Expression Profile of Active Genes in Granulocytes

GRANULOCYTES ARE short-lived cells for body defense, with a half-life of 6 to 7 hours in blood. Because of this short life span, their protein synthetic apparatus is poorly developed,1 2 but they maintain active genes that are responsible for their unique activities. These genes have been the focus of intensive study.

We have initiated a systematic survey of active genes, as well as the relative abundance of mRNA expression, in granulocytes using an expression profiling method that is based on quantitative analysis of mRNA populations.3 This is performed by using 3′-directed cDNA libraries that faithfully represent the mRNA population and by obtaining short base sequences just upstream of polyA, called gene signatures (GSs), by single-pass sequencing of randomly selected clones from such libraries.3-5 Active genes are identified by sequences and the gene activities are identified by their recurrences. The resulting list showing the expressed gene species and the abundance of their transcript is called an expression profile, which illuminates the gene-product–based cellular phenotype.

In the profile are represented several known genes as well as novel genes. These genes can be categorized as those that are commonly expressed in different types of cells (candidate genes for housekeeping functions) and those that are expressed uniquely in granulocytes (candidate genes for granulocyte-specific functions). We used 11 expression profiles obtained with different human cells/tissues for the gene categorization and discovered some genes that are likely to be granulocyte-specific, even though we do not yet know their functions.

A promyelocyte cell line, HL60,6 is converted into granulocytoid cells by treatment with dimethylsulfoxide (DMSO). The cells stopped proliferating, the nucleus became pyknotic and polymorphic, and the cells expressed several genes unique to granulocytes. Thus, because they are morphologically and functionally similar to granulocytes, the cells are referred to as granulocytoids and are widely used as a model system for studies of granulocytes.7-10 We monitored the expression profile of these cells. Comparison of this profile with that of the peripheral granulocytes showed advantages and disadvantages in using the granulocytoids as a model system.

Freshly obtained venous blood was diluted with the same volume of phosphate-buffered saline (PBS), and the suspension was centrifuged on a ficoll step gradient (upper density, 1.077; lower density, 1.119) for 20 minutes at 3,000g.11 12 Granulocytes collected from the interface were washed twice with PBS and resuspended in the same buffer. The purity of the preparation was examined under a microscope upon Giemsa staining. At least 99% of total cells were mature granulocytes.

HL60 cells (Japanese Cancer Research Resource Bank, Osaka, Japan) were grown in RPMI 1640 medium (Nissui, Tokyo, Japan) supplemented with 10% (vol/vol) bovine fetal serum (Hyclone, Logan, UT) and harvested in the logarithmic phase (10⁶ cells/mL). Granulocytoid cells were prepared by seeding HL60 in a plate at a concentration of 2 × 10⁵cells/mL and incubating in the presence of 1.3% (vol/vol) DMSO (Sigma, St Louis, MO) for 72 hours.

RNAs were prepared from the cytoplasmic fraction of DMSO-induced HL60 cells as described3 and from the total cell lysate13 of granulocytes. Purification of polyA RNA was not attempted, because of the presence of RNase. Construction of the 3′-directed cDNA libraries and transformation intoEscherichia coli were performed by synthesizing cDNA using pUC19-based vector primer, digesting with dam-sensitive four-base cutter Mbo I, followed by circularization and transformation into E coli.4 The transformant colonies were randomly selected and cultured in 96-well plates, and the inserted cDNAs were amplified with flanking primers and subjected to cycle sequencing.

The polyA tail was removed from the sequencing data after checking the electropherograms, leaving 3 As as a marker. From the resulting sequence data, those having inserts shorter than 20 bp or those having more than 5 ambiguous bases (N) within the initial 100 bases were discarded. The sequences of the remaining clones were truncated where the N content exceeded 5%, and the final N was replaced by an X to mark the point of truncation. The resulting sequences are referred to as GSs.

The GSs were compared using the FastA program.14 Identical signatures were lumped together, and a representative sequence that had the lowest content of ambiguous bases was selected to represent the group and deposited in the DDBJ, wherein the locus name corresponds to the GS number (such as HUMGS01234 to GS01234). All the representative GSs were searched against GenBank (Re95) using the FastA program,14 and those that had greater than 90% similarity to the 3′ end of the mRNA entries or to the reported terminal exon of genes were regarded as representing the corresponding genes.

From the 3′-directed cDNA library constructed from human peripheral granulocytes, we randomly selected 1,142 independent clones and sequenced them. Among the resulting short sequences called GSs, representing just upstream of the polyA, sequences that were considered essentially identical were lumped together to represent the same gene species. After this treatment, 748 independent GS species resulted. Among them, 216 (28.9%) represented by 493 clones were identified in GenBank (Re95), and the remaining 532 (71.1%), represented by 649 clones, were from novel genes. Table 1A shows an expression profile of active genes as represented by their GSs and their activities with their relative abundance. We listed here only those 64 GSs that appeared 3 times or more in descending order of appearance. Those genes that appeared twice or less can be seen in www bodymapper server (http://www.imcb.osaka-u.ac.jp/bodymap). We believe that this is the first publication describing relative activities of genes in granulocytes that are expressed abundantly. The profile reflects several unique features of the granulocytes physiology. First, it includes several genes that have been well known in peripheral neutrophils, such as genes for β2-microglobulin,15granulocyte colony-stimulating factor receptor,16-18 major histocompatibility complex (MHC) class I, and so on. Genes whose activity has been detected in granulocytes15-36 are marked with asterisks. High activities of genes for spermidine/spermine N1-acetyltransferase, pre-B–cell enhancing factor (PBEF), and B94 protein are also noted. The purity of the source material guarantees that this result reflects the relative activities of those genes.

We categorized the 493 known genes active in the granulocytes into subgroups according to their function and subcellular localization. The results are collectively shown in Table2. The most prominent feature of granulocytes is the high activity with genes for cell surface membrane components. Thirty GS species represented by 114 clones (Table 2J), amounting to 10% of the mRNA population, were of this category. Genes for nuclear DNA binding protein, components for secretory protein, and components for signal transduction were also noticeably active. Genes for energy production, lysosomal proteins, protein synthesis machinery, and cytoskeleton are not so active in the granulocytes, as had been expected.

An expression profile of the granulocytoid cells is represented in Table 1B (column GR). In the same table are collectively displayed the relevant gene activities with HL60 cells (HL) and the monocytoids derived from HL60 by tetradecanoylphorbol-13 acetate (TPA)10 treatment (MO). Comparison of Table 1B with Table1A or column PM versus GR in Table 1A with column GR versus PM in Table1B shows that about 50% (24/48) of the abundantly expressed genes in the DMSO-induced HL60 are also present in peripheral granulocytes, although the abundances differ. Scarcity of highly abundant transcripts is characteristic of the mRNA population in granulocytoid cells. Generally, genes for cytoskeleton and protein synthesis machinery are moderately active, unlike genes for cell surface membrane components in granulocytoid cells. Further discussions will be presented in the Discussion.

We have prepared expression profiles of active genes in 11 other human cells/tissues.3,10 37 Genes listed in Table 1A and B were extracted from each of these profiles, and their activities (abundance of the transcripts among 1,000 mRNA molecules) were compiled. The resulting Table 1A, although incomplete, allows us to categorize genes into those whose expression is peripheral granulocyte-specific, limited to certain types of cells, or ubiquitous. When the genes have been detected in 6 libraries or more, we categorized them as ubiquitous (solid area in column “lib”). Genes known to perform house-keeping functions, such as ubiquitin or ribosomal proteins, are seen in this category. A gene expressed only in granulocytes or in granulocytes and/or granulocytoid cells may be categorized as unique to this type of cell (open area). The rest were categorized as common or intermediate (hatched area). We categorized 22 GSs as unique, among which 12 were identified in GenBank and 10 represent novel genes. Among the 12 known genes are granulocyte colony-stimulating factor receptor, interleukin-8 (IL-8), leukocyte common antigen T200 (CD45), and ICAM-3, which have been known to act mainly in granulocytes. No data have been reported so far as to the cell type specificity of the remaining 8 genes. Thus, at least one third of the genes in the unique category were indeed those that represent specific functions of the granulocytes. We argue that we can extrapolate this finding to the novel genes.

As a result of a rapid expansion in the collection of expressed sequence tags (ESTs), more than 400,000 fragmentary human cDNA sequences from more than 20 tissues have been collected in dbEST. This database can be readily compared with the expression profiles as described here, because the quality of the source cells for this library construction has not been biased and because the cDNA libraries that have been subjected to normalizing protocol do not reflect the composition of the mRNA population in the original source cells/tissues. Nevertheless, they can provide some information as to what RNA species are present in tissues so far examined. We queried the 22 GS sequences that were categorized as unique with dbEST. The results, shown in Table 3, demonstrate that 5 of them, GS08339 (IL-8), GS05242, GS01594 (leukocyte common antigen T200), GS08389 (E4BP4 gene), and GS08424 have not been registered in dbEST. Considering that no granulocytes or related tissues were used for the EST collection, it is not surprising that these sequences failed to appear in dbEST. From an opposite point of view, the absence in dbEST strengthens the idea that such genes are unique to the granulocytes. Five GSs, GS08362 (granulocyte colony-stimulating factor receptor), GS08347, GS08438 (Nramp), GS08336 (secretory granule proteoglycan peptide), and GS08375 (MAD-3) matched ESTs from fetal liver spleen, placenta, lung, and other tissues. This observation shows the limit of the application of dbEST for the categorization under discussion: it may simply show that tissues used in the dbEST data construction contained some granulocytes. As with novel genes, one GS (GS08347) is highly likely to represent a gene that is unique to granulocytes. The other 9 genes were subjected to further examination, because they were found recurrently in tissues not related to granulocytes in the dbEST.

Granulocytes are a major player in the defense of the body against foreign materials. About 90% to 95% of granulocytes are neutrophils, with the remainder being eosinophils and basophils in circulating human blood. Hence, peripheral granulocytes represent the activities of neutrophils.1 The cytoplasm of these cells has highly developed cytomatrixes, as well as granules that contain microbicidal proteins and digestive enzymes. The plasma membranes carry a number of receptors and other structures needed for recognition and disposal of invading pathogens.2 

Although gene activities are not necessarily reflected by the abundance of mRNA, other methods being not available (except for quantitizing two-dimensionally separated protein bands), gene expression profiling3 leads to the best approximation. Table 1A shows several genes well studied in conjunction with the functions in granulocytes. The quantitative ratios should help us understand the regulatory systems acting in the granulocytes. An abundant expression of genes for cell surface membrane proteins drew our attention; eg, genes for β2-microglobulin, MHC class I HLA-Cw, and HLA-E heavy chain, which are components of cell surface receptors. That a lot of genes for cell surface membrane proteins are active in granulocytes supports the notion that granulocyte responses can be evoked by a variety of stimuli caused by particulate and soluble materials. On the other hand, most of the genes for cytoskeleton were not so active, except those for thymosin β4 and β-actin, in accord with the notion that these cells do not maintain a rigid shape.

The list has shown several genes not known to be active in granulocytes. This study points out the importance of elucidating the role of gene products in granulocytes. B94 protein and B4-2 protein are good examples. Expected changes in the expression of genes in association with inflammation or changes in adhesive properties during chemotaxis and phagocytosis remain to be examined. Fibronectins, β2-integrins, and the L-selectins, which are notably associated with adhesiveness as mediators,2 were not detected in our expression profile. Actins, which play important roles in production of pseudopodium for locomotion, were not expressed strongly. Activation of these genes is yet another feature of the activation of granulocytes worth investigating. Genes for chemotactic factor receptors were moderately expressed, including tumor necrosis factor receptor, N-formylpeptide receptor, C5a anaphylatoxin receptor, and IL-8 receptor. On the other hand, expression of receptors for C3b and C3bi were not detected, in line with the fact that our granulocytes were in a resting stage.38 Here again, examination of the profile in induced cells, including the time course of activation and their relative order of activities, is of utmost interest.

Genes for secretory proteins, such as cytokines, are not particularly active in circulating granulocytes and, indeed, only 2 genes, for pre-B–cell colony-enhancing factor and IL-8, were detected. Thus, the relative activities of these important granulocyte-specific genes in resting cells have been determined. As with secretable bactericidal components, there were cathepsin S, neutrophil oxidase factor (NCF2)/p67-phox, proteasome subunit p40, and defensin, in addition to lysosomal proteins. Thus, these proteins are constitutively produced at a level of 4.0% or more of total protein synthesis. Bactericidal/permeability-increasing protein (BPI) was found in granulocytoid cells, but not in the peripheral granulocytes, probably because its expression level is just at the border of the level of detection.

Whereas active expressions of genes for cell surface membrane proteins, including receptors for chemotactic factors as well as genes for bactericidal proteins such as lysosomes, are characteristic to our granulocytes, so is poor expression of components for protein synthesis machinery, as it is for cells in the resting stage.

Among the mRNAs in granulocytes identified in GenBank (Table 1A) are genes for granulocyte colony-stimulating factor receptor, tumor necrosis factor receptor, and T200. These gene products are related to neutrophilic granulopoiesis and their maturation. Thus, these findings strongly suggest that granulocytes in circulating blood wait for stimuli exposing granulopoietic receptors. The list also included IL-8, a neutrophilic chemoattractant and activator. In contrast to IL-8 receptor (Table 2J), IL-8 is highly and specifically expressed. This mRNA has been known to be induced in neutrophils in response to granulocyte/macrophage colony-stimulating factor.31,39Although granulocyte colony-stimulating factor regulates the expression of IL-8 receptor,40 whether granulocyte colony-stimulating factor can induce the expression of IL-8 is not clear. Our results indicate that granulocyte colony-stimulating factor induces the expression of IL-8 mRNA.

Among the 22 genes categorized as unique in Table 1A, 10 were novel genes. It is of utmost importance to characterize these genes, although such categorization can be performed only with abundantly expressed genes, and yet some misleading categorizations are unavoidable due to the limited number of GS collections.

β2-microglobulin and HLA-E heavy chain were commonly expressed in granulocytes and granulocytoid cells. Proteins in lysosomes, BPI, and leukocyte adhesion protein (Mac-1), which are known to be expressed in neutrophils, were also expressed in granulocytoid cells. In total, 20% or more of the mRNAs are commonly expressed in both types of cells (data not shown).

However, their relative proportions in granulocytoids differ from those in granulocytes. In general terms, expression profiles of genes in granulocytes and granulocytoid cells differ from each other qualitatively and quantitatively. In Table 1A, 24 gene species represented by 74 clones were commonly expressed in granulocytes and granulocytoid cells. These observations demonstrate the capacity and limitations of this model system.