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Regulation of Cellular Retinoic Acid Binding Protein (CRABP II) during Human Monocyte Differentiationin Vitro
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
248, 830–834 (1998)
RC989058
Regulation of Cellular Retinoic Acid Binding Protein (CRABP II) during Human Monocyte Differentiation in Vitro Marina Kreutz,1 Jana Fritsche, Reinhard Andreesen and Stefan W. Krause Department of Hematology and Oncology, University of Regensburg, 93042 Regensburg, Germany
Received June 29, 1998
Cellular retinoic acid binding proteins (CRABP) are low molecular weight proteins whose precise function remains unknown. They bind retinoids and may thereby modulate the intracellular steady-state concentration of retinoids. Whereas CRABP I is ubiquitously expressed, CRABP II is mainly detected in various cell types of the skin. By representative difference analysis we found that CRABP II is also strongly expressed in human monocyte-derived macrophages (MAC) but not in freshly isolated monocytes (MO). The CRABP II mRNA was gradually upregulated during differentiation from MO to MAC in the presence of 2% serum. Adherence, which is important for MO differentiation, induced CRABP II expression, but the addition of 1007 M retinoic acid inhibited the upregulation of CRABP II expression during MO/MAC differentiation. As MO can differentiate along the classical pathway not only to MAC but also to dendritic cells we analyzed the expression of CRABP II in MO-derived dendritic cells cultured with 10% FCS, IL-4, and GM-CSF. In contrast to MAC, MO-derived dendritic cells showed an extremely low expression of CRABP II. From these results we conclude (1) that the availability and the metabolism of retinoids may be different in MAC compared to MO and dendritic cells and (2) that this may influence differentiation and activation of those cells. q 1998 Academic Press
Retinoic acid (RA) exerts pleiotropic effects on cellular growth, differentiation and activation both during embryogenesis and in the adult (1). Moreover retinoids can inhibit or reverse the process of malignant transformation (2). RA induces the differentiation of promyelocytic HL-60 cells and primary human leukemia cells into granulocytes (3,4), and induces complete remission in 1 Correspondence should be addressed to Dr. Marina Kreutz, University of Regensburg, Dept. of Hematology & Oncology, 93053 Regensburg, Fax: 0049-941-944 7131, E-mail: Marina.Kreutz@ klinik.uni-regensburg.de.
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patients with acute promyelocytic leukemia (5). Conflicting reports have been published regarding the effects of RA on monocytic differentiation. In the promyelocytic cell line HL-60 RA inhibited monocytic differentiation (6), whereas, in the monoblastic cell line U937 RA was shown to modulate macrophage (MAC) differentiation either positive or negative (7,8). Recently, we could show that the survival and differentiation of primary human monocytes (MO) is inhibited by RA treatment (9). In the skin, as one major target organ of RA, keratinocyte differentiation is also inhibited by RA (10,11). Two classes of proteins play roles in mediating the biological effects of retinoic acid (RA). The nuclear receptors for RA, RAR and RXR, are members of the nuclear receptor superfamily, including receptors for steroid hormones, thyroid hormones and retinoids. They function as ligand-inducible transcription factors and activate target genes after binding as homodimers or heterodimers (12,13). Suppression of the endogenous RA receptor in a multipotent hematopoietic cell line by a dominant negative RA receptor blocked neutrophil differentiation at the promyelocyte stage suggesting a crucial role for RA receptors in the terminal differentiation of neutrophils (14). In addition, skin development is inhibited by targeted expression of a dominant negative retinoic acid receptor (15). Beside the nuclear receptors, a second type of retinoic acid binding proteins, CRABP I and CRABP II, are found in the cytoplasm of different cell types. CRABP I is ubiquitously expressed but the expression of CRABP II seems to be restricted to adult skin in humans and mouse (16,17). In contrast to the nuclear receptors RAR/RXR, the precise functions of CRABP I/II are unknown. Most likely these proteins are involved in the distribution and metabolism of retinoids in the cell and might exert their physiological function by controlling the intracellular levels of free RA. Therefore the relative level of the two types of RA binding proteins in the cell, namely RAR/RXR and CRABP I/II, may determine the responsiveness of a cell to RA treatment. Accordingly, Boylan and Gudas
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FIG. 1. Expression of CRABP II during human monocyte to macrophage differentiation in vitro. (A) Elutriation purified human blood MO were cultured for up to 7 days in the presence of 2% AB serum. At the indicated time periods cells were lysed directly in the plates with guanidine thiocyanate solution. RNA extraction and Northern analysis were performed according to Materials and Methods. (B) Densitometric evaluation of CRABP II expression.
have shown that overexpression of CRABP I results in a reduction of RA-induced gene expression in F9 terarocarcinoma cells (18) indicating that CRABP may sequester RA and thereby block RAR/RXR mediated gene regulation and differentiation. We were interested in genes which may be implicated in the differentiation process of human MO to MAC. Using the Representative Difference Analysis (RDA) technology we found that CRABP II is upregulated during MO differentiation. As RAR/RXR and CRABP I/II seem to be of crucial importance for the differentiation of various cell types, we suggest a role for CRABP II in the differentiation process of human MO. MATERIALS AND METHODS Monocyte separation and culture. Peripheral blood mononuclear cells (MNC) were obtained by leukapheresis of healthy donors, followed by density gradient centrifugation over Ficoll/Hypaque. MO
were isolated from MNC by counter-current elutriation (J6M-E Beckmann centrifuge) using a large-volume chamber (50ml), a JE-5 rotor at 2,500 r.p.m. and a flow rate of 110 ml/min in Hank’s balanced salt solution supplemented with 6% autologous human plasma (19). Elutriated MO were ú90% pure as determined by morphology and expression of the MO antigen CD14. For the generation of MO-derived macrophages, purified MO were cultured on teflon foils (Biofolie 25, Heraeus, Hanau, Germany), or culture plates or under non-adherent conditions in roller bottles, respectively, for up to seven days at a cell density of 106 cells/ml in RPMI 1640 (Biochrom, Berlin, Germany) supplemented with antibiotics (50 U/ml penicillin and 50 mg/ml streptomycin, Gibco, Berlin, Germany), L-glutamine (2 mM, Gibco, Berlin, Germany) and 2% pooled human AB-group serum (Sigma) with or without 9-cis RA (kindly provided by Hoffmann-La Roche, Basel, Switzerland). Cultures containing RA were fed with half of the initial RA dose after two days. At the indicated time periods cells were harvested and viable cells were counted by trypan blue exclusion. For the generation of MO-derived dendritic cells, elutriation-purified MO were cultured for 7 days in RPMI supplemented with 10% FCS, 36 ng/ml rhGM-CSF (Leucomax 400, kindly provided by Sandoz-Essex Pharma, Nu¨rnberg, Germany) and 500 u/ml rhIL4 (Promo cell, Heidelberg, Germany).
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FIG. 2. Regulation of CRABP II expression by adherence. Freshly isolated MO (1) or MO cultured for 24 h in roller bottles (2), on agarose-coated dishes (3), on culture dishes without coating (4), or on Teflon foils (5) in the presence of 2% human AB serum were harvested and RNA extracted. Northern analysis was performed according to Materials and Methods.
Cell lines. All cell lines were cultured in RPMI 1640 with 10 % FCS. Representative difference analysis (RDA). MO-derived macrophages were used as tester and freshly isolated MO as drivers for RDA. Poly-A RNA was purified from total RNA of both cell types by the use of Oligotex beads (Quiagen, Hilden, Germay) and transcribed into dsDNA by the use of a commercially available kit (Stratagene, LaJolla, CA, USA). Generation of representations and three rounds of substraction were performed according to the protocol of Hubank and Schatz (20). PCR products from the third difference product were cloned into the plasmid vector pZErO-2 (Stratagene) for further analysis. Sequencing revealed that three independent cDNA clones analyzed from the third RDA difference product were identical to bp 458-735 of the published cDNA sequence of CRABP II (16,21). RNA extraction. Cells were harvested and lysed with guanidine thiocyanate solution. RNA extraction was performed according to Chomczynski and Sacchi (22). Ten mg of total RNA were run in 1% agarose-formaldehyde gels and transferred to nylon membranes (NT membranes, MSI, Westborough, USA). Northern Analysis. CRABP II mRNA was detected by hybridization of the membranes with a 32P-labeled cDNA probe labeled with gene specific primers I) GCC ATT CCT CT II) CGT CCG AGA GTG (random primed labeling kit, Boehringer Mannheim, Germany). The cDNA probe was identical to bp 458-735 of the published cDNA sequence of CRABP II (16,21). To provide an internal control, membranes were reprobed with an oligonucleotide against 18 S rRNA labeled by T4 kinase.
in vitro in the presence of 2% serum. Accordingly, CRABP II expression is also upregulated during differentiation of keratinocytes in vitro (11). As adherence is important for the survival and differentiation of MO, we investigated the expression of CRABP II in MO/ MAC under adherent vs. non-adherent culture condition. Figure 2 shows again that freshly isolated MO showed no CRABP II expression but after 1 day of adherence, either on teflon foils or on tissue culture plates, CRABP II mRNA could be detected. However, when MO were cultured under non-adherent conditions, either on agarose or in roller bottles, no upregulation of CRABP II was found. This indicates that adherence is important for CRABP II regulation. Retinoic acid (RA) has been shown to regulate CRABP II expression either positively or negatively, dependent on the cell type studied (10,16). As RA is also known to influence human MO differentiation, we analyzed the effect of RA on CRABP II expression during MO differentiation and found that RA dose dependently inhibited CRABP II expression in human MO/MAC (Figure 3). Similar results have been obtained by Astro¨m et al. for keratinocytes. During keratinocyte differentiation CRABP II is upregulated but RA prevents differentiation and thereby CRABP II expression in these cells (16). In contrast, RA induces CRABP II in dermal but not lung fibroblasts (10,16) and topical application of RA on human skin also strongly increases the CRABP II mRNA and protein expression (23). In human neuroblastoma cell lines RA inhibited the proliferation and increased the expression of CRABP II (24). Since retinoic acid response elements, e.g. binding sites for the RA receptor homodimers and heterodimers, are found in the promoter region of the CRABP II gene, it is not surprising that this gene is regulated by RA (25). The divergent results regarding regulation by RA may in part be due to the relative amount of RA receptors and other transcription factors like AP-1 in the respective cell type which are both involved in the regulation of CRABP II. Transcriptional activation by AP-1 has been shown to be negatively regulated by RA (26). In further
RESULTS AND DISCUSSION In order to identify genes which may be involved in MO to MAC differentiation we used the RDA technology with cDNA from human MO as ‘‘driver’’ and cDNA from MO-derived MAC as ‘‘tester’’. Three of the resulting cDNA clones were identical to bp 458-735 of the published human CRABP II sequence (16,21) indicating that the expression of this gene is different in MO vs. MAC. Northern blot analysis revealed that the cDNA fragment detected an approximately 1.2 kb mRNA transcript in human MO-derived MAC which is in accordance with the published CRABP II sequence (Figure 1). Further analysis showed that the CRABP II transcript was not detected in freshly isolated MO but upregulated during differentiation of MO to MAC
FIG. 3. Retinoic acid inhibits CRABP II expression. Elutriation purified human blood MO were cultured in the presence of 2% human serum with and without RA on plastic culture dishes. After 24 h RNA was extracted and Northern analysis performed according to Materials and Methods.
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FIG. 4. Expression of CRABP II in several cell lines and primary cells. All primary cells and cell lines were cultured on plastic culture dishes and directly lysed in the plates with guanidine thiocyanate solution. RNA extraction and Northern analysis were performed according to Materials and Methods.
experiments we were interested whether CRABP II would also be expressed in monocytic cell lines which are often used as a model for MAC differentiation. We found that CRABP II is not expressed in several monocytic cell lines (Figure 4). Even if those cell lines were induced to differentiate with 1,25-dihydroxyvitamin D3 no CRABP II mRNA could be deteceted (data not shown). From the other cell lines we investigated, only HT29, a colon carcinoma cell line, showed a weak expression of CRABP II. Peripheral blood lymphocytes were also negative for CRABP II expression. As MO can not only differentiate along the classical pathway to MAC but also to dendritic cells we analyzed the expression of CRABP II in MO-derived dendritic cells cultured with 10% FCS, IL-4 and GM-CSF (Fig. 5). In contrast to MO-derived macrophages, CRABP II mRNA was nearly not detectable in MO-derived dendritic cells. However, dendritic cells showed a strong expression of RA receptors (manuscript in preparation). Only few data are up to now available regarding
the effect of RA on dendritic cells. The antigen presenting function of dendritic cells tested by the mixed lymphocyte reaction in vitro was found to be inhibited by high doses of RA but lower doses stimulated allogeneic lymphocytes (27). Accordingly, Katz et al. found an increase of the accessory cell function of dendritic cells/ MAC isolated from mice which had been fed with vitamin A acetate or RA (28). Based on these data we suggest that RA modulates the activation and differentiation of cells of the myeloid lineage and that CRABP II may influence this process by regulating the intracellular steady state concentration of retinoids in those cells. ACKNOWLEDGEMENTS The authors thank Ute Ackermann and Lucia SchwarzfischerPfeilschifter for excellent technical assistance. This investigation was supported by Deutsche Forschungsgemeinschaft An111/6-6.
REFERENCES 1. Gudas, L.J. (1994) J. Biol. Chem. 269, 15399–15402. 2. Holdener, E.E., and Bollag, W. (1993) Curr. Opin. Oncol. 5, 1059–1066. 3. Breitman, T.R., Collins, S.J., and Keene, B.R. (1981) Blood 57, 1000–1004. 4. Breitman, T.R., Selonick, S.E., and Collins, S.J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2936–2940. 5. Degos, L., Dombret, H., Chomienne, C., Daniel, M.T., Miclea, J.M., Chastang, C., Castaigne, S., and Fenaux, P. (1995) Blood 85, 2643–2653.
FIG. 5. Comparative analysis of CRABP II expression between MO-derived macrophages and dendritic cells. Elutriation purified human blood MO were cultured either on plastic culture dishes in the presence 10% FCS, GM-CSF, and IL-4 for the generation of MOderived dendritic cells or with 2% pooled AB serum for the generation of macrophages. After 7 days cells were lysed with guanidine thiocyanate solution. RNA extraction and Northern analysis were performed according to Materials and Methods.
6. He, R.Y., and Breitman, T.R. (1991) Eur. J. Haematol. 46, 93. 7. Oberg, F., Botling, J., and Nilsson, K. (1993) J. Immunol. 150, 3487–3495. 8. Taimi, M., Defacque, H., Commes, T., Favero, J., Caron, E., Marti, J., and Dornand, J. (1993) Immunology 79, 229–235. 9. Kreutz, M., Fritsche, J., Ackermann, U., Krause, S.W., and Andreesen, R. (1998) Blood 91. 10. Elder, J.T., Astrom, A., Pettersson, U., Tavakkol, A., Krust, A., Kastner, P., Chambon, P., and Voorhees, J.J. (1992) J. Invest. Dermatol. 98, 36S–41S.
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11. Eller, M.S., Harkness, D.D., Bhawan, J., and Gilchrest, B.A. (1994) J. Invest. Dermatol. 103, 785–790. 12. Glass, C.K. (1994) Endocr. Rev. 15, 391–407. 13. Leid, M., Kastner, P., and Chambon, P. (1992) Trends. Biochem. Sci. 17, 427–433. 14. Tsai, S., and Collins, S.J. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7153–7157. 15. Saitou, M., Sugai, S., Tanaka, T., Shimouchi, K., Fuchs, E., Narumiya, S., and Kakizuka, A. (1995) Nature 374, 159–162. 16. Astrom, A., Tavakkol, A., Pettersson, U., Cromie, M., Elder, J.T., and Voorhees, J.J. (1991) J. Biol. Chem. 266, 17662–17666. 17. Napoli, J.L. (1996) Clin. Immunol. Immunopathol. 80, S52–62. 18. Boylan, J.F., and Gudas, L.J. (1991) J. Cell Biol. 112, 965–979. 19. Andreesen, R., Brugger, W., Scheibenbogen, C., Kreutz, M., Leser, H.G., Rehm, A., and Lohr, G.W. (1990) J. Leukoc. Biol.47, 490–497. 20. Hubank, M., and Schatz, D.G. (1994) Nucleic Acids Res. 22, 5640–5648.
21. Giguere, V., Lyn, S., Yip, P., Siu, C.H., and Amin, S. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6233–6237. 22. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156– 159. 23. Piletta, P., Jaconi, S., Siegenthaler, G., Didierjean, L., and Saurat, J.H. (1994) Exp. Dermatol. 3, 23–28. 24. Plum, L.A., and Clagett Dame, M. (1995) Arch. Biochem. Biophys. 319, 457–463. 25. Durand, B., Saunders, M., Leroy, P., Leid, M., and Chambon, P. (1992) Cell 71, 73–85. 26. Schule, R., Rangarajan, P., Yang, N., Kliewer, S., Ransone, L.J., Bolado, J., Verma, I.M., and Evans, R.M. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6092–6096. 27. Bedford, P.A., and Knight, S.C. (1989) Clin. Exp. Immunol. 75, 481–486. 28. Katz, D.R., Drzymala, M., Turton, J.A., Hicks, R.M., Hunt, R., Palmer, L., and Malkovsky, M. (1987) Br. J. Exp. Pathol. 68, 343–350.
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Regulation of Cellular Retinoic Acid Binding Protein (CRABP II) during Human Monocyte Differentiation in Vitro Marina Kreutz,1 Jana Fritsche, Reinhard Andreesen and Stefan W. Krause Department of Hematology and Oncology, University of Regensburg, 93042 Regensburg, Germany
Received June 29, 1998
Cellular retinoic acid binding proteins (CRABP) are low molecular weight proteins whose precise function remains unknown. They bind retinoids and may thereby modulate the intracellular steady-state concentration of retinoids. Whereas CRABP I is ubiquitously expressed, CRABP II is mainly detected in various cell types of the skin. By representative difference analysis we found that CRABP II is also strongly expressed in human monocyte-derived macrophages (MAC) but not in freshly isolated monocytes (MO). The CRABP II mRNA was gradually upregulated during differentiation from MO to MAC in the presence of 2% serum. Adherence, which is important for MO differentiation, induced CRABP II expression, but the addition of 1007 M retinoic acid inhibited the upregulation of CRABP II expression during MO/MAC differentiation. As MO can differentiate along the classical pathway not only to MAC but also to dendritic cells we analyzed the expression of CRABP II in MO-derived dendritic cells cultured with 10% FCS, IL-4, and GM-CSF. In contrast to MAC, MO-derived dendritic cells showed an extremely low expression of CRABP II. From these results we conclude (1) that the availability and the metabolism of retinoids may be different in MAC compared to MO and dendritic cells and (2) that this may influence differentiation and activation of those cells. q 1998 Academic Press
Retinoic acid (RA) exerts pleiotropic effects on cellular growth, differentiation and activation both during embryogenesis and in the adult (1). Moreover retinoids can inhibit or reverse the process of malignant transformation (2). RA induces the differentiation of promyelocytic HL-60 cells and primary human leukemia cells into granulocytes (3,4), and induces complete remission in 1 Correspondence should be addressed to Dr. Marina Kreutz, University of Regensburg, Dept. of Hematology & Oncology, 93053 Regensburg, Fax: 0049-941-944 7131, E-mail: Marina.Kreutz@ klinik.uni-regensburg.de.
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patients with acute promyelocytic leukemia (5). Conflicting reports have been published regarding the effects of RA on monocytic differentiation. In the promyelocytic cell line HL-60 RA inhibited monocytic differentiation (6), whereas, in the monoblastic cell line U937 RA was shown to modulate macrophage (MAC) differentiation either positive or negative (7,8). Recently, we could show that the survival and differentiation of primary human monocytes (MO) is inhibited by RA treatment (9). In the skin, as one major target organ of RA, keratinocyte differentiation is also inhibited by RA (10,11). Two classes of proteins play roles in mediating the biological effects of retinoic acid (RA). The nuclear receptors for RA, RAR and RXR, are members of the nuclear receptor superfamily, including receptors for steroid hormones, thyroid hormones and retinoids. They function as ligand-inducible transcription factors and activate target genes after binding as homodimers or heterodimers (12,13). Suppression of the endogenous RA receptor in a multipotent hematopoietic cell line by a dominant negative RA receptor blocked neutrophil differentiation at the promyelocyte stage suggesting a crucial role for RA receptors in the terminal differentiation of neutrophils (14). In addition, skin development is inhibited by targeted expression of a dominant negative retinoic acid receptor (15). Beside the nuclear receptors, a second type of retinoic acid binding proteins, CRABP I and CRABP II, are found in the cytoplasm of different cell types. CRABP I is ubiquitously expressed but the expression of CRABP II seems to be restricted to adult skin in humans and mouse (16,17). In contrast to the nuclear receptors RAR/RXR, the precise functions of CRABP I/II are unknown. Most likely these proteins are involved in the distribution and metabolism of retinoids in the cell and might exert their physiological function by controlling the intracellular levels of free RA. Therefore the relative level of the two types of RA binding proteins in the cell, namely RAR/RXR and CRABP I/II, may determine the responsiveness of a cell to RA treatment. Accordingly, Boylan and Gudas
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FIG. 1. Expression of CRABP II during human monocyte to macrophage differentiation in vitro. (A) Elutriation purified human blood MO were cultured for up to 7 days in the presence of 2% AB serum. At the indicated time periods cells were lysed directly in the plates with guanidine thiocyanate solution. RNA extraction and Northern analysis were performed according to Materials and Methods. (B) Densitometric evaluation of CRABP II expression.
have shown that overexpression of CRABP I results in a reduction of RA-induced gene expression in F9 terarocarcinoma cells (18) indicating that CRABP may sequester RA and thereby block RAR/RXR mediated gene regulation and differentiation. We were interested in genes which may be implicated in the differentiation process of human MO to MAC. Using the Representative Difference Analysis (RDA) technology we found that CRABP II is upregulated during MO differentiation. As RAR/RXR and CRABP I/II seem to be of crucial importance for the differentiation of various cell types, we suggest a role for CRABP II in the differentiation process of human MO. MATERIALS AND METHODS Monocyte separation and culture. Peripheral blood mononuclear cells (MNC) were obtained by leukapheresis of healthy donors, followed by density gradient centrifugation over Ficoll/Hypaque. MO
were isolated from MNC by counter-current elutriation (J6M-E Beckmann centrifuge) using a large-volume chamber (50ml), a JE-5 rotor at 2,500 r.p.m. and a flow rate of 110 ml/min in Hank’s balanced salt solution supplemented with 6% autologous human plasma (19). Elutriated MO were ú90% pure as determined by morphology and expression of the MO antigen CD14. For the generation of MO-derived macrophages, purified MO were cultured on teflon foils (Biofolie 25, Heraeus, Hanau, Germany), or culture plates or under non-adherent conditions in roller bottles, respectively, for up to seven days at a cell density of 106 cells/ml in RPMI 1640 (Biochrom, Berlin, Germany) supplemented with antibiotics (50 U/ml penicillin and 50 mg/ml streptomycin, Gibco, Berlin, Germany), L-glutamine (2 mM, Gibco, Berlin, Germany) and 2% pooled human AB-group serum (Sigma) with or without 9-cis RA (kindly provided by Hoffmann-La Roche, Basel, Switzerland). Cultures containing RA were fed with half of the initial RA dose after two days. At the indicated time periods cells were harvested and viable cells were counted by trypan blue exclusion. For the generation of MO-derived dendritic cells, elutriation-purified MO were cultured for 7 days in RPMI supplemented with 10% FCS, 36 ng/ml rhGM-CSF (Leucomax 400, kindly provided by Sandoz-Essex Pharma, Nu¨rnberg, Germany) and 500 u/ml rhIL4 (Promo cell, Heidelberg, Germany).
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FIG. 2. Regulation of CRABP II expression by adherence. Freshly isolated MO (1) or MO cultured for 24 h in roller bottles (2), on agarose-coated dishes (3), on culture dishes without coating (4), or on Teflon foils (5) in the presence of 2% human AB serum were harvested and RNA extracted. Northern analysis was performed according to Materials and Methods.
Cell lines. All cell lines were cultured in RPMI 1640 with 10 % FCS. Representative difference analysis (RDA). MO-derived macrophages were used as tester and freshly isolated MO as drivers for RDA. Poly-A RNA was purified from total RNA of both cell types by the use of Oligotex beads (Quiagen, Hilden, Germay) and transcribed into dsDNA by the use of a commercially available kit (Stratagene, LaJolla, CA, USA). Generation of representations and three rounds of substraction were performed according to the protocol of Hubank and Schatz (20). PCR products from the third difference product were cloned into the plasmid vector pZErO-2 (Stratagene) for further analysis. Sequencing revealed that three independent cDNA clones analyzed from the third RDA difference product were identical to bp 458-735 of the published cDNA sequence of CRABP II (16,21). RNA extraction. Cells were harvested and lysed with guanidine thiocyanate solution. RNA extraction was performed according to Chomczynski and Sacchi (22). Ten mg of total RNA were run in 1% agarose-formaldehyde gels and transferred to nylon membranes (NT membranes, MSI, Westborough, USA). Northern Analysis. CRABP II mRNA was detected by hybridization of the membranes with a 32P-labeled cDNA probe labeled with gene specific primers I) GCC ATT CCT CT II) CGT CCG AGA GTG (random primed labeling kit, Boehringer Mannheim, Germany). The cDNA probe was identical to bp 458-735 of the published cDNA sequence of CRABP II (16,21). To provide an internal control, membranes were reprobed with an oligonucleotide against 18 S rRNA labeled by T4 kinase.
in vitro in the presence of 2% serum. Accordingly, CRABP II expression is also upregulated during differentiation of keratinocytes in vitro (11). As adherence is important for the survival and differentiation of MO, we investigated the expression of CRABP II in MO/ MAC under adherent vs. non-adherent culture condition. Figure 2 shows again that freshly isolated MO showed no CRABP II expression but after 1 day of adherence, either on teflon foils or on tissue culture plates, CRABP II mRNA could be detected. However, when MO were cultured under non-adherent conditions, either on agarose or in roller bottles, no upregulation of CRABP II was found. This indicates that adherence is important for CRABP II regulation. Retinoic acid (RA) has been shown to regulate CRABP II expression either positively or negatively, dependent on the cell type studied (10,16). As RA is also known to influence human MO differentiation, we analyzed the effect of RA on CRABP II expression during MO differentiation and found that RA dose dependently inhibited CRABP II expression in human MO/MAC (Figure 3). Similar results have been obtained by Astro¨m et al. for keratinocytes. During keratinocyte differentiation CRABP II is upregulated but RA prevents differentiation and thereby CRABP II expression in these cells (16). In contrast, RA induces CRABP II in dermal but not lung fibroblasts (10,16) and topical application of RA on human skin also strongly increases the CRABP II mRNA and protein expression (23). In human neuroblastoma cell lines RA inhibited the proliferation and increased the expression of CRABP II (24). Since retinoic acid response elements, e.g. binding sites for the RA receptor homodimers and heterodimers, are found in the promoter region of the CRABP II gene, it is not surprising that this gene is regulated by RA (25). The divergent results regarding regulation by RA may in part be due to the relative amount of RA receptors and other transcription factors like AP-1 in the respective cell type which are both involved in the regulation of CRABP II. Transcriptional activation by AP-1 has been shown to be negatively regulated by RA (26). In further
RESULTS AND DISCUSSION In order to identify genes which may be involved in MO to MAC differentiation we used the RDA technology with cDNA from human MO as ‘‘driver’’ and cDNA from MO-derived MAC as ‘‘tester’’. Three of the resulting cDNA clones were identical to bp 458-735 of the published human CRABP II sequence (16,21) indicating that the expression of this gene is different in MO vs. MAC. Northern blot analysis revealed that the cDNA fragment detected an approximately 1.2 kb mRNA transcript in human MO-derived MAC which is in accordance with the published CRABP II sequence (Figure 1). Further analysis showed that the CRABP II transcript was not detected in freshly isolated MO but upregulated during differentiation of MO to MAC
FIG. 3. Retinoic acid inhibits CRABP II expression. Elutriation purified human blood MO were cultured in the presence of 2% human serum with and without RA on plastic culture dishes. After 24 h RNA was extracted and Northern analysis performed according to Materials and Methods.
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FIG. 4. Expression of CRABP II in several cell lines and primary cells. All primary cells and cell lines were cultured on plastic culture dishes and directly lysed in the plates with guanidine thiocyanate solution. RNA extraction and Northern analysis were performed according to Materials and Methods.
experiments we were interested whether CRABP II would also be expressed in monocytic cell lines which are often used as a model for MAC differentiation. We found that CRABP II is not expressed in several monocytic cell lines (Figure 4). Even if those cell lines were induced to differentiate with 1,25-dihydroxyvitamin D3 no CRABP II mRNA could be deteceted (data not shown). From the other cell lines we investigated, only HT29, a colon carcinoma cell line, showed a weak expression of CRABP II. Peripheral blood lymphocytes were also negative for CRABP II expression. As MO can not only differentiate along the classical pathway to MAC but also to dendritic cells we analyzed the expression of CRABP II in MO-derived dendritic cells cultured with 10% FCS, IL-4 and GM-CSF (Fig. 5). In contrast to MO-derived macrophages, CRABP II mRNA was nearly not detectable in MO-derived dendritic cells. However, dendritic cells showed a strong expression of RA receptors (manuscript in preparation). Only few data are up to now available regarding
the effect of RA on dendritic cells. The antigen presenting function of dendritic cells tested by the mixed lymphocyte reaction in vitro was found to be inhibited by high doses of RA but lower doses stimulated allogeneic lymphocytes (27). Accordingly, Katz et al. found an increase of the accessory cell function of dendritic cells/ MAC isolated from mice which had been fed with vitamin A acetate or RA (28). Based on these data we suggest that RA modulates the activation and differentiation of cells of the myeloid lineage and that CRABP II may influence this process by regulating the intracellular steady state concentration of retinoids in those cells. ACKNOWLEDGEMENTS The authors thank Ute Ackermann and Lucia SchwarzfischerPfeilschifter for excellent technical assistance. This investigation was supported by Deutsche Forschungsgemeinschaft An111/6-6.
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FIG. 5. Comparative analysis of CRABP II expression between MO-derived macrophages and dendritic cells. Elutriation purified human blood MO were cultured either on plastic culture dishes in the presence 10% FCS, GM-CSF, and IL-4 for the generation of MOderived dendritic cells or with 2% pooled AB serum for the generation of macrophages. After 7 days cells were lysed with guanidine thiocyanate solution. RNA extraction and Northern analysis were performed according to Materials and Methods.
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