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Profile of human macrophage transcripts: insights into macrophage biology and identification of novel chemokines David Chantry, Anthony J. DeMaggio, Heather Brammer, Carol J. Raport, Christi L. Wood, Vicki L. Schweickart, Angela Epp, Aaron Smith, Johnny T. Stine, Kim Walton, Larry Tjoelker, Ronald Godiska, and Patrick W. Gray ICOS Corporation, Bothell, Washington

Abstract: High throughput partial sequencing of randomly selected cDNA clones has proven to be a powerful tool for examining the relative abundance of mRNAs and for the identification of novel gene products. Because of the important role played by macrophages in immune and inflammatory responses, we sequenced over 3000 randomly selected cDNA clones from a human macrophage library. These sequences represent a molecular inventory of mRNAs from macrophages and provide a catalog of highly expressed transcripts. Two of the most abundant clones encode recently identified CC chemokines. Macrophage-derived chemokine (MDC) plays a complex role in immunoregulation and is a potent chemoattractant for dendritic cells, T cells, and natural killer cells. The chemokine receptor CCR4 binds MDC with high affinity and also responds by calcium flux and chemotaxis. CCR4 has been shown to be expressed by Th2 type T cells. Recent studies also implicate MDC as a major component of the host defense against human immunodeficiency virus. J. Leukoc. Biol. 64: 49–54; 1998. Key Words: macrophage-derived chemokine · CCR4 · immunoregulation

INTRODUCTION Cells of the monocyte lineage have the potential to differentiate into a diverse array of effector cells with significant phenotypic and functional diversity [1]. This population includes cells able to elaborate a large number of growth factors and cytokines and includes dendritic cells, which play a critical role in antigen presentation and the initiation of the host immune response [2, 3]. Blood monocyte-derived cells are therefore involved in a range of cellular responses that include the regulation and amplification of immune and inflammatory responses, tissue repair and remodeling, and host response to infection [4]. These cells are also the target of many pathogenic organisms, including Leishmania, Listeria, and HIV-1. Traditional approaches to studying the role of a particular cell type in such processes have focused on the generation of cell type-specific antibodies and the purification of cellderived soluble mediators [5]. More recently, DNA sequence

information has facilitated the development of additional approaches to address this question. The profiling of mRNA expression has proven to be a powerful tool for the identification of novel gene products and for the association of particular mRNA subsets with physiological or disease states [6]. Two approaches have been taken for the large-scale analysis of mRNA expression. One involves the use of high-density arrays of gene-specific sequences to which a cDNA of interest is hybridized, and which allows differential measurements in gene expression to be accurately quantitated [7, 8]. The second approach involves the partial sequencing of randomly selected clones from cDNA libraries [9, 10]. Although this strategy will readily identify changes in the levels of expression of abundant mRNAs, the number of clones sequenced (often less than 5000 per cDNA library) limits the ability to detect changes in the expression levels of rare transcripts [6]. The advantage of this approach is that it provides significant amounts of sequence information (up to 500 bp) for each clone, which allows extensive homology-based searching to greatly accelerate the functional characterization of novel cDNAs. Because of the important role that macrophages play in immune and inflammatory responses, we attempted to catalog the repertoire of mRNAs expressed by this cell type. We sequenced 3457 randomly selected clones from a human macrophage cDNA library and compared these to sequences in GenBank. This study provides a profile of the mRNA expression in the macrophage and has led to the identification of a number of novel proteins with potential immunoregulatory properties.

RANDOM SEQUENCING OF HUMAN MACROPHAGE cDNAs During in vitro culture, blood monocytes differentiate into a population of cells with many of the characteristics of tissue

Abbreviations: CCR, CC chemokine receptor; MCP, monocyte chemoattractant protein; MDC, macrophage-derived chemokine; HIV, human immunodeficiency virus; TARC, thymus and activation regulated chemokine; MIP, macrophage inflammatory protein; RANTES, regulated on activation normal T cell expressed and secreted; LARC, liver and activation regulated chemokine; SEAP, secreted alkaline phosphatase; IL-2, interleukin-2; NK, natural killer; NSI, non-syncytium inducing; SI, syncytium inducing. Correspondence: Dr. P. W. Gray, ICOS Corporation, 22021 20th Ave. SE, Bothell, WA 98021. E-mail: [email protected] Received January 21, 1998; accepted January 22, 1998.

Journal of Leukocyte Biology

Volume 64, July 1998

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macrophages, providing a model of in vivo macrophage differentiation [11]. A cDNA library was constructed from macrophages prepared from human monocytes cultured on plastic for 6 days [described in detail in ref. 5]. A total of 3457 clones from this library were subjected to sequence analysis. Plasmid DNA was isolated and 300–500 bp of sequence were generated from each clone by dideoxy sequencing with a plasmid-based primer. Sequences were compared to the GenBank non-redundant database with the use of the BLAST program [12]. These TABLE 1.

A Total of 3457 Partially Sequenced cDNAs from a Human Macrophage Library Were Compared With the GenBank Nonredundant Database Using the BLAST Program [12]

Clone name

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.

sequences were also compared to each other with the Sequencher program (Gene Codes Corp., Ann Arbor, MI). Alignment parameters were set at 95% identity over a minimum of 30-nucleotide overlap. From this random sampling, 370 contiguous sequences were generated, representing cDNA clones that were identified more than once. The fifty most abundant clones identified in this study are presented in Table 1. The highly expressed mRNAs fall into two broad classes: housekeeping genes, such as the elongation factors and b-actin,

Ferritin light chain Ferritin heavy chain Collagenase (type IV) b-Actin Annexin II (Lipocortin II) Chitinase Sphingolipid activator protein p33 (HLA DR invariant chain) b2-Microglobulin Cathepsin D IP-30 LARC/MIP-3a/Exodus Elongation factor 1a-1 Apolipoprotein-E Lysozyme CD63 Granulin Osteopontin MDC Elongation factor 1a-2 Extensin (h) p41 (ARC) Ornithine decarboxylase HLA Class II Cytochrome c oxidase 40S Ribosomal subunit a1-Tubulin Thioredoxin peroxidase Alu warning Novel sequence IL-8 receptor Novel sequence Cathepsin B NADH oxidoreductase ATPase subunit Ficolin 1 60S Ribosomal protein Novel sequence Vimentin KIAA0201 Elongation factor 4a Macrophage capping protein GAPDH a1-Collagen a Enolase Novel sequence Heme oxygenase gp39 F1:6 bisphosphatase Transmembrane protein

Frequency in library

Accession number

Function

126 (3.6%) 46 (1.3%) 45 (1.3%) 33 (0.9%) 25 (0.7%) 17 (0.5%) 16 (0.4%) 16 (0.4%) 15 (0.4%) 15 (0.4%) 15 (0.4%) 13 (0.4%) 13 (0.4%) 11 (0.3%) 10 (0.3%) 10 (0.3%) 10 (0.3%) 10 (0.3%) 9 (0.3%) 9 (0.3%) 9 (0.3%) 9 (0.3%) 8 (0.2%) 8 (0.2%) 8 (0.2%) 8 (0.2%) 8 (0.2%) 8 (0.2%) 8 (0.2%) 8 (0.2%) 7 (0.2%) 7 (0.2%) 7 (0.2%) 7 (0.2%) 7 (0.2%) 7 (0.2%) 6 (0.2%) 6 (0.2%) 6 (0.2%) 6 (0.2%) 6 (0.2%) 5 (0.1%) 5 (0.1%) 5 (0.1%) 5 (0.1%) 5 (0.1%) 5 (0.1%) 5 (0.1%) 5 (0.1%) 5 (0.1%)

M10119 P02794 P14780 P02570 P07355 U29615 M60258 X00497 P01884 P07339 P13284 U77035 P04720 M12592 P00695 P08962 M75161 P10451 U83171 P27706 S22697 AF006084 D87914 P01903 P00395 P23396 P02551 Q06830 NA NA M73969 NA U30877 P03886 D38112 J04942 Q02878 NA M14144 D87433 P29562 M94345 P04406 X15332 P06733 NA P09601 P36222 P09467 Q14956

Iron homeostasis Iron homeostasis Healing/repair Structural Phospholipid binding Host defense Sphingomyelin degradation Antigen presentation HLA class I transport Protease Unknown (IFN-g inducible) Immune regulation Protein synthesis Lipid metabolism Host defense Unknown (transmembrane) Healing/repair Healing/repair Immune regulation Protein synthesis Unknown Structural (actin associated) Metabolism Antigen presentation Metabolism Protein synthesis Structural Redox homeostasis NA Unknown Immune regulation Unknown Protease Metabolism Metabolism Structural Protein synthesis NA Structural Unknown Protein synthesis Structural Metabolism Structural Metabolism Unknown Metabolism Unknown Metabolism Unknown

The 50 most abundant cDNA clones identified are listed above. NA, not applicable; h, homolog of gene identified in another species. Three additional sequences were identified five times, Histone H1 (A56356), HLA Class I (U25971), and Ubiquitin (I52220).

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and a second larger class of cell-specific genes, whose presence reflect the functional roles of macrophages. For example, ferritin heavy chain and ferritin light chain, the most abundant clones, are involved in the uptake and regulation of iron, an important function of macrophages [13]; while the high levels of expression of the proteases cathepsin D and type IV collagenase reflect macrophage involvement in tissue remodeling and repair [4]. Other gene products such as chitinase and lysozyme play important roles in neutralization of pathogens. Two of the most abundant sequences identified in the macrophage library encode chemokines: macrophage-derived chemokine (MDC; 0.3% of cDNAs) and LARC [0.4% of cDNAs, also called EXODUS or macrophage inflammatory protein-3a (MIP-3a)] [14, 15]. Although both of these cDNAs are abundant in our human macrophage library, they each have very different tissue distribution patterns. As shown by Northern blot analysis, MDC is expressed almost exclusively in thymus [14], whereas LARC is expressed highest in liver and lung with almost no thymic expression [15]. The blood-derived monocytes used for our study may have differentiated into macrophages with a broader range of mRNA expression than is seen in vivo. Alternatively, blood-derived monocytes may be heterogeneous and capable of differentiating into different subsets of macrophages (e.g., thymic dendritic cells and liver Kupfer cells). This latter interpretation is supported by the functional and phenotypic heterogeneity seen in monocytes cultured under different conditions [16]. Chemokines are a large family of secreted proteins that are characterized by the presence of four conserved cysteines. The chemokine family has been subdivided into the CXC branch in which there is a non-conserved amino acid separating the first two cysteines, and the CC branch where these residues are juxtaposed. Chemokines are involved in many aspects of the immune and inflammatory response, the best characterized of which is their ability to stimulate the selective accumulation of leukocytes at sites of inflammation [17–19]. The selectivity of chemokines for particular leukocyte subsets is dependent on the repertoire of chemokine receptors expressed on the target cell. CXC chemokines act principally on neutrophils, whereas the CC chemokines are chemotactic for T cells and monocytes [19]. Chemokines are relatively small proteins (70–100 amino acids), whose mRNA is often up-regulated in response to proinflammatory stimuli. Their small coding regions make them well suited to identification using a random sequencing approach. The number of known chemokines has dramatically increased recently, primarily due to their identification by random sequencing programs [19, 20]. Although such an analysis reflects the relative overall abundance of a given chemokine, it is clearly biased by those tissues chosen for large-scale sequence analysis. For example MDC, one of the most abundant clones in our macrophage cDNA library, is not found in the public EST database.

identification of the full-length cDNA clone for this sequence we went on to examine its expression by Northern blotting. As shown in Figure 1, MDC is constitutively expressed at high levels by monocyte-derived macrophages. Expression of MDC is not induced by treatment of fresh blood monocytes with bacterial lipopolysaccharide (Fig. 1). MDC is synthesized late in the macrophage differentiation process: significant levels of expression are seen only after 6 days of macrophage differentiation [14]. This pattern of expression is in contrast to most other chemokines, which are rapidly induced after proinflammatory stimuli. For example, MDC and MCP-1 have distinct expression patterns, as shown in Figure 1. To identify the amino terminus of the mature protein, MDC was expressed in mammalian cells, the CHO cell line. Protein was purified by heparin sepharose chromatography and size exclusion chromatography before amino acid sequencing [14]. The derived amino-terminal sequence (GPYGANMEDSV) begins at amino acid 25 of the predicted protein sequence and is consistent with that predicted by Von Heijne’s rules governing the cleavage of signal sequences [21]. Mature MDC was also prepared by chemical synthesis and shown to behave identically to MDC expressed in CHO cells [14]. The biological effects of MDC have been studied on a range of cell types. As shown in Figure 2, MDC is a potent chemoattractant for dendritic cells, interleukin-2 (IL-2)activated natural killer (NK) cells, and HUT78 cells, a human T cell line [14, 24]. Maximum chemotaxis of these cell types toward MDC was seen at concentrations of MDC as low as 1–10 ng/mL. The chemotactic response of dendritic cells toward MDC is particularly noteworthy because MDC is also expressed at high levels by dendritic cells [14], suggesting an autocrine role for MDC in the accumulation of dendritic cells at sites of

CHARACTERIZATION OF HUMAN MDC One of the most abundant sequences identified in the macrophage cDNA library encoded MDC, a chemokine with about 30% amino acid identity with other CC chemokines [14]. After

Fig. 1. Expression of MDC and MCP-1 was determined by Northern blotting. RNA was prepared from freshly isolated blood monocytes cultured on plastic for 8 h in the presence or absence of 100 ng/mL bacterial lipolysaccharide and from monocytes from the same donor which were cultured on plastic for 6 days to differentiate them to macrophages.

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Fig. 2. Chemotaxis of dendritic cells, IL-2-activated NK cells and the HUT 78 T cell line to MDC (*p , 0.05; **p , 0.01). Cells were isolated and chemotaxis was performed as described [14, 24].

inflammation. Migration of T cells toward MDC may be critical for macrophage/T cell interactions. Analysis of the in vivo expression of MDC by Northern analysis revealed that it is restricted to thymus [14]. This expression pattern is similar to that of several other recently identified chemokines, TARC [22], TECK [25], and PARC (also known as DCCK-1) [26, 27]. TARC is the chemokine most closely related to MDC (37% identity). It is interesting to note that the genes for these two chemokines colocalize to chromosome 16q13 [23, 24], unlike the majority of CC chemokines, which reside on chromosome 17 [19]. The identification of CCR4 as a receptor for TARC led us to examine the interaction of MDC with CCR4 [24]. To facilitate MDC binding studies, we generated a fusion protein in which the MDC coding region was fused to the 5’ of the secreted alkaline phosphatase coding sequence and is described in detail in Imai et al. [24]. As shown in Figure 3, MDC-SEAP binds to CCR4 with high affinity (Kd 5 0.3 nM). Furthermore, CCR4-transfected cells respond to

MDC by calcium flux (Fig. 3) and chemotaxis [24]. Comparison of the binding and chemotaxis of CCR4-expressing cells in response to MDC and TARC revealed that MDC is the more potent ligand for this receptor [24]. Recent studies have shown that CCR4 is expressed selectively on T cells of the Th2 subset [28]. Th2 cells make principally IL-4 and IL-10 and are thought to predominate in the response to parasitic infections and to play a role in allergic inflammation [29]. Chemokines such as MDC and TARC therefore may play a role in the migration of Th2 cells.

CHEMOKINES AND HIV In addition to their well-characterized roles in cell migration, chemokines are an important component in the host response to infection. This is highlighted by the frequency with which chemokines and their receptors have been captured into the

Fig. 3. Binding characteristics of MDC-SEAP to CCR4 and calcium flux of CCR4-expressing cells in response to MDC. Left panel, saturable binding of MDC-SEAP to L1.2 cells expressing CCR4. Cells were incubated with increasing amounts of MDC-SEAP and the cell-associated SEAP activity present after washing was determined enzymatically. Scatchard analysis of the saturable binding data is shown in inset. Right panel, calcium mobilization of CCR4-transfectants stimulated with MDC. L1.2 cells transfected with CCR4 were loaded with Fura-2/AM and then stimulated with 10 nM MDC. Intracellular concentrations of calcium were monitored by the fluorescence ratio (F340/F380). Binding assays and measurements of intracellular calcium concentrations were performed as described in Imai et al. [24].

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genomes of viruses. The genome of the g-herpes virus HHV8 (also known as the Kaposis’ Sarcoma-associated herpes virus, KSHV) encodes three chemokines and one chemokine receptor, suggesting a central role for chemokines in this virus/host interaction [30, 31]. Viruses also may utilize chemokine receptors to enter host cells. In particular, HIV-1 requires the presence of chemokine receptors (along with CD4) to enter a target cell. The cellular tropism of HIV is complex but can be broadly divided into non-syncytium inducing (NSI) or macrophage-tropic and syncytium inducing (SI) or T cell line-tropic strains. NSI viruses make use of CCR5 (a receptor for the CC chemokines MIP-1a, MIP-1b, and RANTES) to enter cells and are capable of infecting both T cells and cells of the monocyte/ macrophage lineage [32, 33]. This form of the virus predominates early in infection and appears to be essential for HIV to become established in the host. Individuals that are homozygous for an inactivating mutation in CCR5 are protected from HIV infection in the face of repeated exposure to the virus [34]. The SI viruses appear during the end stage of the disease, infect primarily T cells, and use CXCR-4 (the receptor for the CXC chemokine SDF) for cell entry [35]. In vitro, the natural chemokine ligands for these receptors can block viral entry by direct competition for the receptor [36]. Recent studies by Gallo and colleagues have identified MDC as a potent HIV suppressive factor secreted by CD81 T cells [37]. In contrast to the CC chemokines MIP-1a, MIP-1b, and RANTES, which selectively inhibit macrophage-tropic strains of HIV through a passive blockade of CCR5, MDC inhibits the replication of both NSI (macrophage-tropic) and SI (T cell line-tropic) isolates of HIV-1 [37]. MDC, which antagonizes a broader spectrum of virus subtypes, may have greater therapeutic potential than strategies directed at inhibiting individual co-receptors. It is noteworthy that MDC is expressed by macrophages and dendritic cells [14], cells that are intimately involved in the initial interaction of the host with HIV at mucosal interfaces. These cells also act as a major reservoir of the virus in asymptomatic individuals [38]. The mechanism of MDC-mediated HIV inhibition has yet to be determined, but in light of the fact that the only receptor for MDC that has so far been identified is CCR4 [24], it seems unlikely to be due to a direct competition with the virus co-receptors.

SUMMARY Macrophages play an important role in both chronic and acute inflammatory responses, as well as serving as scavengers to remove cell debris and toxic materials. Macrophages are also key players in general homeostasis, including iron and lipid regulation. By analyzing the RNA transcripts in macrophages, we have provided a snapshot of the proteins needed for the smooth functioning of these diverse cells. These proteins include many proteins that are known to be essential for established macrophage activities such as ferritin. There are also several novel transcripts that encode previously undescribed proteins such as MDC. Study of these proteins may give us further insight into the function of the macrophage and direct us in new paths for

developing treatments for inflammatory and infectious diseases.

ACKNOWLEDGMENTS We would like to thank Dina Leviten and Marsalina Quiggle for oligonucleotide synthesis and DNA sequencing.

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