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The Journal of Immunology, 2005, 174: 5655-5664.
Copyright © 2005 by The American Association of Immunologists

Multiple Products Derived from Two CCL4 Loci: High Incidence of a New Polymorphism in HIV+ Patients1

Roger Colobran*,{ddagger}, Patricia Adreani*, Yaqoub Ashhab*, Anuska Llano{dagger}, José A. Esté{dagger}, Orlando Dominguez*, Ricardo Pujol-Borrell*,{ddagger} and Manel Juan2,*,{ddagger}

* Laboratory of Immunobiology for Research and Application to Diagnosis, Centre for Transfusion and Tissue Bank, Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, and {dagger} Retrovirology Laboratory, IrsiCaixa Foundation. Hospital Universitari Germans Trias i Pujol, {ddagger} Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Barcelona, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human CCL4/macrophage inflammatory protein (MIP)-1{beta} and CCL3/MIP-1{alpha} are two highly related molecules that belong to a cluster of inflammatory CC chemokines located in chromosome 17. CCL4 and CCL3 were formed by duplication of a common ancestral gene, generating the SCYA4 and SCYA3 genes which, in turn, present a variable number of additional non-allelic copies (SCYA4L and SCYA3L1). In this study, we show that both CCL4 loci (SCYA4 and SCYA4L) are expressed and alternatively generate spliced variants lacking the second exon. In addition, we found that the SCYA4L locus is polymorphic and displays a second allelic variant (hereinafter SCYA4L2) with a nucleotide change in the intron 2 acceptor splice site compared with the one described originally (hereinafter SCYA4L1). Therefore, the pattern of SCYA4L2 transcripts is completely different from that of SCYA4L1, since SCYA4L2 uses several new acceptor splice sites and generates nine new mRNAs. Furthermore, we analyzed the contribution of each locus (SCYA4 and SCYA4L1/L2) to total CCL4 expression in human CD8 T cells by RT-amplified fragment length polymorphism and real-time PCR, and we found that L2 homozygous individuals (L2L2) only express half the levels of CCL4 compared with L1L1 individuals. The analysis of transcripts from the SCYA4L locus showed a lower level in L2 homozygous compared with L1 homozygous individuals (12% vs 52% of total CCL4 transcripts). A possible clinical relevance of these CCL4 allelic variants was suggested by the higher frequency of the L2 allele in a group of HIV+ individuals (n = 175) when compared with controls (n = 220, 28.6% vs 16.6% (p = 0.00016)).


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Chemokines are a large superfamily of small (~8–15 kDa) structurally related molecules that regulate cell trafficking of various types of leukocytes to areas of injury or infection and play different roles in both inflammatory and homeostatic processes (1, 2, 3). According to the arrangement of a structural cysteine motif found near the NH2 terminus of the mature protein, chemokines have been divided into four subfamilies: CXC, CC, CX3C, and C chemokines (4). These chemokines carry out their biological functions through seven-transmembrane domain G protein-coupled receptors, which are expressed on several populations of leukocytes (5, 6, 7). To date, 42 chemokines and 18 chemokine receptors have been identified in humans (8).

A remarkable feature of chemokines and chemokine receptors is their redundancy and binding promiscuity. There are many examples of a single chemokine binding to several receptors, as well as a single chemokine receptor transducing signals for several chemokines. Interestingly, genes encoding the inflammatory chemokines tend to appear in clusters (human CC subfamily in chromosome 17 and the CXC subfamily in chromosome 4). Moreover, an important characteristic of cluster chemokines is that they share many ligands with few receptors (4). Individual members of the chemokine superfamily may contribute to this complex relationship network by two mechanisms of additional variability: 1) pretranslational modifications (such as alternative splicing) (9, 10) and 2) posttranslational modifications (such as NH2-terminal truncations by the dipeptidyl-peptidase (DPP) 3 CD26/DPP IV) (11, 12, 13, 14).

CCL3/macrophage inflammatory protein (MIP)-1{alpha} and CCL4/MIP-1{beta} are two highly related chemokines that belong to a cluster of inflammatory CC chemokines located in chromosome 17 (q11-q21), which are secreted by specific cells after being triggered by Ags or mitogenic signals and attract additional cells involved in immune responses (15). These two chemokines, together with CCL5/RANTES, are the major HIV-suppressive factors produced by CD8+ T cells (16) by binding to CCR5, the coreceptor necessary for the entry of HIV-R5 strains into CD4+ cells (17, 18).

CCL3 and CCL4 were formed by duplication of a common ancestral gene (15) that originated SCYA3/LD78{alpha} and SCYA4/ACT-2 genes which in turn have a second non-allelic copy (SCYA3L1/LD78{beta} and SCYA4L/LAG-1) present in variable numbers in the human genome (19). In the case of CCL3, there is a third gene (SCYA3L2/LD78{gamma}) that is a 5'-truncated pseudogene (20). Therefore, human CCL3 and CCL4 are encoded by two highly related non-allelic isoforms that have been duplicated and mutated to produce two different but highly homologous proteins (>90% between CCL3 (from SCYA3) and CCL3L1 (from SCYA3L1) proteins, and >95% between CCL4 (from SCYA4) and CCL4L (from SCYA4L) proteins) (21, 22, 23). In CCL3, functional differences have been reported between the proteins of two loci. CCL3L1/LD78{beta} has been characterized as a more potent CCR5 agonist and a greater inhibitor of HIV-1 than CCL3/LD78{alpha} (24, 25, 26, 27). Moreover, the potent antiviral activity of CCL3L1/LD78{beta} is increased due to the cleavage by CD26/DPP IV, a dipeptidyl-peptidase that cuts dipeptides from the NH2 terminus of regulatory peptides with a proline or alanine residue in the penultimate position (14, 28). Recently, CCL4 has also been described as a target for CD26/DPP IV, being mainly secreted by stimulated PBLs as a truncated form that lacks the first two amino acids. This posttranslational modification affects receptor specificity but not anti-HIV activity (29, 30).

Our studies point out that, in fact, the word "CCL4" groups a broad quantity of different transcripts coded by the SCYA4 and SCYA4L loci. This high variability is caused by an alternative splicing mechanism and by the presence of a polymorphism in the SCYA4L locus that changes the intron 2 acceptor splice site and creates different mRNA expression patterns. We have assessed the possibility that individuals carrying different genotypes and with different capabilities to exert this diversity of CCL4 variants may respond differently in situations of chronic immunostimulation, such as viral infection or autoimmunity. Therefore, the possible clinical relevance of these CCL4 allelic variants was suggested by the higher frequency of the SCYA4L2 allele in a group of HIV+ individuals when compared with controls.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Isolation and stimulation of human CD8 T cells

PBMCs were isolated from total blood by centrifugation over Lymphoprep (Axis-Shield). CD8 T cells were positively selected using magnetic beads coated with mAbs against CD8 (MACS MicroBeads; Miltenyi Biotec) according to the manufacturer’s instructions. Purity of CD8 T cells was routinely >98%, as determined by flow cytometry.

CCL4 production by purified CD8 T cells was measured at baseline and after PHA stimulation (31). Cells were cultured in RPMI 1640 complete medium (Invitrogen Life Technologies) containing 10% FCS, streptomycin, and penicillin at a cell concentration of 1 x 106 cells/ml in 24-well plates (Corning Costar). To induce CCL4 production, we added 1 µg/ml PHA and 10 ng/ml recombinant human IL-2 (R&D Systems). Cells were harvested and total RNA was extracted after 0, 6, 24, and 48 h of PHA stimulation. Two replicate experiments were performed for each condition.

Human CCL4 amplification

CCL4 was amplified by RT-PCR. RNA was extracted using the RNAqueous-96 kit (Ambion) and was then retrotranscribed with oligo(dT)15 and SuperScript-II (Amersham Biosciences). PCR was performed in 10 µl of total reaction volume. The primers used to amplify all CCL4 variants (from both loci) were 82A/Bex1 5'-GAAGCTCTGCGTGACTGTC-3' and 433A/Bex3 5'-CGGAGAGGAGTCCTGAGTAT-3', and the PCR cycling conditions were 95°C for 30 s, 62°C for 30 s and 72°C for 30 s. The primers used to amplify SCYA4L variants were 124Bex1 5'-TGCCTTCTGCTCTCTAGCA-3' and 615Bex3 5'-TGAAAACACATGGAATTAACG-3', and the PCR cycling conditions were 95°C for 30 s, 59°C for 30 s and 72°C for 30 s. PCR products were visualized by ethidium bromide staining following electrophoresis in 2% Tris borate-EDTA (TBE) high-resolution agarose gels (Sigma-Aldrich).

Restriction digests and densitometric analysis

PCR products were digested with 10 units of MspI (Fermentas) in 15 µl of total reaction volume for 90 min at 37°C. The digested products were evaluated by visualization by ethidium bromide staining following electrophoresis in 2% TBE high-resolution agarose gels. PCR products and subsequent restriction fragments were quantified using the Quantity One software (Bio-Rad) according to the manufacturer’s instructions.

Population study of SCYA4L locus polymorphism

To evaluate the population frequencies of SCYA4L polymorphism, we analyzed 220 healthy donors as well as 175 HIV-seropositive, 80 hepatitis C virus-seropositive, 30 insulin-dependent diabetes mellitus, and 30 autoimmune thyroid disease patients. All individuals are ethnically grouped as Caucasoid Spanish population. The genomic DNA was obtained from total blood of each individual, and by using the primers I2B 5'-GCAGAGGAAGATGCCTACCAC-3' and 503Bex3 5'-AAATAATGGAAATGACACCTAATAC-3', we amplified the junction between intron 2 and exon 3 of the SCYA4L locus under the following PCR cycling conditions: 95°C for 30 s, 59°C for 30 s, and 72°C for 30 s. The PCR product was purified using the GFX PCR DNA and Gel Band purification kit (Amersham Biosciences). Finally, DNA sequencing was performed with both primers on an Applied Biosystems (ABI) Prism 3100 genetic analyzer.

Software tools

To assess the influence of the change in the acceptor splice site sequence in the SCYA4L2 allelic variant, we have used two software applications: 1) GENSCAN (<http://genes.mit.edu/GENSCAN.html>) (32) and 2) HMMgene (v. 1.1) (<http://www.cbs.dtu.dk/services/HMMgene/>) (33).

We used NNSPLICE (v. 0.9) (<http://www.fruitfly.org/seq_tools/splice.html>) (34) to predict the potential acceptor splice sites in the SCYA4L2 allelic variant.

The algorithm to calculate the scores of alternative splice sites found in the SCYA4L2 allelic variant is based on Shapiro and Senepathy (35) (available at <http://www.genet.sickkids.on.ca/~ali/splicesitescore.html>).

A genomic sequence from GenBank was used to establish the distribution and distances among CCL3 and CCL4 gene loci and their duplicates: NT_010799 includes a 9,412,828-bp sequence referred to Homo sapiens chromosome 17. This DNA sequence is part of the second release of the completed human reference genome. It was assembled from individual clone sequences by the Human Genome Sequencing Consortium in consultation with National Center for Biotechnology Information (NCBI) staff.

Real-time PCR and expression studies

To evaluate the total expression of CCL4, we used real-time PCR with primers amplifying all potential variants (from SCYA4 and SCYA4L loci). Standards for CCL4 and GAPDH were obtained by conventional PCR from PHA-stimulated CD8 T cells cDNA. Amplification products were quantified in serial dilutions from 108 to 101 molecules. Real-time PCR from cDNA was performed in a LightCycler (Roche Diagnostics) using the master mix containing 4 mM MgCl2, 0.5 µM primers (82A/Bex1 and 433A/Bex3), and 1 µl of LightCycler Fast Start DNA Master SYBR Green I (Roche Diagnostics). The amount of cDNA was calculated using the second derivate method after confirming the specificity of the amplification with the melting curve profiles. Two replicates of each sample were performed for CCL4 and GAPDH, and a maximum SD of 15% between replicates was accepted. The relative abundance of CCL4 in each sample was calculated by normalizing the mean levels of CCL4 mRNA copies (meanCCL4-sample) with the corresponding mean value for GAPDH (meanGAPDH-sample) using the formula indexsample = (meanCCL4-sample)/(meanGAPDH-sample).

To analyze the contribution of SCYA4 and SCYA4L loci-derived variants to the total CCL4 mRNA, real-time PCR products were recovered and half of the total amount was digested with MspI as described. The digested and non-digested products were visualized in a high-resolution agarose gel electrophoresis and quantified by densitometry as described.

Statistics

Data sets were analyzed using SPSS v. 11.0.1 for Macintosh (SPSS). When necessary, the results were expressed as the mean value ± SD. An independent-samples Student’s t test and a {chi}2 test were applied to the data sets to determine statistically significant differences between groups. Differences were considered significant when p values were <0.05


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The two CCL4 loci generate four mRNAs

CCL4 was amplified from cDNA of human activated CD8 T cells using primers placed in exons 1 and 3. Besides the expected canonical product of the SCYA4 and SCYA4L loci (371 bp), an unexpected low m.w. amplimer was observed (256 bp; Fig. 1, ND lane). The m.w. difference was 115 bp, which was in exact accordance with the length of the second exon of the loci. Cloning and sequencing these two amplimers showed that the 371-bp product included two different sequences corresponding to the CCL4 and CCL4L variants, whereas the unexpected low m.w. band included alternatively spliced variants of CCL4 and CCL4L, thus confirming the lack of exon 2 in 256-bp product. Therefore, the described CCL4 chemokine is codified by two loci that allow at least four different spliced forms: 1) two long, mature mRNAs (full length of 667 bp), with identical size for both loci (codifying for CCL4 and CCL4L proteins) but with 26 nucleotide differences (19 transitions and 7 transversions) and three amino acid changes, two in the signal peptide (V12M and L20P) and the third one (G70S) in the mature protein (Fig. 2); 2) two short spliced mRNAs (full length of 552 bp) also derived from both loci, but lacking the second exon. The predicted amino acid sequences of both forms present a frame shift in the exon 3 reading frame caused by the new junction between exon 1 and 3, and a stop codon appears close to the original one (Fig. 2). Consequently, these mature proteins are shorter (29 amino acids) and only maintain the two initial residues of the complete CCL4 and CCL4L proteins (69 amino acids). Interestingly, due to the frame shift, some of the conservative base substitutions between the two loci become non-conservative in these forms: there are three amino acid differences in the predicted mature protein (A13V, T15I, and S18N). Following commonly accepted nomenclature usage, they will be named CCL4{Delta}2 and CCL4L{Delta}2, respectively.



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  		FIGURE 1. Two canonical products of CCL4 and their variants lacking exon 2. cDNA from stimulated CD8 T cells was amplified using primers in exons 1 and 3. The PCR product was not digested (ND) or digested (D) with MspI. The highest m.w. amplimer in lane ND contains CCL4L and CCL4 products resolved after MspI restriction analysis (lane D). The lowest m.w. amplimer in lane ND contains CCL4L{Delta}2 and CCL4{Delta}2 products resolved after MspI digestion (lane D).

 


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  		FIGURE 2. Alignment of SCYA4 and SCYA4L cDNA with their amino acid sequences. Lanes SCYA4 and SCYA4L are cDNAs, while CCL4/CCL4L are their canonical peptide products and CCL4{Delta}2/CCL4L{Delta}2 are their variants lacking exon 2. Conserved nucleotides are shown as dashes. Exons are separated with vertical bars. Absent amino acids in the variants lacking exon 2 are shown as asterisks (*), and amino acid differences are bold. Amino acids of the signal peptide are in lowercase letters whereas amino acids of the mature protein are in uppercase letters. The initiation codon, the stop codons, and two possible polyadenylation signals are shaded with black. AU-rich sequences that reduce mRNA stability are shaded in gray. The MspI restriction target is underlined.

 
Multiple mRNAs derived from a new allelic variant (SCYAL2) of SCYA4L locus

When we specifically amplified the SCYAL-derived variants (CCL4L and CCL4L{Delta}2) from different individuals, some samples showed an unexpected new pattern of unidentified amplimers. Cloning and sequencing these variants revealed nine new mRNAs formed by the use of different cryptic acceptor splice sites located in the second intron or in the third exon (Fig. 3A, shaded in gray). By sequencing the corresponding genomic DNA of these individuals, we discovered a +590A > G critical change in the original acceptor splice site of the second intron of the SCYA4L locus.



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  		FIGURE 3. Multiple transcripts derived from a polymorphism in SCYA4L locus. A, General description of all detected CCL4 mRNAs. SCYAL1-derived mRNAs are shaded in black and SCYA4L2-derived mRNAs are shaded in gray. The score of acceptor splicing sites is based on Shapiro and Senepathy (35 ). B, Scheme of splicing in SCYA4 and SCYA4L1/SCYA4L2 genes. In SCYA4L2, the original acceptor site is mutated (AG > GG) and the spliceosome is unable to recognize it. Instead, the spliceosome can select another alternative acceptor splice site around the original site. Accession numbers correspond to those provided by GenBank after submission. Gene accession numbers: SCYA4, AY766459; SCYA4L1, AY766460; SCYA4L2, AY766461.

 
Therefore, the SCYA4L locus is a polymorphic gene with two allelic variants: the original SCYA4L locus (hereinafter SCYA4L1) and a second highly related polymorphic variant (hereinafter SCYA4L2); despite the lack of differences between the three exons and the first intron of SCYA4L1 and SCYA4L2, we have found four base substitutions in the second intron of SCYA4L2. Only the fourth of these changes (described above as +590A > G) seems to be critical for the final expression of SCYA4L2. Normally (36, 37), the donor splice site of the second intron in SCYA4L1 shows GT right after the point where exon 2 finishes, whereas the acceptor site has AG just before the point where intron 2 sequence is cleaved (canonical pattern). In SCYA4L2, the sequence of the acceptor splice site in SCYA4L1 experiences a critical change and becomes GG (Fig. 3B). According to the bioinformatic predictions, this polymorphism causes the inability of spliceosomes to recognize the mutated acceptor site (GG), and therefore two splicing options appear: 1) no removal of the second intron (hence generating a mature long mRNA with exon 1 + exon 2 + intron 2 + exon 3), or 2) use of alternative acceptor sites around the original one. Almost all mRNAs derived from SCYA4L2 are generated following this second option (use of an alternative acceptor splice site), but we also observed a few mRNAs caused by lack of splicing in the second intron.

SCYA4 and SCYA4L: from 2 genes to 13 mRNAs

A search in the NCBI nucleotide database for CCL4 (<http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db = Nucleotide>) provided access to a long stretch of the human genomic sequence in chromosome 17 (9,412,828 bp), assembled from individual clone sequences by the Human Genome Sequencing Consortium (NT_010799). In this region we found not only the SCYA3 and SCYA4 loci head to head and separated by 13.7 kb, but also two copies of the alternative forms (SCYA3L1 and SCYA4L) arranged in the same way and separated by a non-coding region of 14.3 kb (Fig. 4, physical map). Although mature mRNAs derived from SCYA4 and SCYA4L have the same length (667 bp), differences in their respective introns resulted in different lengths of the primary transcripts (1795 bp in SCYA4 vs 1816 bp in SCYA4L). A 12-bp deletion in intron 1 and an 11-bp deletion in intron 2 are the most relevant differences between the SCYA4 and the SCYA4L loci.



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  		FIGURE 4. From genomic organization to mRNA products. A, Physical map from 34,550 kb to 34,800 kb, inside the 17q11-q21 region, based on the genomic sequence NT_010799. B, Representative sequences of the junction between intron 2 and exon 3 from SCYA4L1 and SCYA4L2, produced by amplification of genomic DNA from PBMCs by using specific primers for locus SCYA4L. Dinucleotides corresponding to original acceptor splice sites are squared, and the polymorphic position is indicated with an arrow. C, High-resolution agarose gel electrophoresis showing the transcription pattern of each CCL4 gene (cDNA from stimulated CD8 T cells was amplified using locus-specific primers). D, Percentages of expression of each form (SD is <5% in each value). E, All mRNAs derived and cloned from SCYA4 and SCYA4L1/L2 are represented with boxes. F, Alignment of derived mature proteins. Cysteines are underlined.

 
Strikingly, the two copies of the SCYA4L locus present in this sequence are the two allelic variants described in this paper (SCYA4L1 and SCYA4L2). Additional minor differences exist in the 5' and 3' non-coding regions of the two copies of SCYA4L, but we have not addressed their effect on transcription in this study.

SCYA3 and SCYA3L1, as well as SCYA4 and SCYA4L2, are separated by 105 kb. We also found the SCYA3L2 pseudogene close to one of the copies of SCYA3L1. Other non-related loci (e.g., as a member of the TRE17 oncogene family) are duplicated together with the SCYA4L and SCYA3L1 alternative forms. All this suggests repeated duplications of a 120 kb stretch in this region of chromosome 17.

The most abundant mRNA derived from SCYA4L2 (78.2% of total mRNA expression) corresponds to the new CCL4L2 variant of CCL4 generated by the use of an acceptor splice site located 15 nucleotides downstream of the original site (Fig. 4, densitometry of gel electrophoresis). The predicted CCL4L2 mature protein has 64 amino acids and lacks the initial five amino acids codified by the third exon (FQTKR), but the rest of the sequence remains unchanged (Fig. 4, protein sequence alignment).

We have named CCL4L2b, a 41-aa predicted form of CCL4 that is truncated by a premature stop codon and is codified by two mRNAs, CCL4L2b1 and CCL4L2b2, accounting for 6.6 and 2.6% of total message, respectively. The other CCL4 mRNAs detected (CCL4L2c, CCL4L2d, CCL4L2e, each accounting for around 4%) code for three new and highly related CCL4 variants that maintain the frame shift produced in the junction between exon 2 and the new exon 3 of each variant and use the same stop codon. Despite having a non-related amino acid sequence from position 42 to the end compared with the original CCL4L protein, these three proteins show a cysteine at –10 from the C terminus that may maintain the basic chemokine conformation.

Finally, we have cloned three very low frequency mRNAs (not visible by gel electrophoresis): 1) CCL4L2{Delta}2, which is the CCL4L2 variant lacking exon 2; 2) CCL4L2b{Delta}2, which is the CCL4L2b variant lacking exon 2; and 3) CCL4L2f, which uses another different alternative acceptor splice site and has the same reading frame than CCL4L2b{Delta}2. The CCL4L2f predicted protein (80 aa) presents seven cysteines in its sequence (with two CC motifs) (Fig. 4, protein sequences alignment).

In theory, other exon 2-skipped variants of all SCYA4L2-derived proteins (next to CCL4L2{Delta}2 and CCL4L2b{Delta}2) may also exist, but we could not detect them due to their very low frequency.

Expression of CCL4 mRNA variants

The expression of CCL4 mRNA variants was assessed by real-time RT-PCR at 6, 24, and 48 h after PHA-stimulated and control CD8 T lymphocytes from healthy donors of each CCL4 genotype: L1L1, L1L2 and L2L2 (three individuals for each group). To evaluate the contribution of each locus to total mRNA expression, we took advantage of the presence of a MspI restriction site in the products of SCYA4 locus (38). Peak CCL4 expression was observed at 6 h with an average increase of 6- to 7-fold compared with controls in all genotypes (Fig. 5A1, data of 24 and 48 h are only shown in this figure and not in the following because mRNA quantities returned to levels similar to those obtained from non-stimulated samples). Interestingly, the ratio of CCL4 to GAPDH mRNA was higher in L1L1 than in L2L2 individuals (6.2 vs 2.7; Fig. 5A2). Values obtained from heterozygous were intermediate (see graphics in Fig. 5).



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  		FIGURE 5. mRNA expression of CCL4 in CD8 T cells of L1L1, L1L2, and L2L2 individuals. A1, Time course of CCL4 mRNA expression. Expression levels are relative to the housekeeping gene (GAPDH), represented as an index (y-axis), and referred to the basal values (index = 1). A2, Copy number of CCL4 (relative to the GAPDH) in stimulated (6 h) and non-stimulated CD8 T cells. B1, Percentage of mRNAs lacking exon 2 compared with the total CCL4 mRNAs. B2, Relative copy number of mRNAs lacking exon 2. C1, Percentage of SCYA4L locus mRNAs relative to total CCL4 mRNAs. C2, Relative copy number of SCYA4L-derived mRNAs. Each point was performed in duplicate from three samples of different individuals, and the results were represented as the mean ± SD. Differences between the basal and stimulated (6 h) situations were always statistically significant in all genotypes (p < 0.01). *, p < 0.05; ***, p < 0.005.

 
The ratio of short mRNAs lacking the second exon to total CCL4 mRNA was 9.2% in L1L1 individuals (which express the CCL4{Delta}2 and CCL4L{Delta}2 variants), but only 5% in L2L2 individuals (where only CCL4{Delta}2 may be detected) (Fig. 5B1); these ratios decrease to 3.1 and 1.8%, respectively, after 6 h of stimulation with PHA, despite the increase in absolute values (Fig. 5B2).

Finally, we analyzed the relative contribution of the SCYA4L locus to total CCL4 expression. In basal conditions, SCYA4L-derived mRNAs accounted for 29.9% of total CCL4 expression, in L1L1 individuals, but only for 4.6%, in L2L2 individuals (Fig. 5C1,2). After stimulation for 6 h, the percentage of SCYA4L mRNAs was 54% in L1L1 individuals, and 12.1% in L2L2 subjects. Hence, activation seems to shift transcription to the SCYA4L locus in all genotypes. In both conditions, the contribution of SCYA4L2 to the total levels of CCL4 mRNA is very low compared with SCYA4L1.

Population incidence of SCYA4L locus polymorphism and association with HIV infection

To evaluate the incidence of SCYA4L locus polymorphism in the overall population, we sequenced the junction of intron 2 with exon 3 of the SCYA4L locus in healthy individuals. Subjects were classified according to the sequence of acceptor splice site as follows: L1L1 (AG), L2L2 (GG) and L1L2 (double peak –A/G– in the first nucleotide). Of 220 healthy donors tested, 154 were L1L1 (70%), 59 L1L2 (26.8%) and 7 L2L2 (3.2%). If this polymorphism is considered to be a classical biallelic system, derived allelic frequency would be 83.4 and 16.6% for SCYA4L1 and SCYA4L2 alleles, respectively (Table I).


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  		Table I. Poblational distribution of SCYA4L polymorphisma

 
Due to the pivotal role played by CCL4 and its receptor CCR5 in HIV infection we decided to conduct an association study between SCYA4L polymorphism and HIV infection. In a group of 175 HIV+ individuals, 51.4% of them were L1L1 (vs 70% in the controls), 40% were L1L2 (vs 27%), and 8.6% were L2L2 (vs 3%) (p = 0.00039, {chi}2 = 15.71). Therefore, the frequency of allele L2 is clearly higher in the group of HIV+ patients (28.6%) compared with the control group (16.6%) (Table I) (p = 0.00016, {chi}2 = 14.24).

To evaluate the association of the SCYA4L polymorphism with other polymorphisms involved in the progression of HIV, we analyzed the incidence of CCR5{Delta}32 (39, 41, 42) and stromal cell-derived factor (SDF)-1 3'A (40) alleles in a random sample of 138 and 155 individuals from our HIV+ group with CCL4 genotype. (Tables II and III). We did not find any distribution suggesting positive or negative cross-association between the SCYA4L polymorphism and the CCR5{Delta}32 or SDF-1 3'A polymorphisms (Tables II and III). The lack of significant differences between the allelic frequencies of these polymorphisms in our HIV+ and previously reported groups (39, 40, 41, 42) allows us to consider the homogeneity of our group, thus avoiding any unexpected bias in distribution. The allelic frequencies reported in healthy populations (Caucasian and Asian populations) are 74–84% for the wild-type SDF and 16–26% for the SDF 3'A allele (40), and are 86–96% for the wild-type CCR5 and 4–14% for the CCR5{Delta}32 allele (41, 42). We did not find individuals homozygous for SDF 3'A allele due to their potential resistance to AIDS progression in addition to their low theoretical frequency. Similarly we did not find individuals homozygous for CCR5{Delta}32 allele due to their potential resistance to HIV infection in addition to their low theoretical frequency.


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  		Table II. Distribution of CCR5{Delta}32 polymorphisma

 

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  		Table III. Distribution of SDF 3' A polymorphisma

 
To explore the possibility that CCL4 forms may be associated with other diseases characterized by a maintained immune response, we evaluated the SCYA4L polymorphism in groups of patients with hepatitis C virus infection, type-1 diabetes, and autoimmune thyroid diseases. No significant differences were found between healthy donors and patients in the genomic and allelic frequencies of both SCYA4L locus variants, thus suggesting that the observed association with HIV infection is characteristic of this type of infection.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The CCL4 chemokine is encoded by two paralogous genes, SCYA4 and SCYA4L (23), highly related but with different exonic and intronic sequences. We found that these two genes not only express the variants already described (CCL4 and CCL4L), but also alternatively spliced variants lacking exon 2 (CCL4{Delta}2 and CCL4L{Delta}2). Moreover, we report a new polymorphism in the SCYA4L locus that forms nine new mRNAs. In an activated situation, the contribution of the SCYA4L locus to overall CCL4 expression is approximately 50% for the original allelic variant (SCYA4L1), and only 12% for the polymorphic allelic variant (SCYA4L2). The distribution of this polymorphism in HIV+ patients shows an association with the SCYA4L2 allele.

The structural analysis of the protein products of SCYA4 (CCL4) and SCYA4L (CCL4L) revealed the importance for the amino acid at position –4 relative to the fourth conserved cysteine. The amino acid at that position in CCL4 protein (Ser70) forms a hydrogen bond with amino acid Thr67 (located three residues upstream), thus conferring structural stability to the molecule (43). However, the amino acid at that position in the CCL4L protein (Gly70) could not form this hydrogen bond to stabilize the loop defined by the {beta}-turn between the second and third strand of the {beta}-sheet. This loop is believed to be essential for the binding of CCL4 to the glycosaminoglycans (GAGs) (44), and it has been suggested that the immobilization of chemokines by GAGs forms stable solid-phase chemokine foci and gradients necessary for directing leukocyte trafficking in vivo to increase their effective local concentration (thus increasing their binding to cell surface receptors), and potentially influence chemokine t1/2 in vivo (45, 46, 47). Hence, the destabilization of this loop make difficult CCL4L binding to GAGs and therefore modify their functional features in vivo.

We found that both SCYA4 and SCYA4L loci not only produce the expected CCL4 and CCL4L mRNA but also produce alternatively spliced mRNAs that lack the second exon, which give rise to the CCL4{Delta}2 and CCL4L{Delta}2 variants. These two short (29 aa) proteins only maintain the first two amino acids from the CCL4 and CCL4L proteins and lack three of the four cysteine residues critical for intramolecular disulfide bonding. Therefore, CCL4{Delta}2 and CCL4L{Delta}2 may not be structurally considered as chemokines, and despite the difficulty in predicting protein folding, these variants do not seem to be able to bind to CCR5 and thus may have no CCL4 activity. Although CCL3 is closely related to CCL4, we could not detect mRNAs from the two loci (SCYA3 and SCYA3L1) lacking exon 2 (data not shown).

The SCYA4L polymorphism described in this paper reveals that the SCYA4L locus has two allelic variants: the originally described variant (SCYA4L) named SCYAL1 and the new polymorphic variant SCYA4L2. The SCYA4L2 locus has a base substitution in the acceptor splice site of intron 2 that originates a new complex splicing pattern, including the use of six new acceptor splice sites. We notice that the score of alternative acceptor splice sites used in the SCYA4L2 allelic variant does not reflect the quantity of each mRNA generated, because the method used only takes into account the universal dinucleotide and their flanking sequences, although we know that other mechanisms may contribute to the efficiency of recognition of splice sites (48, 49, 50, 51).

CCL4L2 mRNA, the most abundant mRNA derived from SCYA4L2, is characterized by the loss of the first 15 bp in exon 3, being Phe65, Gln66, Thr67, Lys68, and Arg69 (position relative to the initial methionine) the five amino acids deleted in the predicted protein. Critical analysis of the conserved amino acids in CC chemokines show that Phe65, Thr67, and to a lesser degree Lys68, are highly conserved residues in this subfamily. In fact, Phe65 and Gln66 residues are the last two residues of the second strand of the CCL4 {beta}-sheet, and Thr67, Lys68 and Arg69 (next to Gly70, which is characteristic of CCL4L) form the {beta}-turn between the second and third strand. Thus, the deletion of these five amino acids would affect the whole monomer structure by disturbing the formation of the core of the molecule, the {beta}-sheet. It is known that CCL4, as well as CCL3 and CCL5, tends to self-associate and, thereby, form homodimers, tetramers, or high molecular mass aggregates in vitro, and possibly in vivo under certain conditions, in a process that involves residues Lys68 and Arg69 (52); furthermore, it has identified naturally occurring CCL4/CCL3 heterodimers at physiological concentrations (53). We predict that the deletion has a negative effect on the ability of CCL4L2 to form self-aggregates, or heterodimers with CCL3. Additionally, just as in the case of the single amino acid change between CCL4 and CCL4L proteins (Ser70 -> Gly70), we may expect that the GAG binding of CCL4L2 will be seriously affected, if not abrogated.

The folding prediction and the functional features of the other SCYA4L2-derived proteins (CCL4L2b, c, d, e, and f and CCL4L2{Delta}2, L2b{Delta}2) are difficult to establish. The biological relevance of these proteins is unknown and may be related to their low expression level.

Therefore, CCL4 is a highly variable chemokine since a minimum of 12 variants derive from their two codifying loci. These variants are potential targets for CD26/DPP IV and, similar to the originally described CCL4, may be secreted as naturally truncated forms lacking the two NH2-terminal amino acids (29, 30).

Our expression studies show that in activated CD8 T cells (at least in L1L1 individuals), the contribution of the SCYA4L locus (SCYA4L1 allelic variant) is very relevant and accounts for approximately half of CCL4 expression (although in L2L2 individuals the contribution of SCYA4L locus is lower, only 12%), in contrast with most previous descriptions, which did not consider the expression of the two different CCL4 loci and conferred the overall data to the role of the SCYA4 locus. Only recently (54), it was reported that while peripheral blood monocytes predominantly express the SCYA4 locus, peripheral blood B lymphocytes express both SCYA4 and SCYA4L loci in equivalent amounts; however, this study did not take into account the polymorphism of SCYA4L that strongly affects the overall expression of this chemokine.

Concerning the low levels of mRNA derived from SCYA4L2, we consider that although the slight differences found between the 5' and 3' non-coding regions of SCYA4L1 and SCYAL2 allelic variants (data not shown) are probably not responsible for this low expression, a reduced mRNA stability due to a low efficiency of spliceosomes may be a major determinant. This low spliceosome efficiency may be involved in a situation as complex as that found with the SCYA4L2 allele (a minimum of nine different mature mRNAs, eight of them produced by using alternative acceptor splice sites). In other examples, the use of cryptic splice sites was found to reduce the amount of mature mRNA generated (55).

The in vivo relevance of the complex situation produced by this SCYA4L polymorphism was assessed by the analysis of frequencies in disease. Interestingly, a significant difference was only observed in HIV+ patients. Since no definitive results were obtained in the study of other clinical relevant data (e.g., number of CD4 T cells, copies of virus...) and the preliminary experiments (e.g., in vitro analysis of different genotyping infection ability, blocking capability of each form in HIV-1 infection...) are still non-conclusive to suggest any additional pathogenic pathways (data not shown), more extensive studies are undoubtedly needed. Nevertheless, we may hypothesize that the lack of many canonical CCL4L mRNA copies transcribed in L1L2 and L2L2 individuals may reduce the ability of CD8 to protect HIV infection by chemokines, specifically CCL4L. This hypothesis agrees with recent data (56) suggesting a great influence of SCYA3L1 (CCL3L1) gene-containing segmental duplications on HIV-1/AIDS susceptibility. The strong homology between CCL3 and CCL4 and the genetic linkage between SCYA3L1 and SCYA4L genes and their copy number suggest equivalent mechanisms for both genes, remaining to elucidate which one is the gene mainly involved in this susceptibility.

Following the new classification for chemokine ligands established by Zlotnik and Yoshie (4), the formal gene symbols SCYA4 and SCYA4L are currently used to refer to CCL4 and CCL4L. To systematize the complex nomenclature derived from the high number of alternate designations, we suggest naming the two allelic variants of the SCYA4L gene as follows: SCYA4L1 and SCYA4L2 (i.e., as used in this article), or SCYA4L*1 and SCYA4L*2 if we strictly follow the guidelines for human gene nomenclature of HGNC (HUGO Gene Nomenclature Committee). The protein derived from SCYA4L1 is the one currently named CCL4L, but it may alternatively be known as CCL4L1 to combine it with CCL4L2 as the major protein derived from SCYA4L2. Subsequently, the other proteins derived from SCYA4L2 may be named CCL4L2b, c, d, e, and f, whereas the variants lacking exon 2 and derived from SCYA4 and SCYA4L loci may be named like the original protein but adding {Delta}2 (e.g., CCL4{Delta}2, CCL4L{Delta}2, CCL4L2{Delta}2...). To unify this nomenclature with the highly related one for CCL3 (SCYA3, SCYA3L1, and SCYA3L2 currently used to reference CCL3, CCL3L, and LD78{gamma} pseudogene), we also suggest following previous CCL4 nomenclature and the guidelines of HGNC and change the gene symbol for CCL3L to SCYA3L and LD78{gamma} to SCYA3LP, respectively, since LD78{gamma} is a 5'-truncated pseudogene derived from the SCYA3L locus (20).

Data from our study suggest that variants of chemokines, such as CCL4, may be a major feature that determines the final role of these key molecules in immune response, increasing the functional opportunities of these molecules involved in many diseases.


   Acknowledgments
 
We thank Dr. Maria del Pilar Armengol for help in statistical analysis, Dr. Raúl Castaño for critical reading of the manuscript, Rosa Faner, Marco Fernández, and Pepi Caro for general support in the laboratory; we also thank Drs. Jordi Yagüe and Xavier Forns for kindly providing DNA from healthy donors and samples from hepatitis C virus-positive patients, respectively.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Fondo de Investigaciones Sanitarias (project FIS 99/1063 and PI 02/0104), the Ministerio de Educación y Ciencia (project BFI 2003–00405), and the Instituto de Salud Carlos III (RC03/03). Back

2 Address correspondence and reprint requests to Dr. Manel Juan, Laboratory of Immunobiology for Research and Application to Diagnosis, Immunology Unit, Hospital Universitari Germans Trias i Pujol, Ctra. de Canyet s/n, 08916 Barcelona, Spain. E-mail address: mjuan{at}ns.hugtip.scs.es Back

3 Abbreviations used in this paper: DPP, dipeptidyl-peptidase; MIP, macrophage inflammatory protein; GAG, glycosaminoglycan; SDF, stromal cell-derived factor. Back

Received for publication October 13, 2004. Accepted for publication February 18, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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