the control cells.


Figure 1: Level of GM-CSF and IL-1 mRNA in Lf-treated 5637 cell line assay. Subconfluent cultures of 5637, were treated with 0.1 µM human purified Lf (+) for the times indicated. Cytoplasmatic RNA (10 µg) was subjected to Northern analysis. Sequential hybridizations were performed with -P-labeled GM-CSF, IL-1, and -actin probes. See ``Experimental Procedures'' for details of cell culture and mRNA analysis.




Figure 2: Level of GM-CSF and IL-1 mRNA in Lf-transfected 5637 cell line. Cells were transfected with 10 µg of Lf expression vector pLf (lane1) and with an equimolar concentration of the control plasmid pBS (lane2). mRNA was analyzed as described in Fig. 1. Hybridization was also performed with -P-labeled Lf probe. See ``Experimental Procedures'' for details of plasmids and transfection conditions.




Figure 3: Immune-detection of Lf produced in 5637 cells. Samples of transfected 5637 cells were solubilized, and about 100 µg of proteins were subjected to SDS-polyacrylamide gel electrophoresis and Western blot analysis. Lf was immunodetected with a polyclonal anti-human Lf antibody. Lane1, 0.2 µg of human purified Lf; lane2, cells transfected with pBS; lane3, cells transfected with pLf. See ``Experimental Procedures'' for details of immunodetection.



Lf in PEU Cells Can Inhibit GM-CSF Expression in Response to IL-1

We then used PEU cells in which GM-CSF expression is low and can be induced to higher levels by various agents (10) . The induction of GM-CSF expression by tumor necrosis factor-, IL-1, and IL-1 was tested, and IL-1 (15 units/ml) induced the highest levels of GM-CSF mRNA (data not shown). To test whether Lf was able to affect the expression of GM-CSF in PEU cells stimulated by IL-1, we transfected these cells with pLf. Lf in transfected cells was detectable both as mRNA (Fig. 4, lane3) and protein (data not shown). The level of GM-CSF mRNA induced by IL-1, in pLf-transfected cells, was significantly lower than that found in the untransfected cells (Fig. 4, lanes2 and 3). A low secretion of GM-CSF protein was found in PEU cells culture medium (about 0.1 ng/ml), and this protein amount showed a similar trend to mRNA; IL-1 treatment induced a 2-3-fold increase in the GM-CSF protein level, while Lf was able to inhibit such an effect since the protein level was the same as in unstimulated cell culture medium (data not shown).


Figure 4: GM-CSF mRNA decrease induced by Lf in IL-1-stimulated PEU cells. PEU cells transfected (lanes1 and 2) with pBS and with pLf (lane3) were untreated (lane1) and treated (lanes2 and 3) with 15 units/ml of IL-1. mRNAs were analyzed as indicated in Fig. 1. -P-Labeled Lf and GM-CSF probes were used for hybridization. 28 and 18 S rRNA were visualized by ethidium bromide gel staining.



Activity of Endogenous GM-CSF Promoter in Lf-treated 5637 Cells

We investigated whether Lf could down-regulate the rate of mRNA transcription in these cells. As shown in a representative nuclear run-on assay (Fig. 5), GM-CSF transcript appeared more abundant than IL-1 and even more so than -actin. Lf-treated cells showed a 20% reduction in GM-CSF transcription rate. We also observed that transcription of IL-1 gene seems to be affected by Lf in a similar way to GM-CSF.


Figure 5: Activity of endogenous GM-CSF promoter in Lf-treated 5637 cells. 5637 cells were treated without or with 0.1 µM Lf for 18 h. Nuclei were extracted, and the transcriptional rate was measured as described under ``Experimental Procedures.'' We analyzed GM-CSF and IL-1 genes; -actin was used as positive control and pBS as negative control.



Lf Down-modulates GM-CSF Promoter Activity

We next asked whether Lf could mediate a reduction in the transcriptional activity of the GM-CSF promoter. pLf was cotransfected into PEU and 5637 cells together with pPF2000, in which the human GM-CSF promoter is fused to the CAT reporter gene (Fig. 6). We first verified (Fig. 7a) that CAT activity (expressed as mean ± S.E. of four experiments), under the control of the GM-CSF promoter, was increased about 3-fold in IL-1-treated PEU cells compared with untreated cells (4.82 ± 0.18 versus 1.68 ± 0.18). In 5637 cells, in which IL-1 is endogenously expressed (14) , the CAT activity driven by the GM-CSF promoter was high both in treated and in untreated cells (7.18 ± 0.87 versus 6.55 ± 1.84). Co-transfection with pLf (Fig. 7b) caused a significant reduction in CAT activity in both 5637 and PEU cells. A 7.4-fold reduction was achieved in PEU cells stimulated by IL-1, while only a 1.7-fold reduction was observed in IL-1-untreated cells (13.55 ± 2.14% versus 100% and 57.38 ± 1.07% versus 100%, respectively). In the 5637 cell line, Lf caused a 7.3-fold reduction in CAT activity in IL-1-treated cells and a 6.1-fold reduction in untreated cells (13.67 ± 3.95% versus 100% and 16.33 ± 7.31% versus 100%, respectively). We show a representative CAT assay, of 5637 (Fig. 6a) and PEU (Fig. 6b) cells.


Figure 6: Effect of Lf on GM-CSF promoter activity in 5637 and PEU cells. The data show a representative CAT assay. 5637 (a) and PEU (b) cells were transfected with pLf (+) or an equimolar amount of pBS (-) together with 6 µg of pPF2000 (CAT reporter gene containing the GM-CSF promoter). The cells were IL-1-stimulated (+) or unstimulated (-). See ``Experimental Procedures'' for details of plasmids, transfection, and CAT assay.




Figure 7: Kinetics of Lf inhibition of GM-CSF promoter activity. Bars show the mean ± S.E. of the CAT activity of four experiments performed in duplicate. a, to compare CAT activities in two different cell types, percentage chloramphenicol conversion was divided by -galactosidase activity expressed as OD/1 h. Untreated cells (openbars), IL-1-treated cells (stripedbars). b, CAT activity of the pBS transfected cells (openbars) represented 100% activity; percentage CAT activity of pLf-transfected cells (solidbars) was related to respective pBS-transfected cells. Transfections were performed as described in Fig. 6. See ``Experimental Procedures'' for details of plasmids, transfection, and CAT assay.



DISCUSSION

This work was aimed at investigating the effect of Lf on the expression of GM-CSF in various in vitro cultured cells. The results presented here demonstrate that Lf is not able to down-regulate the high levels of GM-CSF and IL-1 expressed in 5637, Mo, and U937 cell lines, but a 20% constant reduction of secreted GM-CSF protein was observed in Lf-treated 5637 cells. By contrast, in PEU cells, Lf leads to a reduction in GM-CSF mRNA level in IL-1-treated cells. The mRNA level, which is 3 times increased by IL-1, was reduced to the basal level by transfected Lf. Reduction in mRNA was followed by a similar reduction in the secreted GM-CSF protein.

In 5637 cells, although Lf does not seem to down-regulate the high level of GM-CSF mRNA, it is able to reduce of about 20% both the activity of the endogenous promoter and the the amount of secreted protein.

The significative mRNA reduction in PEU cells brought about by transfected Lf and the low reduction in GM-CSF gene transcriptional rate in Lf-treated 5637 cells seem to be due, at least in part, to a transcriptional mechanism. In co-transfection experiments down-regulation of GM-CSF promoter activity was significant when PEU cells were treated with IL-1. In 5637 cells, Lf inhibition of the exogenous GM-CSF promoter was significant even in the absence of IL-1 treatment. This can be explained by the endogenous production of IL-1.

GM-CSF mRNA expression is differently regulated in 5637 and PEU cells. In PEU cells, the low basal mRNA level can be increased to relatively high level by induction with several agents. On the contrary, in 5637 cells the constitutive high expression of GM-CSF is not subjected to further increase. A possible explanation is the endogenous production of IL-1 and IL-1, which maintain high level of the GM-CSF through a transcriptional and post-transcriptional regulation (4, 24) . In 5637 cells, mRNA half-life is increased up to 4 h compared with an average of 0.5 h in tumor necrosis factor-stimulated or unstimulated fibroblasts (25) . This different regulation in the two cell types may explain the different effect of Lf on the overall mRNA accumulation. Therefore Northern blot analysis is not so sensitive to indicate a 20% difference in mRNA levels upon Lf-treatment. On the other hand the results obtained by run on assay and by protein assay confirme a Lf action on GM-CSF expression.

We observed a different quantitative effect of Lf-treatment on endogenous and exogenous (pPF2000) GM-CSF promoter in 5637 cells. The endogenous GM-CSF promoter is strongly activated by trans-acting factors. We hypothesize that the ratio between trans-acting factors that strongly activate the GM-CSF promoter in 5637 cells and their related cis-acting elements is such to affect Lf action. The addition of the exogenous promoter, as in co-transfection experiment, is likely to significantly change this ratio, thus allowing Lf to affect promoter activity. Furthermore, CAT activity does not take into account post-transcriptional regulation of GM-CSF mRNA that is, of course, differently regulated with respect to CAT mRNA.

Our results show that IL-1 stimulates GM-CSF promoter activity in embryonic fibroblasts, and provides additional information about the mechanism by which IL-1 regulates GM-CSF expression. Previous results have shown that, in endothelial cells, IL-1 increases GM-CSF expression at the transcriptional level; furthermore, IL-1 activates GM-CSF promoter by acting through a sequence close to the TATA box (26). In WI-38 fibroblasts, IL-1 seems to have no effect on GM-CSF transcription (27) , and other results indicate that, in fibroblasts, GM-CSF expression appears to be regulated both transcriptionally and post-transcriptionally by IL-1 (4, 24) . The cis and trans elements controlling GM-CSF transcription have been partly identified. Of particular interest is the AP1-like recognition site at -53 in human and mouse promoter. It has been demonstrated that AP1, which is induced by IL-1 (28, 29) , binds this sequence (30, 31, 32) . Our co-transfection experiments revealed that Lf down-modulates GM-CSF promoter activity both in PEU and 5637 cells. We believe that, in both cell types, the effect of Lf on GM-CSF promoter is mediated by IL-1.

The molecular mechanism underlying the observed effect of Lf on GM-CSF promoter activity is still unknown. It is tempting to speculate that Lf may interact directly with DNA elements that are located within transcriptional control regions. Such a possibility is consistent with the fact that Lf is able to bind DNA (10, 11) , even in a sequence-specific way (12) . For instance, Lf could modulate the activity of nuclear factors critical to GM-CSF gene activation. Moreover, Lf is very prone to bind other proteins (33, 34) . Lf might be engaged in interactions with factors that are involved in the regulation of GM-CSF transcription. Protein-protein interactions seem to be involved in some repression mechanisms operated through AP1 (35) . However, Lf could participate less directly in transcriptional modulation; it might somehow modify the intranuclear environment in a way that represses the transcription of a set of genes (for example genes modulated by IL-1).

Further work will be necessary to identify more precisely the region of the GM-CSF promoter responsive to the inhibitory effect of Lf and to characterize the molecular mechanism in greater details.

  
Table: GM-CSF protein concentration in Lf-treated and untreated 5637 cells

Cells, at about 70% confluence, were incubated or not with 0.1 µM Lf for 24 h, and GM-CSF protein assay was performed in culture medium by the EASIA method (see ``Experimental Procedures'' for details). The reduction in the protein concentration of Lf-treated cells compared with untreated cells was also expressed as percent of reduction. The three experiments were performed in duplicate.



FOOTNOTES

*
This work was supported in part by grants from MURST (to C. G. and G. B.-S.), Consiglio Nazionale delle Ricerche (CNR) 92.02626CT.04 and 92.02185CT.14 (to C. G.) and CNR 93.01977.14 (to G. B.-S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Inst. of Biology and Genetics, University of Genova, Viale Benedetto XV/6, 16132 Genova, Italy. Tel.: 39-10-353-8949; Fax: 39-10-353-8978.

The abbreviations used are: Lf, lactoferrin; GM-CSF, granulocyte macrophage colony-stimulating factor; IL-1, interleukin; CAT, chloramphenicol acetyltransferase.

The orientation of the Lf cDNA insert in expression vector p91023-B was tested by restriction analysis and limited sequencing.

P. Fiorentini, M. Musso, S. Penco, R. Giuffrida, V. Pistoia, C. Garrè, and G. Bianchi-Scarr, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. M. Musso for helpful advice, G. Bruzzone for photographic assistance, Dr. O. Conneely for the gifts of the p91023-B, and Dr. P. Fiorentini for the pPF2000 used. We also thank Dr. R. Ravazzolo and Dr. B. Patrick for critically reviewing the manuscript.


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