Expression of respiratory syncytial virus-induced chemokine gene networks in lower airway epithelial cells revealed by cDNA microarrays - PubMed (original) (raw)

Expression of respiratory syncytial virus-induced chemokine gene networks in lower airway epithelial cells revealed by cDNA microarrays

Y Zhang et al. J Virol. 2001 Oct.

Abstract

The Paramyxovirus respiratory syncytial virus (RSV) is the primary etiologic agent of serious epidemic lower respiratory tract disease in infants, immunosuppressed patients, and the elderly. Lower tract infection with RSV is characterized by a pronounced peribronchial mononuclear infiltrate, with eosinophilic and basophilic degranulation. Because RSV replication is restricted to airway epithelial cells, where RSV replication induces potent expression of chemokines, the epithelium is postulated to be a primary initiator of pulmonary inflammation in RSV infection. The spectrum of RSV-induced chemokines expressed by alveolar epithelial cells has not been fully investigated. In this report, we profile the kinetics and patterns of chemokine expression in RSV-infected lower airway epithelial cells (A549 and SAE). In A549 cells, membrane-based cDNA macroarrays and high-density oligonucleotide probe-based microarrays identified inducible expression of CC (I-309, Exodus-1, TARC, RANTES, MCP-1, MDC, and MIP-1 alpha and -1 beta), CXC (GRO-alpha, -beta, and -gamma, ENA-78, interleukin-8 [IL-8], and I-TAC), and CX(3)C (Fractalkine) chemokines. Chemokines not previously known to be expressed by RSV-infected cells were independently confirmed by multiprobe RNase protection assay, Northern blotting, and reverse transcription-PCR. High-density microarrays performed on SAE cells confirmed a similar pattern of RSV-inducible expression of CC chemokines (Exodus-1, RANTES, and MIP-1 alpha and -1 beta), CXC chemokines (I-TAC, GRO-alpha, -beta, and -gamma, and IL-8), and Fractalkine. In contrast, TARC, MCP-1, and MDC were not induced, suggesting the existence of distinct genetic responses for different types of airway-derived epithelial cells. Hierarchical clustering by agglomerative nesting and principal-component analyses were performed on A549-expressed chemokines; these analyses indicated that RSV-inducible chemokines are ordered into three related expression groups. These data profile the temporal changes in expression by RSV-infected lower airway epithelial cells of chemokines, chemotactic proteins which may be responsible for the complex cellular infiltrate in virus-induced respiratory inflammation.

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Figures

FIG. 1

Membrane-based array to identify RSV-inducible genes. A Clontech membrane-based cDNA expression array (“cytokine chip”) was used to hybridize radiolabeled RNA from uninfected (control) and pRSV-infected (24 h) cells. Shown is an autoradiogram of the exposure. For orientation, probe set 7F is RANTES (see Table 1 for further orientation information). For each sample, hybridization intensity was determined by exposure to a PhosphorImager cassette for each gene (represented in duplicate spots), normalized to local background, and analyzed by normalizing either to an internal control (thymosin-β10) or to total hybridization signals, with essentially identical results (raw values are given in Table 1).

FIG. 2

Analysis of oligonucleotide-based microarrays. (A) Reproducibility of experimental time courses. Pairwise comparisons of the average differences for the data in time series 1 (Array 1) versus those in time series 2 (array 2) are plotted. The only criterion for inclusion was that the probe set was designated “present” in both time series. Least-squares linear regression was used to determine the fit to a straight line. For the 0-h data set, the regression was described by the equation y = 1.172_x_ − 715.408 (r_2 = 0.935), and for the 36-h data set, the equation was y = 1.198_x − 741.671 (_r_2 = 0.911). (B) Kinetics of changes in total gene expression after pRSV infection. The average difference for each probe set is proportional to its level of expression. Genes whose average difference values differed threefold from those of uninfected cells were determined by algorithm. Shown are individual values for genes upregulated and genes downregulated by RSV infection, taken as means of two independent time courses. The total number of genes influenced was 118 after 6 h of pRSV infection (∼5% of the total genes expressed [scored “present”] at that time and 1% of all the genes on the chip), 267 after 12 h of infection, 796 after 24 h, and 1,200 after 36 h (∼10% of all genes on the chip).

FIG. 3

Changes in chemokine expression after RSV infection. (A) CC chemokines. The hybridization intensity (average difference) for each chemokine is plotted as a function of time of pRSV infection. Data shown here are means for independent RANTES probe set 3 (Affymetrix probe set no. 1403_s_at) and probe set 5 (no. 1405_i_at). At time zero, the absolute call for TARC, RANTES, MDC, and MIP-1α and -1β was “absent,” indicating that the signal hybridization was below the background noise of the microarray. (B) CXC chemokines. Data are plotted as described for Fig. 3A. The CX3C chemokine Fractalkine is included.

FIG. 4

Confirmation of chemokine expression. (A) Multiprobe RPA of CC chemokines. A549 cells were infected with pRSV for the indicated times (3, 6, 12, 24, and 36 h). Uninfected cells treated with sucrose alone served as a control (“0 RSV”). Total RNA was extracted and analyzed for changes in expression of the CC chemokines lymphotactin, RANTES, MIP-1α, MCP-1, IL-8, and I-309 and the L32 and GAPDH housekeeping genes by multiprobe RPA. Shown is a representative autoradiogram with the identities of the undigested CC chemokine probes given. Cont(H), human RNA control; Ltn, lymphotactin. (B) Quantitation of CC chemokine induction. The RPA autoradiogram was exposed to a PhosphorImager cassette, and the intensity of each band was determined relative to local background. For each sample, the signal was normalized to the internal control GAPDH, present in each lane. The normalized signal is plotted as a function of time of pRSV infection. Data are means of triplicate experiments. (C) UV-pRSV is deficient in viral transcription. Shown is a Northern blot of RSV nucleocapsid (N) expression in A549 cells exposed for 36 h to nothing (−−), replication-competent pRSV (++), or UV-pRSV at an MOI of 1. The N transcript is undetectable in UV-pRSV-treated cells. (D). Multiprobe RPA of CC chemokines after UV-pRSV exposure. A549 cells were exposed to replication-competent pRSV (36) or to UV-pRSV (UV) as described for Fig. 4C. Total RNA was extracted and analyzed for changes in CC chemokine expression by RPA. Shown is a representative autoradiogram. First and last lanes, input probe for transcript identification.

FIG. 4

FIG. 5

Profile of novel chemokines induced by pRSV. (A) Northern blot analysis. Total RNA was fractionated by morpholinepropanesulfonic acid (MOPS)-formaldehyde-agarose gel electrophoresis and transferred to nylon membranes. cDNA probes specific for Exodus-1, TARC, MDC, I-TAC, and the internal control thymosin β-10 were used to hybridize individual membranes. Shown is an autoradiographic exposure of the washed membranes. These results were repeated in three independent time courses. (B) RT-PCR for Fractalkine. Total mRNA was reverse transcribed and PCR amplified under linear cycling conditions using Fractalkine-specific primers (top) or primers specific for the internal control β-thymosin (bottom). The specific product is shown.

FIG. 5

FIG. 6

Changes in chemokine expression in type I alveolar cells. SAE cells were cultured and infected with pRSV for 0 to 24 h, and oligonucleotide array analysis was performed. The hybridization intensity (average difference) for each chemokine is plotted as a function of time of pRSV infection. (A) Time course for the CC chemokine set; (B) time course for the CXC chemokine set (also including Fractalkine).

FIG. 7

Clustering analysis of chemokine expression patterns. (A) Hierarchical clustering, performed by AGNES on data for 19 probe sets belonging to the chemokine group. The “agglomerative coefficient,” a figure of merit that measures the amount of clustering structure found, was 0.74, indicating the best cluster relationship of the methods tried. For each gene, the dissimilarity value is plotted. The degree of dissimilarity is indicated by the height of a common line which connects the two nodes. In the array, RANTES was detected by three independent probe sets; the data for RANTES probe set 3 (no. 1403_s_at), and probe set 5 (no. 1405_i_at) correspond to the behavior of the endogenous gene (compare Fig. 3A and 4A). (B) PCA of the chemokine gene set. Shown is the result of iterative convergence of a k means PCA where three centroids (represented by plus signs) are specified on the chemokine gene set. Dashed ovals each contain the members of a group.

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Expression of respiratory syncytial virus-induced chemokine gene networks in lower airway epithelial cells revealed by cDNA microarrays - PubMed (original) (raw)