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(Journal of Leukocyte Biology. 2001;69:361-372.)
© 2001 by Society for Leukocyte Biology

Retinoic acid up-regulates myeloid ICAM-3 expression and function in a cell-specific fashion—evidence for retinoid signaling pathways in the mast cell lineage

Magda Babina, Kerstin Mammeri and Beate M. Henz

Department of Dermatology, Charité, Humboldt-Universität zu Berlin, Germany

Correspondence: Magda Babina, Department of Dermatology, Charité, Campus Virchow, Humboldt-Universität zu Berlin, Augustenburger Platz 1, D-13344 Berlin, Germany. E-mail: magda.babina{at}charite.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Investigation of mast cell responsiveness toward retinoic acid (RA) revealed selective promotion of ICAM-3 expression in the human mast cell line HMC-1. This process was dose- and time-dependent and detectable by flow cytometry, Western blot analysis, ELISA, and Northern blot analysis. ICAM-3 modulation was found to be cell-type dependent, detectable also for HL-60 cells and monocytes but not U-937 and only weakly for KU812 cells. Terminally differentiated skin mast cells also failed to up-modulate their ICAM-3, suggesting the requirement for some degree of immaturity for the process. RA-mediated effects on ICAM-1 expression, studied in parallel, were clearly distinct from those on ICAM-3. Investigation of retinoid receptor expression, known to mediate intracellular RA signaling, revealed presence of RAR{alpha}, RAR{gamma}, RXRß, and RXR{gamma} transcripts in all cell lines studied, and HMC-1 cells were the only line lacking RXR{alpha}. RARß, not expressed at baseline, was induced by RA in a fashion obviously correlating with ICAM-3 up-regulation. Increased ICAM-3 expression was of functional significance, such that processes stimulated or co-stimulated via ICAM-3 (homotypic aggregation, IL-8 secretion) were clearly enhanced upon RA pretreatment, suggesting that RA may contribute via hitherto unrecognized pathways to immune function and host defense.

Key Words: vitamin A metabolites • adhesion molecules • homotypic aggregation • interleukin-8


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intercellular adhesion molecule (ICAM)-3, an adhesion molecule of the immunoglobulin (Ig) superfamily, represents a cellular counter-receptor for the ß2 integrins, lymphocyte function-associated antigen-1 (LFA-1) and {alpha}dß2, as well as for the recently discovered lectin, DC-SIGN [1 2 3 4 5 6 ]. Although highly homologous to ICAM-1 and ICAM-2, its expression profile and regulatory potential are quite distinct from that of the other two ICAM receptors. ICAM-1 is present on a broad spectrum of cells and up-regulated by a variety of exogenous factors, especially by pro-inflammatory cytokines such as interleukin-1 (IL-1), interferon-{gamma} (IFN-{gamma}), and tumor necrosis factor {alpha} (TNF-{alpha}) [7 , 8 ] as well as by retinoic acid (RA) [9 , 10 ], and ICAM-2 expression is constitutive and found predominantly on endothelial cells and certain leukocyte subsets [11 , 12 ]. Conversely, ICAM-3 is expressed exclusively among cells of hematopoietic origin under nonpathological conditions [1 2 3 , 13 ]. Although already high at baseline, enhanced ICAM-3 expression was found to be associated with lymphoid differentiation and/or activation [14 , 15 ].

ICAM-3 is suggested to mediate various processes in addition to its function as adhesion receptor. Cross-linking of ICAM-3 by monoclonal antibodies (mAbs) directed against certain epitopes triggers intracellular signaling involving calcium mobilization and tyrosine phosphorylation [15 16 17 18 19 ]. The cellular consequences thereof vary from cell type to cell type but involve co-activation of lymphocytes, changes in cell morphology, chemokine production, homotypic and heterotypic aggregation, as well as cellular adhesion to immobilized integrin ligands [18 19 20 21 22 23 24 25 ]. Furthermore, the ability of ICAM-3 to enhance T-cell activation by antigen-presenting cells corroborates its importance for the initial phase of an immune response [6 , 26 27 28 ]. Thus, ICAM-3 is equipped with the property of mediating and regulating cellular adhesion as well as other physiological processes and therefore appears to be of key relevance to immune function.

RA, active metabolites of vitamin A, are implicated in a broad array of biological processes such as development, cell growth, and differentiation [reviewed in 29]. However, it becomes increasingly clear that RA also possess potent immunomodulatory capacity, although the underlying processes at the cellular level are only incompletely understood [29 30 31 ]. The predominant mode of RA action at the molecular level is based on its activation of nuclear receptors, which belong to the family of steroid/thyroid/retinoid-activated transcriptional regulators [32 , 33 ]. Two classes of retinoid receptors have been described, the RA receptors (RAR{alpha}, RARß, RAR{gamma}) and the retinoid X receptors (RXR{alpha}, RXRß, RXR{gamma}), with each receptor subtype being encoded by a separate gene. Genes responsive to RA contain RA response elements (RARE) within their promoter/enhancer regions consisting of two half-sites of a PuG(G/T)TCA or related motif [32 , 33 ]. In most cases, an efficient transduction of the RA signal requires heterodimerization between RAR and RXR [34 ].

Although ICAM-3 up-regulation by RA has been shown before for the acute promyelocytic leukemia (APL) cell line NB-4 by flow cytometry, neither the functional consequences thereof nor the cell-type dependence of the process has been addressed [35 ]. In addition, a strong distinction between individual cell types with regard to retinoid-induced processes is widely appreciated. In contrast to most other myeloid cell lines, effects of RA on cells of the mast cell lineage have been poorly investigated so far. We have, however, demonstrated previously that expression of mast cell adhesion receptors is modulated by RA treatment in a highly selective manner [36 , 37 ]. We show herein that several RA isoforms also affect ICAM-3 expression and function by immature HMC-1 cells and certain other cell types, thereby supporting and enlarging the previous observation that pathways regulating ICAM-3 expression prior to translation also exist for myeloid cells [35 ]. Furthermore, the presence of several retinoid receptors in the mast cell line, shown for the first time by the present study, offers a rational explanation for the susceptibility of these cells to RA action.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and reagents
The murine anti-ICAM-3 mAbs CAL3.10 (IgG1), recognizing an epitope within IgSF domain I [23 ], CAL3.34 (IgG1, IgSF domain IV) [23 ], and ICAM-3.3 (IgG1, IgSF domain I; information from the supplier) were purchased from R&D Systems (Wiesbaden, Germany). The mAbs 25.3.1 (anti-LFA-1{alpha}, IgG1), 3E8 (anti-LFA-1ß, IgG1), and D2.10 (anti-glycophorin A, IgG1) were supplied by Immunotech (Marseille, France). mAb RR1/1, recognizing human ICAM-1 and mAb CBR-IC3/1 against ICAM-3 (binding to IgSF domain I [20 ]), were from Bender MedSystems (Vienna, Austria). Streptavidin-peroxidase was from Boehringer-Mannheim (Mannheim, Germany). Fluorescein isothiocyanate (FITC)-conjugated, affinity-pure F(ab')2 fragment goat anti-mouse was from Jackson Immuno Research Laboratories (West Grove, PA). The calcium ionophore A23187 was supplied by Calbiochem (San Diego, CA). Phorbol 12-myristate 13-acetate (PMA), 9-cis, 13-cis, and all-trans RA were purchased from Sigma (München, Germany). The RAR-specific agonists CD336, CD437, and CD2019 were a kind gift of Dr. U. Reichert (CIRD, Galderma, France).

Cell culture and treatment
The human mast cell line HMC-1 [38 ] was kindly provided by Dr. Butterfield (Rochester, MN). Cells of the HMC-1 subclone 5C6 were used throughout [39 ]. They were grown in basal Iscove’s medium and supplemented with 10% heat-inactivated fetal calf serum (FCS) and 10 µM monothioglycerol (Sigma). The human basophilic precursor cell line KU812 [40 ] was kindly provided by the Borstel Research Institute (Germany) and cultured in RPMI 1640 medium containing 15% FCS and 50 µM ß-mercaptoethanol (Sigma). The promyeloid cell line HL-60 [41 ] and the promonocytic cell line U-937 [42 ] were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and maintained in RPMI 1640 medium/10% FCS. All cell culture media contained 4 mM L-glutamine, streptomycin, and penicillin (all from Seromed, Berlin, Germany). Monocytes were isolated from buffy coats of healthy donors by Ficoll-Hypaque (Seromed) density-gradient centrifugation, followed by one step of adherence to plastic culture dishes at 37°C for 30–40 min. Adherent cells were >95% positive for CD14, as determined by flow cytometry. Isolated monocytes were kept in RPMI 1640 medium/10% FCS, 4 mM L-glutamine, and antibiotics.

Isolation of skin mast cells was performed as described previously with several modifications [37 ]. Briefly, foreskins were cut into stripes and treated with dispase (Boehringer-Mannheim) at 0.5 mg/ml and 4°C overnight. The epidermis was then removed from the dermis, the latter chopped in small pieces and treated with collagenase at 5 mg/ml (type 4, Worthington, Lakewood, NJ) for 1 h at 37°C. Cells were separated from remaining tissue by three steps of filtration. Mast cells were further purified by positive selection using anti-c-kit mAb YB5.B8 (kindly provided by Dr. L. K. Ashman), goat anti-mouse Ig-coated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany), and a MACS separation device. Mast cell purity in these preparations ranged from 85% up to 95% typically, as assessed by toluidine blue staining.

Cells were treated in standard growth medium containing 9-cis, 13-cis, all-trans RA, or RAR-specific agonists at 37°C for the periods and at the concentrations indicated. Control cells were kept in medium alone for the same time.

Flow cytometry
Approximately 5 x 105 cells were washed twice in Ca2+/Mg2+-free phosphate-buffered saline (PBS), reacted with saturating concentrations of primary mAb diluted in human AB-serum (Biotest, Dreieich, Germany) for 30 min at 4°C, washed, and stained with FITC-conjugated F(ab')2 fragment of goat anti-mouse Ig for 20 min at 4°C. After being washed, cells were analyzed by an EPICS XL flow cytometer (Coulter Electronics, Krefeld, Germany). A minimum of 10,000 cells were investigated/sample. Mean channel fluorescence (MCF) was considered to correlate with surface-Ag density. The MCF value of untreated cells was considered as 100%. Cell-surface molecule density of differently treated cells was determined as MCF (treated cells)/MCF (medium control) x 100. An isotype-matched, nonbinding mAb (R&D Systems) was used as negative control.

In case of skin mast cells, 2 x 105 cells were used per assay and stained with FITC-coupled, anti-ICAM-3 mAb CBR-IC3/1 or isotype-matched, nonbinding mAb.

Immunoprecipitation, SDS-PAGE, and Western blot
Proteins at the exterior cell surface of HMC-1 cells were biotinylated specifically using the BM cellular labeling and immunoprecipitation kit (Boehringer-Mannheim), following the manufacturer’s instructions. After labeling, cells were lysed at 1 x 107/ml for 1 h at 0°C in 1% Nonidet P-40, 0.5% sodium deoxycholate, 50 mM sodium borate, pH 8.0, 150 mM NaCl, 100 µg/ml phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml aprotinin, and 1 µg/ml leupeptin. Protein concentration was determined using the bicinchoninic acid (BCA) protein determination kit (Pierce, Rockford, IL), and samples were adjusted to the same protein concentration (600 µg/ml) prior to being precleared overnight using the irrelevant mAb D2.10 and 50 µl anti-mouse IgG1 (heavy chain-specific) agarose beads (Sigma) per 1 ml lysate. The supernatants were reacted with 3 µg mAb followed by addition of 100 µl beads, and mixtures were incubated overnight under gentle rocking at 4°C. Beads were collected after brief centrifugation and washed, and proteins were eluted with 2 x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 200 mM dithiothreitol, and 0.2% bromphenol blue) prior to being heated at 95°C for 5 min. Immunoprecipitated proteins were then resolved by a discontinuous SDS-PAGE using a 6% separating gel and a 5% stacking gel and subsequently blotted onto polyvinylidene difluoride transfer membrane (Du Pont de Nemours, Bad Homburg, Germany) using a semi-dry transfer cell (Bio-Rad Laboratories, Richmond, CA). The membrane was saturated with 5% nonfat dried milk and incubated with streptavidin-peroxidase (Boehringer-Mannheim) at 50 mU/ml for 30 min at room temperature. After extensive washing, immunoprecipitated proteins were visualized with 3,3',5,5'-tetramethylbenzidine (TMB, insoluble; Calbiochem).

Enzyme-linked immunosorbent assay (ELISA)
A commercially available ELISA kit specific for human soluble ICAM-3 (R&D Systems) was used for quantification of ICAM-3 expression. Differently treated HMC-1 cells were lysed at 1 x 107/ml in 1% Triton X-100, 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 100 µg/ml PMSF, 1 µg/ml aprotinin, and 1 µg/ml leupeptin. Protein concentrations were determined with the BCA protein determination kit as above. The lysates were diluted 1:5 with assay diluent during the assay. ICAM-3 concentration was determined relative to a standard curve and is expressed as ng ICAM-3/mg total protein.

For quantification of IL-8 in the supernatants of HMC-1 cells, cells were pretreated with or without RA for three days and then washed three times in serum- and RA-free medium. Cells from the different groups were plated in culture dishes at 1 x 106/ml under serum-free conditions, preincubated or not with different anti-ICAM-3 mAbs (30 min at 37dgC), and then stimulated by PMA (at 25 ng/ml) alone or in combination with the calcium ionophore A23187 (at 500 nM). After 24 h, supernatants were removed and kept at -80°C until use. IL-8 content was detected by a specific ELISA kit (R&D Systems) following the manufacturer’s instructions.

Northern blot
For comparison of ICAM-3-specific mRNA content, total cellular RNA was extracted using the RNeasy Midi Kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. RNA concentration was assessed by the RiboGreen RNA quantitation kit (Molecular Probes, Göttingen, Germany). Samples of 15 µg RNA were electrophoresed through a 1% agarose/2.2 M formaldehyde gel, transferred to GeneScreen Plus nylon membrane (NEN, Zaventem, Belgium) by capillary transfer, and fixed by ultraviolet light.

Hybridization with peroxidase-labeled DNA probe (generated with the primer pairs given in Table 1 ) at 10 ng/ml and specific detection of messenger RNAs were performed with the North2South labeling and detection system, according to the manufacturer’s protocol (Pierce). Specific transcripts were visualized by enhanced chemiluminescence. After drying, the same membrane was rehybridized with a peroxidase-labeled glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe as control of equal RNA loading.


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  		Table 1. Primer Pairs Used for PCR Amplifications

 
Reverse transcriptase-polymerase chain reaction (RT-PCR)
For determination of RAR and RXR expression, RNA from approximately 3 x 105 cells/assay (differently treated cell lines) was reverse-transcribed in a 25 µl vol with a first-strand synthesis kit (Boehringer-Mannheim) using random priming, as detailed by the manufacturer, and the resulting cDNA was used at a dilution of 1:20 or 1:40 in PCR amplifications. Specific primers for the different nuclear receptors are given in Table 1 . Reactions were run on an Omni Gene thermocycler with the following cycle program: 94°C/5 min (1 cycle), 94°C/1 min, 61–72°/1 min, 72°C/1 min (35 cycles), and 72°C/10 min (1 cycle). The following annealing temperatures were applied: RAR{alpha}, 61°C; RARß and RXR{alpha}, 63°C; RXRß and RXR{gamma}, 70°C; and RAR{gamma}, 72°C. RNA at the same dilution as cDNA was used as negative control and run under the same conditions.

Aggregation assay
Quantitative aggregation assays were performed in 96-well microtiter plates (Greiner, Frickenhausen, Germany) essentially as described [37 ]. In brief, cells treated without (medium control) or with 9-cis RA, 13-cis RA, or all-trans RA (all at 10 µM) for 4 days were plated in culture dishes at 5 x 105/ml and stimulated with anti-ICAM-3 mAbs (each at 1 µg/ml) in a total reaction vol of 100 µl at 37°C for the times indicated. After incubation, suspensions of each sample were mixed by pipetting up and down 20 times and were transferred to a hemocytometer. The number of free cells and total cell number were assessed, and percent aggregation was determined as (1 number of free cells/total cell number) x 100.

Statistics
Statistical analysis was carried out with the unpaired Student’s t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Modulation of ICAM-3 and ICAM-1 expression by 9-cis, 13-cis, and all-trans RA reveals cell-type-specific differences
We [45 ] and others [46 ] have recently demonstrated ICAM-3 expression on human mast cells. To analyze whether incubation with RA modulates ICAM-3 expression, surface density of the molecule was evaluated by indirect immunofluorescence and flow cytometry. Treatment of HMC-1 cells with 9-cis, 13-cis, or all-trans RA (at 1 or 10 µM) for three days indeed resulted in enhancement of cellular ICAM-3 density, which is already high at baseline (Fig. 1 and Table 2A ), and expression of the homologous receptor ICAM-1 was not influenced by the treatment (Table 2B) . CD11a and CD18 expression on HMC-1 cells also remained unchanged upon RA administration (unpublished results), in accordance with data previously shown [36 ].



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  		Figure 1. Cell-specific ICAM-3 regulation in response to all-trans RA. Different myeloid cell types were cultured in the presence or absence of all-trans RA (all at 1 µM) for 3 days and investigated concerning surface expression of ICAM-3 by flow cytometry. Representative histograms of at least three separate tests are shown. Black line, cells kept in medium only; gray line, all-trans RA-treated cells. Very similar results were obtained when 9-cis or 13-cis RA were used instead of all-trans-RA.

 

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  		Table 2. Modification of ICAM-3 and ICAM-1 Expression on Different Myeloid Cells Following RA Treatment

 
As shown in Figure 1 and Table 2A and in contrast to what was observed for HMC-1 cells, no increase in ICAM-3 expression was detected for U-937 and only a minor up-modulation on prebasophilic KU812 cells. The effect achieved for the HL-60 line, however, was of an even higher extent compared with HMC-1 cells. Freshly isolated blood monocytes from 10 different donors up-regulated ICAM-3 significantly upon culture in the presence of RA, but the effect was strongly donor-dependent, ranging from no increase at all (three donors), to up-regulation to an intermediate degree of maximally 40% (five donors), to ICAM-3 induction in the range of up to 80% (two donors). In contrast to HMC-1 cells, which resemble an immature mast cell phenotype, no alteration of ICAM-3 expression was found for mature skin mast cells (Fig. 1 and Table 2A ). This might be a consequence of the much lower ICAM-3 expression by cutaneous mast cells, suggesting down-regulation of the molecule upon mast cell maturation [45 ].

Conversely, ICAM-1 expression was increased substantially on KU812, HL-60 cells, and blood monocytes but not on HMC-1 or U-937 cells (Table 2B) . In contrast to ICAM-3, RA-mediated effects on ICAM-1 obviously depended on the precise RA isoform, with all-trans RA being lowest throughout in potency. Also, the maximal response was lower for ICAM-1 than for ICAM-3.

Dose-response studies revealed concentration dependency of ICAM-3 up-regulation over a very wide concentration range from 0.0001 up to 10 µM (Fig. 2A ). Slight differences among the three RA isoforms could be detected: Although all-trans RA appeared most potent at very low doses, 9-cis RA was most effective in the range of 0.1–1 µM. At 10 µM, there were no significant differences among the RA isoforms. Because 9-cis RA represents the only RA isoform interacting efficiently with RXR in addition to its binding to the RAR [47 48 49 ], this suggests that ICAM-3 up-modulation occurs independently from transactivation through RXR, fitting the concept that RXR does not necessarily need to be ligand-activated on its own and functions as a silent partner in RXR-RAR heterodimers [32 , 33 ]. The anticipated EC50 values (corresponding to the RA concentration that exerts half-maximal ICAM-3 induction) were approximately 0.03 µM (9-cis RA), 0.08 µM (13-cis RA), and 0.01 µM (all-trans RA). Time-course studies revealed that ICAM-3 induction was slow in onset, first detectable after 2 days, and further increasing up to day 5 after treatment (Fig. 2B) .



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  		Figure 2. Concentration and time dependency of ICAM-3 up-regulation on HMC-1 cells by 9-cis, 13-cis, and all-trans RA. (A) Cells were treated with various concentrations of 9-cis, 13-cis, and all-trans RA for 72 h at 37°C or (B) kept in the presence or absence (control) of either RA for the indicated time periods. ICAM-3 surface density was quantitated by flow cytometry using CAL3.10 as anti-ICAM-3 mAb. Data are expressed in relation to the medium control, which was normalized at 100%. Results are means ± SD of three (A) or four (B) independent assays.

 
To confirm RA-mediated ICAM-3 modulation at the protein level by an independent method, Western blot analysis was performed. As shown in Figure 3A , up-regulation of ICAM-3 in response to RA could be demonstrated clearly upon immunoprecipitation with two different anti-ICAM-3 mAbs, and there were no changes in the apparent molecular weight of the molecule, indicating that its glycosylation pattern was preserved in the presence of RA. ICAM-3 concentration was detected additionally by ICAM-3-specific ELISA. The lysates of nontreated HMC-1 cells contained approximately 13.1 ± 2.1 ng ICAM-3/mg total protein (Fig. 3B) , which is largely in accordance with our previous data [45 ]. Treatment with either RA resulted in remarkable up-regulation of ICAM-3 in the same range as could be detected by flow cytometry, demonstrating that cell-surface expression represents or at least reflects total cellular ICAM-3 expression.



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  		Figure 3. Up-modulation of ICAM-3 expression by RA as detected by Western blot analysis (A) and ELISA (B). HMC-1 cells were cultured in the presence or absence of 9-cis, 13-cis, or all-trans RA (all at 10 µM, 72 h), after which time, cells were harvested. (A) The cell surface was biotinylated, the cells were lysed, and ICAM-3 was immunoprecipitated using the mAbs CAL3.34 (lanes 1–4), ICAM-3.3 (lanes 5–8), or D2.10 (negative control; lanes 9–12). Untreated cells: lanes 1, 5, and 9; 9-cis RA: lanes 2, 6, and 10; 13-cis RA: lanes 3, 7, and 11; all-trans RA: lanes 4, 8, and 12. (B) ICAM-3 content in lysates of untreated or RA-treated (10 µM, 72 h) HMC-1 cells was determined by an ICAM-3-specific ELISA. Protein concentration was quantitated by the BCA protein determination kit. Data are presented as ng ICAM-3/mg total protein and are the mean ± SD of three independent experiments.

 
Northern blot analyses revealed constitutive expression of the ICAM-3-specific mRNA at approximately 1.7 kb [2 ] in KU812 and HMC-1 cells (Fig. 4 ). ICAM-3 transcript in relation to the housekeeping gene GAPDH was clearly increased in HMC-1 (but not in KU812) following treatment with 9-cis and all-trans RA, implying that enhanced protein expression was at least in part a consequence of increased steady-state level of ICAM-3 mRNA. Results from Northern blot analyses were confirmed perfectly by semi-quantitative RT-PCR (unpublished results).



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  		Figure 4. RA treatment results in an augmented steady-state level of ICAM-3-specific transcript in HMC-1 but not in KU812 cells. Total RNA was extracted from untreated or RA-treated (all-trans and 9-cis RA, each at 1 µM, 72 h) HMC-1 and KU812 cells, and equal amounts thereof were subjected to Northern blot analysis as described in Materials and Methods. Lanes 1 and 4: Cells kept in medium only; lanes 2 and 5: cells treated with 9-cis RA; lanes 3 and 6: cells treated with all-trans RA. The Northern blot was hybridized sequentially with ICAM-3 and GAPDH probes, as indicated in the figure. Blots are representative of four independent tests.

 
Enhanced ICAM-3 expression alters homotypic cell adhesion triggered through ICAM-3
We have shown recently [45 ] that human mast cells exhibit the phenomenon of homotypic adhesion when treated with mAbs directed against certain epitopes of ICAM-3. Therefore, we sought to determine whether the extent of cluster formation induced via ICAM-3 could be affected by altered ICAM-3 cell-surface density.

As shown in Table 3 , significant differences between RA-pretreated and control cells could be detected after 2 h and 24 h with the inducing anti-ICAM-3 mAbs CAL3.10 or CBR-IC3/1, and spontaneous aggregation and aggregation in the presence of inert mAb CAL3.34 were unaffected by RA treatment. Very similar results were obtained when 9-cis and 13-cis RA were used instead of all-trans RA (unpublished results).


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  		Table 3. Up Regulation of ICAM-3 by RA Leads to Enhanced Homotypic Aggregation Induced via ICAM-3

 
Correlation between the level of ICAM-3 expression and IL-8 secretion co-stimulated by anti-ICAM-3 mAb
Signaling via ICAM-3 leads to modulated release of certain cytokines from HMC-1 cells [45 ]. For example, secretion of IL-8 is enhanced by the agonistic ICAM-3 mAb CBR-IC3/1, and CAL3.34 is without such effect. Therefore, we tested next whether the increase in ICAM-3 expression is accompanied by higher release of IL-8 upon ICAM-3-mediated co-stimulation. As presented in Figure 5 , it is very interesting that pretreatment of HMC-1 cells with all-trans RA led by itself to an increase in PMA and PMA + A23187-stimulated IL-8 secretion from 8.6 to 19.8 ng/ml and 28.4 to 51.9 ng/ml, respectively, in the absence of anti-ICAM-3 mAb. The influence of CBR-IC3/1 on IL-8 secretion was, however, of greater magnitude for all-trans, RA-pretreated cells than for control cells, resulting in 384% versus 234% of the respective control in the case of PMA-mediated stimulation and 362% versus 160% in the case of PMA + A23187-stimulated IL-8 release, clearly substantiating that newly synthesized ICAM-3 is functionally active and capable of transducing signals across the plasma membrane.



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  		Figure 5. Effects of RA pretreatment on IL-8 release from HMC-1 cells co-stimulated via ICAM-3. HMC-1 cells were cultured in the presence or absence of all-trans RA (10 µM, 72 h), after which time cells from both groups were washed in serum-free medium and plated in culture dishes at 1 x 106/ml. Preincubation (30 min, 37°C) was done in the presence or absence of the anti-ICAM-3 mAbs CBR-IC3/1 or CAL3.34 (each at 2 µg/ml) prior to stimulation by PMA (at 25 ng/ml) or PMA + A23187 (25 ng/ml and 500 nM, respectively) for 24 h. IL-8 concentration in the cell-free supernatants was measured by an IL-8-specific ELISA (R&D Systems). Data from one single experiment performed in duplicate are shown. Similar results were obtained in two further tests.

 
Expression patterns of RAR and RXR in myeloid cell lines
Because RA influence on cellular behavior is mediated by the activation of retinoid receptors and because no data regarding expression of these receptors were available for mast or basophilic cells, it appeared essential from our present and previous data to investigate the basal and RA-induced expression patterns of RAR and RXR in these cells and compare them with other cells of the myeloid lineage.

As determined by RT-PCR technique and summarized in Table 4 , HMC-1, KU812, U-937, and HL-60 cells all expressed RAR{alpha}, RAR{gamma}, RXRß, and RXR{gamma} at baseline and following treatment with either RA isoform. In contrast to the other cell lines, it is interesting that HMC-1 cells did not exhibit appreciable RXR{alpha} mRNA levels, irrespective of treatment. The only receptor whose expression was strongly influenced by RA was RARß. None of the cell lines expressed RARß at baseline, but its transcript was clearly detected in RA-treated HMC-1 and HL-60 cells, very weakly in KU812, and not at all in U-937 cells. Expression patterns of RAR and RXR in HMC-1 and KU812 cells are presented in Figure 6 .


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  		Table 4. Expression Profiles of RAR and RXR in HMC-1, KU812, HL-60, and U-937 Cells

 


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  		Figure 6. Expression pattern of RAR and RXR in HMC-1 and KU812 cells. Representative RT-PCR amplifications using RNA of untreated cells (lane 1) and cells treated with 9-cis RA (lane 2), 13-cis RA (lane 3), and all-trans RA (lane 4) are shown. Lanes 5 and 6: negative controls using RNA from untreated and RA-treated cells instead of cDNA. RT-PCR reactions were carried out as described in Materials and Methods using a minimum of three different cell preparations. Note that RARß expression in RA-treated KU812 cells is undetectable in this form of presentation, although a faint band was visible in the original gel in three out of three experiments.

 
ICAM-3 modulation by RAR-selective agonists
To elucidate the role of the different RAR subtypes in ICAM-3 up-regulation, HMC-1 cells were cultured in the presence of specific RAR{alpha}, RARß, and RAR{gamma} agonists (all at 1 µM) for three days, after which time ICAM-3 expression was investigated by flow cytometry and by ELISA. As illustrated in Figure 7 , the highest effect on ICAM-3 surface density was obtained with the RAR{gamma}-selective compound CD437, followed by the RAR{alpha}-specific retinoid CD336. Conversely, the RARß agonist CD2019 produced a weak effect only. CD437, however, also possessed potent influence on morphological features and viability of HMC-1 cells (unpublished results), and CD336 and CD2019 displayed no such effects and were thus better comparable to all-trans RA.



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  		Figure 7. ICAM-3 modulation by RAR-selective agonists. HMC-1 cells were cultured in the presence of the retinoids all-trans RA, CD336, CD2019, CD437, or CD336 + CD2019 (all at 1 µM for 72 h) or kept in medium alone. Cells were then investigated concerning their ICAM-3 expression by the use of flow cytometry (A) or ELISA (B). Data are the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus the medium control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Vitamin A and its derivatives are suggested to participate in a broad range of immune function, and their effects on different leukocyte subsets, including mast cells, have not been clearly determined yet. In the present study, we demonstrate that three RA isoforms up-modulate ICAM-3 expression in the human mast cell line HMC-1, thereby further substantiating that cells of the mast cell lineage are RA-responsive, in analogy to other hematopoietic cells with the effects, however, highly selective thus far, affecting only CD43 and ICAM-3 expression [36, 37, and this study].

In addition to HMC-1 cells, ICAM-3 regulation by RA was found to be a more generalized phenomenon, with strong differences between individual cell types, however. Although the highest effect was observed for HL-60 cells, responsiveness of KU812 and U-937 cells was low or absent, and strong donor-dependence was revealed for blood monocytes. Although HL-60 and HMC-1 cells vary remarkably with regard to morphology and phenotype, the positive influence of RA on their ICAM-3 expression suggests that a certain degree of myeloid immaturity (HL-60) or mast cell commitment (HMC-1) might be necessary prerequisites for the process.

Our data are in line with previous observations made by Larson et al. [35 ], who showed up-regulation of ICAM-3 expression in response to RA for the APL cell line NB-4, further proving that ICAM-3 is an RA-inducible molecule. Conversely, our study shows that the process of ICAM-3 up-regulation is clearly cell-type-dependent affecting some but not all myeloid cells. Based on the correlation between steady-state level of ICAM-3 transcript (clearly shown to increase in HMC-1 but not in unresponsive KU812 cells) and ICAM-3 protein in our study, the regulatory process is likely to occur pretranslationally by enhanced transcription or selective mRNA stabilization. Whether additional mechanisms (such as enhanced translation and/or reduced protein degradation) also contribute to ICAM-3 up-regulation cannot be completely ruled out, however.

It is interesting that ICAM-3 expression of fully differentiated skin mast cells was not modulated by RA, suggesting that some degree of cellular immaturity is a necessary prerequisite for the response. ICAM-3 expression on mast cells most likely decreases in a differentiation-dependent manner, as discussed previously [45 ]. Therefore, terminally differentiated mast cells express ICAM-3 to a much lower degree, with the dominant percentage of cells negative for ICAM-3 expression (Fig. 1) , and mature cells are obviously unable to re-express it following administration of exogenous stimuli like RA. Also, the HMC-1 line was established from a patient with mast cell leukemia [38 ], and there are differences with regard to responsiveness of leukemic cells and their normal counterparts toward RA also in other cellular systems such as monocytes [50 ]. Because committed precursors of the mast cell lineage have not been identified so far in the circulation, these cells thus cannot be isolated for study [51 ]. Immature mast cells have, however, been shown to increase in various types of inflammatory conditions [52 , 53 ], including psoriasis, an RA-responsive disease; ICAM-3 may well be involved in the increased influx into the tissue and subsequent increased mediator production of these cells.

The expression of the homologous ICAM-1 receptor was also cell-specifically affected by RA in a fashion that did not correlate to ICAM-3 modulation. HMC-1 cells, for example, did not up-modulate their surface ICAM-1 after a 72-h incubation (Table 2B) , and an increase was detected for monocytes, KU812, and HL-60 cells, with the strongest effect being, however, lower for ICAM-1 than for ICAM-3. ICAM-1 expression has been shown in different studies to be up-regulated by RA [9 , 54 ], and the ICAM-1 promoter is known to harbor a degenerate RARE involved in its transcriptional regulation [10 ]. Given the high homology between ICAM-1 and ICAM-3, it is tempting to speculate that the ICAM-3 promoter may also possess potential RARE(s), but clarification of the precise mechanism by which RA up-regulates ICAM-3-specific transcript will await characterization of the organization and regulatory sequences of the ICAM-3 gene.

The selective up-regulation of ICAM-3 versus that of ICAM-1 as observed for the HMC-1 cell line (and given it also happens in vivo) could lead to a shift in a cell’s preference to bind to LFA-1 by these two counter-receptors, thereby favoring ICAM-3-mediated signal transduction leading to aggregation and cytokine production. These processes appear selective for ICAM-3 because we have not been able to induce any of them by mAbs directed against various epitopes of ICAM-1 (unpublished results), which is in accordance with the hypothesis that ICAM-3 is the predominant signal transducer among the ICAM subfamily.

Because our previous [36 , 37 ] and present data strongly implied the presence of retinoid receptors in mast and basophilic cells, this issue was addressed in greater detail, revealing expression of RAR{alpha}-, RAR{gamma}-, RXRß-, and RXR{gamma}-specific mRNA in HMC-1 and KU812 cells (in addition to HL-60 and U-937 cells). Our data for HL-60 cells are largely in accordance with previous studies: Although expression of RAR{alpha} and RXR{alpha} in these cells was shown by several groups, slight differences were obtained for the other receptor subtypes, possibly because of differences in the precise subline used or the detection limit of the method applied [55 56 57 58 59 60 61 62 ]. It is of interest, however, that HMC-1 cells represented the only cell line lacking detectable RXR{alpha} expression. Because RA induction of ICAM-1 at the transcriptional level has been demonstrated to be mediated by RARß in combination with RXR{alpha} [63 ], the inefficiency of RA to modulate ICAM-1 expression in HMC-1 cells might be a result of the lack of RXR{alpha} expression. Because RXR{alpha} is expressed by all other hematopoietic cell types thus far investigated [60–62, 64, 65, and this study], the absence of RXR{alpha} in HMC-1 cells makes them an attractive model with which to delineate processes selectively dependent on RXR{alpha} action.

With regard to RARß, none of the cell lines under investigation expressed detectable levels of its transcript prior to RA treatment. However, its induction by RA was observed in HMC-1 and HL-60 and very weakly in KU812 cells. RARß has been shown previously to be induced vigorously by retinoids in different cells [56 , 66 67 68 ], and the RARß gene is known to harbor a functional RARE in its P2 promoter involved in this process [69 ].

The fact that RARß induction, as shown here, occurred in a cell-specific manner, obviously correlating with RA-mediated modulation of ICAM-3, suggested a functional role for this receptor in myeloid ICAM-3 regulation or the requirement for additional factors responsible for RARß and ICAM-3 induction. We tested the first hypothesis by the use of selective RAR{alpha}, RARß, and RAR{gamma} agonists. Although RAR{gamma}-specific CD437 produced the highest response with regard to ICAM-3, it additionally possessed very pronounced effects on proliferation and viability of HMC-1 cells (in contrast to RA and the other retinoids used), making it difficult to ascertain whether its positive influence on ICAM-3 expression was a direct effect of the compound or secondary to other changes. Further, CD437 has been shown previously to act by other mechanisms in addition to those mediated by RAR [70 , 71 ]. Because RARß-selective CD2019 was lowest in potency by far, not enhancing ICAM-3 expression further even when combined with CD336, these experiments, however, largely ruled out the possibility that RARß might itself be involved in ICAM-3 regulation. Therefore, it appears more likely that RARß induction and ICAM-3 up-regulation in myeloid cells depend on the same prerequisites, one of which may be presence of COUP-TF, which has been shown very recently to be involved in RARß expression [72 ]. Studies aimed at assessing expression of this transcription factor in the cell lines we used are underway currently.

ICAM-3 is known to mediate and regulate cellular adhesion and to be capable of signal transduction. Therefore, two experimental approaches were used to elucidate the functional significance of enhanced ICAM-3 expression. First, ICAM-3 surface density was found to correlate with the level of HMC-1 aggregation triggered through ICAM-3. Second, ICAM-3 up-regulation resulted in enhanced IL-8 production by HMC-1 cells co-stimulated via ICAM-3. It is of interest that RA pretreatment of HMC-1 cells exerted by itself a significant effect on stimulated secretion of the chemokine, implying that RA is possibly able to regulate other mast cell functions in addition to those involving adhesion receptors. However, the enhancing effect of anti-ICAM-3 mAb was clearly more striking for RA-pretreated cells (i.e., cells expressing a higher level of ICAM-3) than for control cells, and this was detected with both activation regimens used, namely PMA alone and PMA in combination with A23187, further substantiating a role for newly synthesized ICAM-3 for mast cell cytokine production. It remains to be established whether enhanced ICAM-3 expression on leukocytes may also be viewed in the context of their clearance by macrophages following apoptosis. ICAM-3 expressed by apoptotic cells was demonstrated recently to function as ligand for a macrophage counter-structure, possibly CD14, and to be involved in intercellular interactions relevant to removal of apoptotic cells [73 ]. It should be noted that CD437, producing the highest effect with regard to ICAM-3, was (in contrast to RA) also a potent inducer of HMC-1 apoptosis (unpublished results). Thus, increased ICAM-3 expression by RA may also be exploited experimentally to delineate further functional aspects of the molecule.

ICAM-3 appears to be especially important for mediation (by binding to LFA-1, {alpha}dß2, and the very recently described counter-receptor, DC-SIGN) and regulation of adhesion (by its property to induce homotypic and heterotypic adhesion events and adhesion of leukocytes to components of the extracellular matrix, the latter event being mediated by ß1 integrins).

RA is used therapeutically in different disease states, most importantly for the treatment of APL. An undesired, adverse effect in those settings is the RA syndrome, which is characterized by fever, progressive hypoxemia, and multiorgan failure [74 ]. The basic mechanisms underlying this syndrome are poorly understood but appear to involve extravasation of leukemic cells and other leukocytes into tissues and organs, and cytokine release. Therefore, RA-mediated alterations in expression and/or function of adhesion molecules are likely involved. Thus, the high levels of RA achievable in serum during RA therapy could in fact up-regulate ICAM-3 expression by leukemic and normal myeloid cells, thereby not only strengthening cellular binding to LFA-1-expressing cells but also increasing the processes stimulable through this receptor, such as activation state of other adhesion receptors and, very importantly, production of inflammatory or immunomodulatory cytokines, as shown in this study (Fig. 5) and substantiated by previous studies [25 , 45 ]. Because ICAM-3 is such a versatile molecule, its up-modulation on different cells (including APL cells, as shown previously [35 ]) may be of relevance to the events observed during RA syndrome, such as migration of leukocytes into respiratory tissue and kidney.

Together with our very recent study of 1,25-dihydroxyvitamin D3-mediated, ICAM-3 up-modulation by HMC-1 cells [75 ], the present study thus established a regulatory pathway for ICAM-3 expression used by some but not all myeloid cells. Pronounced ICAM-3 expression on HMC-1 cells correlated with certain functional properties of the molecule, and others (such as clearance of apoptotic leukocytes) need to be addressed in the future. The detailed analysis of retinoid receptor expression patterns in mast (and basophilic) cells is thought to contribute to a better understanding of the observed effects exerted by RA. In addition to ICAM-3 up-regulation, primarily addressed in this study, RA indeed exerted other profound effects on HMC-1 cells such as RARß induction and enhanced IL-8 secretion. Thus, these cells may represent a further interesting model system for investigation of the basic principles of RA action on growth, differentiation, and immune function. Some of these studies are currently underway.


   ACKNOWLEDGEMENTS
 
This work was supported by the Deutsche Forschungsgemeinschaft (grants WE1568/3-6 and HE2686/14-1). The authors thank Dr. J. H. Butterfield (Mayo Clinics, Rochester, MN) and the Borstel Research Institute (Germany) for providing cell lines, Dr. U. Reichert (CIRD Galderma, Valbonne, France) for supplying us with the RAR-selective agonists, and Dr. L. K. Ashman for donating the YB5.B8 mAb. We are grateful to Dr. C. S. Shelley (Renal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA) for critical reading of the manuscript and valuable discussion.

Received April 3, 2000; revised October 29, 2000; accepted November 20, 2000.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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J. Wang and A. Yen
A Novel Retinoic Acid-Responsive Element Regulates Retinoic Acid-Induced BLR1 Expression
Mol. Cell. Biol., March 15, 2004; 24(6): 2423 - 2443.
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