HCC-1 Theodora W. Salcedo* Human Genome Sciences, Inc., 9410 Key West Avenue, Rockville, MA 20850, USA * corresponding author tel: 301-309-8504, fax: 301-294-4843, e-mail:
[email protected] DOI: 10.1006/rwcy.2000.11014.
SUMMARY
Alternative names
Hemofiltrate CC chemokine (HCC-1/NCC-2/CK -1/ M-CIF) is a chemokine family member that shares highest structural similarity with macrophage inflammatory protein (MIP)-1 . Cross-desensitization experiments suggest that HCC-1 and MIP-1 share a common receptor. CCR1 was identified as a functional HCC-1 receptor upon analysis of transfectants expressing various cloned chemokine receptors. Functionally, HCC-1 displays calcium mobilization and chemotactic activity toward monocytes, but with 100-fold reduced potency compared with MIP-1 . HCC-1 fails to activate T lymphocytes, neutrophils, and eosinophils, but displays activity on myeloid progenitors. Unlike most CC chemokines, it is constitutively expressed in many tissues without activation and is present in nanomolar concentrations in normal human plasma.
HCC-1 was first identified as hemofiltrate CC chemokine (Schulz-Knappe et al., 1996). This chemokine was also discovered independently by other groups and was named new CC chemokine 2 (NCC-2) and CK -1/M-CIF. The designation HCC-3 corresponds to an uncharacterized splice variant of HCC-1.
BACKGROUND
Discovery HCC-1 was initially described as a hemofiltrate CC chemokine after the protein was purified from hemofiltrate collected from patients with chronic renal failure and the HCC-1 cDNA was isolated from a human bone marrow cDNA library (Schulz-Knappe et al., 1996). In another study, a novel expressed sequence tag (EST) for this chemokine was mapped to the CC chemokine cluster on chromosome 17, and named NCC-2 for new CC chemokine 2 (Naruse et al., 1996). A third group identified a cDNA encoding CK -1 in the HGS/TIGR database as part of a largescale sequencing effort and subsequently named the chemokine monocyte colony inhibitory factor (MCIF) to reflect functional activity (Kreider et al., 1996).
Structure HCC-1 is a member of the CC or chemokine family. The complete sequence encodes for a 93 amino acid chemokine with a putative N-terminal 19 amino acid leader sequence followed by a mature protein of 74 amino acids. Signal sequence cleavage is predicted to occur at residue 19, with the N-terminal amino acid being methionine (HCC-1(20±93)). This is the form of the protein isolated from renal patient hemofiltrate (Schulz-Knappe et al., 1996). Two additional Nterminally truncated variants of HCC-1 have been generated and activity compared with HCC-1(20±93). The shorter forms (HCC-1(23±93) and HCC-1(25± 93)) were significantly more potent in chemotaxis and cAMP assays compared to HCC-1(20±93). Four conserved cysteines typical of the CC chemokine family are present in HCC-1 at amino acid positions 35, 36, 59, and 75. The observed molecular mass for HCC-1 purified from hemofiltrate is 8673.
Main activities and pathophysiological roles HCC-1 displays functional activity towards monocytes, but not T lymphocytes, eosinophils or neutrophils. On monocytes, it induces a rise of intracellular
1266 Theodora W. Salcedo Ca2 and a slight release of N-acetyl- -D-glucosaminidase (Schulz-Knappe et al., 1996). The amount of HCC-1 needed for these responses is 100 and 1000 nM, respectively. While in one report, HCC-1 failed to stimulate monocyte chemotaxis (SchulzKnappe et al., 1996), a second study demonstrated significant chemotactic activity for monocytes (Tsou et al., 1998). In line with the potency of HCC-1 in calcium flux and degranulation assays, the optimal concentration of HCC-1 needed to induce monocyte chemotaxis is 100 nM relative to 1 nM for MIP-1 . On monocytes, cross-desensitization experiments suggest that HCC-1 acts through a shared receptor with MIP-1 . Pretreatment of monocytes with MIP-1 or RANTES, but not MIP-1 , inhibited a subsequent intracellular calcium response to HCC-1 (SchulzKnappe et al., 1996; Tsou et al., 1998). In the reciprocal experiment, HCC-1 reduced the calciummobilizing activity of MIP-1 and RANTES, but not MIP-1 (Schulz-Knappe et al., 1996; Tsou et al., 1998). In experiments with HEK-293 cells transfected with CCR1, HCC-1 competed for MIP-1 binding, mobilized calcium, and induced chemotaxis, but with 100-fold lower affinity/potency compared with MIP1 . Specificity of HCC-1 for CCR1 was demonstrated using HEK-293 cells transfected with other known chemokine receptors (CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, or CXCR1) which fail to mobilize calcium or induce chemotaxis in response to HCC-1 (Tsou et al., 1998). HCC-1 also displays activity toward bone marrow cells. It enhances the proliferation of CD34 cells and very early progenitor/stem cells of CD34/CD38ÿ phenotype (Schulz-Knappe et al., 1996). On both mouse and human bone marrow progenitor cells, HCC-1 inhibits M-CSF-mediated colony formation (Kreider et al., 1996).
GENE AND GENE REGULATION
Accession numbers Human gene: AC004675, AF088219, Z49269 Human mRNA: Z49270, Z70292, Z70293 HCC-3, uncharacterized splice variant of HCC-1: Z70293
Chromosome location The human HCC-1 gene maps to chromosome 17q 11.2 (Naruse et al., 1996; Pardigol et al., 1998). By YAC contig-based mapping, the HCC-1 gene was
localized in a cluster on chromosome 17 that contains several CC chemokines, including MCP-3, MCP-1, NCC-1, I-309, and RANTES (Naruse et al., 1996).
Relevant linkages By analyzing the 50 flanking region of the gene encoding HCC-1, it was found that HCC-1 is arrayed in tandem with the gene for HCC-2 (Pardigol et al., 1998). These two genes are separated by 12 kb and lie in a head-to-tail orientation. HCC-2 displays a four exons and three intron structure, while HCC-1, like other CC chemokines, has a three exonz±two intron structure. The HCC-2/HCC-1 gene complex encodes for the expression of both bicistronic and monocistronic transcripts (Pardigol et al., 1998). While HCC-1 monocistronic mRNA is expressed highly in all tissues except brain, placenta, and leukocytes, HCC-2 monocistronic and HCC-1/HCC-2 bicistronic transcripts were more restricted in expression in colon and liver (Pardigol et al., 1998).
Regulatory sites and corresponding transcription factors The promoter for HCC-1 was identified by primer extension and RT-PCR. Several putative binding sites were identified for transcriptional factors, including Myc-Max, E47, and AP-2 (Pardigol et al., 1998).
Cells and tissues that express the gene By northern analysis monocistronic mRNA for HCC1 is detected in many tissues (spleen, colon, small intestine, liver, skeletal, and heart muscle). Little or no expression is detected in kidney, brain, placenta, leukocytes, monocyte cell lines (U937, THP1), and HL-60 or Jurkat cells (Schulz-Knappe et al., 1996; Pardigol et al., 1998).
PROTEIN
Accession numbers SwissProt: Human: Q16627
HCC-1 1267 Figure 1 Amino acid sequence for HCC-1. Signal peptide is underlined. MKISVAAIPF FLLITIALGT KTESSSRGPY HPSECCFTYT TYKIPRQRIM DYYETNSQCS KPGIVFITKR GHSVCTNPSD KWVQDYIKDM KEN
that CCR1 is a functional receptor for HCC-1 (Pardigol et al., 1998). HCC-1 competes with MIP-1 binding to CCR1-transfected cells, but binds with reduced affinity compared to MIP-1 (IC50=93 nM versus 1.3 nM for MIP-1 ) (Pardigol et al., 1998).
Sequence
IN VITRO ACTIVITIES
See Figure 1.
Regulatory molecules: Inhibitors and enhancers
Important homologies HCC-1 is a member of the CC or chemokine family. It displays highest structural homology to MIP-1 (46% amino acid sequence identity), MPIF1 (44% identity), HCC-4 (42% identity), and HCC-2 (36% identity).
Posttranslational modifications Based on amino acid and nucleotide sequence analysis, no apparent N- or O-linked glycosylation sites are present in HCC-1.
CELLULAR SOURCES AND TISSUE EXPRESSION
Cellular sources that produce Expression of HCC-1 has been detected in many tissues at the level of mRNA (spleen, colon, small intestine, liver, skeletal, and heart muscle). Little or no expression is detected in kidney, brain, placenta, and leukocytes (Schulz-Knappe et al., 1996; Pardigol et al., 1998). There is little information regarding the regulation of the protein at the cellular level, except that high concentrations of HCC-1 are found in normal plasma. Using western blotting of plasma samples, HCC-1 migrates as a single band of approximately 8 kDa, with no degradation products (Schulz-Knappe et al., 1996).
Although HCC-1 is constitutively expressed by many tissues and exists in high concentration in normal serum, little is known about the regulation of this gene.
Bioassays used Several bioassays have been used for study of HCC-1 functional activity. These include calcium mobilization, chemotaxis, enzyme release, and adenyl cyclase activity.
PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY
Normal levels and effects The levels of HCC-1 in human plasma range from 1 to 10 nM in healthy subjects and 2±80 nM in patients with chronic renal failure (Schulz-Knappe et al., 1996). This high expression level of HCC-1 is unique within the human chemokine family and the significance of this observation is unknown.
IN THERAPY
RECEPTOR UTILIZATION
Preclinical ± How does it affect disease models in animals?
Based on calcium cross-desensitization studies, HCC1 shares a functional receptor with MIP-1 (SchulzKnappe et al., 1996; Pardigol et al., 1998). Experiments using a panel of cloned chemokine receptors (CCR1, CCR2, CCR3, CCR4, CCR5, or CXCR1) revealed
The sepsis syndrome is a life-threatening systemic inflammatory response to microbial infection and may be complicated by the development of shock and multiple organ failure. Bacterial products such as endotoxin are believed to contribute to sepsis by
1268 Theodora W. Salcedo causing the release of inflammatory cytokines from activated macrophages. Prophylactic administration of recombinant human HCC-1/M-CIF by the intraperitoneal route substantially reduced lethality in endotoxin-challenged mice (Zhang et al., 1996, 1997). In the HCC-1/M-CIF-treated groups, serum levels of IL-10 were significantly increased; however levels of the proinflammatory cytokines were not markedly altered. Downregulation of proinflammatory cytokine production did not contribute to the protective effects of HCC-1/M-CIF against endotoxemia. Interestingly, although HCC-1/M-CIF and the chemokine MCP-1 differ markedly in chemoattractant and other biological properties, MCP-1 was reported to elicit protective effects in murine endotoxemia that were similar to those of HCC-1/M-CIF (Zisman et al., 1997). MCP-1 protected against lethality when administered prophylactically to mice and caused similar changes in the pattern of circulating cytokine levels (increased IL-10). As with HCC-1/MCIF, however, no clear explanation for the protection could be determined. Other chemokines such as MIP2 (Standiford et al., 1995a), MIP-1 (Standiford et al., 1995b), and RANTES (VanOtteren et al., 1995), which have robust proinflammatory properties, were reported to be expressed during murine, baboon, and human endotoxemia. Results using various neutralization strategies suggest that these chemokines may mediate the influx of inflammatory cells into the lungs and other target organs after endotoxin challenge. Thus, HCC-1/M-CIF, similar to other members of the chemokine family, appears to contribute to the regulation of both pro- and anti-inflammatory responses during experimental endotoxemia.
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