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(Journal of Leukocyte Biology. 2001;70:357-366.)
© 2001 by Society for Leukocyte Biology

Hemofiltrate CC chemokines with unique biochemical properties: HCC-1/CCL14a and HCC-2/CCL15

Ulf Forssmann, Hans-Jürgen Mägert, Knut Adermann, Sylvia E. Escher and Wolf-Georg Forssmann

IPF PharmaCeuticals GmbH, Institute of the Medical School of Hanover, Section of Pharmacology, D-30625 Hanover, Germany

Correspondence: Wolf-Georg Forssmann, M.D., Ph.D., Feodor-Lynen-Strasse 31, D-30625 Hanover, Germany. E-mail: wgforssmann{at}gmx.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MOLECULAR FORMS AND STRUCTURES
 ORGANIZATION AND EXPRESSION...
 BIOLOGICAL PROPERTIES
 PATHOPHYSIOLOGICAL ROLES OF...
 CONCLUSIONS
 REFERENCES
 
The hemofiltrate CC chemokines CCL14a (formerly HCC-1), CCL14b (formerly HCC-3), and CCL15 (formerly HCC-2) are encoded by mono- as well as bicistronic transcripts from a tandem gene arrangement on human chromosome 17q11.2. The transcription and splicing into several mono- and bicistronic transcripts of this gene complex are unique for human genes. No corresponding mechanism is known in nonprimate mammalian species such as mice and rats. The extremely high concentration of CCL14a in human plasma is exceptional for chemokines and led to the identification of this chemokine. Several molecular forms of CCL14a have been isolated and investigated. The mature propeptide CCL14a(1–74) is a low-affinity agonist of CCR1 which is converted to a high-affinity agonist of CCR1 and CCR5 on proteolytic processing by serine proteases. In contrast, CCL15 is characterized using molecular forms deduced from the mRNA/cDNA and shown to activate cells via CCR1 and CCR3, also dependent on the amino-terminal length. Hemofiltrate CC chemokines are chemoattractants for different types of leukocytes including monocytes, eosinophils, T cells, dendritic cells, and neutrophils. In this review, we emphasize the genomic organization, expression patterns, and biochemical properties of CCL14a, CCL14b, and CCL15. We report results of significance for the development of therapeutic strategies, especially concerning HIV infection and inflammatory diseases.

Key Words: chemokine receptor • bicistronic gene • chemotaxis • HIV


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MOLECULAR FORMS AND STRUCTURES
 ORGANIZATION AND EXPRESSION...
 BIOLOGICAL PROPERTIES
 PATHOPHYSIOLOGICAL ROLES OF...
 CONCLUSIONS
 REFERENCES
 
Chemokines are small chemotactic peptides that represent the largest subfamily of cytokines. According to the arrangement of the amino-terminal first two cysteine residues of the molecule, which can be adjacent or separated by one amino acid residue, chemokines are subdivided into CX/C{alpha} and CC(ß) chemokines. These cysteine residues, together with two or four additionally occurring conserved cysteine residues, form disulfide bonds. As yet, two exceptions to these structures are known: fractalkine, a CX3C chemokine, and lymphotactin, a C chemokine containing only one disulfide bond. Chemokines are small peptides with a molecular mass between 7 and 13 kDa and sequence identities ranging from 20 to 90%. Up to now, >40 human chemokines have been described. They play a major role in the recruitment, navigation, and activation of different leukocytes. With respect to pathophysiology, the regulation of myelopoiesis, antiviral effects, and angiogenetic potential, chemokines exhibit important functions. They display their biological activity via activation of seven-transmembrane-domain G-protein-coupled receptors. To date, 19 of these receptors interacting with chemokines as specific ligands have been discovered: 11 CC, 6 CXC, 1 C, and 1 CX3C chemokine receptors. The majority of these receptors interact with more than one chemokine. Chemokine biology and structure-function relations have been reviewed comprehensively [1 2 3 4 5 6 ].

Various approaches have led to the identification and isolation of chemokines: database mining [7 , 8 ], isolation from tissue of diseased individuals [9 ], or isolation from supernatants of cell cultures [10 ]. Attempts to isolate chemokines from blood plasma have not been successful, because chemokines usually occur in extremely low concentrations. Thus, the discovery of the chemokine CCL14a represents an exception. Due to its extremely high plasma levels, CCL14a was first detected in human hemofiltrate during the systematic extraction and isolation of peptides [11 ]. Based on its origin and structure, CCL14a was initially designated as hemofiltrate CC chemokine 1 (HCC-1) [12 ]. According to the new chemokine terminology, HCC-1 and HCC-3 are now termed CCL14 [3 ], and because they represent products of two splice variants, we refer to CCL14a and CCL14b, respectively.

At the genomic level, another chemokine, formerly termed HCC-2 (new CCL15), was discovered by analyzing the upstream DNA sequence of the CCL14 gene [13 ]. In the course of our molecular biological investigations, surprisingly, a bicistronic mRNA encoding both CCL15 (upstream) and CCL14a (downstream) was identified and later verified by RT-PCR experiments. Bi- or polycistronic mRNAs are usually generated by bacterial and some viral microbes, and their occurrence is rare in higher animal species. Thus, this bicistronic CCL14a- and CCL15-specific transcript represents a novel mechanism of gene expression in mammals. Further characterization of the CCL14-CCL15 gene complex and its transcripts led to the identification of CCL14 gene-specific mRNA species exhibiting an insertion of 48 bp, which corresponds to an additional exon of the CCL14 gene. This insertion was detected within the monocistronic as well as the bicistronic transcripts of the CCL14 gene, leading to at least five different mRNAs generated by the CCL14-CCL15 gene complex. The product of this splice variant was initially termed HCC-3 (now CCL14b). Here, we review the biochemical and genomic features of chemokines CCL14a and CCL15 with particular reference to the different molecular forms investigated, and we discuss possible roles in pathophysiology.


   MOLECULAR FORMS AND STRUCTURES
TOP
 ABSTRACT
 INTRODUCTION
 MOLECULAR FORMS AND STRUCTURES
ORGANIZATION AND EXPRESSION...
 BIOLOGICAL PROPERTIES
 PATHOPHYSIOLOGICAL ROLES OF...
 CONCLUSIONS
 REFERENCES
 
CCL14a was first isolated from human hemofiltrate during a systematic search for novel bioactive factors [11 ], as an 8.7-kDa peptide of 74 amino acids (Fig. 1 a) [12 ]. The occurrence of this peptide is in agreement with a predicted secretory signal peptide contained in the CCL14a precursor [14 ]. CCL14a(1–74) can thus be considered as the secreted mature propeptide of CCL14a. It occurs in high concentrations (1.6–10 nM) in plasma of healthy subjects and in patients with chronic renal failure (2.5–80 nM) [12 ].



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  		Figure 1. (a) Amino acid sequence alignment of CCL14a, its splice variant CCL14b, CCL15, and related CC chemokines. The primary structures of predicted proproteins are shown. Cysteine residues are marked by gray boxes. (b) A phylogenetic tree was generated using the AlignX program of the Vector NT suite 6 (InforMax, Bethesda, MD) and applying the blosum62mt2 score matrix with Clustal W. All known human CC chemokines were considered.

 
Additional studies revealed other forms of circulating CCL14a (Table 1 ). Glycosylated CCL14a(1–74), with an O-glycosylation at Ser-7, has been detected in plasma by Western blot analysis, isolated, and characterized [15 ]. The following structure of the glycosyl chain moiety has been recently proposed: {alpha}-D-neu5-ac-(2->3)-ß-D-gal-(1->3)-{{alpha}-D-gal N ac[{alpha}-D-neu5 ac-(2->6)]-(1->Ser)}. This tetrasaccharide is abundant among glycoproteins and also occurs bound to other cytokines such as interleukin (IL)-2 [16 ]. However, little is known about the functional importance of the glycosylation of CCL14a. Two amino terminally truncated forms, CCL14a(3–74) and CCL14a(4–74), were isolated from chromatographic fractions of a human hemofiltrate peptide library [15 ]. These truncated forms circulate in blood plasma in low concentrations. An analysis of CCL14a peptide concentrations based on the yield by purification suggests that only ~3% of total CCL14a is represented by CCL14a(3–74) and 1% by CCL14a(4–74). Tsou et al. investigated other forms of CCL14a based on the score obtained from the prediction of signal sequence cleavage [17 ]. The chemokines CCL14a(4–74) and CCL14a(6–74) were considered to be further potential cleavage products. These peptides were chemically synthesized, and their receptor interaction was studied in detail.


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  		Table 1. Overview of Molecular Forms of CCL14a, CCL14b, and CCL15

 
Recently, another CCL14a variant lacking the first eight amino acids was isolated using a functional assay with a cell line overexpressing CCR5 [18 ]. CCL14a(9–74) was biochemically characterized and, in contrast to other CCL14a variants described above, is a highly potent ligand of the chemokine receptors CCR1 and CCR5. It occurs at a 100- to 1,000-fold-lower concentration in human hemofiltrate than CCL14a(1–74), when estimated from relative recovery during isolation.

We observed that alternative splicing of the CCL14 gene primary transcript can occur, resulting in the CC chemokine named CCL14b. The insertion of 48 nucleotides in the CCL14a open reading frame (ORF) results in a variant exhibiting 16 additional amino acid residues within the amino-terminal region of the sequence introduced at position 9 of mature CCL14a (A. Pardigol, unpublished results) (GenBank no. Z70293). In this variant, the amino acid residue Gln is substituted for Arg-8, due to the exon/intron border in the corresponding codon.

CCL14a exhibits considerable sequence homology when compared with CCL3 (46%), CCL15 (39%), and CCL16 (38%). The latter chemokine has been designated HCC-4, although it was not isolated from hemofiltrate [19 20 21 ]. An alignment of the amino acid sequences of CCL14a and CCL15 with other closely related CC chemokines and a phylogenetic analysis of the human CC chemokine family are depicted in Figure 1 .

Up to now, using nuclear magnetic resonance (NMR)-spectroscopic analysis with different CCL14a variants, no structural data could be obtained. This can be attributed to the oligomerization tendency of this protein. Only cursory data regarding the main secondary structural elements are known from X-ray crystallography using synthetic CCL14a(1–74), in which the protein was found to be octameric (D. Alexeev, R. Ramage, unpublished results).

Because the naturally occurring CCL15 peptides have not yet been isolated, the amino acid sequence has been deduced either from the cDNA of an expressed sequence tag (EST) clone [22 ], by molecular cloning [23 , 24 ], or from the bicistronic CCL14a-CCL15 mRNA [13 ]. CCL15 exhibits the typical CC motif but contains two additional cysteine residues, in contrast to most other CC chemokines (Fig. 1a) . The signal peptide prediction suggests cleavage of a 21-residue secretory leader sequence resulting in a pro-CCL15 molecule of 92 amino-acid residues. Ongoing work in our laboratory shows that several circulating forms of CCL15 exist [25 ]. According to the results obtained so far, a similar proteolytic processing to that described for CCL14a is likely.

Up to now, three molecular forms of CCL15 with different amino-terminal lengths (1–92, 25–92, and 27–92) have been studied to characterize the biological activity of this chemokine (Table 1) . CCL15 was expressed in Pichia pastoris [13 ], in which the predominantly secreted form corresponds to CCL15(27–92), a protein containing 66 amino acid residues and having a molecular mass of 7.2 kDa. An elongated CCL15(25–92) derivative having a length of 68 amino acid residues was chemically synthesized [22 ]. This larger CCL15(25–92) was assumed based on the corresponding amino-terminal length of other chemokines isolated. CCL15(1–92) was expressed in Escherichia coli and HEK-293 cells, having a molecular mass of ~12 kDa [23 , 24 ]. It is interesting that in this communication, the use of an insect cell system for the recombinant expression resulted also in the partial formation of CCL15(25–92). It was reported that CCL15(25–92) exhibits significantly less activity on calcium flux assays than CCL15(1–92) [23 ]. Because the amino terminus of natural CCL15 has not yet been determined and the role of the length of the amino-terminal region is critical for the activity of chemokines, the isolation of the natural CCL15 protein is most important. Minor amino-terminal truncation or elongation of chemokines frequently results in a significant enhancement or loss of activity, the appearance of antagonistic activity, or a change in chemokine receptor specificity [26 ].

CCL15 exhibits high sequence identity with CCL23 [68% (Fig. 1a) ] [27 , 28 ]. Similarly to CCL14a and CCL15, several variants have been described for CCL23, namely the truncated forms CCL23(24–99) and (25–99) [29 ] and the product of a splice variant with 116 amino acid residues [30 ]. It is interesting that both CCL15 and CCL23 contain a third disulfide bond formed by two additional conserved cysteines: one between Cys-2 and Cys-3 of the classical chemokine cysteine pattern and the other exocyclic to the carboxy-terminal cysteine (Fig. 1a) . Similar murine CC chemokines with two additional cysteines have been described. Other human CC chemokines with two additional cysteine residues are CCL1 [31 ], showing a structural array corresponding to CCL15 and CCL23, and CCL21, which, in contrast, contains the additional disulfide bridge at the carboxy terminus [32 33 34 ]. As murine orthologues of CCL15 and CCL23, the chemokines CCL6 [35 , 36 ] and CCL9,10 [37 38 39 ] have been identified. These chemokines exhibit a rather long amino terminus preceding the typical CC motif. Murine CCL9,10 also resembles CCL14a because it circulates in healthy mice at concentrations of ~90 nM.

To investigate a possible functional importance of the third disulfide bond of CCL15, the three-dimensional structures of the biologically active chemokine and related molecules were examined by homonuclear two-dimensional NMR and circular-dichroism (CD) spectroscopy. For this study, CCL15(27–92) and a chemically synthesized mutant of the same length, in which alanine residues substitute for the two additional cysteine residues, have been used [40 ]. Both proteins contain identical secondary structural elements also present in other CC chemokines such as CCL5, i.e., a triple-stranded, antiparallel ß-sheet covered by a carboxy-terminal {alpha}-helix (Fig. 2 ). Far-UV CD spectra confirmed the type and extent of secondary structural elements for both CCL15 derivatives [41 ]. The structure of CCL15(27–92) is well defined, exhibiting average root-mean-square deviations of 0.58 and 0.96 Å for the backbone heavy atoms and all heavy atoms of residues 31–90, respectively. In addition to the disulfide bonds, the tertiary fold of the molecule is mainly stabilized by a hydrophobic core formed by residues from the ß-sheet and the carboxy-terminal {alpha}-helix. A particular property of CCL15(27–92) is that it is monomeric even at millimolar concentrations. This allows a detailed determination of structure without quaternary interactions, and consequently it is accepted that CCL15 binds to its receptors as a monomer. In contrast to other chemokines, it appears that chemokines containing six cysteines generally tend to form monomers in solution [42 , 43 ].



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  		Figure 2. Schematic ribbon drawing of the NMR structure of CCL15(27–92) showing the elements of regular secondary structure. The view on the left was obtained by rotating the righthand view by 90° around the horizontal axis. Cysteine residues and disulfide bonds are highlighted in yellow [40]._art>

 
A comparison of CCL15(27–92) with the related dimeric chemokines CCL4 and CCL5 demonstrates that their secondary structural elements and spatial arrangements are highly comparable [40 ]. The absence of the third disulfide bond of CCL15(27–92) does not lead to major structural changes, e.g., in the orientation of the {alpha}-helix to the ß-sheet. In CCL15 mutants lacking the third disulfide bond, only slight changes of the side-chain orientation of Ile-15, most likely leading to a destabilization of the loop formed by amino acids 15–20, have been observed. It has been proposed that the third disulfide bond compensates for the absence of Trp-53, which is responsible for stabilization of this loop in other chemokines [44 ]. As in CCL15, the third disulfide bond of CCL23 also has no significant influence on the tertiary structure [43 ]. In contrast, the additional disulfide bond of CCL1 results in significant structural changes, such as the disruption of the carboxy-terminal helix and the formation of a short ß-strand expanding perpendicularly to the plane of the three-stranded ß-sheet [42 ].


   ORGANIZATION AND EXPRESSION PATTERNS OF THE GENES
TOP
 ABSTRACT
 INTRODUCTION
 MOLECULAR FORMS AND STRUCTURES
 ORGANIZATION AND EXPRESSION...
BIOLOGICAL PROPERTIES
 PATHOPHYSIOLOGICAL ROLES OF...
 CONCLUSIONS
 REFERENCES
 
Chromosomal location and organization of the genes encoding CCL14a and CCL15
It is well established that the genes encoding most of the currently known human CC chemokines are clustered on chromosome 17q11–q21 [1 ]. A more detailed analysis of this region has revealed that at least 14 CC chemokine genes are located at 17q11.2 in two groups, which can be distinguished according to their sequence similarities [45 ]. One group, the monocyte chemoattractant protein (MCP)-like group, includes the genes for CCL2, CCL7, CCL13, and CCL1. The second, the RANTES/macrophage-inflammatory protein (MIP)-like group located in closer proximity to the telomere, includes the genes for CCL3, CCL4, CCL5, CCL14, CCL15, and CCL16. The gene encoding CCL23 also maps to the position of the second group close to CCL15 [30 ]. Comprehensive genomic investigations performed by our group have revealed that the genes encoding CCL14 and CCL15 reside in a head-to-tail orientation with the CCL15 gene located upstream of the CCL14 gene [13 ]. A recent analysis shows that CCL14, CCL15, CCL16, and CCL23 are clustered in an identical orientation within a region spanning ~40 kbp [46 ]. Sequence comparisons between the CCL15 and CCL23 genes imply that one of them may have been generated by a relatively recent gene duplication effect. The two genes are more closely related to one another than those for the murine CC chemokines CCL6 and CCL9,10. These proteins show high sequence identity with CCL15 and CCL23, including the six cysteine residues. However, murine counterparts of CCL15 and CCL23 have not been determined unambiguously. A murine orthologue of CCL14 has not been identified, whereas the murine CCL16 is encoded by a pseudogene [47 ].

Human CC chemokines like CCL14a exhibit a typical organization of their genes. The first exon encodes the signal peptide and the first amino acids of the propeptide. The second and third exons include the codons for the four conserved cysteines forming the characteristic disulfide bonds. In contrast, the CCL15 gene contains an additional exon in position two, which in a similar way has been described for the genes encoding CCL6 and CCL23 [13 , 30 , 36 ], both chemokines having an identical arrangement of six cysteines. In these cases, the regions of the proteins that contain the disulfide bond-forming cysteines are encoded by exons 3 and 4. From the information presently available, it is not clear whether the second exon of the CCL15 gene encodes part of the secretory signal peptide [22 ] or exclusively parts of the propeptide [30 ]. Several CXC chemokine genes also contain a fourth exon, encoding a small part of the carboxy-terminal region [1 ].

Within the promoter region of the CCL14 gene, putative binding sites for the transcription factors Myc-Max, E47, and activator protein (AP)-2 have been determined, whereas the CCL15 gene promoter contains putative binding sites for interferon regulatory factor 2, ets-like protein 1, and YY-1 [13 ]. The promoter of the closely related CCL23 gene was reported to be very similar to that of the CCL15 gene [46 ]. However, sequence analysis of both promoters revealed the occurrence of several potential binding sites for factors involved in inflammatory processes and regulation of the immune system (Fig. 3 ), including nuclear factor (NF)-{kappa}B, Ikaros factors, NF of activated T-cells (NFAT), synergistic NFATp/AP-1 sites, AP-1 fos/jun sites, and a signal transducer and activator of transcription site (within the CCL15 gene promoter). Although the functionality of these regions still must be verified experimentally, their occurrence is compatible with the biological roles of CCL14a and CCL15 in the context of the immune system and inflammation.



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  		Figure 3. Scheme of the promoter regions of the genes encoding CCL14 and CCL15. Positions of potential regulatory elements as determined by the MatInspector program (http://genomatix.gsf.de/free_services/) are shown. It is in accordance with chemokine function that both promoters contain a large number of potential binding sites for transcription factors related to the immune system and inflammation such as NF-{kappa}B, Ikaros factors, nuclear factor of activated T-cells (NFAT), synergistic NFATp/AP-1 sites, AP-1 fos/jun, and STAT.

 
Expression of the genes encoding CCL14a and CCL15 in human tissues and cell lines
The entire CCL14a cDNA (GenBank no. Z49270) has been cloned from a human bone marrow cDNA library using a specific partial cDNA fragment generated by RT-PCR as a hybridization probe. It contains a 279-bp ORF exactly encoding the isolated CCL14a(1–74) plus an amino-terminal extension of 19 residues showing the characteristics of a secretory signal peptide [12 ]. Database searches for related ESTs led to the discovery of a 5' terminally elongated CCL14a-specific cDNA (GenBank no. R99672). This EST differs from the cloned CCL14a cDNA, because it contains 150 additional nucleotides but lacks the first 16 nucleotides. Sequence comparison of this extension surprisingly reveals that it represents a part of the CCL15-specific cDNA [13 ]. The nucleotide sequence of the EST clone R99672 together with the arrangement and nucleotide sequences of the genes encoding CCL14a and CCL15 indicated the existence of a bicistronic CCL15-CCL14a gene transcript. Successful amplification of full-length cDNA from adult liver using primers specific for CCL15 (sense primer) and CCL14a (antisense) finally confirmed the existence of the bicistronic mRNA. The entire 925-bp fragment (EMBL no. Z70292) includes the CCL15 and CCL14a ORFs and a spacer sequence [13 ], representing a potential internal ribosomal entry (IRE) site. Further RT-PCR analysis led to the detection of a CCL14a-related cDNA containing an insertion of 48 bp. These additional nucleotides correspond to an additional exon positioned within intron 1 of the CCL14 gene. Based on the designations HCC-1 (now CCL14a) and HCC-2 (now CCL15), we termed the translation product HCC-3 (now CCL14b) (Pardigol, unpublished results). As demonstrated by RT-PCR, the extended splicing variant not only occurs in the monocistronic but also in the bicistronic mRNA (GenBank no. Z70293), which results in a total of five different transcripts of the CCL14-CCL15 gene complex (Fig. 4 ). To verify the putative IRE site, in vitro translation experiments or alternatively the construction and analysis of dual reporter gene constructs still remains to be performed. The existence of monocistronic CCL15 gene transcripts was demonstrated by RT-PCR analysis with cDNA first strand from HUH-7 cells (Pardigol, unpublished results). Further confirmation of these results has been provided by two EST clones (GenBank no.s R16807 and H62013), representing the monocistronic CCL15 mRNA. In addition, monocistronic CCL14a and CCL15 mRNA as well as the bicistronic CCL14a/15 gene transcripts were detected by Northern blot hybridization [13 ].



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  		Figure 4. Scheme of the CCL14/15 gene complex on human chromosome 17q11.2 and its transcripts. The promoters (P) of the genes are indicated by arrows. Corresponding regions of exons and mRNAs are hatched (untranslated regions) and shaded, respectively, in an identical way. Five different transcripts are generated by the CCL14-CCL15 gene complex. Regular monocistronic as well as bicistronic mRNAs including a splice variant of the CCL14 gene primary transcript encode three different CC chemokines designated as CCL14a, CCL15, and CCL14b. Exons (E) {delta}' and E1' are exclusively represented in the monocistronic transcripts. The mono- and bicistronic CCL15 gene transcripts were detected in liver and the intestine only, whereas monocistronic transcripts specific for CCL14a and CCL14b were detected in nearly all human tissues.

 
The tissue-specific distributions of monocistronic CCL14a and CCL15 mRNAs exhibit remarkable differences. The CCL14 gene is constitutively expressed in various tissues such as spleen, heart, and skeletal muscle, lung, liver, gut, thymus, prostate, ovary, testis, and pancreas [12 , 13 ]. As determined by RT-PCR analysis, mRNA of the splice variant CCL14b is located in all tissues expressing the CCL14 gene except skeletal muscle and pancreas. In contrast, CCL15 expression occurs only in liver, gut, and lung leukocytes [13 , 22 ]. Also, the occurrence of the bicistronic transcripts is limited to these tissues. However, another group additionally detected high levels of CCL15 gene expression in heart and skeletal muscle and also in the adrenal gland [30 ].

Because the gene encoding the murine CC chemokine CCL9,10 is expressed constitutively in a wide variety of tissues, its expression is comparable to that of the human CCL14 gene. The level of expression in kidney tissue is very low, like that for CCL14a, the mRNA of which is virtually not detectable in this organ. No expression of either of these genes occurs in the brain. Similar expression patterns were found for the genes encoding human CCL15 and the structurally related chemokine CCL23. Specific transcripts are distinctly detectable in liver, colon, and small intestine but not in other human tissues examined [13 , 28 ].


   BIOLOGICAL PROPERTIES
TOP
 ABSTRACT
 INTRODUCTION
 MOLECULAR FORMS AND STRUCTURES
 ORGANIZATION AND EXPRESSION...
 BIOLOGICAL PROPERTIES
PATHOPHYSIOLOGICAL ROLES OF...
 CONCLUSIONS
 REFERENCES
 
CCL14a
The biological activity of naturally occurring CCL14a(1–74), mobilization of intracellular calcium, and chemotaxis require higher concentrations compared with other chemokines such as CCL3 or CCL5, which act in the nanomolar range on peripheral blood leukocytes. CCL14a(1–74) induces the mobilization of intracellular calcium at doses of 100 nM or higher and causes a slight but significant release of lysosomal N-acetyl-ß-D-glucosaminidase at 1,000 nM [12 ]. Receptor desensitization studies in monocytes to assess the receptor usage of CCL14a(1–74) showed that it shares receptors with CCL3 and CCL5, indicating that CCL14a(1–74) acts on CCR1. The dose needed to desensitize the response to 30 nM CCL5 is also in the lower micromolar range (1,000 nM), suggesting a much lower affinity of CCL14a(1–74) for the shared receptors. Experiments with glycosylated CCL14a(1–74) did not result in significant differences in calcium mobilization [15 ]. In agreement with these results, CCR1 has been identified as a specific receptor for CCL14a(1–74) using stably transfected cells [17 ]. Cells expressing CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, or CXCR1 failed to respond to CCL14a(1–74). Three variants of CCL14a (1–74, 4–74, and 6–74) with potential signal cleavage sites were compared for their potential to inhibit the generation of cyclic AMP. Stimulation of CCR1-transfected cells showed that the shortest form tested (6–74) is the most potent, whereas the longest form of CCL14a, which circulates in high levels in human plasma, is the least potent. CCL14a(1–74) and (6–74) are able to induce migration of CCR1-transfectants, whereas the shorter form is also more potent.

Recently, the screening of human hemofiltrate fractions for novel natural ligands of CCR5 led to the identification of another CCL14a variant lacking the first eight amino acids at the amino terminus [18 ]. This variant, CCL14a(9–74), is a potent agonist inducing the release of intracellular calcium through activation of CCR1 (50% effective concentration (EC50), 2.8 nM; CCL5, 6.3 nM) and CCR5 (EC50, 4.8 nM; CCL5, 2.4 nM), and a reasonably potent agonist of CCR3 (EC50, 78 nM; CCL11, 2.4 nM). It is inactive on CCR2, CCR4, CCR6, CC7, CCR8, CCR9, CXCR1, and CX3CR1. These properties were confirmed by competition binding assays on CCR1 (Ki, 0.023 nM), CCR3 (Ki, 2.7 nM), and CCR5 (Ki, 0.04 nM). In contrast, full-length CCL14a is only a weak agonist of CCR1 (Ki, >100 nM) and inactive on CCR5. However, CCL14a(1–74) competes partially for CCL11 binding to CCR3, despite the absence of a functional response of this receptor. Moreover, CCL14a(9–74) is an efficient and potent chemoattractant (peak activity ~10 nM) for monocytes, eosinophils, and T lymphoblasts, whereas CCL14a(1–74) acts only in the micromolar range on monocytes. Low doses of CCL14a(9–74) completely abrogate the response of monocytes to CCL5, of eosinophils to CCL3 but not to CCL11, and of T lymphoblasts to CCL4, indicating that CCR1 and CCR5 are the principal receptors of CCL14a(9–74). These data clearly show that truncation of the molecule at the amino terminus dramatically increases its potency and that CCL14a(1–74) can be considered as the secreted proprotein of CCL14a, which is activated by specific proteolytic processing.

CCL15
In contrast to CCL14a, naturally occurring forms of CCL15 have not yet been described. Different forms of CCL15 have been characterized biologically by several groups (Table 1) with some conflicting data [13 , 22 23 24 ]. CCL15(27–92) as an efficient chemoattractant for monocytes is also able to attract eosinophils with high potency corresponding to that of CCL3 but with a moderate efficacy compared with CCL11 [13 ]. Lymphocytes cultured in the presence of IL-2 are only weakly attracted by CCL15(27–92), and neutrophils do not migrate on stimulation by CCL15(27–92), although mobilization of intracellular calcium can be detected. Similar observations on neutrophils have been made for CCL3 [48 ]. CCL15(25–92) shows a marked chemotactic activity on freshly isolated monocytes, dendritic cells, and T lymphocytes but a moderate chemotactic effect on eosinophils, and it fails to mobilize polymorphonuclear leukocytes (PMNs) [22 , 49 ]. The activity of the potential proprotein CCL15(1–92) has been studied by two groups [23 , 24 ]. Both have shown that CCL15(1–92) induces chemotaxis of freshly isolated monocytes and T lymphocytes, similar to the truncated forms CCL15(25–92) and (27–92). It has also been reported that CCL15(1–92) induces migration of PMNs with the same efficacy and potency as CXCL8, resulting in a maximum stimulus for these cells [23 ].

The receptor usage by CCL15(27–92) has been studied in cross-desensitization experiments. On eosinophils, CCL15(27–92) desensitizes CCR1 after CCL3 stimulation but not CCR3, because CCL11 still induces cytosolic calcium changes. Stimulation of neutrophils, monocytes, and lymphocytes with CCL15(27–92) also abolishes the responsiveness to CCL3, indicating that CCL15(27–92) acts mainly via CCR1 on peripheral blood leukocytes [13 ], because CCR5, the other CCL3-receptor, is not expressed in PMNs and barely detectable on freshly isolated monocytes. CCL15(25–92) in contrast has been shown to induce calcium fluxes on CCR1- (EC50, 13 nM) and CCR3-transfected cell lines (EC50, 1 nM), although CCR3-transfected cells respond only very weakly to CCL11. Full cross-desensitization of CCL15(1–92) with CCL3 has been observed for CCR1 transfectants, and partial cross-desensitization with CCL11 has been observed for CCR3 transfectants [23 ]. Another group found that CCL15(1–92) induces calcium fluxes in CCR1 transfectants but not in CCR2-, CCR3-, CCR4-, or CCR5-transfected cells [24 ]. In addition, CCL15(1–92) mobilizes calcium in murine PMNs [50 ]. The same cells from CCR1-/- mice do not respond to CCL15(1–92), indicating that calcium mobilization depends on CCR1. In agreement, injection of CCL15(1–92) into mouse peritoneum leads to infiltration of murine neutrophils, monocytes, and lymphocytes. Only binding of CCL15(25–92) has been assessed on stably transfected Chinese hamster ovary cells, showing that it displaces CCL3 with a 50% inhibitory concentration (IC50) of 12 nM on CCR1 and displaces CCL7 with an IC50 of 2.5 nM on CCR3, whereas it does not bind to CCR5 [22 ].

Because CCL15 contains a third disulfide bond [13 ], the influence of this additional bond on biological activity has been investigated. CCL15(27–92) has been compared with a mutant in which the third and the sixth cysteines are both replaced by alanine residues [40 ]. In agreement with the three-dimensional structure in which replacement of the residues resulted in minimal changes, the biological activities of both forms are indistinguishable (S. E. Escher, unpublished results).

The present results suggest that, on natural cells, CCL15 displays its effects mainly via CCR1. The different forms of CCL15 exert effects similar to those of CCL3 via CCR1 but less prominent effects on CCR3 in contrast to CCL11. The differences reported for the effects on neutrophils might depend on isolation procedures or on the state of activation. For example, an up-regulation of CCR1 and CCR3 can be found when PMNs are stimulated with interferon {gamma}. This up-regulation also results in the ability of stimulated PMNs to migrate in a dose-dependent manner to CCL3, CCL11, and CCL15, whereas unstimulated PMNs are unresponsive [51 ].


   PATHOPHYSIOLOGICAL ROLES OF CCL14a AND CCL15
TOP
 ABSTRACT
 INTRODUCTION
 MOLECULAR FORMS AND STRUCTURES
 ORGANIZATION AND EXPRESSION...
 BIOLOGICAL PROPERTIES
 PATHOPHYSIOLOGICAL ROLES OF...
CONCLUSIONS
 REFERENCES
 
Both CCL14a and CCL15 have been shown to modulate the proliferation of stem cells and myeloid progenitors. The first chemokine shown to exhibit such properties was CCL3. It was reported to enhance the proliferation of more mature myeloid progenitors, although it inhibits the growth of more immature forms and stem cells [6 , 52 ]. However, the role of CCL3 in blood cell development was not evident in mice deficient for CCL3 [53 ]. CCL3 was also reported to enhance the proliferation of hematopoietic stem cells [54 ]. Later on, CCL14a(1–74) was shown to enhance the proliferation of CD34+ bone marrow cells in the presence of stem cell factor [12 ]. It is less potent than CCL3 (1,000 vs. 10 ng/mL) but has similar efficacy. Corresponding effects have been observed for very early progenitor/stem cells (CD34+/CD38- bone marrow cells). In agreement with this, CCL14a(1–74) does not inhibit colony formation of immature myeloid progenitor cells [6 ]. In contrast, CCL15(1–92) like CCL23 has a suppressive effect on human immature myeloid progenitor cells, such as GM-CFU and CFU-granulocyte-erythrocyte-monocyte-macrophage [23 ]. The ability to inhibit stem cell proliferation implies a therapeutic potential for decreasing the toxicity of high-dose chemotherapy, although this has been shown in animal studies but not as yet in clinical trials [6 ]. Therefore, it is possible that CCL15 or CCL15 agonists may prove useful in myeloprotection. The attraction of stem cells or progenitor cells, as reported for CXCL12 [55 ], might play an important role in the homing of stem cells and thus in their use in transplantation. To our knowledge, no reports regarding chemotactic properties of CCL14a and CCL15 on stem cells or progenitor cells have been published.

The most significant findings on CCL14a are related to the identification of the naturally processed form of CCL14a(9–74), which has anti-HIV properties [18 ]. The novel profile of receptor specificity [CCR1, CCR3, and CCR5, rather than CCR1 only (compare also Table 1 )] gives rise to this interesting aspect. It is well established that CXCR4 and CCR5 are the major coreceptors for lymphocyte-tropic and macrophage-tropic strains of HIV [56 ]. Given this role for CCR5, the antiviral activity of CCL14a(9–74) has been investigated in HIV infection assays. CCL14a(9–74) inhibits the infection of the macrophage-tropic strains YU2 and JR-CSF with similar efficiency, whereas infection of the T-tropic strain JR-CSF is not inhibited. Infection of human PBMCs is also inhibited by CCL14a(9–74). Thus, structural modification of the amino terminus of this molecule, as performed with CCL5, may generate derivatives with comparable anti-HIV properties [57 ]. Nevertheless, chemokines and derivatives have considerable limitations as therapeutic agents given the size of the molecule, the difficulties in the application, and the rapid clearance from the circulatory system. Despite this, inducing the formation of highly active CCL14a(9–74) from the abundant but inactive CCL14a(1–74) is a promising approach to inhibit HIV infection.

Initial investigations of the proteolytic processing of CCL14a(9–74) suggest a role in inflammation, tissue remodeling, and tumorigenesis. Limited proteolysis in vitro of full-length CCL14a by trypsin generates CCL14a(9–74). Further investigations using conditioned media derived from tumor cell lines, which are known to release a number of proteases, have also shown evident processing activities on CCL14a(1–74) [18 ]. Maximum CCR5-stimulatory activity, implying the generation of CCL14a(9–74), can be obtained by incubating CCL14a(1–74) in the medium from monolayer cell cultures of prostate carcinoma or osteosarcoma cell lines. This proteolytic processing of inactive CCL14a is inhibited by coincubation with 4-[2-aminoethyl]-benzylsulfonylfluoride, a serine protease inhibitor, and by leupeptin, a serine and cysteine protease inhibitor, suggesting that this activity is mediated by a serine protease. Because serine proteases are known to be activated in the course of inflammation and tumorigenesis, a role for CCL14a(9–74) can be expected during these processes. The processing of CCL14a(1–74) is as unique as it is abundant in plasma, and its proteolytic processing transforms a virtually inactive precursor into a wide-ranging chemoattractant. Such a mechanism could represent an alternative to transcriptional control for the generation of inflammatory cytokines.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MOLECULAR FORMS AND STRUCTURES
 ORGANIZATION AND EXPRESSION...
 BIOLOGICAL PROPERTIES
 PATHOPHYSIOLOGICAL ROLES OF...
 CONCLUSIONS
REFERENCES
 
Further work on the physiological functions of CCL14a is still required. However, the current data indicate that CCL14a is mainly involved in the trafficking of inflammatory cells, although high concentrations of this chemokine are required for cellular activation. The chemokine CXCL13 attracts B cells in vitro only at concentrations of >100 nM [58 ]. Nevertheless, there is strong evidence that this relatively weak chemokine is important for the development of B-cell areas of secondary lymphoid tissues. Consistent with these findings, a similar pivotal role could exist for CCL14a, which might be responsible for a basic level of circulating leukocytes by mobilizing them into the plasma. The high plasma levels of low-affinity-binding CCL14a(1–74) might define a certain threshold for further mobilization of CCR1+ cells by more potent ligands. Thus, CCL14a(1–74) would retain CCR1+ cells in the blood, being unsusceptible to nonspecific, low doses of chemotactic stimuli. In this fashion, CCR1+ cells would circulate constantly in the body and could act very rapidly in the required compartments on specific chemotactic stimuli generated during inflammation or other scenarios. Therefore, CCL14a(1–74) might represent a factor for homeostasis of CCR1+ inflammatory cells in the blood stream.

CCL15 is most likely a classical inflammatory chemokine, attracting almost all types of inflammatory cells, especially those involved in the innate immune response. CCL3, which has an activity pattern similar to CCL15, was recently shown to recruit mainly neutrophils, monocytes, and lymphocytes after intradermal injection in allergic human subjects [59 ]. Because CCL15 attracts the same type of cells after intraperitoneal injection in mice, identical effects can be expected for humans [50 ].

However, to define the exact biological role of CCL14a and CCL15 in detail, identification of the murine orthologues that would facilitate experiments with genetically manipulated mice is of great importance. Disruption of CCL9,10, which competes for receptors with MIP-1{alpha}-like CCL14a and CCL15, is of particular interest. This murine chemokine displays some of the biological characteristics of CCL14a (high plasma levels and broad expression pattern) and characteristics of CCL15, i.e., six conserved cysteines and suppression of bone marrow myeloid colony formation [38 , 39 ].


   ACKNOWLEDGEMENTS
 
This work was supported in part by a grant from the Bundesministerium für Bildung und Forschung (FKZ 0311815).

Received April 2, 2001; revised May 20, 2001; accepted May 24, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MOLECULAR FORMS AND STRUCTURES
 ORGANIZATION AND EXPRESSION...
 BIOLOGICAL PROPERTIES
 PATHOPHYSIOLOGICAL ROLES OF...
 CONCLUSIONS
 REFERENCES
 

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