B cell- and monocyte-activating chemokine (BMAC), a novel non-ELR
-chemokine
Matthew A. Sleeman,
Jonathon K. Fraser,
James G. Murison,
Sharon L. Kelly,
Ross L. Prestidge,
David J. Palmer,
James D. Watson and
Krishnanand D. Kumble
Genesis Research and Development Corp. Ltd, PO Box 50, Auckland, New Zealand
Correspondence to:
M. Sleeman
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Abstract
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A novel
-chemokine, designated KS1, was identified from an EST database of a murine immature keratinocyte cDNA library. The EST has 94% similarity to a recently cloned human gene, BRAK, that has no demonstrated function. Northern analysis of mouse and human genes showed detectable mRNA in brain, intestine, muscle and kidney. Tumour panel blots showed that BRAK was down-regulated in cervical adenocarcinoma and uterine leiomyoma, but was up-regulated in breast invasive ductal carcinoma. KS1 bound specifically to B cells and macrophages, as well as two B cell lines, CESS and A20, and a monocyte line, THP-1. KS1 showed no binding to naive or activated T cells. In addition, KS1 stimulated the chemotaxis of CESS and THP-1 cells but not T cells. The s.c. injection of KS1 creates a mixed inflammatory response in Nude and C3H/HeJ mice. The above data indicates that KS1 and its human homologue represents a novel non-ELR
-chemokine that may have important roles in trafficking of B cells and monocytes. We propose the name B cell- and monocyte-activating chemokine (BMAC) for this molecule to reflect the described biological functions.
Keywords: chemotaxis, inflammation, migration, nude mice, tumour
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Introduction
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Chemokines are a large family of small peptides that are involved in the trafficking of leukocytes around the body. They consist of proteins between 8 and 12 kDa in size with a number of conserved cysteines that form two disulphide bridges (13). The chemokine superfamily is currently classified with respect to the number and position of the first cysteines, CC, CXC, CX3C and C. The two main groups are (i) the CXC or
-chemokines, defined by a single amino acid separating the first two cysteines, and (ii) the CC or ß-chemokines, with the first two cysteines being contiguous. The
-chemokines can be further subdivided into two groups depending on whether they contain a GluLeuArg (ELR) motif immediately prior to the first cysteine (1). Initially, these molecules were thought to only be involved in stimulating an inflammatory response by promoting chemotaxis of leukocytes from the peripheral blood to sites of inflammation. IL-8 was one of the first chemokines identified and was shown to promote neutrophil migration (46). Since then the chemokine family has grown to >50 members (http://cytokine.medic.kumamoto-u.ac.jp/CFC/CK/chemokine.html) with every leukocyte population having its own particular subset of chemokines and chemokine receptors. The non-ELR
-chemokines currently consist of six members whose chemotactic functions are highly diverse (713), in contrast to the neutrophil migration-promoting ELR chemokines, and are of great interest for their therapeutic potential in areas other than leukocyte migration. PF-4, IP-10 and Mig have all been shown to have anti-angiogenic properties in a range of tumour models (1416). SDF-1
has been shown to competitively block viral entry in HIV strains that uniquely use CXCR4 as their co-receptor for infection (17,18). The potential of this therapeutic approach is supported by the observation that high circulating levels of ß-chemokines can confer a degree of immunity on those exposed to HIV (19). Recently, a non-ELR
-chemokine, BRAK, was identified by screening human EST databases (20). Function has yet to be assigned for this molecule, although it has been postulated to have a role in oncogenesis. We have identified the murine homologue of this gene, the responding cell types for this new chemokine and propose a new name that reflects its biological activity.
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Methods
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Chemicals and reagents
Recombinant human stromal derived factor-1
and human IL-2 were purchased from PeproTech (Rocky Hill, NJ). The following primary anti-murine antibodies were obtained from PharMingen (San Diego, CA), I-Ak (A
k) biotin (clone 11-5.2), CD19FITC (Clone 1D3), CD4FITC (clone RM4-5), CD8aFITC (clone 53-6.7), rat IgG2aFITC (R35-95) and mouse IgG2bbiotin (clone 49.2). The secondary antibody goat anti-human IgGphycoerythrin (PE) and streptavidinPE were purchased from Southern Biotechnology Associates (Birmingham, AL), and the streptavidinalexa 488 from Molecular Probes (Eugene, OR).
Bioinformatic analysis
An oligo-d(T)-primed directionally cloned murine immature keratinocyte cDNA library was constructed from poly(A)+ RNA using a ZAP express cDNA kit (Stratagene, La Jolla, CA) following the manufacturer's protocol. The library was mass excised and colonies randomly selected for sequencing. High-throughput single-pass sequence from the 5' end of the clones was obtained on ABI377 sequencers (Perkin Elmer, Foster City, CA). Novel sequences were analysed using BLAST (21), Prosite (Swiss Institute of Bioinformatics, University of Geneva) and SignalP V1.1 (Center for Biological Sequence Analysis, Technical University of Denmark), and the Phylip package (University of Washington) to define similarities to known gene families or motifs.
Sequence and cloning of KS1
The full-length sequence of KS1 was obtained by subcloning and sequence primer walking. The coding region, without the predicted signal sequence, was PCR amplified using Klentaq polymerase (Clontech, Paolo Alto, CA) and KS1 as template, using the following sequences 5'-CATGCCATGGCGTCCAAGTGTAAGTGTTCCCGGAAGGGG-3' and 5'-CATGCCATGGCTAATGGTGGTGATGGTGATGTTCTTCGTAGACCCTGCGCTTCTC-3' as forward and reverse oligonucleotides respectively. The product was purified using a PCR purification kit (Qiagen, Valencia, CA), digested with NcoI and ligated into pET16B (Novagen, Madison, WI) to obtain the sequence in frame with the C-terminal (His)6 tag. In addition to this we cloned the full-length coding region into a eukaryotic expression vector, pIGFc, using 5'-GGAATTCCATGAGGCTCCTGGCGGCCGCGCTGCTC-3' and 5'-ACGGATCCACTTACCTGTTTCTTCGTAGACCCTGCGCTTCTCGTT-3' as forward and reverse primers respectively. PCR products were prepared as above and ligated into pIGFc to obtain the sequence in-frame with human IgG1 Fc present in the vector. All constructs were confirmed by automated sequencing.
Northern analysis
KS1 probe was PCR amplified using Taq polymerase (Qiagen) with 5'-ACGCGTCGACATGAGGCTCCTGGCGGC-3' and 5'-TCGTCCAGATCTTTCTTCGTAGACCCTGCGCTT-3' as forward and reverse oligonucleotides respectively. BRAK probe was PCR amplified from human keratinocyte cDNA using Taq polymerase (Qiagen) with 5'-ACGCGTCGACATGAGGCTCCTGGCGGCCGCGCTGCTC-3' and 5'-ATAAGATCTTTCTTCGTAGACCCTGCGCTTC-3' as forward and reverse oligonucleotides respectively. Probe identity was confirmed by sequencing. PCR products were labelled with [
-32P]dCTP (3000 Ci/mmol, NEN/Life Science products, Boston, MA) using 25 ng of DNA in a Rediprime II random-primed labelling system (Amersham Pharmacia, Piscataway, NJ). Human multiple tissue northern blots (Clontech) were hybridized with a 300 bp PCR product (nucleotides 1300 bp of the BRAK coding sequence) following the manufacturer's protocol (Clontech). A human tumour panel blot (Invitrogen, Calsbad, CA) was hybridized with the probe prepared as described above, in 6xSSC buffer, 2xDenhardt reagent, 2% SDS, 120 µg heparin and 100 µg yeast tRNA (Boehringer Mannheim, Mannheim, Germany) at 65°C for 18 h. RNA for mouse tissue blots was isolated using Trizol reagent (Life Technologies, Grand Island, NY) and 20 µg total RNA loaded per lane in a 1% formaldehyde agarose gel, transferred to Hybond N+ membrane (Amersham) and hybridized with the radiolabelled PCR product (nucleotides 1300 bp of the KS1 coding sequence). Mouse tissue blots were hybridized as described for the tumour panel blots. All blots were washed under stringent conditions as specified by the manufacturers or by standard protocols (22). Northern blots were exposed to X-ray film at 80°C and developed at various times up to 7 days. Both the tumour panel blot and human tissue blots were re-probed, as described previously, with a 500 bp ß-actin probe as a loading control.
Expression and purification of recombinant KS1
A C-terminal (His)6 tag fusion protein of KS1 was expressed in BL21(DE3) Escherichia coli cells (Novagen). One litre cultures were induced at an OD600 of 0.5 with 1 mM IPTG and harvested after 3 h. All subsequent procedures were performed on ice. The pellet was re-suspended in lysis buffer (20 mM TrisHCl, pH 8.0, 1 mM PMSF, 10 mM ß-mercaptoethanol, 1% NP-40) and sonicated using a Virsonic ultrasonicator (Virtis, Gardiner, NY) fitted with the miniprobe at 20% output for 4x15 s bursts with 15 s intervals. The sonicate was centrifuged in a JA20 rotor at 18,000 r.p.m. for 10 min at 4°C. The resultant pellet was washed twice for 1 h each in lysis buffer containing 0.5% CHAPS and solubilized in 20 mM TrisHCl, pH 8.0, containing 6 M guanidineHCl and 0.5 M NaCl. The (His)6 fusion protein was isolated by chromatography using nickel chelating Sepharose FF resin (0.5 ml column; Pharmacia). After loading, the column was washed sequentially with 20 volumes of binding buffer (6 M urea, 0.5 M NaCl and 20 mM TrisHCl, pH 8.0), 20 volumes of 0.5% sodium deoxycholate in binding buffer and 20 volumes of binding buffer containing 20 mM imidazole. The protein was eluted with 10 volumes 300 mM imidazole in binding buffer. The eluate was dialysed against binding buffer and re-chromatographed as above. Fusion protein in the eluate was then refolded by dialysis against 1 l of 4 M urea, 20 mM TrisHCl, pH 7.5, overnight while 1 l of 20 mM TrisHCl, pH 7.5, was pumped into the dialysis beaker at a rate of 1 ml/min. The refolded protein was finally dialysed against 20 mM TrisHCl, pH 7.5, containing 10% (w/v) glycerol. Preparations obtained were >95% pure as determined by SDSPAGE using FragmeNT Analysis Package (Molecular Dynamics, Sunnyvale, CA). Endotoxin contamination of purified KS1 was determined using a limulus amebocyte lysate assay kit (Biowhittaker, Walkersville, MD). Endotoxin levels were <0.1 ng/µg of protein. Internal amino acid sequencing was performed on tryptic peptides of KS1 by the Protein Sequencing Unit at the University of Auckland, New Zealand.
An Fc fusion protein was produced by expression in HEK 293 T cells. Using 35 µg of KS1pIGFc DNA to transfect 6x106 cells/flask, 200 ml of KS1 Fc-containing supernatant was produced. The Fc fusion protein was isolated by chromatography using an Affiprep Protein A resin (0.3 ml column; BioRad, Hercules, CA). After loading, the column was washed with 15 ml of PBS, followed by a 5 ml wash of 50 mM Na citrate, pH 5.0. The protein was then eluted with 6 column volumes of 50 mM Na citrate, pH 2.5, collecting 0.3 ml fractions in tubes containing 60 µl of 20 mM TrisHCl, pH 7.5. Fractions were analyzed by SDSPAGE and pooled.
Cell isolation and culture
Murine spleens, thymus, peripheral lymph node and bone marrow cells for flow cytometric analysis were obtained from C3H/HeJ mice, erythrocytes were lysed using ACK lysis buffer (0.15 M NH4Cl, 1 mM KHCO3 and 0.1 mM Na2EDTA). Peritoneal exudate cells (PEC) were obtained by i.p. lavage from C3H/HeJ mice. In brief, euthanized mice were injected with 2x4 ml volumes of 0.02% EDTA/PBS into the peritoneal cavity using an 18 gauge needle. Cells were then drawn out from the peritoneal cavity, pelleted and washed in PBS prior to further analysis. Murine IL-2-activated T cells were cultured as described below. Briefly, splenocytes were activated with 2 µg/ml concanavalin A (Con A) (Sigma, St Louis, MO) in the presence of 5% FBS in DMEM supplemented with 2 mM L-glutamine (Sigma), 1 mM sodium pyruvate (Life Technologies), 0.77 mM L-asparagine (Sigma), 0.2 mM arginine (Sigma), 160 mM penicillin G (Sigma), 70 mM dihydrostreptomycin sulfate (Boehringer Mannheim) and 50 µM 2-mercaptoethanol, for 3 days followed by addition of recombinant human IL-2 (PeproTech) at 10 ng/ml. Cytokine was added at 3 day intervals for 921 days. Peripheral blood mononuclear cells (PBMC) were isolated in heparin (10U/ml) containing tubes from human donors and purified on a Ficoll-Hypaque (Pharmacia) gradient by centrifugation at 900 g for 20 min with no brake. PBMC were aspirated from the interface, washed and re-suspended in HBSS, 20 mM HEPES, 0.5% BSA and used directly for assays. Human IL-2-activated T cells were cultured as described below. Briefly, PBMC were activated with 0.1% phytohemagglutinin (PHA) (Gibco/BRL) in the presence of 5% FBS in RPMI supplemented with 2 mM L-glutamine (Sigma), 160 mM penicillin G (Sigma), 70 mM dihydrostreptomycin sulphate (Boehringer Mannheim) and 50 µM 2-mercaptoethanol, for 3 days followed by addition of recombinant human IL-2 (PeproTech) at 10 ng/ml. CESS, THP-1 and Jurkat cells were maintained in complete RPMI as described previously, whereas A20 cells were grown in 5% FBS in DMEM supplemented with 2 mM L-glutamine (Sigma), 1 mM sodium pyruvate (Life Technologies), 0.77 mM L-asparagine (Sigma), 0.2 mM arginine (Sigma), 160 mM penicillin G (Sigma) and 70 mM dihydrostreptomycin sulphate (Boehringer Mannheim).
Flow cytometric binding studies
Binding of KS1 to cells was tested in the following manner. Cells (5x105) were resuspended in 3 ml of wash buffer (2% FBS and 0.2% sodium azide in PBS) and pelleted at 4°C, 200 g for 5 min. Ig Fc receptors were blocked with 1% goat serum in wash buffer for 30 min on ice. Cells were washed, pelleted, re-suspended in 50 µl of KS1Fc at 10 µg/ml and incubated for 30 min on ice. After incubation the cells were prepared as before and resuspended in 50 µl of goat anti-human IgGPE at 1 µg/ml and incubated for 30 min on ice. Cells were washed and resuspended in 250 µl of wash buffer containing 40 ng/ml propidium iodide (Sigma) to exclude any dead cells. A purified Fc tagged plant protein (EGBFc) was used, at 10 µg/ml, as a negative control in place of KS1Fc to determine non-specific binding. For two-colour staining, cells were incubated with one of the following antibodies prior to staining with KS1Fc or EGBFc, anti-CD4FITC, anti-CD8aFITC, anti-CD19FITC and rat IgG2aFITC at 10 µg/ml. Biotinylated Iak and its isotype control, mouse IgG2bbiotin, were used to identify MHC class II+ cells and detected using streptavidinalexa 488. Ten thousand gated events were analysed on a log scale using a FITC, PE and propidium iodide filter arrangement with peak transmittance at 525, 575 and 675 nm respectively with a bandwidth of 10 nm on an Elite cell sorter (Coulter, Hileah, FL). To determine KS1Fc binding to human cell lines CESS, THP-1 and Jurkat, and to reduce level of non-specific binding, both KS1Fc and control protein EGBFc were biotinylated using the Sigma biotinylation kit (Sigma BK-101) as described in the manufacturer's protocols. Human cells were labelled with KS1Fcbiotin or EGBFcbiotin as described previously and then detected with streptavidinPE. Cold competition was performed by adding various concentrations of (His)6KS1 at 4°C as a competitor prior to labelling with KS1Fc. An equivalent concentration of (His)6GV14B, an identically expressed unrelated bacterial protein, was used as control in competition experiments.
Chemotaxis assays
Cell migration in response to KS1 was tested using a 48-well Boyden chamber (Neuro Probe, Cabin John, MD) as described in the manufacturer's protocol. In brief, agonists were diluted in HBSS, 20 mM HEPES, 0.5% BSA and added to the bottom wells of the chemotactic chamber. Cells were re-suspended in the same buffer at 3x105 cells/50 µl. Top and bottom wells were separated by a PVP-free polycarbonate filter with a 5 µm pore size for CESS and THP-1 cells or 3 µm pore size for splenocytes and lymphocytes. Cells were added to the top well and the chamber incubated for 2 h for THP-1 and 4 h for CESS cells, splenocytes and lymphocytes in a 5% CO2 humidified incubator at 37°C. After incubation the filter was fixed and cells scraped from the upper surface. The filter was then stained with Diff-Quik (Dade Behring Diagnostics, Deerfield, IL) and the number of migrating cells counted in five randomly selected high-power fields. The results are expressed as a migration index defined as: migration index = no. of test migrated cells/no. of control migrated cells. Assays were repeated in triplicate.
In vivo experiments
BalbcByJ Hfh11 nu/nu (Nude) and C3H/HeJ inbred mice strains used for all experiments were maintained in house. C-terminal (His)6KS1 (20 µg) was injected into the left footpads of either Nude or C3H/HeJ mice in triplicate. The right foot of each animal was injected with an equal volume of 20 mM TrisHCl, pH 7.5. Mice were sacrificed after 18 h, and feet dissected and fixed in 3.7% formol saline. All tissues were sectioned and stained with haemotoxylin & eosin. Histology was performed at AgroQuality (Auckland, NZ). Photomicrography was performed on a Leica compound microscope and images prepared using Adobe Photoshop.
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Results
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Identification of KS1 cDNA sequence
A directionally cloned cDNA library was constructed from immature murine keratinocytes and submitted for high-throughput sequencing. Sequence data from a clone designated KS1 showed 35% identity over 74 amino acids with rat macrophage inflammatory protein (MIP)-2B and 32% identity over 72 amino acids with its murine homologue. The insert of 1633 bp (Fig. 1A
) contained an open reading frame of 300 bp with a 5' untranslated region of 202 bp and a 3' untranslated region of 1161 bp (this sequence is available from GenBank under accession no. AF144754). A poly-adenylation signal of AATAAA is present 19 bp upstream of the poly(A) tail. The predicted mature polypeptide is 77 amino acids in length containing four conserved cysteines with no ELR motif. The putative signal peptide cleavage site between Gly22 and Ser23 was predicted by the hydrophobicity profile. The full-length sequence was then screened against the EMBL database using the BLAST program, and showed 92.6, 94 and 93.6% identity at the nucleotide level with human EST clones AA643952, AA865643 and HS1301003 respectively. A recently described human
-chemokine, BRAK, has 94% identity with KS1 at the protein level (20). The alignment of KS1, BRAK and other murine
-chemokines is shown in Fig. 1B
. The phylogenetic relationship between KS1 and other
-chemokine family members was determined using the Phylip package (Fig. 1C
). KS1 and BRAK demonstrate a high degree of divergence from the other
-chemokines supporting the relatively low homology shown in the multiple alignment.



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Fig. 1. (A) The nucleotide sequence of KS1 cDNA is shown along with the deduced amino acid sequence using the single letter code. The 5' untranslated region is indicated by negative numbers. The underlined N-terminal amino acids represent the predicted leader sequence and the stop codon is denoted by `***'. The poly-adenylation signal is marked by a double underline. The sequence data is available from GenBank under accession no. AF144754. (B) Comparison of the complete open reading frame of KS1 with its human homologue BRAK and with the mouse -chemokines mCrg-2, mMig, mSDF-1, mBLC, mMIP-2, mKC and mLIX. An additional five residues are present in KS1 and BRAK between cysteine 3 and cysteine 4 that have not previously been described for chemokines. (C) A phylogenetic tree of KS1 was constructed against current murine -chemokines using Phylip software version 3.57c, and programs protdist and neighbour joining. The figure represents the degree of divergence between each of the family members. The branch lengths are proportional to the numbers of substitutions, based on the amino acid homology, the level of conservation between the different amino acid residues and the rate of evolution. GenBank accession nos for the sequences are (from top to bottom): L12030, M34815, M86829, U27267, J04596, X53798, AF044196, AF073957 and AF144754.
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Tissue expression of KS1 and BRAK
Tissue distribution of KS1 by Northern hybridization showed high expression in brain, ovary, lung and muscle, with low levels of expression in bone marrow. The transcript size on the Northern blot of 1.6 kb was the same size as the full-length cDNA sequence. BRAK was highly expressed in small intestine, colon and kidney, with moderate to low levels in liver, spleen, thymus, placenta, brain and pancreas. BRAK mRNA could also be detected in skeletal muscle and heart. Expression could not be detected in ovary, testis or prostate. The transcript size of BRAK was ~1.8 kb, which is similar to KS1 (Fig. 2B
). As non-ELR
-chemokines have been implicated as having angiostatic properties, BRAK expression levels were tested in a variety of tumours and compared to normal tissue. BRAK was expressed in normal uterine and cervical tissue, whereas it was completely down-regulated in their respective tumours, uterine leiomyoma and cervical adenocarcinoma (Fig. 2C
). Conversely, BRAK was expressed in breast tissue but was up-regulated in breast invasive ductal carcinoma (Fig. 2C
).
Recombinant expression of KS1
Recombinant C-terminal (His)6KS1 was a homogenous protein with an apparent molecular mass of 15 kDa (Fig. 3
). Internal sequencing of the 15 kDa protein gave the peptide sequence WYNAWNEK, confirming that the observed sequence is identical to that predicted from the cDNA sequence. The isoelectric point was predicted to be 10.26 using DNASIS (Hitachi Software Engineering, Yokohama, Japan). Recombinant KS1Fc, expressed and purified using Protein A-affinity column chromatography, revealed a protein with a molecular mass of 43 kDa corresponding to the predicted size plus the Fc fusion tag (data not shown).

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Fig. 3. Analysis of purified (His)6KS1 fusion protein by SDSPAGE. Protein was resolved on a 12% acrylamide denaturing gel and stained with Coomassie blue. Lane 1, mol. wt standards; lane 2, 5 µg of purified (His)6KS1 protein.
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Flow cytometric analysis of KS1 binding
Fc tagged KS1 (KS1Fc) was used to determine the cell types which express the receptor for this chemokine. KS1Fc bound to 54% of splenocytes and 9% peripheral lymph node cells (Fig. 4A and B
). No positive binding was identified in thymocytes (Fig. 4C
). Dual labelling experiments with antibodies to cell surface antigens showed that KS1Fc bound B cells in spleen (Fig. 4D
) but not CD4 or CD8 T cells (Fig. 4E and F
). KS1Fc also bound to the B cells in peripheral lymph node cells but not the T cells (data not shown). The matched isotype control for CD19, CD8 and CD4, rIgG2aFITC, showed no positive labelling (data not shown).

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Fig. 4. Flow cytometric analysis of KS1Fc binding to murine splenocytes, peripheral lymph node cells and thymocytes. Cells were labelled with KS1Fc or negative control protein EGBFc and visualized with a two-step staining procedure using goat anti-humanPE. Ten thousand gated events were analysed for each of the experiments. (A) Binding of KS1Fc was detected on murine splenocytes and (B) peripheral lymph node cells as compared with the negative control, EGBFc. (C) Alternatively, KS1Fc showed no binding to thymocytes when compared to negative control. The phenotype of the KS1Fc+ splenocytes was determined using two-color analysis with the following antibody markers. (D) Murine splenocytes were double positive for KS1Fc and CD19 (D) but not for CD4 (E) or CD8a (F) cells. KS1Fc (solid line), EGBFc (dotted line).
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Additionally, we screened peritoneal exudate cells (PEC) to determine whether KS1Fc bound monocytes. Forward and side scatter histograms from PEC were used to identify the monocyte population in region H (Fig. 5A
). Cells in region H were CD19 (Fig. 5B
), but were MHC class II+ (Fig. 5C
) indicating that they were monocytes and not B cells. Dual labelling experiments showed that all the cells in region H were double positive for MHC class II and KS1Fc (Fig. 5D
). The control protein, EGBFc, showed no binding to the MHC class II+ cells from region H (Fig. 5E
). The matched isotype control for MHC class II, mIgG2abiotin, showed no positive labelling (Fig. 5F
).

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Fig. 5. Flow cytometric analysis of KS1Fc binding to monocytes from PEC. Ten thousand gated events were analysed for each of the experiments. Forward (FS) versus side scatter (SS) histogram of PEC (A). All subsequent histograms were gated on region H. Cells from this region were CD19 (B) [CD19 (solid line), rIgG2a (dotted line)] and MHC class II+ (C) [MHC class II (solid line), mIgG2a (dotted line)]. Two-colour analysis shows that MHC class II+ cells are positive for KS1Fc (D) and not the control protein, EGBFc (E). The matching isotype for MHC class II, mIgG2a, showed no non-specific binding (F).
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As many non-ELR chemokines stimulate activated T cells we analysed KS1Fc binding to Con A-activated splenocytes grown in the presence of 10 ng/ml IL-2 for 9 days. All cells were positive for the activation marker CD69, and consisted of 63% CD4 cells and 37% CD8 cells (data not shown). KS1Fc showed no positive binding to these cells (Fig. 6D
). KS1Fc also bound to the murine B cell line, A20 (Fig. 6A
), and the human B lymphoblastoid cell line, CESS (Fig. 6B
). Additionally, KS1Fc bound to the monocyte leukemia cell line THP-1 (Fig. 6C
) but not Jurkat T cells (Fig. 6E
). Preliminary analysis identifies B cells and monocytes as responsive cells for KS1. To demonstrate specificity (His)6KS1 was used in cold competition with KS1Fc against murine splenocytes. Increasing concentrations of (His)6KS1 reduced the level of binding of KS1Fc (Fig. 7
), as demonstrated by a decrease in the mean channel fluorescence, to murine splenocytes. An equivalent concentration of a non-specific (His)6-tagged protein, GV14B, showed no decrease in mean channel fluorescence when co-incubated with KS1Fc (Fig. 7
). The ability of (His)6KS1 to competitively inhibit the binding of KS1Fc validates the hypothesis that this reagent bound via the KS1 receptor.

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Fig. 6. Flow cytometric analysis of KS1Fc binding to murine and human cell lines. Murine cells were labelled with KS1Fc or negative control protein EGBFc and visualized with a two-step staining procedure using goat anti-human IgGPE. Human cell lines were labelled with KS1Fcbiotin or negative control EGBFcbiotin and visualized with a two-step staining procedure using streptavidinPE. Ten thousand gated events were analysed for each of the experiments. Enhanced KS1Fc binding was detected on A20 (A), CESS (B) and THP-1 (C) cells but not on Con A IL-2-activated T cells (D) or Jurkat T cells (E). KS1Fc (solid line), EGBFc (dotted line).
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Fig. 7. Cold competition of KS1Fc binding with increasing concentrations of (His)6KS1. KS1Fc binding on murine splenocytes was inhibited by increasing concentrations of (His)6KS1 protein, whereas it was not influenced by the negative control protein, (His)6GV14B. Ten thousand gated events were analysed for each experiment. Values are the geometric mean channel fluorescence ± SD obtained for duplicate samples and are representative of two individual experiments.
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(His)6KS1 induces chemotaxis in B cells and monocytes
Flow cytometric analysis revealed that KS1 specifically bound to B cells and monocytes. We determined whether KS1 could stimulate the chemotaxis of these cells using a modified Boyden chamber. (His)6KS1 induced a concentration-dependant migration in murine splenocytes, with optimal activity at 100 ng/ml (Fig. 8A
). In addition, (His)6KS1 stimulated the migration of the B lymphoblastoid cell line, CESS, and the monocyte line, THP-1 (Fig. 8B and C
). However, unlike SDF-1
, KS1 did not stimulate the migration of either human or murine activated T cells (Fig. 8D and E
).
(His)6KS1 induces inflammation in vivo
To determine whether KS1 was active in vivo and whether T cells are required for an inflammatory response we injected Nude mice s.c. with (His)6KS1. Histological examination of mouse footpads injected s.c. with (His)6KS1 showed a leukocyte infiltrate (Fig. 9A
). The inflammation was of a mixed phenotype with evidence of mononuclear cells and polymorphonuclear cells (Fig. 9C and D
). No obvious inflammation was apparent in the feet of mice injected with Tris, the buffer excipient (Fig. 9B
). To confirm that this inflammation was due to (His)6KS1 and not endotoxin we repeated the experiment in LPS-insensitive C3H/HeJ mice. (His)6KS1-injected footpads from these mice showed a similar inflammatory response to the Nude mice (Fig. 9E
) with the buffer excipient-injected footpads having no marked inflammation (Fig. 9F
).

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Fig. 9. Inflammatory response of Nude and C3H/HeJ mice upon s.c. injection of KS1. Each group of three mice was injected with (His)6KS1 per group. Mice were injected in the left footpad with (His)6KS1. Equal volumes of Tris buffer were injected into the right footpads as controls. Feet were biopsied after 18 h and haemotoxylin & eosin sections prepared. (A) Nude mice demonstrate a mixed inflammatory response upon injection with KS1, as indicated by an arrow. (B) No inflammation is apparent in the footpad injected with Tris buffer control. (C and D) High-power magnification (x100 objective) of cells in Nude mouse inflammation indicates the presence of monocytes, mononuclear cells and polymorphonuclear cells. (E) A mixed inflammatory response was also present in C3H/Hej mice as denoted by an arrow. (F) No inflammation was detectable in footpads injected with the negative control. Abbreviations: mu, muscle; v, vein; e, epidermis; d, dermis; m , monocyte; mn, mononuclear; and pmn, polymorphonuclear cells. Scale bar = 50 µm. B cell- and monocyte-activating chemokine (BMAC) B cell- and monocyte-activating chemokine (BMAC) B cell- and monocyte-activating chemokine (BMAC) B cell- and monocyte-activating chemokine (BMAC)
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Discussion
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We have identified some of the biological activities of a novel non-ELR
-chemokine, KS1, and described its tissue distribution. The cDNA was similar to a recently cloned human gene called BRAK (20). Homology of KS1 to BRAK was 94% at the protein level, indicating it as the murine homologue of this gene. To date no known function has been described for BRAK. KS1 and BRAK appear to be distant relatives of the non-ELR
-chemokines as was shown by their phylogenetic relationship. There are five additional residues (KS1, SMSRY and BRAK, SVSRY), between cysteines 3 and 4 of the conserved cysteines, which is not consistent with the predicted Prosite motif for
-chemokines. The predicted N-terminus of the mature protein upstream of the first cysteine has only two residues in contrast to other non-ELR
-chemokines. Furthermore, KS1 has a lysine in place of an arginine immediately prior to the first cysteine. The conservation of a highly basic residue, typically arginine, prior to the first conserved cysteine has been postulated as a requirement for binding to the receptor (1). The amino acid substitutions between KS1 and BRAK in the mature peptide are conservative, indicating that these differences are likely to be insignificant.
KS1 tissue distribution in mouse and BRAK in human is unusual for
-chemokines in that it is highly expressed in normal non-lymphoid tissues. Although expression levels are different between mouse and human, KS1 and BRAK are expressed in brain and muscle. Differences between mouse and human expression profiles have been described for other chemokines (2325), and are thought to reflect pathological changes of the particular donor. In contrast to the reported expression profile for BRAK (20), we found BRAK was expressed at higher levels in small intestine, colon and kidney. Additionally, the predominant band ran at 1.8 kb rather than 2.5 kb as reported earlier (20), raising the possibility of splice variance. Alternative splicing has previously been described as a property of some chemokines, for example LARC/MIP3
(23).
As non-ELR
-chemokines have been shown to have angiostatic function (15,16,26,27), we investigated BRAK expression in normal versus tumour tissue. The blot revealed that BRAK is expressed in non-malignant breast, uterine and cervical tissues. With good expression in human breast and kidney it is not surprising that BRAK was identified from breast and kidney EST. From the tumour expression data we saw two patterns emerge, either BRAK was up-regulated in breast invasive ductal carcinoma or, in the case of uterine leiomyoma and cervical adenocarcinoma, BRAK mRNA was undetectable. The reason for the disparate trends in expression levels from the different tumours is unclear at this stage and may be related to differences in cancer pathology. Furthermore, these biopsies were likely derived from a single patient and may not reflect the majority of cases. Nevertheless, it would be of interest to determine whether BRAK added directly to tumour models of these cancers could alter malignancy.
KS1 had a predicted size of 9.4 kDa; however, the purified (His)6KS1 protein had an apparent size of 15 kDa which could not be accounted for by the additional histidine residues. This discrepancy between the predicted and apparent size has previously been reported for a number of chemokines (28,29), and is thought to be due to the highly basic nature of these proteins.
The high degree of homology between KS1 and BRAK suggested that they would be active on both mouse and human cell types. This was demonstrated by flow cytometric analysis which showed that KS1 binds directly to mouse and human B cells and monocytes. Furthermore, KS1 induced chemotaxis on both mouse and human cells. This phenomenon of rodent chemokines stimulating human cells has been previously described for a number of different chemokines (3032). We clearly defined B cells and monocytes, and not T cells, as target cells for KS1 by binding studies. The only other non-ELR
-chemokine to bind to these cells is SDF-1
; however, SDF-1
also binds to T cells (17,18,33). We were able to confirm that KS1 stimulates B cells and monocytes but not T cells in migration assays using a range of different cell types. In the case of splenocytes and THP-1 cells, SDF-1
and KS1 stimulated equivalent levels of migration; however, CESS cells were 15-fold more responsive to SDF1
than KS1.
As the in vitro data indicated that B cells and monocytes respond to KS1, we tested its inflammatory properties by injecting Nude mice with the protein. Mice injected s.c. with (His)6KS1 showed a mixed inflammatory response. As Nude mice have no T cells this supported the in vitro data that (His)6KS1 promotes extravasation of cells other than T cells. As seen with the Nude mice, C3H/HeJ also had an inflammatory response to (His)6KS1, demonstrating that the response in the Nude mice was (His)6KS1 specific and not due to endotoxin. Although we have demonstrated the ability of KS1 to stimulate chemotaxis of B cells and monocytes, we do not rule out the possibility that other haemopoetic or non-haemopoetic cells might respond to KS1.
The majority of non-ELR
-chemokines have been shown to be chemotactic for activated T cells; however, KS1 did not cause the migration of these cells. Therefore, this raises the question of which receptor does KS1 utilize? There are currently only three known chemokine receptors that bind non-ELR
-chemokines: CXCR3, the receptor for I-TAC (12), Mig and IP-10 (34); CXCR4, the receptor for SDF-1
(17,18); and CXCR5, the receptor for BCA-1 (13). As we have shown that KS1 does not stimulate T cells it is unlikely that it is binding via CXCR4. Furthermore, it is unlikely to bind via CXCR3, a receptor on activated T cells, as we can demonstrate no activity on Con A IL-2-activated T cells. This then leaves CXCR5; however, this receptor has only been demonstrated on B cells and not monocytes. Therefore, the likelihood of KS1 acting via a novel receptor merits further investigation.
The biological function of a novel chemokine, initially identified as KS1, is described. KS1 has a broad expression in non-lymphoid tissue, altered expression levels in tumours and a role in trafficking of B cells and monocytes. Therefore, we propose the name B cell- and monocyte-activating chemokine (BMAC) for this molecule to reflect its described biological functions
 |
Acknowledgments
|
---|
We are grateful to Dr Paul Tan for his helpful comments with the manuscript, Dr Matthew Glenn and Dr Ilkka Havukkala for maintenance of the EST database, and Stewart Whiting for managing the animal facility. We are also grateful to Dr Annette McGrath for assistance with the bioinformatic analysis. We also appreciate the support of the Functional Genomics group at Genesis Research and Development Corp. Ltd, Auckland, NZ and Immunex Corp., Seattle, WA.
 |
Abbreviations
|
---|
BMAC B cell- and monocyte-activating chemokine |
Con A concanavalin A |
ELR GluLeuArg |
MIP macrophage inflammatory protein |
PBMC peripheral blood mononuclear cells |
PE phycoerythrin |
PEC peritoneal exudate cells |
PHA phytohaemagglutinin |
 |
Notes
|
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Transmitting editor: M. Feldmann
Received 10 September 1999,
accepted 28 January 2000.
 |
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