MATERIALS AND METHODS

Animals

BALB/C Ann mice were bred in our animal facilities from breeding pairs originally obtained from the Zentralinstitut für Versuchstierkunde (Hannover, Germany). The mice were used at 2–5 mo of age.

Monoclonal antibody (MoAb)

Clone 145–2C11 [anti-mouse CD3, hamster immunoglobulin (Ig) G2b] (^{Leo et al. 1987}) was kindly provided by Dr J.A. Bluestone (Ben May Institute, Chicago, IL). Clone 2G9 (anti-mouse I-A^d,b, I-E^d, rat IgG2a) (^{Becker et al. 1992}) was a kind gift from Dr M. Mohamadzadeh at the Department of Dermatology (Mainz, Germany). Clone MK-D6 (anti-mouse I-A^d, mouse IgG2a) (^{Kappler et al. 1981}) and clone 14.4.4S (anti-mouse I-E^k/d/p/r, mouse IgG2a) (^{Ozato et al. 1980}) were obtained from the American Type Culture Collection (Rockville, MD).

Cytokines

As a source of recombinant interleukin (IL)-2 supernatants of X63–Ag8-653 cells transfected with the murine IL-2 gene (^{Karasuyama & Melchers 1988}) were used. The cells were a kind gift by Dr F. Melchers (Basel Institute of Immunology, Basel, Switzerland). Human recombinant macrophage-colony stimulating factor (rM-CSF) and mouse recombinant granulocyte-M-CSF were kindly provided by Dr F. Seiler and D. Krumwieh (Behringwerke AG, Marburg, Germany). Mouse recombinant interferon- was kindly given to us by Dr H. Kirchner (Institut für Immunologie und Transfusionsmedizin, Lübeck, Germany). Mouse recombinant IL-4 was a kind donation by DNAX (Palo Alto, CA). Mouse TNF- for the cultivation of bone marrow-derived DC was obtained from R&D Systems (Minneapolis, MN). RANTES was purchased from RDI Inc. (Flanders, NJ).

Cells

Epidermal cell suspensions were prepared from pelts as described previously (^{Becker et al. 1991}). They were used for Langerhans cell enrichment either directly (fLC) or following in vitro cultivation for 1–3 d (cLC) (^{Ross et al. 1998a}). For mRNA isolation Langerhans cells were enriched from epidermal cells by immunomagnetic separation with Dynabeads M-450 (Dynal, Oslo, Norway) loaded with anti-major histocompatibility complex (MHC) class II MoAb 2G9 as described (^{Ross et al. 1998a}). MHC class II-positive, bead-coupled Langerhans cells and cells without attached beads were counted using a Neubauer hemocytometer. A purity of Langerhans cells of approximately 92–95% was obtained as determined by the ratio of bead-rosetted to nonrosetted cells. Langerhans cell viability, determined by Trypan Blue exclusion, was > 95%. For chemotaxis assays Langerhans cells were enriched using the CELLection Pan Mouse IgG kit (Dynal). Mouse anti-MHC class II MoAb MK-D6 and 14.4.4.S were bound to anti-mouse immunoglobulin, which in turn were connected to beads with a DNA link. After assessment of purity and viability of Langerhans cells, which was performed as described above with comparable results, beads were detached by incubating the cells in medium containing releasing buffer (Dynal), including DNAse I. Bone marrow-derived dendritic cells were cultured in the presence of GM-CSF and TNF- as described by^{Scheicher et al. (1992)} and^{Lutz et al. (1999)} and were enriched likewise.

For migration assays T lymphocytes and B lymphocytes were purified from spleen cells using Mouse T Cell Enrichment Columns (R&D Systems) and the Mouse B Cell Recovery Kit (Cedarlane, Hornby, Canada), respectively. For mRNA preparation, B cells were enriched using anti-mouse IgM paramagnetic beads (Dynal). Purity as assessed by fluorescence-activated cell sorter analysis was > 95%.

To generate T lymphoblasts, splenocytes were cultivated in the presence of 5 g per ml concanavalin A (Con A) (Sigma, St Louis, MO) in Iscove's modified Dulbecco's medium (Life Technologies, Eggenstein, Germany) supplemented with 5% fetal calf serum (Sigma), 2 mM L-glutamine, 5 10^-5 M 2-mercaptoethanol, 100 IU penicillin, and 100 g streptomycin per ml (complete medium). After 48 h the cells were washed thoroughly and were expanded with recombinant IL-2 for 2 d. Alternatively, splenocytes were cultured in complete medium in 24-well tissue culture plates (Costar, Cambridge, MA) previously coated with anti-mouse CD3 MoAb by overnight incubation with 3 g per ml anti-CD3 MoAb 145–2C11.

To generate B cell blasts, B cells isolated from splenocytes were cultured in the presence of 25 g LPS per ml (Sigma) in complete medium for 2–3 d.

Bone marrow-derived macrophages were grown for 10 d in medium containing rM-CSF (50 ng per ml) as described (^{Fischer et al. 1988}). For the last 48 h of culture cells were grown in rM-CSF-containing medium alone, with recombinant interferon- (10 U per ml), recombinant IL-4 (50 U per ml) or recombinant GM-CSF (200 ng per ml), respectively.

Clone BK-OVA-1R was cultivated as described (^{Reske-Kunz et al. 1986}). Mast cells, grown from bone marrow precursors in the presence of IL-3 were kindly given to us by Dr E. Schmitt (Institut für Immunologie, Mainz, Germany). Cells of the melanocyte line B78 (^{Thayer & Weintraub 1990}), the keratinocyte line PAM 212 (^{Yuspa et al. 1980}), the macrophage line P388D1 (^{Koren et al. 1975}), the B cell lymphoma A20.2J (^{Kim et al. 1979}), and the fibroblast line WEHI-164 (^{Ziegler-Heitbrock & Riethmüller 1984}) were cultured in complete medium.

cDNA library construction

A cDNA library derived from cLC was constructed in ZAP II (^{Ross et al. 1998a}). mRNA was isolated from cLC (purity 95% rosetted cells) using the QuickPrep mRNA Purification Kit (Amersham Pharmacia Biotech, Uppsala, Sweden). Double-stranded cDNA was prepared from 5 g mRNA using the Time Saver cDNA Synthesis Kit (Amersham Pharmacia Biotech) and ligated with EcoRI/NotI adapters (Amersham Pharmacia Biotech) according to the recommendations of the manufacturer. The adaptor-flanked cDNA fragments were ligated with dephosphorylated, EcoRI-digested ZAP II vector arms (Stratagene, La Jolla, CA) and were in vitro packaged using the in vitro packaging kit Gigapack II Gold (Stratagene).

Preparation and amplification of cDNA probes

cDNA probes were prepared as we described earlier (^{Ross et al. 1997b}) or as follows. Poly(A)+ RNA (200 ng) was reverse transcribed using 200 U of SuperScript II Reverse Transcriptase from Life Technologies GmbH, and cDNA was amplified in a Model 480 DNA Thermal Cycler (Perkin-Elmer, Foster City, CA) using the SMART polymerase chain reaction (PCR) cDNA Synthesis Kit (Clontech Laboratories, Palo Alto, CA) following the recommendations of the manufacturer. Probes were labeled radioactively as described (^{Benton & Davis 1977}) or, alternatively, aliquots representing 2 g DNA were labeled by incubation with 0.1–2 l DIG-Chem Link (Boehringer-Mannheim, Mannheim, Germany) in a 20 l volume at 85°C for 30 min.

Plaque filter-screening and characterization and sequencing of cDNA clones

Bacteria and recombinant phages were plated on 24.5 24.5 cm culture dishes (Nunc, Roskilde, Denmark). For detection using radioactively labeled probes, phages were transferred to nitrocellulose filters, and replicate filters from each plate were treated and hybridized according to^{Benton & Davis (1977)}. For chemiluminescent detection, neutral nylon membranes (Boehringer-Mannheim) were used according to the recommendations of the manufacturer. The latter membranes were also used for southern hybridizations (^{Southern 1975}). The filters were air-dried and processed like plaque filters. Membranes were prehybridized with DIG Easy Hyb (Boehringer-Mannheim) at 42°C for 1–3 h under slight agitation. The solution was replaced by the hybridization solution containing the denatured probe, and membranes were incubated under slight agitation at 37°C overnight in a water-bath. Final concentrations of 100 ng per l of complex cDNA probes were used. The blots were washed twice with 2 sodium citrate/chloride buffer, 0.1% (wt/vol) sodium dodecyl sulfate for 10 min at room temperature and then twice with prewarmed 0.5 sodium citrate/chloride buffer, 0.1% (wt/vol) sodium dodecyl sulfate for 15 min at 68°C. Hybridizations with radioactively labeled probes were carried out and detected as described (^{Ross et al. 1997b}). Chemiluminescent detection was performed according to the recommendations of the manufacturer using CDP-Star (Boehringer-Mannheim) with subsequent exposure to X-ray film.

The internal pBluescript SK(–) plasmid containing the cLC-specific cDNA was recovered from pure, cLC-specific, recombinant ZAP II phages by in vivo excision. The plasmid DNA was purified using the Plasmid Kit Midi (Qiagen, Valencia, CA), restricted and subjected to southern hybridization (^{Southern 1975}) or to sequencing on an automated sequencer, model 373A (Applied Biosystems, Foster City, CA).

Reverse transcription–PCR

Poly(A)⁺ RNA was isolated from cell pellets using the mRNA Isolation Kit from Boehringer-Mannheim. Aliquots were used for reverse transcription–PCR as described previously (^{Ross et al. 1995}) using a Model 480 DNA Thermal Cycler (Perkin-Elmer). The RNA concentration and quality were assessed by reverse transcription–PCR with primers specific for 3-phosphoglycerate kinase (PGK-1: CCT GGC TAT CTT GGG AGG CG and PGK-2: CCC ATC CAG CCA GCA GGT AT). DC/B-CK specific primers were chosen to amplify a 378 bp fragment of DC/B-CK cDNA (DC/B-CK-1: ATG TGA GGC CAA ATA GAC GA and DC/B-CK-2: CTG GCA CTG TCA ATC CCT G). The primers were generated by MWG-Biotech (Ebersberg, Germany). The cDNA was amplified in 35 cycles with 1 min denaturation at 94°C, 1 min annealing at different temperatures and 1 min extension at 72°C. Following the 35 cycles a final extension was performed for 7 min at 72°C. The annealing temperature was decreased during the first 20 cycles from 65°C to 60.5°C in steps of 0.5°C (two cycles at each temperature step) followed by 15 cycles at 60°C.

Production of recombinant DC/B-CK

A 204 bp fragment encoding the core region of the DC/B-CK cDNA (amino acids 25–92) was cloned into the XbaI and PstI sites of the Pichia pastoris expression vector pPICZ B (Invitrogen, San Diego, CA) such that the -factor prepropeptide of Saccharomyces cerevisiae (^{Brake et al. 1984;Scorer et al. 1993}) encoded by the vector was connected to the N-terminus of DC/B-CK core protein. The plasmid was transfected into Pichia pastoris strain GS 115 by electroporation (Gene Pulser, Bio-Rad Laboratories, Hercules, CA) according to the recommendations for P. pastoris (Invitrogen). Transformants were screened for Zeocin (Invitrogen) resistance and Mut+ phenotype.

Positive clones were cultured in histidine-containing minimal medium, and methanol as the sole source of carbon was added at 0, 24, 48, and 72 h. After 96 h the culture supernatant containing the secreted heterologous protein was collected and freeze-dried for storage.

For purification, the lyophilized supernatant was reconstituted in sterile water and desalted using a Sephadex PD 10 column (Amersham Pharmacia Biotech). DC/B-CK was purified by passage through a Heparin HiTrap affinity column (Amersham Pharmacia Biotech) at 4°C. The column was washed with 0.2 M NaCl, 20 mM Tris, pH 8.0, and eluted with 0.6 M NaCl, 20 mM Tris, pH 8.0. The supernatant of a vector-transfected Pichia pastoris clone was treated identically.

Chemotaxis assay

Cell migration was evaluated using a chemotaxis microchamber methodology and 5 m pore size polycarbonate filters (Neuro Probe Inc., Cabin John, MD). Cells were washed once, were resuspended at 1 10⁶ cells per ml in complete medium and were incubated at 37°C in 10% CO₂ for 10 min. Chemokines were diluted in prewarmed complete medium and were added to the lower wells of a 48 well microchamber (Neuro Probe). Cells were seeded in the upper wells of the chamber. An identically treated preparation obtained from a vector-transfected P. pastoris clone served as a control. Following incubation at 37°C in 10% CO₂ for 3 h, cells in the lower wells were counted using a Neubauer hemocytometer. Migration assays were performed in triplicate. The number of migrated cells induced by the control preparation was subtracted from the number of migrated cells induced by the DC/B-CK preparation. Results are presented as the percentage of migrated cells to the total number of cells seeded. In selected experiments cells were incubated with pertussis toxin (Calbiochem, La Jolla, CA) at 100 and 500 ng per ml, respectively, for 90 min at 37°C in 10% CO₂ prior to the assay.

RESULTS

Isolation of differentially expressed genes from a cLC-derived cDNA library and identification of DC/B-CK

Approximately 40,000 cDNA clones of a cLC-derived cDNA library (^{Ross et al. 1998a,1999a}) in ZAP II were plated, replicate filters were taken and hybridized as reported with in vitro amplified cDNA probes derived from fLC and cLC (^{Ross et al. 1999a}). Clones yielding signals with cLC-derived probes but not with fLC-derived probes were isolated, plated and re-screened. Differential expression was confirmed for 112 of 180 clones isolated from the first screening procedure. The pBluescript phagemids containing the cLC-specific cDNAs were recovered by in vivo excision from all 112 verified clones. Plasmid DNA was isolated and digested with EcoRI. The restriction analysis revealed similar banding patterns for a number of clones. Therefore, one of them was selected, labeled, and subjected to southern hybridization with blots of all 112 clones. Thirty-five clones yielded strong signals indicating that homologous or identical cDNAs had been cloned. Several of the hybridizing clones and the 76 remaining nonhybridizing cDNAs were sequenced from the ends with vector-specific primers. Comparison of the sequences obtained with the Geall databases (DKFZ, Heidelberg) revealed that known as well as unknown cDNAs had been detected.

The most abundant cDNA species was analyzed in more detail. The restriction patterns of all clones were compared and the restriction sites turned out to be conserved. Overlapping sequences were found to be identical. Sequence analysis of full-length cDNA (pcLC112) revealed an open reading frame comprising 276 bp. In order to confirm the nucleotide sequence by an independent cloning strategy nearly the complete cDNA (positions 196–1625) was amplified from cLC-derived mRNA by reverse transcription–PCR using Pfu polymerase (Stratagene), subcloned, and sequenced. The open reading frame codes for a protein of 92 amino acids with a molecular weight of 10.3 kDa (EMBL database accession number AJ238238).

The nucleotide sequence of pcLC112 was further analyzed by DNA database searches at the EBI (Hinxton, U.K.). Several nearly identical sequence fragments including a full-length cDNA obtained by random sequencing of murine cDNA libraries were identified. With respect to already characterized genes the best homology was found to the human CC chemokine MDC, also named STCP-1, indicating that pcLC112 coded for a novel CC chemokine, which we named DC/B-CK due to its expression pattern (see below). Sequence identity between DC/B-CK and MDC/STCP-1 within the coding region is 74% on the DNA level and 64% on the protein level. The long untranslated 3'-regions of the cDNAs show no significant sequence identity between human MDC/STCP-1 and mouse DC/B-CK. An alignment of the deduced amino acid sequences of DC/B-CK and MDC/STCP-1 is displayed in Figure 1(a). Taking similar amino acids into account (indicated by single dots in Figure 1a) 94% of the amino acid sequences of DC/B-CK and MDC/STCP-1 are similar to each other. The first 24 amino acids constitute a leader sequence, mediating secretion of the mature protein which consists of 68 amino acids. The mature protein contains the characteristic four cysteine motif of CC chemokines in addition to 10 other residues that are highly conserved within the family of CC chemokines Figure 1(b)). A nearly identical sequence (99.4% identity) corresponding to the murine CC chemokine ABCD-1 (^{Schaniel et al. 1998}) identified in activated B cells by suppression subtractive hybridization, was added to the databases during progress of this work.

Figure 1.

Alignment of DC/B-CK with other CC chemokines. (A) Alignment of DC/B-CK with MDC/STCP-1, the best fitting human homolog. Identical amino acids are indicated by double dots, similar amino acids by single dots. The cysteine residues, characteristic for CC chemokines, are underlined. The first 24 amino acids, comprising a signal peptide, are written in bold face letters. (B) Multiple alignment of DC/B-CK (bold face, arrow) with the homolog regions of other murine and human CC chemokines. The first letter indicates human (h) or murine (m) origin of the sequence. All sequences were obtained from the EMBL DNA database at EBI (Hinxton, U.K.). Standard abbreviations were used. Alignment was performed using the program clustal of the GCG software at DKFZ (Heidelberg, Germany). Conserved amino acids including the characteristic cysteines are boxed. Only the parts corresponding to the complete DC/B-CK sequence are shown. (C) Phylogenetic tree of CC chemokines including DC/B-CK set up by using the GCG software at DKFZ according to^{Kimura (1980)}. Sequence identity to DC/B-CK/ABCD-1 is shown in brackets behind the name of the chemokines.

Full figure and legend (65K)

A phylogenetic tree of CC chemokines including DC/B-CK, created by comparison of the CC chemokine amino acid sequences using the GCG software at Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany, is shown in Figure 1(c)). The calculation, based on the two parameter model by^{Kimura (1980)}, clearly indicates that MDC/STCP-1 and DC/B-CK/ABCD-1 occupy a new branch within the group of CC chemokines. All other known murine or human chemokines show 40% (human RANTES) or less sequence identity on the amino acid level.

Expression of DC/B-CK mRNA during maturation of Langerhans cells

Differential expression of DC/B-CK mRNA was confirmed and analyzed in more detail by semiquantitative reverse transcription–PCR. We isolated mRNA from equal numbers of fLC and from cLC cultivated for 1, 2, and 3 d, respectively. Equal amounts of mRNA were subjected to reverse transcription, and aliquots were used for PCR with specific primers for the housekeeping gene 3-phosphoglycerate kinase. Signals of equal intensity were obtained indicating comparable amounts of mRNA Figure 2a. Aliquots of the same reverse transcription–reaction were used for PCR with DC/B-CK-specific primers. As shown in Figure 2(a), no signals were obtained with fLC, whereas strong signals were obtained with cLC cultivated for 1, 2, or 3 d. The signal intensities on days 1–3 were similar.

Figure 2.

Analysis of DC/B-CK expression by semiquantitative reverse transcription–PCR. Equal amounts of mRNA as assessed by reverse transcription–PCR with primers specific for the housekeeping gene 3-phosphoglycerate kinase (lower panels) were subjected to reverse transcription–PCR with DC/B-CK-specific primers (upper panels). M, marker X174 HaeIII; –, negative control without RNA. (A) DC/B-CK expression during maturation of Langerhans cells. From left to right: fLC (1), cLC on day 1 of culture (2), cLC on day 2 of culture (3), cLC on day 3 of culture (4). (B) DC/B-CK expression in primary murine cells. From left to right: cLC (1), LPS-activated, splenic B cells (2), freshly isolated B cells (3), mast cells (4), bone marrow-derived macrophages stimulated with interferon- (5), or IL-4 (6), or GM-CSF (7) or kept without stimulus (8). (C) DC/B-CK expression in murine cell lines and tissues. From left to right: macrophage line P388D1 (1), B cell line A20.2J (2), keratinocyte line PAM 212 (3), fibroblast line WEHI 164 (4), melanocyte line B78 (5), Th cell line BK-OVA-1R (6), cell suspensions from brain (7), liver (8), kidney (9), spleen (10), DC/B-CK plasmid DNA (11).

Full figure and legend (30K)

Expression of DC/B-CK is primarily restricted to mature DC and activated B cells

DC/B-CK expression was further analyzed using a panel of cell types and tissues. Equal numbers of cells were subjected to mRNA isolation. Quality and amount of mRNA were assessed by reverse transcription–PCR with specific primers for 3-phosphoglycerate kinase and equal mRNA amounts were subjected to reverse transcription–PCR with DC/B-CK-specific primers Figure 2b/c. Strong signals were obtained with cLC (Figure 2b, lane 1) and DC/B-CK plasmid DNA (Figure 2c, lane 11) as positive controls and with cultured epidermal cells and DC grown from bone-marrow precursors in the presence of GM-CSF (not shown). B cells were isolated from splenic cell suspensions with anti-mouse IgM paramagnetic beads and were activated with LPS for 3 d in vitro. Strong DC/B-CK expression was observed in these activated B cells (Figure 2b, lane 2) whereas DC/B-CK was not expressed in unstimulated B cells Figure 2b, lane 3) and in the B cell lymphoma A20.2J (Figure 2c, lane 2). The homolog human CC chemokine MDC/STCP-1 is expressed in macrophages (^{Godiska et al. 1997}). Therefore we analyzed bone marrow-derived, M-CSF-cultured macrophages following stimulation with various cytokines. Neither macrophages cultured with M-CSF alone, nor stimulated on day 8 of culture for 48 h by addition of GM-CSF, IL-4, or interferon-, expressed DC/B-CK mRNA (Figure 2b, lanes 5–8). Mast cells did not express DC/B-CK either (Figure 2b, lane 4). Likewise, no signals were obtained with mRNA derived from the Th line BK-OVA-1R, the melanocyte line B78, the keratinocyte line PAM 212, the macrophage line P388D1 and from spleen, kidney, liver, and brain Figure 2c. The fibroblast line WEHI-164 showed a weak signal (Figure 2c, lane 4).

Recombinant DC/B-CK is a chemoattractant for activated T cells

Recombinant DC/B-CK protein was produced by P. pastoris after transfection with an expression vector comprising DC/B-CK-derived DNA sequences (corresponding to the mature protein, amino acids 25–92) and the heterologous -factor from S. cerevisiae as a secretion signal. DC/B-CK was purified from supernatant by affinity to heparin (Heparin HiTrap affinity columns, Amersham Pharmacia Biotech). The purified protein yielded a single band on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (data not shown). Supernatant derived from cells transfected with the vector alone was treated likewise to serve as a negative control. The recombinant DC/B-CK protein was subjected to microchamber migration assays.

Because mature DC interact with naïve and activated T cells, we analyzed chemoattractant activity of DC/B-CK for T cells. T cells were purified from splenocytes using T Cell Enrichment Columns (R&D Systems). Cells were > 95% positive for Thy1, they were CD44^low and L-selectin^high. Migration induced by DC/B-CK was weak Figure 3a. In contrast, Con A-activated, IL-2 expanded T lymphoblasts showed extensive migratory activity to DC/B-CK Figure 3a). Migratory activity was concentration-dependent with an optimum at 25 g per ml DC/B-CK. The chemotactic response to DC/B-CK was even stronger than that to an optimal concentration of RANTES Figure 4a.

Figure 3.

DC/B-CK is a chemoattractant for activated T cells. Cells (5 10⁵/50 l) were seeded in the upper wells of a microchamber, and purified DC/B-CK was added to the lower wells. An identically treated preparation obtained from a vector-transfected P. pastoris clone served as a control. Chambers were incubated at 37°C in 10% CO₂ for 3 h. The percentage of migrated cells to the total number of cells was calculated. Each test was performed in triplicate and the mean value ( SEM) is presented. (A) Migration of Con A-activated/IL-2-expanded T lymphocytes (squares) and freshly isolated T lymphocytes (triangles). (B) Migratory responsiveness of LPS-activated B lymphocytes (squares) and freshly isolated B lymphocytes (triangles). (C) Migration of cLC (squares), fLC (diamonds), and bone marrow-derived dendritic cells (triangles).

Full figure and legend (18K)

Figure 4.

Migration of Con A-activated/IL-2-expanded T lymphocytes. The chemotaxis assay was performed as described in Figure 3. Each test was performed in triplicate and the mean value ( SEM) is presented. (A) Migratory responsiveness of Con A-activated/IL-2-expanded T lymphocytes to optimum concentrations of DC/B-CK and RANTES as well as to supernatant of 3 d cultured epidermal cells used undiluted. (B) Inhibition by pertussis toxin (PTX) of T lymphoblast migration induced by DC/B-CK. Con A-activated/IL-2-expanded T lymphocytes were incubated with the indicated concentrations of PTX for 90 min prior to the assay.

Full figure and legend (17K)

In order to provide a more ''physiologic'' polyclonal T cell stimulation, we cultured splenic cells for 3 d on culture plates previously coated with anti-CD3 MoAb 145–2C11. Anti-CD3-treated T lymphoblasts, expanded for 48 h with IL-2 exhibited a clear chemotactic response towards DC/B-CK as well. Total numbers of migrating cells at optimal DC/B-CK concentrations, however, were approximately 20% reduced as compared with Con A-activated cells (data not shown).

DC were reported to form clusters with CD40-activated B cells, to enhance their proliferation and to induce their differentiation (^{Dubois et al. 1997}). We therefore tested whether DC/B-CK acts as a chemoattractant for B cells. B cells, isolated via B cell columns (Mouse B Cell Recovery Kit, Cedarlane) were subjected to migration assays either directly or following activation by LPS for 3 d. Neither freshly isolated nor activated B cells showed DC/B-CK-induced migratory activity Figure 3b).

MDC/STCP-1 was shown to attract monocyte-derived DC. To test for a migratory response of DC towards DC/B-CK, fLC and cLC were isolated by immunomagnetic separation, using MHC class II MoAb MKD6 and 14.4.4.S combined with anti-IgG antibodies coupled via DNAse I-labile bonds to paramagnetic beads (CELLection beads, Dynal). Following isolation of Langerhans cells, beads were detached by DNAse I treatment and removed by immunomagnetic separation. Neither fLC nor cLC exhibited DC/B-CK-induced migratory activity Figure 3c, whereas a significant response (12% migrated cells) of fLC was observed using recombinant RANTES at optimal concentrations (not shown).

As another source of DC we tested DC cultivated from bone marrow precursors in the presence of GM-CSF and TNF-. Following culture, DC were positive for the costimulatory molecules CD80, CD86 and ICAM-1 and the DC markers NLDC145 and N418, but negative for B220 and Thy 1 (not shown). No DC/B-CK-dependent migratory activity of cultured DC was observed Figure 3c.

All chemokine receptors known so far are seven transmembrane domain proteins coupled with guanosine triphosphate-binding proteins, which are sensitive to pertussis toxin (^{Murphy 1994}). Migratory response of Con A-activated, IL-2 expanded T lymphoblasts to DC/B-CK at various concentrations was inhibited by pertussis toxin in a concentration-dependent manner Figure 4b.

Supernatant of cultured epidermal cells attracts activated T lymphocytes in vitro

Epidermal cells were cultured for 3 d. The supernatant was tested for the capacity to induce migration of Con A-activated, IL-2-restimulated T lymphoblasts. When used undiluted, a migratory response of the T cells was observed, which was slightly lower compared with the response induced by an optimal concentration of RANTES Figure 4a.

DISCUSSION

In this study we show that mature Langerhans cells, but not freshly isolated Langerhans cells, express a CC chemokine, that specifically attracts activated T cells. This chemokine is also produced by activated B cells, prompting us to designate the chemokine DC/B-CK.

The strategy of differential screening of a cLC-derived cDNA library with fLC- and cLC-specific probes was used to identify differential gene expression in maturing epidermal Langerhans cells. Comparison of the hybridization patterns following plaque-filter hybridization using probes derived from these two well-defined cell populations allowed us to isolate 112 cLC-specific cDNAs from 40,000 clones, representing 0.3% of the clones. Only clones showing drastic differences in signal intensity were taken into account. We estimate that approximately 1–3% of total cDNAs might be expressed differentially in fLC and cLC. Previously, we confirmed differential expression of several genes and related these findings to cellular functions of maturing Langerhans cells (^{Ross et al. 1998a,b,1999b}). Furthermore, the chemokine receptor CCR7, that we identified to become upregulated during maturation of Langerhans cells (unpublished result), was recently thoroughly analyzed in DC by several groups, showing its differential expression during maturation of DC (^{Dieu et al. 1998;Sallusto et al. 1998;Sozzani et al. 1998}).

Here we describe that the chemokine DC/B-CK is the most highly expressed gene among the differentially expressed genes of maturing Langerhans cells. The cDNA library constructed from cLC was validated to be representative, and the majority of differentially expressed genes were isolated by this screening only once or twice (^{Ross et al. 1998a,1999a}). This includes even highly expressed genes like the actin-bundling protein fascin (^{Ross et al. 1998a}), which was isolated twice. In contrast, DC/B-CK cDNAs were isolated 36 times, indicating its extraordinarily high expression level. Strong expression already on day 1 of Langerhans cell culture, which was maintained on day 2 and 3 of culture, suggests an early onset of DC/B-CK expression following activation. Analysis of DC/B-CK expression by reverse transcription–PCR in a large panel of cell types revealed specific expression in bone marrow-derived DC and activated B cells besides expression in cLC. These data confirm and extend findings reported by^{Schaniel et al. (1998)} who had identified a chemokine with a nearly identical sequence, named ABCD-1, in anti-CD40 and IL-4 stimulated B cells by suppression subtractive hybridization. ABCD-1 was shown to be expressed in CD11c+ DC isolated from mesenteric lymph nodes in addition to activated B cells.

Restricted expression to activated, professional antigen-presenting cells suggested a role of DC/B-CK in attraction of T cells. A strong migratory activity of anti-CD3 activated or Con A-activated, IL-2-expanded T lymphoblasts in response to DC/B-CK was observed. DC/B-CK concentrations inducing optimal chemotaxis were much higher as compared with optimal concentrations of the T cell-attracting CC chemokines RANTES Figure 4 and MIP-1 (not shown), but higher total migratory activity was observed as well. This finding suggests that DC/B-CK exerts its effects on T cells over short distances. This notion seems reasonable in the light of the probable function of DC/B-CK. DC and B cells are in close vicinity to T cells within lymph nodes when DC/B-CK is expressed, whereas inflammatory chemokines like MIP-1 and RANTES attract effector cells to remote inflammatory sites over longer distances.

Given the spatial proximity of T cells and DC in lymph nodes, the minimal chemoattractant activity of DC/B-CK observed for resting T cells might be sufficient to mediate a first contact between T cell and DC. It cannot be excluded, however, that the few migrating cells represented contaminating activated T cells. The first contact between naïve T cells and DC might be completely mediated by other chemokines such as DC-CK1/AMAC-1, which is known to attract naïve T cells (^{Adema et al. 1997}). In the case of DC-CK1/AMAC-1, data from^{Kodelja et al. (1998)} imply that expression is downregulated during TNF--induced maturation of DC in vitro, but the expression levels detected in lymphoid organs by^{Adema et al. (1997)} might be sufficient for attracting naïve T cells. Even if the first contact between T cells and DC proceeds without involvement of DC/B-CK, this chemokine might play a part in primary activation of T cells by enhancement of DC/T cell contact once T cells are activated.

Several findings suggest that DC/B-CK is the murine homolog to the human CC chemokine MDC/STCP-1, the most obvious being the sequence homology of 64% on the amino acid level. The homology can be extended to 94% if similar amino acids are included in this calculation Figure 1a). All other known CC chemokines show far less homology to MDC/STCP-1 and DC/B-CK Figure 1b/c. The independent position of human MDC/STCP-1 is stressed by the fact that MDC/STCP-1 is located on chromosome 16q13 together with TARC and the CX₃C chemokine fractalkine (^{Imai et al. 1998;Nomiyama et al. 1998}) whereas the classical, closely related CC chemokines are clustered on chromosome 17.^{Schaniel et al. (1998)} showed that the exon/intron structure of the ABCD-1 gene is identical to that of the MDC/STCP-1 gene (^{Chang et al. 1997}), and that probes derived from murine ABCD-1 and human MDC/STCP-1 co-hybridize on human northern blots under low stringency. Functional aspects coincide as well. MDC/STCP-1 is expressed by DC (^{Godiska et al. 1997}) and activated B cells (^{Schaniel et al. 1998}) and attracts activated, but not resting T cells (^{Chang et al. 1997}). The same holds true for ABCD-1/DC/B-CK as shown by^{Schaniel et al. (1998)} and in this study. Nevertheless, there are important differences as well. We did not detect DC/B-CK expression in bone marrow-derived macrophages with or without further stimulation by various cytokines nor in the macrophage line P388D1, whereas MDC/STCP-1 is highly expressed in macrophages (^{Chang et al. 1997;Godiska et al. 1997}). Furthermore, we did not observe a migratory response of fLC, cLC nor of bone marrow-derived DC upon stimulation by DC/B-CK, whereas^{Godiska et al. (1997)} found MDC to attract DC generated from precursors by culture with GM-CSF and IL-13 in the human system.

Further studies will be needed to elucidate the impact of these differences on the murine and human immune response.

References

1. Adema, GJ, Hartgers, F, Verstraten, R, et al.: A dendritic-cell-derived C-C chemokine that preferentially attracts naive T cells. Nature, 1997 387:713–717,  | Article | PubMed | ISI | ChemPort |
2. Baggiolini, M: Chemokines and leukocyte traffic. Nature, 1998 392:565–568,  | Article | PubMed | ISI | ChemPort |
3. Bazan, JF, Bacon, KB, Hardiman, G, et al.: A new class of membrane-bound chemokine with a CX3C motif. Nature, 1997 385:640–644,  | Article | PubMed | ISI | ChemPort |
4. Becker, D, Reske-Kunz, A, Knop, J, Reske, K: Biochemical properties of MHC class II molecules endogenously synthesized and expressed by mouse Langerhans cells. Eur J Immunol, 1991 21:1213–1220,  | PubMed | ISI | ChemPort |
5. Becker, D, Mohamadzadeh, M, Reske, K, Knop, J: Increased level of intracellular MHC II molecules in murine Langerhans cells following in vivo and in vitro administration of contact allergens. J Invest Dermatol, 1992 99:545–549,  | Article | PubMed | ISI | ChemPort |
6. Benton, WD, Davis, RW Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science, 1977 196:180–182,  | PubMed | ISI | ChemPort |
7. Brake, AJ, Merryweather, JP, Coit, DG, et al.: Alpha-factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae. Proc Natl Acad Sci USA, 1984 81:4642–4646,  | PubMed | ChemPort |
8. Chang, M-S, McNinch, J, Elias, C, et al.: Molecular cloning and functional characterization of a novel CC chemokine, stimulated T cell chemotactic protein (STCP-1) that specifically acts on activated T lymphocytes. J Biol Chem, 1997 272:25229–25237,  | Article | PubMed | ISI | ChemPort |
9. Dieu, MC, Vanbervliet, B, Vicari, A, et al.: Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. J Exp Med, 1998 188:373–386,  | Article | PubMed | ISI | ChemPort |
10. Dubois, B, Vanbervliet, B, Fayette, J, et al.: Dendritic cells enhance growth and differentiation of CD40-activated B lymphocytes. J Exp Med, 1997 185:941–951,  | Article | PubMed | ISI | ChemPort |
11. Fischer, HG, Frosch, S, Reske, K, Reske-Kunz, AB: Granulocyte-macrophage colony-stimulating factor activates macrophages derived from bone marrow cultures to synthesis of MHC class II molecules and to augmented antigen presentation function. J Immunol, 1988 141:3882–3888,  | PubMed | ISI | ChemPort |
12. Godiska, R, Chantry, D, Raport, CJ, et al.: Human macrophage-derived chemokine (MDC), a novel chemoattractant for monocytes, monocyte-derived dendritic cells, and natural killer cells. J Exp Med, 1997 185:1595–1604,  | Article | PubMed | ISI | ChemPort |
13. Hart, DN: Dendritic cells: unique leukocyte populations which control the primary immune response. Blood, 1997 90:3245–3287,  | PubMed | ISI | ChemPort |
14. Imai, T, Chantry, D, Raport, CJ, et al.: Macrophage-derived chemokine is a functional ligand for the CC chemokine receptor4. J Biol Chem, 1998 273:1764,  | Article | PubMed | ISI | ChemPort |
15. Kappler, JW, Skidmore, B, White, J, Marrack, P: Antigen-inducible, H-2-restricted, interleukin-2-producing T cell hybridomas. Lack of independent antigen and H-2 recognition. J Exp Med, 1981 153:1198–1214,  | Article | PubMed | ISI | ChemPort |
16. Karasuyama, H, Melchers, F: Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors. Eur J Immunol, 1988 18:97–104,  | PubMed | ISI | ChemPort |
17. Kennedy, J, Kelner, GS, Kleyensteuber, S, et al.: Molecular cloning and functional characterization of human lymphotactin. J Immunol, 1995 155:203–209,  | PubMed | ISI | ChemPort |
18. Kim, KJ, Kanellopoulos-Langwin, C, Merwin, RM, Sachs, DH, Asofsky, R: Establishment and characterization of BALB/C-lymphoma lines with B cell properties. J Immunol, 1979 122:549,  | PubMed | ISI | ChemPort |
19. Kimura, M: A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol, 1980 16:111–120,  | Article | PubMed | ISI | ChemPort |
20. Kodelja, V, Muller, C, Politz, O, Hakij, N, Orfanos, CE, Goerdt, S: Alternative macrophage activation-associated CC-chemokine-1, a novel structural homologue of macrophage inflammatory protein-1 alpha with a Th2-associated expression pattern. J Immunol, 1998 160:1411–1418,  | PubMed | ISI | ChemPort |
21. Koren, HS, Handwerger, BS, Wunderlich, JR: Identification of macrophage-like characteristics in a cultured murine tumor line. J Immunol, 1975 114:894–897,  | PubMed | ISI | ChemPort |
22. Leo, O, Foo, M, Sachs, DH, Samelson, LE, Bluestone, JA: Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc Natl Acad Sci USA, 1987 84:1374,  | PubMed | ChemPort |
23. Lutz, MB, Kukutsch, N, Ogilvie, ALJ, Rössner, S, Koch, F, Romani, N, Schuler, G: An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J Immunol Methods, 1999 223:77–92,  | Article | PubMed | ISI | ChemPort |
24. Murphy, PM: The molecular biology of leukocyte chemoattractant receptors. Annu Rev Immunol, 1994 12:593–633,  | Article | PubMed | ISI | ChemPort |
25. Nomiyama, H, Imai, T, Kusuda, J, Miura, R, Callen, DF, Yoshie, O: Human chemokines fractalkine (SCYD1), MDC (SCYA22) and TARC (SCYA17) are clustered on chromosome 16q13. Cytogenet Cell Genet, 1998 81:10–11,  | Article | PubMed | ISI | ChemPort |
26. Ozato, K, Mayer, N, Sachs, DH: Hybridoma cell lines secreting monoclonal antibodies to mouse H-2 and Ia antigens. J Immunol, 1980 124:533–540,  | PubMed | ISI | ChemPort |
27. Pan, Y, Lloyd, C, Zhou, H, et al.: Neurotactin, a membrane-anchored chemokine upregulated in brain inflammation. Nature, 1997 387:611–617,  | Article | PubMed | ISI | ChemPort |
28. Reske-Kunz, AB, Steldern, D, Rüde, E, Diamantstein, T: Regulation of interleukin 2 receptor expression by interleukin 2. Scand J Immunol, 1986 23:693–701,  | PubMed | ChemPort |
29. Ross, R, Kleinz, R, Reske-Kunz, AB: A method for rapid generation of competitive standard molecules for RT-PCR avoiding the problem of competitor/probe cross-reactions. PCR Methods Applic, 1995 4:371–375,  | ISI | ChemPort |
30. Ross, R, Schwing, J, Kumpf, K, Endlich, A, Reske-Kunz, AB: Isolation of differentially expressed genes in epidermal Langerhans cells. Adv Exp Med Biol, 1997a 417:481–486,  | ISI | ChemPort |
31. Ross, R, Kumpf, K, Reske-Kunz, AB: PCR-amplified cDNA probes for verification of differentially expressed genes. Biotechniques, 1997b 22:894–897,  | ISI | ChemPort |
32. Ross, R, Ross, X, Schwing, J, Längin, T, Reske-Kunz, AB: The actin-bundling protein fascin is involved in the formation of dendritic processes in maturing epidermal Langerhans cells. J Immunol, 1998a 160:3776–3782,  | PubMed | ISI | ChemPort |
33. Ross, R, Endlich, A, Kumpf, K, Schwing, J, Reske-Kunz, AB: Maturation of epidermal Langerhans cells in vitro is accompanied by downregulation of 4F2 (CD98) as determined by differential display. J Invest Dermatol, 1998b 110:57–61,  | Article | ISI | ChemPort |
34. Ross, R, Ross, X-L, Rueger, B, Laengin, T, Reske-Kunz, AB: Non-radioactive detection of differentially expressed genes using complex RNA or DNA hybridization probes. Biotechniques, 1999a 26:150–155,  | PubMed | ISI | ChemPort |
35. Ross, R, Ross, X-L, Längin, T, Reske-Kunz, AB: Maturation of epidermal Langerhans cells: Increased expression of beta- and gamma-actin isoforms as a basis of specialized cell functions. Exp Dermatol, 1999b in press,
36. Rossi, DL, Hardiman, G, Copeland, NG, Gilbert, DJ, Jenkins, N, Zlotnik, A, Bazan, JF: Cloning and characterization of a new type of mouse chemokine. Genomics, 1998 47:163–170,  | Article | PubMed | ISI | ChemPort |
37. Sallusto, F, Schaerli, P, Loetscher, P, et al.: Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation. Eur J Immunol, 1998 28:2760–2769,  | Article | PubMed | ISI | ChemPort |
38. Schaniel, C, Pardali, E, Sallusto, F, et al.: Activated murine B lymphocytes and dendritic cells produce a novel CC chemokine which acts selectively on activated T cells. J Exp Med, 1998 188:451–463,  | Article | PubMed | ISI | ChemPort |
39. Scheicher, C, Mehlig, M, Zecher, R, Reske, K: Dendritic cells from mouse bone marrow: in vitro differentiation using low doses of recombinant granulocyte-macrophage colony-stimulating factor. J Immunol Methods, 1992 154:253–264,  | Article | PubMed | ISI | ChemPort |
40. Scorer, CA, Buckholz, RG, Clare, JJ, Romanos, MA: The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris. Gene, 1993 136:111–119,  | Article | PubMed | ISI | ChemPort |
41. Southern, EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol, 1975 98:503–517,  | PubMed | ISI | ChemPort |
42. Sozzani, S, Sallusto, F, Luini, W, et al.: Migration of dendritic cells in response to formyl peptides, C5a, and a distinct set of chemokines. J Immunol, 1995 155:3292–3295,  | PubMed | ISI | ChemPort |
43. Sozzani, S, Luini, W, Borsatti, A, et al.: Receptor expression and responsiveness of human dendritic cells to a defined set of CC and CXC chemokines. J Immunol, 1997 159:1993–2000,  | PubMed | ISI | ChemPort |
44. Sozzani, S, Allavena, P, D'Amico, G, et al.: Differential regulation of chemokine receptors during dendritic cell maturation: a model for their trafficking properties. J Immunol, 1998 161:1083–1086,  | PubMed | ISI | ChemPort |
45. Steinman, RM: The dendritic cell system and its role in immunogenicity. Annu Rev Immunol, 1991 9:271–296,  | Article | PubMed | ISI | ChemPort |
46. Thayer, MJ, Weintraub, H: Activation and repression of myogenesis in somatic cell hybrids: evidence for trans-negative regulation of MyoD in primary fibroblasts. Cell, 1990 63:23–32,  | Article | PubMed | ISI | ChemPort |
47. Vicari, AP, Figueroa, DJ, Hedrick, JA, et al.: TECK. a novel CC chemokine specifically expressed by thymic dendritic cells and potentially involved in T cell development. Immunity, 1997 7:291–301,  | Article | PubMed | ISI | ChemPort |
48. Xu, LL, Warren, MK, Rose, WL, Gong, W, Wang, JM: Human recombinant monocyte chemotactic protein and other C-C chemokines bind and induce directional migration of dendritic cells in vitro. J Leukoc Biol, 1996 60:365–371,  | PubMed | ISI | ChemPort |
49. Yuspa, SH, Hawley-Johnson, P, Koehler, B, Stanley, JR: A survey of transformation markers in differentiating epidermal cell lines in culture. Cancer Res, 1980 40:4694–4703,  | PubMed | ISI | ChemPort |
50. Ziegler-Heitbrock, HW, Riethmüller, G: A rapid assay for cytotoxicity of unstimulated human monocytes. J Natl Cancer Inst, 1984 72:23–29,  | PubMed | ChemPort |

Acknowledgments

We gratefully acknowledge the expert technical assistance of Ms. Tina Laengin. We thank Drs Bluestone, Kirchner, Krumwieh, Melchers, Mohamadzadeh, Seiler and Schmitt for their kind gift of reagents and cells. This work was supported by the Deutsche Forschungsgemeinschaft, Kn 120/6-3 and by Stiftung Rheinland-Pfalz für Innovation, 8312-386261/280.