Early high expression of IP-10 in F344 rats resistant to Sendai virus-induced airway injury

Xuezhong Cai and William L. Castleman

Department of Pathobiology, University of Florida, Gainesville, Florida 32610

Submitted 8 August 2002 ; accepted in final form 30 July 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Weanling F344 and BN rats differ markedly in their susceptibility to Sendai virus-induced airway injury. Early gene expression that controls their differences in susceptibility remains poorly understood. In this study we combined suppressive subtractive hybridization and cDNA library array hybridization to identify genes differentially expressed in virus-susceptible BN and virus-resistant F344 rats during the first 3 days after inoculation. Differential expression of selected clones was further verified by quantitative RT-PCR. Seven virus-induced gene segments were identified. Of them, interferon-{gamma}-inducible protein 10 (IP-10), Mx1, and guany-late-binding protein-2 mRNA abundance in infected F344 rats was 201.5, 188.2, and 281.7% higher, respectively, than that of infected BN rats at 2 days after inoculation. In situ hybridization indicated that virus-induced IP-10 was expressed mainly in airway epithelial cells of F344 rats. Sendai virus infection can directly induce IP-10 expression in rat tracheal epithelial cells in vitro. IP-10 early high expression might contribute to the resistance to virus-induced airway disease in F344 rats by promoting Th1 responses and increasing antiviral activity.

asthma; parainfluenza type 1; interferon-{gamma}-inducible protein 10; suppressive subtractive hybridization; airway epithelial cells


RESPIRATORY VIRAL INFECTION during early life has been identified as an important risk factor in the development of asthma (10, 25). Asthma is characterized by variable and episodic airflow obstruction, airway inflammation, and bronchial hyperresponsiveness (10, 25, 36). There is strong evidence that asthma is controlled by multiple genes. However, the specific genes and their functions have not been completely characterized (36).

Parainfluenza type 1 (Sendai) virus infection in in-bred rat strains has been developed as an experimental model of asthma induced by viral infection during early life (5, 6, 15, 19, 26, 27, 32, 33). Virus infection during early life in susceptible BN rats results in acute bronchiolitis and airway injury that is followed by persistent airway inflammation and airway remodeling. Virus-resistant F344 rats develop acute airway inflammation following infection that resolves rapidly and is not accompanied by airway remodeling or airway dysfunction (32). BN rats develop chronic airway dysfunction in association with airway remodeling that is characterized by increased airway resistance and hyperresponsiveness to methacholine (15).

Comparative viral pathogenesis studies between BN and F344 rats have demonstrated that F344 rats have slightly more rapid clearance of Sendai virus from lungs than BN rats (27). Virus-susceptible BN rats develop persistent lymphocytic infiltrates in bronchioles following infection and have Th2-dominated cytokine responses that are associated with low interferon-{gamma} (IFN-{gamma}) responses (5, 27, 33). The greater susceptibility of BN rats to developing bronchiolar fibrosis and other remodeling changes that are associated with airway dysfunction is related to higher expression of tumor necrosis factor-{alpha} (TNF-{alpha}) and transforming growth factor-{beta} (TGF-{beta}) genes in bronchioles during chronic inflammation (32, 33). We hypothesize that other genes that are differentially expressed in the first 1-3 days after infection are critical in controlling resistance and/or susceptibility to virus-induced airway injury and development of subsequent asthma-like disease.

In this study, we used suppressive subtractive hybridization (SSH) (8) and cDNA library array hybridization to identify mRNA transcripts that were differentially expressed between BN and F344 rats from 1 to 3 days after Sendai virus inoculation. Quantitative RT-PCR assay and Northern blot analysis were performed to confirm the differential expression of target genes. In situ hybridization was used to localize expression of target genes in the lung.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Experimental strategy to identify susceptibility and resistance genes. This study focused on genes differentially expressed from 1 to 3 days after viral inoculation. Subtracted cDNA libraries were constructed for F344 and BN rats at 1, 2, and 3 days after virus inoculation in BN and F344 rats. Control BN and F344 cDNAs were used from uninoculated rats at day 0 in the study. The cDNA analysis in this study can be simplified as the following steps:

  1. infected BN rat (BI) cDNAs - control BN rat (BC) cDNAs = virus-induced genes in BN rats + Sendai viral cDNAs [A]
  2. infected F344 rat (FI) cDNAs - control F344 rat (FC) cDNAs = virus-induced genes in F344 rats + Sendai viral cDNAs [B]
  3. A - B = virus-induced, differentially expressed susceptibility genes in BN rats
  4. B - A = virus-induced, differentially expressed resistance genes in F344 rats.

Steps 1 and 2 were accomplished by SSH. Steps 3 and 4 were accomplished with cDNA library array hybridization to identify potential susceptibility and resistance genes.

Rats and virus infection. Male weanling BN/RijHsd and F344/NHsd rats (22 days old) were purchased from Harlan Sprague Dawley (Indianapolis, IN). Eight rats from each infection group were inoculated at 25 days of age via aerosol exposure in a Tri-R aerosol exposure apparatus with strain P3193 Sendai virus [1-3 plaque-forming units (PFU)/ml gas] for 15 min as described (4, 6). On the study day (0, 1, 2, and 3 days after inoculation) rats were deeply anesthetized with pentobarbital sodium and killed by exsanguination via intra-cardiac bleeding. Right lungs were pooled by group for poly(A)+ RNA isolation, and left lungs were individually frozen for total RNA isolation, which were used for RT-PCR analysis. Four FC rats and four FI rats at 5 days after inoculation were processed for in situ hybridization study. Fifteen-week-old male F344 rats were used to isolate rat tracheal epithelial cells.

Construction of subtracted cDNA libraries. Poly(A)+ RNA was isolated with oligo(dT) cellulose (Pharmacia, Piscataway, NJ) as described (7, 21). The contaminating genomic DNA was eliminated by DNase I treatment (Promega, Madison, WI). cDNA synthesis and SSH were performed using a PCR-Select cDNA Subtraction kit (Clontech, Palo Alto, CA) according to the manufacturer's protocol. Two micrograms of poly(A)+ RNA from both infected and control lung samples were used to synthesize cDNAs. cDNA samples from BI and FI rats (tester cDNAs) were subtracted by cDNA samples from BC and FC rats (driver cDNAs), respectively. Subtracted cDNAs were ligated to A/T cloning vector pT-Adv (Clontech) to construct subtracted cDNA libraries for BI and FI rats at 1, 2, and 3 days after virus inoculation. Finally, the ligation product was transformed into TOP10F' Escherichia coli-competent cells (Clontech).

Screening of differentially expressed genes in BN and F344 rats. Screening of differentially expressed mRNAs was performed using the method of cDNA library array hybridization with a PCR-Select Differential Screening kit. For each subtracted library, ~400 recombinants were randomly picked, and inserts were separately amplified by PCR. PCR product from each colony was arrayed onto eight identical pieces of positively charged nylon membranes for hybridization. Then each membrane was hybridized with one of eight different cDNA probes (BI forward-subtracted, BI reverse-subtracted, FI forward-subtracted, FI reverse-subtracted, BI unsubtracted, BC unsubtracted, FI unsubtracted, and FC unsubtracted probes). Probes were prepared by labeling cDNAs (double-strand tester or driver cDNAs were used to make unsubtracted probes, and subtracted cDNAs were used to make subtracted probes) with [{alpha}-32P]dCTP. Reverse-subtracted probes were used as a nonspecific control for the forward-subtracted probe. Hybridization was performed at 72°C. Only array elements demonstrating apparent differences in signal intensity between BI and FI rats were selected for further studies.

DNA sequencing of expression sequence tags and BLAST analysis. To obtain sequence information of expression sequence tag (EST) inserts in 63 selected positive clones, the sequencing reaction was performed at the sequencing core laboratory of the Interdisciplinary Center for Biotechnology Research, University of Florida. Sequence data were submitted for sequence similarity search [basic local alignment search tool nucleotide (BLAST) search] at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) by use of the GenBank database. ESTs that had high (>98%) similarity with rat reported DNA sequences were regarded as known genes; others were considered as new rat genes.

Confirmation of positive clones by quantitative RT-PCR. Differential expression of all positive clones selected in cDNA array hybridization was verified by quantitative RT-PCR. Relative mRNA expression levels were compared among BI, FI, BC, and FC rats. Primers were designed with the Prime program in the Genetics Computer Group (GCG, University of Wisconsin, Madison, WI) and synthesized by Sigma-Genosys (The Woodlands, TX). mRNA/cDNA concentration of samples was normalized by {beta}-actin. Semiquantitative RTPCR was performed according to the modified method of Zipris et al. (38). Briefly, 4 µg of total RNA were diluted in 10-fold series with H2O before RT-PCR. The relative abundance of target mRNA was determined by end points, which are the highest dilution yielding visible target DNA bands under UV light. Competitive quantitative RT-PCR was performed with the modified method of Siegling et al. (24). Briefly, the competitive DNA fragment for each EST clone was prepared by a PCR reaction with a composite primer plus the respective sense or antisense primer. Twofold serial dilution of competitive DNA fragments was used to determine the relative mRNA concentration of target genes. The possibility of contamination was ruled out, as no PCR product was observed in the negative control.

Northern blot analysis and in situ hybridization. Riboprobes were generated by T7 and SP6 RNA polymerase with digoxigenin (DIG)-labeled UTP (Roche Diagnostics, Indianapolis, IN). One microgram of pooled poly(A)+ RNA sample from each group was separated on a 1.5% formaldehyde gel and blotted onto a positive charged nylon membrane (Roche Diagnostics). Blotted membrane was fixed and then hybridized at 68°C in DIG Easy Hyb (antisense riboprobe concentration ~100 ng/ml). The hybrid signal was detected with chemiluminescent disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2-(5-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate (Roche Diagnostics). DIG-labeled {beta}-actin riboprobe (Roche Diagnostics) was used as an internal control to normalize mRNA concentration. Band intensities were compared between samples by ChemiImager 4400 (Alpha Innotech, San Leandro, CA). In situ hybridization was performed as described by Uhl et al. (33). Lung sections (from FC and FI rats 5 days after virus inoculation) were incubated with DIG-labeled IP-10 antisense riboprobe at 37°C. IP-10 sense riboprobe served as a negative control in this experiment.

Sendai virus infection in cultured rat tracheal epithelial cells. Rat tracheal epithelial cells were isolated from F344 rats and cultured as described by Kaartinen et al. (14). After cultures had been established for 2 days, tracheal epithelial cells were inoculated with Sendai virus at a multiplicity of infection of 1 PFU/cell. Plates were kept in an incubator at 37°C and 5% CO2 for 84 h; then cells were harvested and stored at -70°C for RT-PCR analysis. Untreated tracheal epithelial cells were used as control. Assays were run in triplicate.

IFN-{gamma} was used as a positive control for IP-10 induction in tracheal epithelial cells. Medium in the established 2-day culture of rat tracheal epithelial cells was removed and replaced with fresh rat tracheal culture medium containing various doses of rat IFN-{gamma} (0, 10, 100, or 1,000 ng/ml; Research Diagnosis, Flanders, NJ). Plates were incubated at 37°C and 5% CO2 for 8 h. Cells were collected and stored at -70°C for RT-PCR analysis. Untreated tracheal epithelial cells served as control. Assays were run in triplicate.

IP-10 abundance in cell samples was analyzed by PCR assay. Total RNA of each cell sample was isolated with TRIzol (Life Technologies, Rockville, MD). Forward primer 5'-AAGCACCATGAACCCAAGTG-3'and reverse primer 5'-TGCATGTCTAGGTTCCTGTG-3' were used in a regular PCR assay. The housekeeping gene {beta}-actin was used as an internal control. To relatively quantify IP-10 mRNA abundance in these samples, competitive quantitative RT-PCR assay was performed using the method described in Confirmation of positive clones by quantitative RT-PCR.

Data analysis. Mean values between groups were compared by ANOVA in SigmaStat (SigmaStat for Windows; Jandel Scientific, San Rafael, CA). Differences between individual means were analyzed by the Student-Newman-Keuls method of all pairwise multiple comparison in SigmaStat.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Construction of subtracted cDNA libraries and differential screening. With SSH, subtracted lung cDNA libraries were constructed for virus-inoculated BN and F344 rats at 1, 2, and 3 days after Sendai virus inoculation. PCR assay indicated that >90% of the clones in libraries carried a cDNA insert. A total of 63 clones were identified as positive from 2,406 subtracted cDNA clones by cDNA library array hybridization (Table 1).


View this table:
[in this window]
[in a new window]
 
Table 1. Summary of results in differential screening

 

Identification of cDNA inserts. To identify cDNA inserts in the 63 positive clones, recombinants were sequenced and analyzed by BLAST search (1). Twenty-eight (44.4%) of them were identifiable as segments of cDNAs contained in GenBank, and 35 (55.6%) were unknown and submitted to the GenBank database. However, RT-PCR analysis indicated that most of the 63 clones were false positive (no significant difference between infected and control rats). Only seven clones were confirmed as inducible by Sendai virus infection and are summarized in Table 2.


View this table:
[in this window]
[in a new window]
 
Table 2. List of differentially expressed clones in rats after virus inoculation

 

Magnitude of differential expression determined by quantitative RT-PCR. The magnitude of difference in mRNA abundance of differentially expressed EST inserts among FI, BI, BC, FC rats was further examined by quantitative RT-PCR. Semiquantitative RT-PCR analysis showed that, at 3 days after inoculation, interferon-regulatory factor 7 (IRF-7) mRNA expression was induced by Sendai virus in both BN and F344 rats. However, there was no difference in levels of expression between FI and BI rats (data not shown). Similarly, expression of rat RNA helicase induced by virus 1 (RHIV-1) was upregulated by virus infection in both BN and F344 rats at 2 days after inoculation (data not shown). Bone-expressed sequence tag 5 (Best5) was also induced in a similar manner between rat strains at 2 days after virus inoculation (data not shown). Mx1 expression was induced by virus infection as early as 2 days after virus inoculation in both BN and F344 rats. At 2 days after virus inoculation, Mx1 mRNA abundance in FI rats was 188.2% greater than that in BI rats (Fig. 1A). Expression of guanylate-binding protein-2 (GBP-2) peaked at 2 days after virus inoculation. Competitive quantitative RT-PCR indicated that GBP-2 mRNA levels in FI rats were 281.7% higher than those in BI rats at 2 days after inoculation (Fig. 1B). Although differential expression of IP-10 was initially identified at 3 days after virus inoculation, its mRNA expression was already elevated 100-fold at 2 days after inoculation in both BN and F344 rats. Quantitative RT-PCR demonstrated that IP-10 mRNA in F344 rats was 201.5% greater than that in BN rats at 2 days after virus inoculation (Fig. 1C). Virus-induced IP-10 mRNA abundance was still at high levels at 5 days after virus inoculation in both BN and F344 rats (data not shown).



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 1. Mx1 (A), guanylate-binding protein-2 (GBP-2; B), and interferon (INF)-{gamma}-inducible protein 10 (IP-10; C) mRNA expression in parainfluenza type 1 (Sendai)-infected F344 (FI) and BN (BI) rats. mRNA levels of Mx1, GBP-2, and IP-10 were assayed by competitive quantitative RT-PCR. Differences in means were analyzed with ANOVA and Student-Newman-Keuls method. *Significant difference in mRNA expression levels between infected BN and F344 rats (P < 0.05).

 

IP-10 differential expression indicated by Northern blot. Differential expression of IP-10 was further verified by Northern blot. As seen in Fig. 2, IP-10 baseline expression in both BN and F344 rats was undetectable by Northern blot analysis. At 3 days after virus infection, IP-10 mRNA expression levels were dramatically upregulated in both F344 and BN rats. Comparison of band intensities normalized by {beta}-actin indicated that FI rats had 213.0% greater IP-10 mRNA than BI rats. This result was consistent with the findings from quantitative RT-PCR assay.



View larger version (50K):
[in this window]
[in a new window]
 
Fig. 2. Northern blot analysis of IP-10 mRNA. Approximately 1 µgof poly(A)+ RNA was loaded onto each lane, separated, and blotted onto a membrane hybridized with digoxigenin (DIG)-labeled IP-10 riboprobe. {beta}-Actin was used to normalize mRNA abundance in samples. FC, control F344 rats; BC, control BN rats; FI3, FI rats at 3 days after virus inoculation; BI3, BI rats at 3 days after virus inoculation.

 

Airway epithelial cells highly express IP-10 mRNA at 5 days after virus inoculation. Localization of IP-10 mRNA expression was performed by in situ hybridization. Only a very weak signal was detected in normal rat lung. However, the signal was much stronger in airway epithelial cells of F344 rats at 5 days after virus inoculation (Fig. 3). This showed that rat airway epithelial cells had been induced to express abundant IP-10 mRNA after Sendai virus infection.



View larger version (134K):
[in this window]
[in a new window]
 
Fig. 3. Virus-induced IP-10 mRNA expression in airway epithelial cells (magnification x720). Five-micrometer-thick paraffin lung sections were prepared from Sendai virus (A and B)- or sham (C and D)-inoculated F344 rats at 5 days after virus inoculation. Lung sections were hybridized with DIG-labeled rat IP-10 sense (A and C) or antisense (B and D) riboprobes. There is strong binding of antisense probe to airway epithelial cells in virus-infected rats (B). There is residual nonspecific alkaline phosphatase activity in mononuclear cells infiltrating in sections hybridized with sense probe.

 

Sendai virus infection can directly induce IP-10 expression in cultured tracheal epithelial cells. Increased IP-10 mRNA abundance was detected in virus-infected tracheal epithelial cells at 84 h after inoculation (Fig. 4). Competitive quantitative RT-PCR further showed that IP-10 mRNA abundance was increased 10-fold at 84 h after Sendai virus inoculation (Fig. 5). As a positive control, IFN-{gamma} (1,000 ng/ml) increased the IP-10 mRNA abundance ~100-fold at 8 h after the addition of IFN-{gamma} (Figs. 4 and 5). Induction of IP-10 expression was activated even at a low dose (10 ng/ml) of IFN-{gamma} (Figs. 4 and 5).



View larger version (61K):
[in this window]
[in a new window]
 
Fig. 4. RT-PCR of IP-10 mRNA in IFN-{gamma}-stimulated or Sendai virus-infected rat tracheal epithelial cells. Lane 1, 0 ng/ml IFN-{gamma} (control); lane 2, 10 ng/ml IFN-{gamma}; lane 3, 100 ng/ml IFN-{gamma}; lane 4, 1,000 ng/ml IFN-{gamma}; lane 5, no virus infection; lane 6, Sendai virus-inoculated cells; lane 7, IP-10-positive control (lungs from F344 rats at 2 days after virus inoculation); N, negative control; M, 100-bp DNA ladder. Representative assay from 3 replicates.

 


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 5. Quantification of IP-10 mRNA in IFN-{gamma}-stimulated and Sendai virus-inoculated tracheal epithelial cells. IP-10 mRNA levels were measured by competitive quantitative RT-PCR. Representative assay from 3 replicates. On average, both IFN-{gamma} and Sendai virus induced increases in IP-10 74.2- and 17.0-fold, respectively.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Previous studies demonstrated that the greater susceptibilities of BN rats to Sendai virus-induced chronic airway inflammation, airway remodeling, and airway dysfunction were associated with low IFN-{gamma} and high interleukin (IL)-4, TNF-{alpha}, and TGF-{beta} compared with responses of virus-resistant F344 rats (5, 32, 33). These differences were detected by Northern blot analysis and quantitative RT-PCR between 3 and 14 days after virus inoculation. In the present study, we reasoned that gene differential expression at times between 1 and 3 days after inoculation was probably important in initiating the cascade of resistance/susceptibility genes that we had detected to date. We combined SSH and cDNA library array hybridization, which have been used successfully by others (16, 20), to identify differentially expressed mRNAs in F344 and BN rats during the early time following virus inoculation. We found that Sendai virus infection increased expression of IRF-7, Best5, RHIV-1, GBP-2, Mx1, and IP-10 in BN and F344 rats at 2 days after inoculation. IP-10, Mx1, and GBP-2 are induced by virus at higher levels in F344 rats than in BN rats. Airway epithelial cells are a major cellular source of virus-induced IP-10. Sendai virus infection directly induced IP-10 expression in the culture of rat tracheal epithelial cells. Higher virus-induced expression of IP-10 in virus-resistant F344 rats was of particular interest because of the potential role that it might play in the resistance to virus-induced airway injury and dysfunction.

IP-10 is a non-ELR CXC chemokine (22). IP-10 participates in many disease processes with two basic functions: promotion of Th1 cell responses and angio-static activity. First, as a CXC chemokine, IP-10 can selectively recruit activated T cells, monocytes, and natural killer cells into inflammatory sites (9). T cells recruited by IP-10 are dominated by Th1 cells, since its receptor (CXCR3) is expressed at high levels on Th1 lymphocytes and at low levels on Th2 lymphocytes (23). In addition, in vivo and in vitro studies have also indicated that IP-10 can promote expression of the Th1 cytokine IFN-{gamma} (11, 35). Th1 responses, therefore, are selectively enhanced in IP-10-dominant inflammatory sites. In this study, we found that the IP-10 gene was expressed to higher levels in infected F344 rats than in BN rats at the early time after virus inoculation. This higher expression of IP-10 might contribute to Th1-dominant cytokine expression in infected F344 rats observed in the previous studies (5, 28). Second, IP-10 is implicated in the inhibition of endothelial cell proliferation and angiogenesis and furthers the development of remodeling (2). This inhibitory activity has been characterized extensively in idiopathic pulmonary fibrosis and bleomycin-induced pulmonary fibrosis (2, 9). Our results make it reasonable to speculate that higher expression of IP-10 in F344 rats may be an important mechanism in their resistance to Sendai virus-induced chronic airway inflammation and airway fibrosis (32, 33) through regulation of recruitment of activated T cells and related regulation of airway inflammation and repair.

In a separate series of studies (3), we tested whether blocking IP-10 early in the course of Sendai virus infection would alter the development of airway inflammation and fibrosis. F344 rats treated with a neutralizing rabbit antibody against IP-10 recruited fewer lymphocytes into lung than rats treated with normal rabbit serum at 7 days after virus inoculation (3). Neutralizing IP-10 activity resulted in a paradoxical increase in IFN-{gamma} protein production in the lung at 7 days after inoculation (3). However, no difference in severity of chronic airway inflammation and fibrosis was associated with IP-10 neutralization. If IP-10 plays an important role in regulating susceptibility to virus-induced airway damage, it is probably a complex interaction with other cytokines such as IFN-{gamma}.

Although IP-10 can be induced by IFN-{gamma} in human monocytic cells (17), IP-10 is more than the downstream product of IFN-{gamma}, since that expression of IP-10 is also induced by lipopolysaccharide, IL-1{alpha}, IFN-{alpha}, IFN-{beta}, IL-6, and TNF-{alpha} (9) and since IP-10 can also promote Th1 responses and IFN-{gamma} production (11, 35). Therefore, a feedback loop between IP-10 and IFN-{gamma} could potentially influence disease processes. In this study, the possibility that differential expression of IP-10 is the result of the difference on IFN-{gamma} expression between F344 rats and BN rats cannot be completely excluded. However, we found that Sendai virus infection in airway epithelial cells can directly induce IP-10 expression without the concurrent presence of IFN-{gamma}.

Cultured human tracheal epithelial cells can express IL-6, IL-1{beta}, IL-8, TNF-{alpha}, and intercellular adhesion molecule-1 in response to viral infection. (30, 31) Our study shows for the first time that cultured rat tracheal epithelial cells are induced to express IP-10 after Sendai virus inoculation.

Mx1 was identified as an INF-inducible protein that confers resistance to rhabdovirus and influenza virus (29). Previous studies of our rat model (26) showed that F344 rats have a slightly more rapid clearance of virus from lungs compared with BN rats. It is possible that early higher Mx1 protein expression in F344 rats may enhance the clearance of pulmonary virus and lessen the induction of exaggerated repair processes that lead to airway fibrosis and airway remodeling, eventually.

Mouse GBP-2 was initially identified from INF-{gamma}-induced bone marrow-derived macrophages. Except for GTPase activity in vitro, little information on its function is available. Because the induction of GBPs is common to many cell types that respond to IFNs, it is possible that GBPs may mediate some responses induced by IFNs in inflammatory and autoimmune diseases (34).

IRF-7 is a transcription factor that regulates expression of IFN-{alpha} and RANTES (regulated upon activation, normal T cell expressed and secreted) (12, 18). RHIV-1 is homologous to human RIG-I. It was evident that infection of another RNA virus, porcine reproductive and respiratory syndrome virus, could also induce expression of RNA helicase (37). Best5 has been documented primarily as an INF-inducible gene expressed during osteoblast differentiation and bone formation (13).

In conclusion, we used suppressive subtractive hybridization, cDNA library array hybridization, and quantitative RT-PCR to identify seven mRNA transcripts induced by Sendai virus infection in young F344 and BN rats in this study. Of them, IP-10, GBP-2, and Mx1 were expressed to much higher levels in infected F344 than in infected BN rats at 2 days after virus inoculation. The higher expression of these three genes might play an important role in the development of Th1 responses, rapid virus clearance, and resistance to virus-induced chronic airway inflammation and fibrosis in F344 rats following Sendai virus inoculation.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grant HL-61018.


    ACKNOWLEDGMENTS
 
We are grateful to Karen Dukes for technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: W. Castleman, Dept. of Pathobiology, Univ. of Florida, PO Box 110880, Gainesville, FL 32610-0880 (E-mail: Castlema{at}ufl.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

  1. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, and Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389-3402, 1997.[Abstract/Free Full Text]
  2. Belperio JA, Keane MP, Arenberg DA, Addison CL, Ehlert JE, Burdick MD, and Strieter RM. CXC chemokines in angiogenesis. J Leukoc Biol 68: 1-8, 2000.[Abstract/Free Full Text]
  3. Cai X, Castleman WL. Increased IFN{gamma} protein in bronchoalveolar lavage fluid of anti-IP-10 antibody-treated F344 rats following Sendai virus infection. J Interferon Cytokine Res 22: 1175-1179, 2002.[ISI][Medline]
  4. Castleman WL, Brundage-Anguish LJ, Kreitzer L, and Neuenschwander SB. Pathogenesis of bronchiolitis and pneumonia induced in neonatal and weanling rats by parainfluenza (Sendai) virus. Am J Pathol 129: 277-286, 1987.[Abstract]
  5. Castleman WL, Busse WW, Sorden SD, and Dukes KR. Hyperresponsiveness of BN rats to virus induced persistent lung dysfunction is associated with delayed viral clearance, high IL-4 and IL-5, and low CD8 cell and gamma-interferon response (Abstract). Am J Respir Crit Care Med 153: A866, 1996.
  6. Castleman WL, Sorkness RL, Lemanske RF, and McAllister PK. Viral bronchiolitis during early life induces increased numbers of bronchiolar mast cells and airway hyperresponsiveness. Am J Pathol 137: 821-831, 1990.[Abstract]
  7. Celano P, Vertino PM, and Casero RA Jr. Isolation of polyadenylated RNA from cultured cells and tissues. Biotechniques 15: 26-28, 1993.[ISI][Medline]
  8. Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, and Siebert PD. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA 93: 6025-6030, 1996.[Abstract/Free Full Text]
  9. Farber JM. Mig and IP-10: CXC chemokines that target lymphocytes. J Leukoc Biol 61: 246-257, 1997.[Abstract]
  10. Folkerts G, Busse WW, Nijkamp FP, Sorkness R, and Gern JE. Virus-induced airway hyperresponsiveness and asthma. Am J Respir Crit Care Med 157: 1708-1720, 1998.[ISI][Medline]
  11. Gangur V, Simons FE, and Hayglass KT. Human IP-10 selectively promotes dominance of polyclonally activated and environmental antigen-driven IFN-{gamma} over IL-4 responses. FASEB J 12: 705-771, 1998.[Abstract/Free Full Text]
  12. Genin P, Algarte M, Roof P, Lin R, and Hiscott J. Regulation of RANTES chemokine gene expression requires cooperativity between NF-kB and IFN-regulatory factor transcription factors. J Immunol 164: 5352-5361, 2000.[Abstract/Free Full Text]
  13. Grewal TS, Genever PG, Brabbs AC, Birch M, and Skerry TM. Best5: a novel interferon-inducible gene expressed during bone formation. FASEB J 14: 523-531, 2000.[Abstract/Free Full Text]
  14. Kaartinen L, Nettesheim P, Adler KB, and Randell SH:. Rat tracheal epithelial cell differentiation in vitro. In Vitro Cell Dev Biol 29A: 481-492, 1993.
  15. Kumar A, Sorkness RL, Kaplan MR, and Lemanske RF Jr. Chronic, episodic, reversible airway obstruction after viral bronchiolitis in rats. Am J Respir Crit Care Med 155: 130-134, 1997.[Abstract]
  16. Luong A, Hannah VC, Brown MS, and Goldstein JL. Molecular characterization of human acetyl-CoA synthetase, an enzyme regulated by sterol regulated by sterol regulatory element-binding proteins. J Biol Chem 275: 26458-26466, 2000.[Abstract/Free Full Text]
  17. Luster AD, Unkeless JC, and Ravetch JV. Gamma-interferon transcriptionally regulates an early-response gene containing homology to platelet proteins. Nature 315: 672-676, 1985.[ISI][Medline]
  18. Mamane Y, Heylbroeck C, Genin P, Algarte M, Servant MJ, Lepage C, Deluca C, Kwon H, Lin R, and Hiscott J. Interferon regulatory factors: the next generation. Gene 237: 1-14, 1999.[ISI][Medline]
  19. Mikus LD, Rosenthal LA, Sorkness RL, and Lemanske RF Jr. Reduced interferon-{gamma} secretion by natural killer cells from rats susceptible to postviral chronic airway dysfunction. Am J Respir Cell Mol Biol 24: 74-82, 2001.[Abstract/Free Full Text]
  20. Porkka KP and Visakorpi T. Detection of differentially expressed genes in prostate cancer by combining suppression subtractive hybridization and cDNA library array. J Pathol 193: 73-79, 2001.[ISI][Medline]
  21. Puissant C and Houdebine LM. An improvement of the single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Biotechniques 8: 148-149, 1990.[ISI][Medline]
  22. Rollins BJ. Chemokines. Blood 90: 909-928, 1997.[Free Full Text]
  23. Sallusto F, Lenig D, Mackay CR, and Lanzavecchia A. Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes. J Exp Med 187: 875-883, 1998.[Abstract/Free Full Text]
  24. Siegling A, Lehmann M, Platzer C, Emmrich F, and Volk HD. A novel multispecific competitor fragment for quantitative PCR analysis of cytokine gene expression in rats. J Immunol Methods 177: 23-28, 1994.[ISI][Medline]
  25. Sigurs N, Bjarnason R, Sigurbergsson F, Kjellman B, and Bjorksten B. Asthma and immunoglobulin E antibodies after respiratory syncytial virus bronchiolitis: a prospective cohort study with matched controls. Pediatrics 95: 500-505, 1995.[Abstract]
  26. Sorden SD and Castleman WL. Brown Norway rats are high responders to bronchiolitis, pneumonia, and bronchiolar mastocytosis induced by parainfluenza virus. Exp Lung Res 17: 1025-1045, 1991.[ISI][Medline]
  27. Sorden SD and Castleman WL. Virus-induced increases in airway mast cells in Brown Norway rats are associated with enhanced pulmonary viral replication and persisting lymphocytic infiltration. Exp Lung Res 21: 197-213, 1995.[ISI][Medline]
  28. Sorkness RL, Castleman WL, Kumar A, Kaplan MR, and Lemanske RF Jr. Prevention of chronic postbronchiolitis airway sequelae with IFN-gamma treatment in rats. Am J Respir Crit Care Med 160: 705-710, 1999.[Abstract/Free Full Text]
  29. Staeheli P, Haller O, Boll W, Lindenmann J, and Weissmann C. Mx protein: constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus. Cell 44: 147-158, 1986.[ISI][Medline]
  30. Suzuki T, Yamaya M, Kamanaka M, Jia YX, Nakayama K, Hosoda M, Yamada N, Nishimura H, Sekizawa K, and Sasaki H. Type 2 rhinovirus infection of cultured human tracheal epithelial cells: role of LDL receptor. Am J Physiol Lung Cell Mol Physiol 280: L409-L420, 2001.[Abstract/Free Full Text]
  31. Terajima M, Yamaya M, Sekizawa K, Okinaga S, Suzuki T, Yamada N, Nakayama K, Ohrui T, Oshima T, Numazaki Y, and Sasaki H. Rhinovirus infection of primary cultures of human tracheal epithelium: role of ICAM-1 and IL-1{beta}. Am J Physiol Lung Cell Mol Physiol 273: L749-L759, 1997.[Abstract/Free Full Text]
  32. Uhl EW, Castleman WL, Sorkness RL, Busse WW, Lemanske RF Jr, and McAllister PK. Parainfluenza virus-induced persistence of airway inflammation, fibrosis, and dysfunction associated with TGF-beta 1 expression in brown Norway rats. Am J Respir Crit Care Med 154: 1834-1842, 1996.[Abstract]
  33. Uhl EW, Moldawer LL, Busse WW, and Castleman WL. Increased tumor necrosis factor-{alpha} (TNF-{alpha}) gene expression in parainfluenza type 1 (Sendai) virus-induced bronchiolar fibrosis. Am J Pathol 152: 513-522, 1998.[Abstract]
  34. Vestal DJ, Buss JE, McKercher SR, Jenkins NA, Copeland NG, Kelner GS, Asundi VK, and Maki RA. Murine GBP-2: a new IFN-gamma-induced member of the GBP family of GTPases isolated from macrophages. J Interferon Cytokine Res 18: 977-985, 1998.[ISI][Medline]
  35. Wiley R, Palmer K, Gajewska B, Stampfli M, Alvarez D, Coyle A, Gutierrez-Ramos J, and Jordana M. Expression of the Th1 chemokine IFN-{gamma}-inducible protein 10 in the airway alters mucosal allergic sensitization in mice. J Immunol 166: 2750-2759, 2001.[Abstract/Free Full Text]
  36. Xu J, Meyers DA, Ober C, Blumenthal MN, Mellen B, Barnes KC, King RA, Lester LA, Howard TD, Solway J, Langefeld CD, Beaty TH, Rich SS, Bleecker ER, and Cox NJ. Genomewide screen and identification of gene-gene interactions for asthma-susceptibility loci in three U.S. populations: collaborative study on the genetics of asthma. Am J Hum Genet 68: 1437-1446, 2001.[ISI][Medline]
  37. Zhang X, Wang C, Schook LB, Hawken RJ, and Rutherford MS. An RNA helicase, RHIV-1, induced by porcine reproductive and respiratory syndrome virus (PRRSV) is mapped on porcine chromosome 10q13. Microb Pathog 28: 267-278, 2000.[ISI][Medline]
  38. Zipris D, Greiner DL, Malkani S, Whalen B, Mordes JP, and Rossini AA. Cytokine gene expression in islets and thyroids of BB rats. IFN-gamma and IL-12p40 mRNA increase with age in both diabetic and insulin-treated nondiabetic BB rats. J Immunol 156: 1315-1321, 1996.[Abstract]




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
285/6/L1263    most recent
00274.2002v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Cai, X.
Articles by Castleman, W. L.
Articles citing this Article
PubMed
PubMed Citation
Articles by Cai, X.
Articles by Castleman, W. L.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2003 by the American Physiological Society.