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Originally published online as doi:10.1189/jlb.0507273 on August 20, 2007

Published online before print August 20, 2007
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(Journal of Leukocyte Biology. 2007;82:1353-1360.)
© 2007 by Society for Leukocyte Biology

Novel interferon-β-induced gene expression in peripheral blood cells

M. R. Sandhya Rani*, Jennifer Shrock*, Swathi Appachi*,1, Richard A. Rudick*,{dagger}, Bryan R. G. Williams{ddagger} and Richard M. Ransohoff*,{dagger},2

* Neuroinflammation Research Center, Department of Neurosciences, Lerner Research Institute, and
{dagger} Mellen Center for MS Treatment and Research, Cleveland Clinic, Cleveland, Ohio, USA; and
{ddagger} Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia

2 Correspondence: Neuroinflammation Research Center, Department of Neurosciences, Lerner Research Institute, NC30, Mellen Center for MS Treatment and Research, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA. E-mail: ransohr{at}ccf.org


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ABSTRACT
 
Type I IFNs are used for treating viral, neoplastic, and inflammatory disorders. The protein products encoded by IFN-stimulated genes (ISGs) likely mediate clinical effects of IFN in patients. Macroarray assays, used for studying ISG induction in IFN-treated patients, comprise genes identified predominantly through analysis of long-term cell lines. To discover genes induced selectively by IFN-β in PBMC, we exposed whole blood to physiological concentrations of IFN-β. PBMC were prepared, and RNA was extracted, reverse-transcribed, and hybridized to cDNA microarrays, and microarray analysis identified 39 ISGs and 20 IFN-repressed genes (IRGs). Thirty-three ISGs were known previously, and six ISGs were novel. New ISGs included GTP cyclohydrolase 1; hypothetical protein LOC129607; hypothetical protein FLJ38348; leucine aminopeptidase 3; squalene epoxidase; and GTP-binding protein overexpressed in skeletal muscle. Twenty IRGs included IL-1β and CXCL8, which had been identified earlier. CXCL1 was a novel IRG identified in the current study. PCR analysis demonstrated the regulation of six novel ISGs and CXCL1 as an IRG in PBMC and astrocytoma cells. Results were validated using RNA obtained ex vivo from blood of patients after injection with IFN-β. Identification of new ISGs and IRGs in primary PBMC will enhance macroarray assays for monitoring IFN responsiveness.

Key Words: microarray • IFN-stimulated genes • IFN-repressed genes • multiple sclerosis


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INTRODUCTION
 
Type I IFNs are used for treatment of a wide spectrum of infectious, neoplastic, and inflammatory diseases [1 2 3 4 ].

In many instances, IFN therapy is partially effective. Therapeutic efficacy may be limited by disease-specific factors such as the hepatitis C virus (HCV) genotype. The host response to IFN might also vary among individuals, and differential induction of IFN-stimulated genes (ISGs) could contribute to the spectrum of clinical effects [5 ].

Progress in defining individual responses to IFN was notably accelerated by Schlaak, Kerr, and colleagues [6 , 7 ], who developed and validated a robust, convenient, quantitative macroarray assay for ISG characterization. One limitation is that ISGs incorporated in this assay were identified by microarray analysis in long-term, cultured epithelial cell lines, whereas the target cells for IFN response in vivo are largely blood cells [7 8 9 ].

The current study was undertaken to identify ISGs induced in primary PBMC by IFN-β. We used a cDNA microarray platform for analysis of gene expression [10 ]. A novel approach of treating whole blood in vitro with IFN-β followed by separation of PBMC was developed. Six novel ISGs and one IFN-repressed gene (IRG) were identified and validated by PCR analysis. Variations in the expression of these genes were observed in ex vivo blood of multiple sclerosis (MS) patients after IFN-β injection, suggesting differences in the molecular signatures of individual responses to IFN-β.


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MATERIALS AND METHODS
 
Blood collection and RNA extraction
Blood was collected from healthy donors into heparinized tubes and exposed to IFN-β in vitro. Eight tubes of blood (8 mL each) were obtained from each of three healthy donors. Two tubes were untreated and reserved as controls. Blood samples were treated for 2, 6, or 12 h with recombinant IFN-β (rIFN-β; 200 U/mL) and placed in the tissue-culture incubator at 37ºC. At the end of treatment, PBMC were isolated from heparinized blood by density centrifugation using a lymphocyte separation medium (Mediatech, Herndon, VA, USA) as described previously [11 ]. RNA was extracted from the PBMC using the TRIzol reagent (Promega, Madison, WI, USA) as described earlier [12 ].

Microarray analysis
The cDNA microarray (Genomics Core, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA) was used to analyze gene expression for the purpose of finding new ISGs and IRGs. We used the human IAD-v2 array, which has approximately 3000 spotted, unique cDNAs, 850 IFN-regulated genes, 130 dsRNA-induced genes, 1400 genes containing AU-rich regions in the 3'-UTR, and 750 p53-regulated, cancer-related genes.

The current study used cDNA microarrays containing DNA fragments of 500–1500 bp. Two cDNA samples (control and experimental) were labeled with fluorophores Cy5 and Cy3 and competitively hybridized to a single microarray slide. The cDNA microarray output data provided a ratio of the gene expression levels for each RNA in the experimental versus the control sample, directly measured on a single slide. The induction ratio (IR) was calculated by dividing the actual fluorescence intensity numbers of the sample by the control. ISG up-regulation is any ISG with an IR >2.0, and IRGs have an IR <1.0.

PCR analysis
RNA was reverse-transcribed and PCR-amplified using gene-specific primers. The DNA sequences for the primer pairs for ISGs and IRGs analyzed are shown in Table 1 . The cycling conditions were 94ºC, 1 min (denaturation); 57–60ºC, 1 min (annealing); and 72ºC, 2 min (extension). The number of cycles of amplification and the expected size of the PCR products are also shown in Table 1 . GAPDH mRNA was analyzed by RT-PCR in each sample to confirm the presence of intact mRNA.


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  		Table 1. The Primer Sequences and PCR Conditions Used for Analysis of ISGs and IRGs Induced/Repressed by IFN-β in PBMC

Extraction of RNA from whole blood
Blood (20 ml) was collected directly into PAXgeneTM tubes, according to the manufacturer’s instructions from five newly diagnosed MS patients 12 h before and 12 h after a first intramuscular injection of 6 million IU IFN-β1a. The Institutional Review Board of the Cleveland Clinic approved the study, and written, informed consent was obtained from all individuals enrolled in the study. RNA was extracted ex vivo from blood using the PAXgene RNA blood extraction kit (PreAnalytix, Switzerland), as per the manufacturer’s instructions and concentrated by ethanol precipitation.

Isolation of RNA from astrocytoma cells
The CRT astrocytoma cell line was described previously and was derived from a Grade IV human astrocytoma [13 ]. Cells were maintained in RPMI-1640 medium supplemented with 5 mM glutamine and 10% FBS. The cells were treated with and without rIFN-β (1000 U/mL) for 6 h, and RNA was extracted using the TRIzol reagent (Promega) as described earlier [12 ].


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RESULTS
 
cDNA microarray analysis of IFN-β-treated PBMC
To identify ISGs induced in PBMC by IFN-β, we incubated whole blood obtained from a healthy control with 200 U/ml IFN-β. This approach was intended to mimic in vivo conditions in blood after an IFN injection and preserves cell–cell interactions, which occur in whole blood, incubated with IFN-β for 2, 6, or 12 h at 37ºC in an incubator. PBMC were prepared, and total RNA was extracted.

Identification of novel ISGs induced in PBMC by IFN-β
cDNA microarray analysis identified 39 ISGs to be induced in all three healthy controls (Table 2 ) and an additional 10 ISGs (not shown), which were differentially induced. The kinetic analysis and the average fold induction and SDs of several ISGs are shown in Table 2 . The fold induction and kinetics of mRNA accumulation varied from gene to gene as described previously [14 ]. Subsequent experiments used a treatment time of 6 h (in vitro) or 12 h (ex vivo).


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  		Table 2. cDNA Microarray Analysis of ISGs Induced in IFN-β-Treated Blood Cells

The results obtained by array analysis for novel genes shown in bold in Table 2 were validated using RT-PCR analysis. RNA was isolated from IFN-β-treated PBMC or astrocytoma cells. CD69 was robustly induced in IFN-β-treated PBMC but not in astrocytoma cells (Fig. 1A and 1B ). This gene is also induced by polyinosine:polycytidylic acid, a double-stranded, RNA mimetic, which induces IFNs [15 ]. The CD69 glycoprotein is an early activation antigen of T and B lymphocytes, but its expression is induced in vitro on cells of most hematopoietic lineages [16 ]. Our results using astrocytoma cells are consistent with the restriction of CD69 expression to hematopoietic cells.


Figure 1
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  		Figure 1. Gene expression in PBMC treated in vitro with IFN-β. (A) Novel ISGs induced in PBMC treated with IFN-β. Whole blood was incubated in vitro with 200 U/mL IFN-β (β) or without IFN-β (C) for 6 h, and RNA was extracted from PBMC. The astrocytoma CRT cells (Astro) were also incubated with 1000 U/mL IFN-β or without IFN-β for 6 h, and RNA was extracted. RT-PCR was performed for CD69, FLJ, GCH1, GEM, LAP3, LOC1, SQLE, and GAPDH. The primer sequences and PCR conditions used are listed in Table 1 . The figure shows ethidium bromide staining of the PCR-amplified fragments separated on a 1% agarose gel from one representative experiment out of three. The first lane is a negative control for PCR, which has primers and DNA polymerase but no template. (B) Quantitation of ISG induction in IFN-β-treated PBMC. The intensity of the ISG and GAPDH bands induced in PBMC, shown in A, was quantitated on a StormImager using Imagequant software (Molecular Dynamics, Sunnyvale, CA, USA). The densitometric readout for individual ISG is divided by the densitometric readout for GAPDH. The IFN-β-induced, ISG-normalized readout divided by the untreated, control-normalized readout constitutes the IR for that particular ISG. Results are expressed as fold induction compared with untreated controls, set at 1 (C). (C) Quantitation of ISG induction in IFN-β-treated astrocytoma cells. The intensity of the ISG bands induced in the astrocytoma cells shown in A was quantitated as explained for B.

Two hypothetical proteins, LOC1 and FLJ, were induced in IFN-β-treated PBMC and astrocytes (Fig. 1A 1B 1C) . National Center for Biotechnology Information (NCBI) Blast analysis using the nucleotide sequence of the above two proteins did not yield homology to known mRNAs. Open-reading frame analysis of the nucleotide sequences of these two genes generated partial protein sequences. Blast analysis using the partial protein sequence of LOC1 revealed extensive homology to thymidine kinase (TK) [17 ]. The partial protein sequence of FLJ did not reveal homology to known proteins.

The GEM was induced by IFN-β in PBMC but was constitutively expressed in the astrocytoma cells (Fig. 1A 1B 1C) .

Three of the mRNA transcripts induced (GCH1, LAP3, and SQLE) encoded enzymes, and PCR analysis confirmed their induction in IFN-β-treated PBMC and astrocytoma cells (Fig. 1A 1B 1C) . Unlike other enzymes, whose expression was tightly controlled by IFN-β, SQLE was constitutively expressed in the astrocytoma cells but not PBMC.

Down-regulation of IRGs in PBMC treated with IFN-β
Among 20 IRGs repressed by IFN-β, we noted CXCL8 and IL-1β, which were reported by others (Table 3 ). The average fold repression of IRGs with SDs is shown in Table 3 . It is interesting that we observed down-regulation of CXCL1, which is functionally related to CXCL8 (Table 3) . PCR analysis confirmed the down-regulation of CXCL1 by IFN-β in PBMC (Fig. 2A and 2B ). The repression of CXCL1 by IFN-β in PBMC is more impressive than that observed in microarray experiments (Fig. 2A and 2B , and Table 3 ). In astrocytoma cells, varying sizes of CXCL1 transcripts were detected, possibly as a result of differential splicing (Fig. 2A) . Further studies are required to confirm the above observations. Also, we observed that the larger form of CXCL1 mRNA showed repression by IFN-β in astrocytoma cells, and the smaller isoform was induced (Fig. 2A) . Hence, only the expression of CXCL1 in PBMC from three independent experiments was quantitated, and the results are shown in Figure 2B . In the IFN-β-treated PBMC, the repression was 30% compared with untreated controls and was statistically significant (P<0.05; paired t-test; Fig. 2B ). Unlike quantitative PCR, the RT-PCR assay used in the current study is only semiquantitative and is sufficient for confirming differences between untreated and treated samples. Quantitation of the ethidium bromide-stained agarose gel DNA bands from our assay will yield lower induction numbers as a result of quantitation of DNA bands after completion of a definite number of PCR cycles when the differences are not as dramatic compared with quantitation during the linear range of PCR amplification.


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  		Table 3. cDNA Microarray Analysis of IRGs in IFN-β-Treated Blood Cells


Figure 2
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  		Figure 2. CXCL1 repression in PBMC treated in vitro with IFN-β. (A) CXCL1 was repressed in PBMC treated with IFN-β. The figure shows an ethidium bromide staining of PCR-amplified CXCL1 and GAPDH, separated on a 1% agarose gel. NCBI Blast analysis of the protein sequence of the hypothetical protein LOC1 showed homology to TKs, which play key roles in the synthesis of DNA and in cell division [18 ]. The mRNA for LOC1 was induced by IFN-β in PBMC and astrocytoma cells (Fig. 1A 1B 1C) . The lanes are as explained in Figure 1A , and the primer sequence and PCR conditions used for CXCL1 are shown in Table 1 . (B) Quantitation of CXCL1 expression in PBMC. The intensity of the bands was quantitated as explained in Figure 1B . The results are expressed as fold repression compared with untreated controls set at 1 (C). The figure shows the average repression of CXCL1 derived from three independent experiments and was statistically significant (*, P<0.05; paired t-test).

Ex vivo confirmation of novel ISGs and IRGs after IFN-β injection
To address whether injections of IFN-β regulated these ISGs and IRGs in blood cells, we analyzed whole blood RNA extracted directly ex vivo from five MS patients, newly beginning therapy with IFN-β.

All five MS patients responded to IFN-β, as revealed by the induction of ISGs (Fig. 3A and 3B ). It is interesting that there was differential induction of novel genes in MS patients. LOC1 was induced in all five patients. Four of five patients showed induction of FLJ, GCH1, and LAP3 (Fig. 3A and 3B) ; CD69 was induced in three of five patients; and SQLE was induced in two patients (Fig. 3A and 3B) . Variable responses were also observed for CXCL1, which was repressed in four of five MS patients (Fig. 4A and 4B ). These results are in agreement with our hypothesis that regulation of ISGs and IRGs will vary among patients and that the molecular responses to IFN-β might account for individual therapeutic responses.


Figure 3
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  		Figure 3. Ex vivo confirmation of novel ISGs in whole blood RNA obtained from five MS patients. (A) Induction of ISGs in RNA prepared from whole blood of MS patients newly beginning therapy with IFN-β. Blood was drawn 12 h before (C) and 12 h after injection with IFN-β (β). RNA was extracted ex vivo from blood, reverse-transcribed, and amplified by PCR using gene-specific primers and separated by electrophoresis on a 1% agarose gel. PCR analysis was performed for CD69, FLJ, GCH1, LAP3, LOC1, SQLE, and GAPDH. The first lane is a negative control for PCR. The five MS patients are referred to as 2, 13, 22, 26, and 28. The results shown are representative of one experiment out of three. (B) Quantitation of ISG induction in whole blood RNA from MS patients. The intensity of the bands shown in A was quantitated on a StormImager using Imagequant software (Molecular Dynamics). Quantitation was done as explained in Figure 1B . The results are expressed as fold increase compared with untreated controls set at 1 (C).


Figure 4
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  		Figure 4. Ex vivo confirmation of repression of CXCL1 in whole blood RNA obtained from five MS patients. (A) CXCL1 was repressed in whole blood RNA from MS patients after treatment with IFN-β. The lanes are as explained in Figure 3A , and the expression of CXCL1 is shown. (B) Quantitation of CXCL1 expression in whole blood RNA from MS patients. The intensity of the bands from the gels shown in A was quantitated as explained in Figure 1B . The results are expressed as fold repression compared with untreated controls set at 1 (C).


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DISCUSSION
 
The goal of the current study was to expand the number of ISGs on a custom cDNA macroarray (which already has 200 ISGs) used to characterize the molecular response to IFN-β in MS patients. We focused on genes induced in PBMC, the elements in whole blood, which are most relevant in MS and are direct targets of IFN-β treatment. High-density oligonucleotide gene expression arrays have been used to identify ISGs from long-term, treated, tissue-culture cell lines derived mainly from epithelial or fibroblast cells [8 ]. Using two different microarray formats, over 300 ISGs have been identified [7 , 10 ].

Our long-term goal is to identify molecular biomarkers of the therapeutic response to IFN-β in MS patients. MS was selected for these studies according to several considerations. In particular, although patients with HCV or HBV or cancers, such as chronic myelogenous leukemia or hairy cell leukemia, are also treated with IFNs, MS patients typically have fewer comorbidities, making the interpretation of results more straightforward. Also, the clinical response to IFN for HCV or HBV can be followed by monitoring for virus load. Tumor burden can be monitored in the case of neoplasia. However, in MS, there are no known biomarkers for predicting the response to IFN.

MS is an inflammatory CNS disorder of unknown pathogenesis. IFN-β injections are partially effective for treating MS, and the therapeutic mechanism is unknown. We reasoned that relating patterns of ISG induction to therapeutic response would provide insight into MS pathogenesis and identify biomarkers of the therapeutic response to IFN for this disorder. Using the protocol and gene array described by J. F. Schlaak, C. M. Hilkens, A. P. Costa-Pereira, B. Strobl, F. Aberger, A. M. Frischauf, and I. M. Kerr (unpublished results), we constructed a customized cDNA macroarray consisting of ~200 ISGs. We are studying the ex vivo molecular response to IFN-β in a longitudinal cohort, beginning at the time that patients initiate IFN-β therapy. The genes included on our cDNA macroarray were identified originally from microarray analysis of fibrosarcoma or endothelial cell lines treated with IFN-β [7 8 9 ].

The features of interest in the present study were using a focused human cDNA microarray containing a comprehensive compilation of reported ISGs, dsRNA-induced genes, and genes with 3' UTR, AU-rich regions, many of which are cytokines and treating whole blood with IFN-β (in vitro) and then isolating the PBMC, which helped preserve the cell–cell interactions and aimed to mimic authentic treatment responses; the novel ISGs identified in the blood cells have been confirmed in ex vivo blood of MS patients on IFN-β by PCR analysis.

Our gene expression analysis using cDNA microarray in PBMC isolated from whole blood treated in vitro with IFN-β revealed 39 ISGs (Table 2) . These included well-characterized ISGs such as IFIT1/p56, IFIT2/p54, 6–16, 9–27, 2',5'-oligoadenylate synthetase, TRAIL, and CXCL10/IFN-inducible protein 10, lending confidence to the analysis [8 ]. We also identified ~20 genes, which were significantly down-regulated in blood after treatment with IFN-β and are candidate IRGs (Table 3) . These included genes such as IL-1β and CXCL8/IL-8, which have been shown to be repressed by IFNs [19 , 20 ].

The induction/repression of genes could be secondarily regulated by IFN-induced cytokines. Our experiments were designed to avoid this confound by focusing on genes, which were equally induced or repressed at 2, 6, or 12 h of IFN exposure, as shown in Tables 2 and 3 .

We confirmed using RT-PCR analysis, the induction of seven genes in RNA obtained from PBMC and astrocytoma cells treated with IFN-β (Fig. 1A) ; of these, CD69 expression was restricted to hematopoietic cells. The induction of these genes could be physiologically relevant, as we have also demonstrated their induction in RNA obtained ex vivo from blood of five MS patients after injection with IFN-β (Fig. 3A) . Differential responses to IFN-β, with respect to gene induction, were observed among the five MS patients (Fig. 3A) . These results are consistent with our hypothesis that differences in therapeutic response to type I IFNs could be related to differential gene expression responses between individuals.

The induction of CD69, a type II transmembrane protein in PBMC after treatment with IFN-β, was not unexpected. CD69 was induced by IFN-{alpha}/β on T cells in vitro and promoted retention of lymphocytes in lymphoid organs [15 ]. CD69 was also induced in neutrophils and eosinophils by IFN-{alpha} [16 , 21 ].

The induction of a number of functionally important enzymes in the PBMC by IFN-β was noted. GCH1 is the rate-limiting enzyme for synthesis of tetrahydrobiopterin (BH4), a cofactor required for NO synthesis, which is a key signaling molecule in vascular homeostasis [22 ].

Antigenic peptides presented to MHC class I molecules are processed to mature epitopes by aminopeptidases [23 ]. LAP3 was identified as a gene induced by IFN-{gamma} in human fibroblast and HeLa cells [24 , 25 ]. This is the first report of LAP3 gene induction in response to IFN-β. SQLE catalyzes the conversion of squalene to 2,3-oxidosqualene in cholesterol biosynthesis, and the expression of the gene is regulated by sterols [26 , 27 ]. NCBI Blast analysis of the protein sequence of the hypothetical protein LOC1 showed homology to TKs, which play key roles in the synthesis of DNA and in cell division [18 ]. The mRNA for LOC1 was induced by IFN-β in PBMC and astrocytoma cells (Fig. 1A 1B 1C) .

We also noted induction of nonenzymatic ISGs. GEM belongs to a family of small, Ras-related GEMs and was induced in human peripheral blood T cells by mitogens, oncogenic kinases, and inflammatory cytokines [28 29 30 ]. It functions as a potent inhibitor of voltage-dependent calcium channels [31 ]. Regulatory and pore-forming calcium channels expressed on T lymphocytes are essential for T cell receptor-mediated calcium response, nuclear translocation of transcription factors, and cytokine production [32 ]. However, there is no report of its induction by IFNs. GEM was induced in PBMC treated with IFN-β but constitutively expressed in astrocytoma cells (Fig. 1A 1B 1C) .

CXCL8 is a chemokine, which promotes accumulation of neutrophils in areas of inflammation. IFN-β mediated repression of this gene in vitro and in vivo [33 34 35 ]. Consistent with previous reports, we observed a decrease in mRNA expression of CXCL8 in IFN-β-treated PBMC (Table 3) .

CXCL1 is functionally related to CXCL8. CXCL1 was detected on hypertrophic astrocytes in active MS lesions, and its receptor CXCR2 was expressed in the periphery of active MS lesions [36 ]. CXCL1/CXCR2 interactions were proposed to have the potential to inhibit MS lesion repair [37 ]. IFN-β treatment resulted in decreased levels of CXCL1 expression in peripheral blood leukocytes (Fig. 2A and 2B) .

In summary, we report a new method to identify novel ISGs in PBMC after in vitro treatment with IFN-β. Induction of a subset of the ISGs was confirmed in RNA extracted from whole blood of five MS patients after injection with IFN-β. Differential responses to IFN-β were also observed. The functional implications for the induction or noninduction of these genes in MS patients are not yet clear. These results need to be confirmed in well-designed, longitudinal studies using a larger cohort of MS patients. Current research is being focused on defining leukocyte subtype-specific expression of these ISGs.


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ACKNOWLEDGEMENTS
 
This research is supported by National Institutes of Health (NIH)/National Institute of Neurological Disorders and Stroke (NINDS) (PO1 NS38667 to R. M. R., Program Project PI; Project #4 leader, R. A. R.; R. M. R., coinvestigator) and National Multiple Sclerosis Society Grant RG 3604A6/1 to R. M. R. The procurement of patient samples was supported in part by the NIH, National Center for Research Resources, General Clinical Research Center, Grant MO1 RR-018390. We give special thanks to Barbara Tucky for help with isolation of PBMC from blood and to George R. Stark for helpful discussions.


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FOOTNOTES
 
1 Current address: Duke University, Durham, North Carolina, USA. Back

Received May 3, 2007; revised June 26, 2007; accepted July 30, 2007.


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