CK11, a Teleost Chemokine with a Potent Antimicrobial Activity.

CK11 is a rainbow trout (Oncorhynchus mykiss) CC chemokine phylogenetically related to both mammalian CCL27 and CCL28 chemokines, strongly transcribed in skin and gills in homeostasis, for which an immune role had not been reported to date. In the current study, we have demonstrated that CK11 is not chemotactic for unstimulated leukocyte populations from central immune organs or mucosal tissues but instead exerts a potent antimicrobial activity against a wide range of rainbow trout pathogens. Our results show that CK11 strongly inhibits the growth of different rainbow trout Gram-positive and Gram-negative bacteria, namely Lactococcus garvieae, Aeromonas salmonicida subsp. salmonicida, and Yersinia ruckeri and a parasitic ciliate Ichthyophthirius multifiliis Similarly to mammalian chemokines and antimicrobial peptides, CK11 exerted its antimicrobial activity, rapidly inducing membrane permeability in the target pathogens. Further transcriptional studies confirmed the regulation of CK11 transcription in response to exposure to some of these pathogens in specific conditions. Altogether, our studies related to phylogenetic relations, tissue distribution, and biological activity point to CK11 as a potential common ancestor of mammalian CCL27 and CCL28. To our knowledge, this study constitutes the first report of a fish chemokine with antimicrobial activity, thus establishing a novel role for teleost chemokines in antimicrobial immunity that supports an evolutionary relationship between chemokines and antimicrobial peptides.

A scalable low-cost cGMP process for clinical grade production of the HIV inhibitor 5P12-RANTES in Pichia pastoris.

In the continued absence of an effective anti-HIV vaccine, approximately 2 million new HIV infections occur every year, with over 95% of these in developing countries. Calls have been made for the development of anti-HIV drugs that can be formulated for topical use to prevent HIV transmission during sexual intercourse. Because these drugs are principally destined for use in low-resource regions, achieving production costs that are as low as possible is an absolute requirement. 5P12-RANTES, an analog of the human chemokine protein RANTES/CCL5, is a highly potent HIV entry inhibitor which acts by achieving potent blockade of the principal HIV coreceptor, CCR5. Here we describe the development and optimization of a scalable low-cost production process for 5P12-RANTES based on expression in Pichia pastoris. At pilot (150 L) scale, this cGMP compliant process yielded 30 g of clinical grade 5P12-RANTES. As well as providing sufficient material for the first stage of clinical development, this process represents an important step towards achieving production of 5P12-RANTES at a cost and scale appropriate to meet needs for topical HIV prevention worldwide.

Efficient production of FAM19A4, a novel potential cytokine, in a stable optimized CHO-S cell system.

FAM19A4 is a novel potential cytokine identified by our group, which can chemoattract macrophages, promote phagocytosis against zymosan and increase reactive oxygen species (ROS) release. To further explore the role of FAM19A4 in immune system, abundant recombinant protein with high quality is indispensable. For efficient production of FAM19A4, we used an improved CHO-S cell expression system on the basis of pMH3 vector containing GC-rich regions which were novel ubiquitous chromatin opening elements (UCOEs). We selected CHO-S cells stably expressing FAM19A4 with G418 and screened cell clones with high level of FAM19A4 expression by immune blot and his-ELISA, adapted cell clones to serum-free suspension culture. Afterwards, we obtained the highest FAM19A4 expressing cell clone (2#) through 40 ml batch culture. We optimized the fed-batch culture condition and discovered the final cell viability was critical for FAM19A4 production successfully. Then we scaled 2# clone up to 3 L in fed-batch culture and obtained 22 mg (7.33 mg/L, averagely) endotoxin free FAM19A4 protein with purity over 95% using Ni affinity chromatography and size exclusion chromatography. The final yield was increased 3.6-folds compared to that of our previously reported transient system. Besides, the purified FAM19A4 protein showed chemotactic activity on macrophages. In summary, we developed a stable optimized fed-batch CHO-S cell system to produce FAM19A4, which not only provided sufficient bioactive FAM19A4 protein for further research but also offered an efficient strategy for other recombinant protein production.

Molecular cloning and functional characterization of a mouse ccl6 analog gene in the rat.

Suppression subtractive hybridization was used to analyze differential expression of genes in rat peritoneal macrophages after granulocyte macrophage colony-stimulating factor treatment. We identified and cloned the mouse C10 analog gene in the rat, and named it as ccl6. The full-length cDNA of rat ccl6 was 467 bp, which contains a single-open reading frame and encodes 116 amino acid residues. Compared with other C-C chemokines, the rat ccl6 gene had an unusual four-exon genome structure instead of the typical three exons, it had the highest homology with murine ccl6. The rat ccl6 gene was localized on chromosome 10, where most of the C-C chemokine superfamily members are located. The recombinant rat C-C chemokine ligand 6 (CCL6) protein was expressed by the pGEX4T-1 plasmid in Escherichia coli BL21. The purified recombinant protein had bioactivity similar to that of mouse CCL6, which is a chemoattractant for macrophages and lymphocytes, but not for neutrophils.

A rapid and efficient way to obtain modified chemokines for functional and biophysical studies.

Chemokines and their receptors control cell migration associated with routine immune surveillance, inflammation and development. They are also implicated in a large number of inflammatory diseases, cancer and HIV. Here we describe a rapid and efficient way to express and purify milligram quantities of multiple chemokine ligands (CCL7/MCP-3, CCL14/HCC-1, CCL3/MIP-1α and CXCL8/IL-8) containing C-terminal modifications to enable coupling to fluorescent dyes or small molecules such as biotin, in vitro. These labeled chemokines display wild-type behavior in both receptor binding and calcium mobilization assays. The ability to rapidly and inexpensively produce labeled chemokines opens the way for their use in many applications, including non-traditional chemokine-receptor interaction studies, both on intact cells and with purified receptor reconstituted in artificial membranes in vitro. Furthermore, the ability to immobilize chemokines to obtain ligand affinity columns aids in efforts to purify chemokine receptors for structural and biophysical studies, by facilitating the separation of functional proteins from their non-functional counterparts.

Expression of soluble proteins in Escherichia coli by linkage with the acidic propiece of eosinophil major basic protein.

An expression method has been developed to produce soluble cationic polypeptides in Escherichia coli while avoiding inclusion body deposition. For this technique the recombinant product is linked through a thrombin or factor Xa susceptible bond to the amino-terminal domain of the precursor of eosinophil major basic protein (MBP). This N-terminal domain is strongly acidic and is apparently able to shield eosinophils from the potentially injurious activities of MBP. It was reasoned that constructs of this acidic domain with small heterologous cationic proteins expressed in E. coli could result in soluble expression while preventing trafficking and packaging into insoluble inclusion bodies. This has been demonstrated using four examples: complement C5a, CCL18, fibroblast growth factor-ß, and leukemia inhibitory factor, whose isoelectric points range from 8.93 to 9.59. Further general applicability of this technique has been shown by using two different expression systems, one which encodes an amino-terminal oligo-histidine leash, and another that codes for an amino-terminal glutathione-S-transferase. Thus the utility of coupling MAP to cationic polypeptides for the purpose of soluble heterologous protein expression in E. coli has been demonstrated.

Human macrophage-derived chemokine (MDC), a novel chemoattractant for monocytes, monocyte-derived dendritic cells, and natural killer cells.

A cDNA encoding a novel human chemokine was isolated by random sequencing of cDNA clones from human monocyte-derived macrophages. This protein has been termed macrophage-derived chemokine (MDC) because it appears to be synthesized specifically by cells of the macrophage lineage. MDC has the four-cysteine motif and other highly conserved residues characteristic of CC chemokines, but it shares <35% identity with any of the known chemokines. Recombinant MDC was expressed in Chinese hamster ovary cells and purified by heparin-Sepharose chromatography. NH2-terminal sequencing and mass spectrophotometry were used to verify the NH2 terminus and molecular mass of recombinant MDC (8,081 dalton). In microchamber migration assays, monocyte-derived dendritic cells and IL-2-activated natural killer cells migrated to MDC in a dose-dependent manner, with a maximal chemotactic response at 1 ng/ml. Freshly isolated monocytes also migrated toward MDC, but with a peak response at 100 ng/ml MDC. Northern analyses indicated MDC is highly expressed in macrophages and in monocyte-derived dendritic cells, but not in monocytes, natural killer cells, or several cell lines of epithelial, endothelial, or fibroblast origin. High expression was also detected in normal thymus and less expression in lung and spleen. Unlike most other CC chemokines, MDC is encoded on human chromosome 16. MDC is thus a unique member of the CC chemokine family that may play a fundamental role in the function of dendritic cells, natural killer cells, and monocytes.

Inhibition of HIV-1 infection by the beta-chemokine MDC.

CD8(+) T lymphocytes from individuals infected with human immunodeficiency virus-type 1 (HIV-1) secrete a soluble activity that suppresses infection by HIV-1. A protein associated with this activity was purified from the culture supernatant of an immortalized CD8(+) T cell clone and identified as the beta-chemokine macrophage-derived chemokine (MDC). MDC suppressed infection of CD8(+) cell-depleted peripheral blood mononuclear cells by primary non-syncytium-inducing and syncytium-inducing isolates of HIV-1 and the T cell line-adapted isolate HIV-1IIIB. MDC was expressed in activated, but not resting, peripheral blood mononuclear cells and binds a receptor on activated primary T cells. These observations indicate that beta-chemokines are responsible for a major proportion of HIV-1-specific suppressor activity produced by primary T cells.

Isolation of cDNA encoding a novel human CC chemokine NCC-4/LEC.

We have determined the entire sequence of human cDNA encoding a novel CC chemokine NCC-4 by 5' and 3' RACE methods. Two types of transcripts, 579 bp and 1503 bp long, respectively, are generated through alternative polyadenylation sites. Both species contain an open reading frame encoding 120 amino acids with 19-38% identity to other human CC chemokines. The short and long transcripts are expressed highly selectively in the liver at nearly equivalent levels. There seems to be one copy of the gene per haploid genome. We now designate NCC-4 as LEC from liver-expressed chemokine.

CC and CXC chemokines in breastmilk are associated with mother-to-child HIV-1 transmission.

INTRODUCTION: CC and CXC chemokines may play a role in mother-to-child HIV-1 transmission by blocking HIV-1 binding to chemokine receptors and impeding viral entry into cells. METHODS: To define correlates of breastmilk chemokines and associations with infant HIV-1 acquisition, chemokines in breastmilk and infant HIV-1 infection risk were assessed in an observational, longitudinal cohort study. We measured MIP-1alpha, MIP-1beta, RANTES, and SDF-1 in month 1 breastmilk specimens from HIV-1-infected women in Nairobi and HIV-1 viral load was calculated in maternal plasma and breastmilk at delivery and 1 month postpartum. Infant infection status was determined at birth and months 1, 3, 6, 9, and 12. RESULTS: Among 281 breastfeeding women, 60 (21%) of their infants acquired HIV-1 during follow-up, 39 (65%) of whom became infected intrapartum or after birth. MIP-1alpha, MIP-1beta, RANTES, and SDF-1 were all positively correlated with breastmilk HIV-1 RNA (P<0.0005). Women with clinical mastitis had 50% higher MIP-1alpha and MIP-1beta levels (P<0.001 and P=0.006, respectively) and women with subclinical mastitis (breastmilk Na(+)/K(+)>1) had approximately 70% higher MIP-1alpha, MIP-1beta and RANTES (P<0.002 for all) compared to women without mastitis. Independent of breastmilk HIV-1, increased MIP-1beta and SDF-1 were associated with reduced risk of infant HIV-1 (RR=0.4; 95% CI 0.2-0.9; P=0.03 and RR=0.5; 95% CI=0.3-0.9; P=0.02, respectively) and increased RANTES was associated with higher transmission risk (RR=2.3; 95% CI 1.1- 5.3; P=0.04). CONCLUSIONS: These observations suggest a complex interplay between virus levels, breastmilk chemokines, and mother-to-child HIV-1 transmission and may provide insight into developing novel strategies to reduce infection across mucosal surfaces.

Identification of NH(2)-terminal amino acid residues essential for the biological activity of leukotactin-1.

Leukotactin-1 (Lkn-1), a human CC chemokine that binds to both CC chemokine receptor (CCR)1 and CCR3, is distinct from other human CC chemokines in that it has long amino acid residues preceding the first cysteine at the NH(2)-terminus. Serial deletion studies showed that at least three amino acid residues, alanine-alanine-aspartic acid (A-A-D), preceding the first cysteine at the NH(2)-terminus are essential for the biological activity of Lkn-1. Point mutation and deletion studies for the three amino acids were performed in the present study. Substitutions of the first alanine residue with other amino acids did not cause significant loss of biological activities. Deletion of the third amino acid, aspartic acid, resulted in more than 100-fold loss of the activity. Deletion of two amino acids, alanine-alanine (A-A) or alanine-aspartic acid (A-D), resulted in almost complete loss of the activity. Loss of agonistic activity by deletion of two amino acids was due to impaired binding to CCR1. These results identify that alanine-aspartic acid residues preceding the first cysteine at the NH(2)-terminus are essential for the binding and biological activity of Lkn-1.

Molecular cloning of a novel CC chemokine, interleukin-11 receptor alpha-locus chemokine (ILC), which is located on chromosome 9p13 and a potential homologue of a CC chemokine encoded by molluscum contagiosum virus.

Molluscum contagiosum virus (MCV) encodes a CC chemokine MC148R which is likely to have been acquired from the host. By a homology search employing MC148R as a probe, we have identified a novel CC chemokine whose gene exists next to the IL-11 receptor alpha (IL-11Ralpha) gene in both humans and mice. Thus, this chemokine maps to chromosome 9p13 in humans where IL-11Ralpha has been assigned. We term this novel chemokine IL-11Ralpha-locus chemokine (ILC). ILC has the highest homology to MC148R among the known human CC chemokines. Furthermore, ILC is strongly and selectively expressed in the skin where infection of MCV also takes place. Thus, ILC is likely to be the original chemokine of MC148R.

Semi-quantification of human C-C chemokine mRNAs with reverse transcription/real-time PCR using multi-specific standards.

A reverse transcription/real-time polymerase chain reaction (PCR) assay was established to semi-quantify the mRNA levels of the human C-C chemokines RANTES, MIP-1beta and MCP-1 relative to the housekeeping gene beta-actin. The assay showed a high sensitivity (below 60 cDNA molecules/10 microl reaction) and dynamic range (8 log units); both within-assay and inter-assay variability were below 0.06 log units and the accuracy was +/-0.06 log units for all four chemokines. Moreover, it is demonstrated that a multi-specific DNA fragment, which had previously been constructed for competitive PCR, can be used as a reliable external standard. This allows a direct semi-quantitative comparison of different chemokine mRNA levels and is a convenient alternative to the use of different sets of homologous external standards. The method was successfully applied to the semi-quantification of chemokines in human liver specimens and should be useful in further studies on steady state mRNA levels of C-C chemokines from low cell numbers or small tissue specimens.

CCL19 and CCL21 induce a potent proinflammatory differentiation program in licensed dendritic cells.

Dendritic cells (DCs) are key instigators of adaptive immune responses. Using an alphaviral expression cloning technology, we have identified the chemokine CCL19 as a potent inducer of T cell proliferation in a DC-T cell coculture system. Subsequent studies showed that CCL19 enhanced T cell proliferation by inducing maturation of DCs, resulting in upregulation of costimulatory molecules and the production of proinflammatory cytokines. Moreover, CCL19 programmed DCs for the induction of T helper type (Th) 1 rather than Th2 responses. Importantly, only activated DCs that migrated from the periphery to draining lymph nodes, but not resting steady-state DCs residing within lymph nodes, expressed high levels of CCR7 in vivo and responded to CCL19 with the production of proinflammatory cytokines. Migrating DCs isolated from mice genetically deficient in CCL19 and CCL21 (plt/plt) presented an only partially mature phenotype, highlighting the importance of these chemokines for full DC maturation in vivo. Our findings indicate that CCL19 and CCL21 are potent natural adjuvants for terminal activation of DCs and suggest that chemokines not only orchestrate DC migration but also regulate their immunogenic potential for the induction of T cell responses.

Differential regulation of CCL22 gene expression in murine dendritic cells and B cells.

The activated T cell-attracting CC chemokine CCL22 is expressed by stimulated B cells and mature dendritic cells (DC). We have cloned and sequenced the complete mouse gene, including 4 kb of the 5'-flanking promoter region, and detected two distinct sites for initiation of transcription by 5'-RACE. Reporter gene assays indicate that the promoter reflects the specificity of the endogenous gene. Within the proximal promoter region, we identified potential binding sites for NF-kappaB, Ikaros, and a putative GC box. All three regions bind proteins. The NF-kappaB site was shown to specifically bind NF-kappaB subunits p50 and p65 from nuclear extracts of LPS-stimulated B cells, B cell line A20/2J, TNF-alpha-stimulated bone marrow-derived DC, and DC line XS106. Furthermore, promoter activity was affected by targeted mutagenesis of the NF-kappaB site and transactivation with p50 and p65. The region harboring the putative Ikaros site contributes to promoter activity, but the binding protein does not belong to the Ikaros family. The GC box was shown to specifically bind Sp1 using extracts from LPS-stimulated B cells and A20/2J but not from DC and DC line XS106. Additionally, Sp1 transactivated the promoter in A20/2J but not in XS106 cells, and mutation of the Sp1 site diminished transactivation. Furthermore, binding of the protein complex at the GC box is required for NF-kappaB activity, and the spatial alignment of the binding sites is of critical importance for promoter activity. Thus, identical and distinct proteins contribute to expression of CCL22 in DC and B cells.

Multiple products derived from two CCL4 loci: high incidence of a new polymorphism in HIV+ patients.

Human CCL4/macrophage inflammatory protein (MIP)-1beta and CCL3/MIP-1alpha 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)).