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American Journal of Respiratory and Critical Care Medicine Vol 167. pp. 779-786, (2003)
© 2003 American Thoracic Society

Original Article

Increased IP-10 and MIG Expression after Intra-amniotic Endotoxin in Preterm Lamb Lung

Suhas G. Kallapur, Alan H. Jobe, Machiko Ikegami and Cindy J. Bachurski

Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio

Correspondence and requests for reprints should be addressed to Suhas G. Kallapur, M.D., Cincinnati Children's Hospital Medical Center, Division of Pulmonary Biology, 3333 Burnet Avenue, Cincinnati, OH 45229–3039. E-mail: kalls0{at}chmcc.org


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Subtraction hybridization was performed to explore changes in gene expression in the fetal lung after 20 mg of intra-amniotic (IA) endotoxin. Interferon-{gamma}–inducible 10-kd protein (IP-10) and monokine induced by interferon-{gamma} (MIG) constituted 20% of 102 endotoxin-induced clones identified in the preterm lamb lung. IP-10 (CXCL10) and MIG (CXCL9) are T-cell chemoattractants that have angiostatic properties. Both IP-10 and MIG mRNA were induced 30- to 40-fold in the fetal lung at 1 to 2 days after IA endotoxin. Intense IP-10 mRNA expression was detected by in situ hybridization in the bronchiolar and peribronchiolar areas and the vascular endothelium after IA endotoxin at all time points tested. MIG mRNA expression was detected initially focally in infiltrating neutrophils (15 hours after IA endotoxin) and later in the bronchiolar and peribronchiolar areas and vascular endothelium (1 day after IA endotoxin). In contrast to endotoxin, IA tumor necrosis factor-{alpha} or interleukin-1{alpha} did not induce IP-10 or MIG mRNA in the lung. IA endotoxin also caused a modest induction of IP-10 and MIG mRNA in the jejunum, liver, and spleen. The IP-10 and MIG receptor CXCR3 was detected in the bronchiolar epithelium of preterm lambs by immunostaining. IP-10 and MIG are potent angiostatic chemokines that may contribute to lung injury and altered pulmonary vascular development in the preterm exposed to chorioamnionitis.

Key Words: bronchopulmonary dysplasia • chemokines • vascular development • lung inflammation • chorioamnionitis

Microbial invasion of the amniotic fluid occurs in approximately 10–20% of women in preterm labor with intact membranes and in about 30–60% of patients with preterm premature rupture of membranes (1–3). Chorioamnionitis resulting in preterm birth is associated with an increased incidence of bronchopulmonary dysplasia (BPD) and adverse neurodevelopmental outcomes such as intraventricular hemorrhage and periventricular leukomalacia (1, 4, 5). BPD occurs in approximately 50% of preterm infants who weigh less than 1,000 g at birth and is a major cause of morbidity and mortality (6).

BPD in very low birth weight infants (less than 1,000 g at birth) is associated with an arrest in lung development at the saccular stage characterized by decreased lung microvascular development and decreased alveolarization (7–9). Arrested lung development has also been demonstrated experimentally in baboons and sheep that were mechanically ventilated after preterm delivery (10, 11). We have shown that intra-amniotic (IA) injection of endotoxin as either a single dose of 20 mg at 118 days or a continuous infusion of 0.6 mg/day from 80 to 108 days of gestation impaired alveolarization in lambs delivered at 125 days of gestation (12, 13). These results demonstrate that perinatal inflammation can influence lung development.

Overexpression of proinflammatory cytokines tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin (IL)-6 (14, 15) in transgenic mice interferes with alveolarization, and these mediators are present in the lungs of preterm infants developing BPD (16, 17). In preterm lambs given IA endotoxin, IL-1ß, IL-6, IL-8, and TNF-{alpha} mRNA were maximally induced in the lung at 1 to 2 days after IA endotoxin (18). To ask what other genes are induced by IA endotoxin in the lung, a subtractive hybridization experiment was performed using tissues from preterm lambs given IA endotoxin versus saline 1 day before preterm delivery at 119 days of gestation. Two C-X-C chemokines, interferon-{gamma}–inducible 10-kd protein (IP-10) and monokine induced by interferon-{gamma} (MIG) were the most abundant differentially expressed cDNA clones identified in the library.

IP-10 (CXCL10) and MIG (CXCL9) are closely related chemokines that are chemotactic for T cells and are potent angiostatic agents (19). Both IP-10 and MIG are ligands for the CXCR3 receptor (20). IP-10 and MIG are induced in adult animals by {gamma}-interferon and endotoxin (19). In the developing lung, inhibition of vascular development can block alveolarization (21). However, the potential role of IP-10 and MIG in the pathogenesis of lung diseases of premature infants has not been explored. This study was performed to characterize the IP-10 and MIG responses to IA inflammatory mediators associated with chorioamnionitis and BPD.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Sample Collection
Singleton preterm merino lambs at 119 to 125 days of gestation were delivered by cesarean section, and tissue samples were collected as described (see the online supplement for an expanded method) (18). Approval was obtained from the animal care and use committees at the Department of Agriculture (Western Australia) and at Cincinnati Children's Hospital (Ohio).

Treatments
The following IA injections were given as described (22): saline (control); Escherichia coli 055:B5 endotoxin 4, 10, or 20 mg (Sigma, St. Louis, MO); synthetic sheep cytokines TNF-{alpha} 150 µg; or IL-1{alpha} 150 µg (23). There are no differences in the lung inflammatory response to IA endotoxin dose ranging from 4 to 20 mg (18, 24). Sheep TNF-{alpha} and IL-1{alpha} were synthesized by Protein Express (Cincinnati, OH) and were negative for endotoxin contamination by the amebocyte lysate assay (23).

Subtraction Hybridization
Complimentary DNA derived from pooled poly A+ mRNA (poly A+ mRNA purification kit; Ambion, Austin, TX) from the lungs of three endotoxin-exposed lambs was hybridized against cDNA from pooled poly A+ lung mRNA of three saline control lambs (PCR select cDNA hybridization kit; Clontech, Palo Alto, CA). The differentially expressed cDNAs were amplified by suppression PCR (PCR select cDNA hybridization kit; Clontech), cloned into pGEMT vector (Promega, Madison, WI) and arrayed in 96-well plates. A test of subtraction was performed with cDNA probes from saline and endotoxin-exposed animals sequentially hybridized to dot blots representing individual clones. The differentially expressed cDNAs were analyzed by high-throughput sequencing (MWG Biotech, High Point, NC). Identities of the cDNAs were established using the standard nucleotide Basic Local Alignment Search Tool (BLAST) from the National Center for Biotechnology Information (NCBI).

IP-10 and MIG Ribonuclease Protection Assay
Total RNA was isolated using a modified Chomzynski method (25), and 10 µg of total RNA was used for the RNase protection assays as described (18). A 360-bp cDNA clone representing the 3' untranslated region of the IP-10 mRNA (psIP-10[1D12]) (Figure 1A) and a 686 cDNA clone representing the 3' untranslated region of the MIG mRNA (psMIG[1H9]) were used for RNAse protection assay and in situ hybridization. L32 (ribosomal protein mRNA) was used as a loading control for RNAse protection assay.



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  		Figure 1. (A) Cloning of sheep IP-10: diagram of the relative alignment of the sheep IP-10 cDNA clones to the human IP10 cDNA (NM_001565). The coding sequence is depicted as a gray shaded box. The riboprobes for in situ hybridization and RNAse protection assay were transcribed from the clone psIP10(1D12), and the protein sequence was translated from psIP10(1B3). The clone psIP10(pcr7) was generated by polymerase chain reaction using primers derived from psIP10(1B3) and psIP10(1D12) to verify that all the clones represent sheep IP10 cDNAs. (B) Sheep IP-10 amino acid sequence. Deduced amino acid sequence of sheep IP-10 compared with human IP-10 and mouse crg-2 (murine homologue of IP-10) (NM_021274). Identical amino acids between the species are shaded in gray. The propeptide is indicated by a dashed line (amino acids 1–21), and the characteristic C-X-C motif is indicated by the solid line (amino acids 30–32).

 
IP-10 and MIG In Situ Hybridization
In situ hybridization was performed as described (26). [35]S-UTP–labeled riboprobes were used in a final concentration of 3 x 104 cpm/µl for IP-10 and 2 x 104 cpm/µl for MIG. Control for specificity of riboprobe binding included the use of lung tissues obtained from lambs exposed to IA saline or the use of a homologous (sense) probe.

CXCR3 Immunostaining
The immunostaining was performed using goat polyclonal anti-CXCR3 antibody (sc 9901; Santa Cruz Biotechnology, Santa Cruz, CA) as described (27). Antigen retrieval was performed by incubating tissue sections with 0.1% trypsin and 0.1% calcium chloride, pH 7.6, at 37oC for 5 minutes.

Statistical Analysis
The mRNA quantitation was expressed as median with 25th- and 75th- percentile range, and comparisons between endotoxin-exposed animals and control subjects were made with the two-tailed Mann-Whitney nonparametric test. Significance was accepted at p < 0.05. Tissues from some of the animals used for this study were used for studies reported previously (18).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Subtraction Hybridization
Suppressive subtraction hybridization was performed using lung tissues from 119-day gestation lambs. Sheep cDNAs selectively expressed in the lung 1 day after IA endotoxin versus control subjects were cloned. Subtraction was confirmed by screening the subtracted cDNA pool for selective enrichment of the genes that we have previously shown to be induced by IA endotoxin (IL-1ß) (18) and for the absence of constitutively expressed genes such as L32 (ribosomal protein mRNA). In an initial evaluation, a total of 102 clones were screened. Nearly all of the 102 clones were differentially expressed by endotoxin treatment, as detected by hybridization of replica dot blots with cDNA probes from control and endotoxin-exposed animals. The endotoxin-induced clones were then sequenced and identified by the standard BLAST analysis. The high-throughput sequence data on IP-10 and MIG clones were further confirmed by an additional double-stranded sequencing. IP-10 and MIG comprised 17% and 3%, respectively, of the cDNAs identified in the suppression subtraction library.

Cloning and Analysis of Sheep IP-10 and MIG
The subtraction library contained clones representing three regions of the IP-10 cDNA (Figure 1A). The clones containing the 3' untranslated region sequence were more divergent from the human sequence. To verify the identity of these clones, oligonucleotides complimentary to the coding sequence and the far 3' untranslated region were used to amplify and clone cDNA spanning all three clones psIP10(pcr7) (Figure 1A). The sheep IP-10 cDNA clone representing the entire coding sequence (psIP-10[1B3]) was translated and aligned with the human and mouse protein (Figure 1B). The deduced sheep IP-10 amino acid sequence was 75% identical to human IP-10. MIG cDNA clone representing the 3' untranslated region was identified by BLAST analysis (psMIG[1H9]). The clone was verified as sheep MIG by cloning and sequencing a reverse transcription-polymerase chain reaction product, which was generated using a 5' primer derived from the coding sequence of human MIG and a 3' primer derived from psMIG(1H9) (data not shown).

IP-10 and MIG mRNA Expression in the Lung
The time course of induction of IP-10 and MIG mRNA was studied by RNAse protection assay of preterm lamb lung mRNA after IA endotoxin injection. There was minimal expression of these chemokines in saline-treated control subjects. At 15 hours after endotoxin, IP-10 and MIG mRNAs were increased in the lung relative to control subjects, with maximal 30- to 40-fold increases 1 to 2 days after endotoxin (Figures 2A and 2B) . The increased chemokine mRNA values returned to control levels at 4 to 7 days after endotoxin.



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  		Figure 2. The time course of IA endotoxin-induced lung IP-10 and MIG mRNA expression. Box plot with median and 25th- to 75th-percentile range showing induction of IP-10 and MIG mRNA in the lung of preterm lambs after 20 mg of IA endotoxin. The chemokine mRNA values were normalized to L32 (ribosomal protein RNA). The mean chemokine mRNA signal in control animals was given value of 1, and levels at each time point for individual animals were expressed as mRNA values relative to the mean control value. (A) IP-10. (B) MIG. This shows a maximal 30- to 40-fold induction of IP-10 and MIG at 1 to 2 days after IA endotoxin with a return to control levels by 4 to 7 days. RNA from six animals for control and three animals for each time point was used. The inset shows representative RNase protection assay using 10 µg of total RNA from lung of three animals exposed to IA saline (control subjects) or IA endotoxin (Endo) at 1 day after treatment (*p <= 0.05 vs. control subjects).

 
Expression Pattern of IP-10 mRNA in the Lung After IA Endotoxin
The cellular localization of IP-10 mRNA in the endotoxin-exposed fetal lung was evaluated by in situ hybridization. IP-10 mRNA was not detected in the control animals (Figures 3A and 3B) and in lung sections from endotoxin-treated animals hybridized to the sense IP-10 probe (data not shown). Similar intense focal expression patterns of IP-10 mRNA were detected in the bronchiolar epithelium, vascular endothelium, and peribronciolar interstitial cells, likely comprising smooth muscle cells and the fibroblasts at 5 (data not shown), 15, and 24 hours after IA endotoxin (Figures 3C–3H). Infiltrating inflammatory cells were devoid of IP-10 mRNA expression at all time points tested (data not shown), although some peribronchiolar inflammatory cell expression of IP-10 mRNA could not be excluded.



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  		Figure 3. IP-10 mRNA expression in endotoxin-exposed lung. In situ hybridization was performed with [35]S–labeled antisense sheep IP-10 riboprobe on lung sections from control and preterm lambs exposed to 4 mg of IA endotoxin. Saline control lung tissue (A, B), at 15 hours after IA endotoxin (C, D), and at 1 day after IA endotoxin (E–H) is shown. The pattern of IP-10 expression at 15 hours (C, D) and at 1 day after endotoxin exposure (data not shown) was similar. Intense focal expression of IP-10 was seen in bronchioles (Br) and peribronchiolar regions and the vasculature (V). Higher magnification shows IP-10 expression in vascular endothelium (E, F) and bronchiolar epithelial, smooth muscle, and peribronchiolar areas (G, H). A, C, E, and G are dark-field images, and B, D, F, and H are corresponding bright-field images (bar represents 50 µm).

 
Expression Pattern of MIG mRNA in the Lung After IA Endotoxin
MIG mRNA signal was not detected by in situ hybridization in saline-treated control animals (Figures 4A and 4B) or in lung sections from endotoxin-treated animals using the sense MIG probe (data not shown). In contrast to IP-10 mRNA, at 15 hours after IA endotoxin, intense focal MIG mRNA expression was detected in the infiltrating neutrophils identified by their lobed nuclei (Figures 4C and 4D and inset in 4D). Only a subset of the infiltrating neutrophils expressed MIG at 15 hours after endotoxin. Occasional MIG mRNA expression was also detected in the parenchymal lung cells at 15 hours after IA endotoxin (data not shown). At 1 day after IA endotoxin, intense focal expression of MIG mRNA was detected in the bronchiolar epithelium and peribronciolar interstitial cells, likely comprising smooth muscle cells and fibroblasts (Figures 5C and 5D) and vascular endothelium (Figures 5E and 5F). This pattern was similar to the expression pattern of IP-10 mRNA at 1 day after IA endotoxin. The infiltrating inflammatory cells were devoid of MIG mRNA 1 day after IA endotoxin (arrow in Figure 5D), although some peribronchiolar inflammatory cell expression of MIG mRNA could not be excluded.



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  		Figure 4. MIG mRNA expression in endotoxin-exposed lung at 15 hours after IA endotoxin. In situ hybridization was performed with [35]S–labeled antisense sheep MIG riboprobe on lung sections from control (A, B) and 15 hours after 4 mg of IA endotoxin exposure (C, D). Intense focal expression of MIG was seen in neutrophils (N shown in inset in D) with no signal seen in the parenchymal lung cells at this time point (C, D) (Br = bronchiole, V = blood vessel). A and C are dark-field images, and B and D are corresponding bright-field images (bar represents 50 µm).

 


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  		Figure 5. MIG mRNA expression in endotoxin-exposed lung at 1 day after IA endotoxin. In situ hybridization was performed with [35]S–labeled antisense sheep MIG riboprobe on lung sections from preterm lambs 1 day after 4 mg of IA endotoxin exposure. Intense focal expression of MIG was seen in bronchioles (Br) and peribronchiolar regions and the vasculature (V) (A, B). Higher magnification is seen of the bronchiolar epithelial, smooth muscle, and peribronchiolar expression of MIG (C, D) and the vascular endothelial expression of MIG (E, F). Note the absence of MIG expression in inflammatory cells (arrow) in D. A, C, and E are dark-field images, and B, D, and F are corresponding bright-field images (bar represents 50 µm).

 
Expression of CXCR3 in the Bronchiolar Epithelium
To test whether the IP-10 and MIG receptor CXCR3 is expressed in the fetal lung, immunostaining was performed with a polyclonal antihuman CXCR3 antibody. CXCR3 expression was localized to the bronchiolar epithelium in control as well as endotoxin-exposed preterm lambs (Figure 6A) . The specificity of the staining was confirmed by the absence of staining when the primary antibody was incubated with excess blocking peptide (Figure 6B) and when no primary antibody was used (data not shown). Within the limits of immunostaining, CXCR3 expression did not change after endotoxin treatment (data not shown).



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  		Figure 6. CXCR3 expression in fetal bronchial epithelium. (A) Control preterm lamb lung section stained with polyclonal goat CXCR3 (sc 9901) showing specific staining in the bronchial epithelium Br. The inset shows a higher magnification of the bronchial epithelium. (B) Specificity of the staining was demonstrated by abolition of the staining with a blocking peptide (L-17) (bar represents 50 µm).

 
Effects of IA TNF-{alpha} and IL-1{alpha}
We have previously shown that the proinflammatory cytokines IL-1ß and TNF-{alpha} are induced in the preterm lamb lung at 5 hours after IA endotoxin (18). These cytokines were previously shown to induce IP-10 in adult animals (28). To test whether endotoxin-mediated IP-10 and MIG mRNA induction in the lung was secondary to TNF-{alpha} or IL-1{alpha}, we evaluated the response to these recombinant cytokines in 110-day gestation preterm lambs. IP-10 and MIG mRNA were not induced by IA TNF-{alpha} (150 µg) or IL-1{alpha} (150 µg), suggesting that endotoxin induction of IP-10 and MIG mRNA was not mediated by either cytokine (Figure 7) .



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  		Figure 7. Selective induction of IP-10 and MIG mRNA in the lung by endotoxin: representative RNase protection assay using 10 µg of total RNA from lung of two animals exposed to IA endotoxin (10 mg), TNF-{alpha} (150 µg), or IL-1{alpha} (150 µg). IP-10 and MIG mRNA were induced in the lungs of the preterm lambs given IA endotoxin but not TNF-{alpha} or IL-1{alpha}. IL-{alpha} was given 1 day before delivery, and endotoxin and TNF-{alpha} were given 2 days before delivery (n = three to six animals per group).

 
IP-10 mRNA expression in Liver, Spleen, and Jejunum
To determine whether there was a generalized induction of IP-10 in fetal tissues in response to IA endotoxin, we measured IP-10 mRNA in the amnion–chorion membrane, placenta, jejunum, liver, and spleen. IP-10 mRNA was not detected in either amnion–chorion membrane or placenta in either control or endotoxin-treated animals (data not shown). The jejunum of control animals had detectable expression of IP-10 mRNA. At 5 to 24 hours after endotoxin, there was a threefold increase of IP-10 mRNA in the jejunum, which decreased to control levels by 4 days (Figure 8A) . The fetal liver of control lambs had a very low expression of IP-10 mRNA. IA endotoxin induced a maximum sixfold increase in IP-10 mRNA 2 days after endotoxin (Figure 8B). Compared with the jejunum, the timing of induction of IP-10 in the fetal liver was delayed in response to IA endotoxin. The control lambs had detectable IP-10 mRNA expression in the spleen. A maximum fivefold increase in IP-10 mRNA expression was detected in the spleen 24 hours after IA endotoxin (Figure 8C). In contrast to the response seen in the lung, IA endotoxin caused smaller increases of IP-10 mRNA in the fetal jejunum, liver, and spleen and no change in amnion–chorion membrane and the placenta.



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  		Figure 8. Systemic induction of IP-10 mRNA after IA endotoxin: induction of IP-10 mRNA in preterm lamb jejunum (A), liver (B), and spleen (C). Jejunum and liver IP-10 mRNA induction was measured after 20 mg of IA endotoxin and in the spleen after 4 mg of IA endotoxin. The chemokine mRNA values were normalized to L32 (ribosomal protein RNA) and plotted as mean ± SE. The mean chemokine mRNA signal in control animals was designated as 1 (shown as a closed box), and levels at each time point were expressed as relative mRNA increase over control subjects. This shows a modest systemic induction of IP-10 mRNA after IA endotoxin (n = 6 animals for control and three to five animals for each time point) (*p <= 0.05 vs. control animals).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study was conducted to find changes in gene expression that have not been previously identified with chorioamnionitis in the preterm lung. We performed suppressive subtraction hybridization with mRNA from control versus lambs 1 day after IA endotoxin at the peak of the lung inflammatory response (18, 24). IP-10 and MIG were the most abundant IA endotoxin-induced and differentially expressed cDNAs identified in the library. Both IP-10 and MIG belong to the C-X-C class of chemokines, are T-cell chemoattractants, and have angiostatic properties (19). There is an emerging view that the BPD in the very preterm is characterized by decreased alveolar development, decreased angiogenesis, and less fibrosis than described previously (8, 29). The preterm lambs in this study were not exposed to mechanical ventilation, and thus, the lung inflammation in this model was due to fetal exposure to a proinflammatory stimulus without the confounding effects of postnatal inflammation. This study is the first report of the induction of IP-10 and MIG in a preterm fetal lung injury model.

IP-10 and MIG in Lung Inflammation
Systemic administration of endotoxin or staphylococcal enterotoxin caused maximal induction of IP-10 and MIG mRNA in adult mouse lungs at 4 to 8 hours after treatment (30). Intraperitoneal administration of staphylococcal enterotoxin also induced IP-10 and MIG mRNA in adult mouse lungs (31). Our results show that in a preterm lamb animal model, IA endotoxin caused induction of IP-10 and MIG mRNA 1 to 2 days after treatment. Therefore, both Gram-negative as well as Gram-positive bacterial cell wall components can cause IP-10 and MIG mRNA induction in the lung.

In preterm lambs exposed to IA endotoxin, MIG mRNA in the lung was detected first in a subset of infiltrating neutrophils. IP-10 and MIG mRNA expression was detected focally in the bronchiolar epithelium and the vascular endothelium. Similar patterns of IP-10 and MIG expression have been described in an adult mouse Th1 lung injury model (32) and in adult humans with tuberculosis (33). The bronchiolar and vascular expression of IP-10 and MIG may serve to amplify the inflammatory response and recruit activated lymphocytes and monocytes as in the pathogenesis of bronchiolitis obliterans syndrome after lung transplantation (34). However, the role of lymphocytes and monocytes in the pathogenesis of BPD is not known.

Alternatively, IP-10 and MIG may modulate pulmonary remodeling and repair after injury without significant inflammatory effects. For example, in mice with bleomycin-induced pulmonary fibrosis, systemic injection of IP-10 did not alter pulmonary lymphocyte or natural killer cell populations but decreased pulmonary fibrosis and decreased pulmonary angiogenesis. (35). In this study, IP-10 and MIG mRNA expression was also detected in the peribronciolar interstitial cells, consistent with expression in fibroblasts and smooth muscle cells. Interstitial and adventitial fibroblast and smooth muscle cells have been implicated in the pathogenesis of hypoxia and stress-induced vascular remodeling and idiopathic pulmonary fibrosis (35, 36). The precise roles of chorioamnionitis-induced IP-10 and MIG in the preterm lung and their possible effects on the function of different cell types in the lung need to be determined. Thus, after an antenatal proinflammatory stimulus, IP-10 and MIG may modulate lung inflammation and remodeling contributing to the pathogenesis of BPD.

IP-10 and MIG Receptor in Preterm Lung
CXCR3 was originally identified as an IP-10 and MIG receptor in activated T cells (37). Subsequently, other cell types, including vascular pericytes (38) and dividing microvascular endothelial cells (39, 40), were shown to express CXCR3. However, in vivo, the mechanism of IP-10 and MIG mediated angiostatic activity is not fully understood. IP-10 has also been shown to bind and activate signal transduction via an as yet undefined receptor (41). IP-10 binds to heparan sulfate proteoglycan with high affinity, but this interaction does not result in signal transduction and may serve to sequester IP-10 locally (42). Another receptor similar to CXCR3 has been identified; however, the function of this receptor and the ligand binding specificity is not known (43). The expression and relative abundance of any of these receptors are not known in preterm animals. In our experiments in the fetal lambs, CXCR3 was found to be expressed constitutively in the bronchiolar epithelium. We could not detect CXCR3 expression in the vascular endothelium in the lung of preterm lambs by immunohistology. Some endothelial cells such as human umbilical vein endothelial cells express no detectable CXCR3, although IP-10 has direct effects on endothelial cells (44–46). The mechanisms of the potential angiostatic effects of IP-10 and MIG in the preterm lung remain to be defined.

IP-10 and MIG Induction by Cytokines
IA endotoxin induces IL-1, IL-6, IL-8, and TNF-{alpha} mRNA expression in the fetal lung (18). In the preterm lamb model, IA injection of either TNF-{alpha} or IL-1{alpha} did not induce IP-10 and MIG mRNA in the lung. The ability of IL-6 and IL-8 to induce IP-10 and MIG in the fetal lung was not tested. In the TNF null mice, inflamed lymph nodes had decreased MIG expression compared with lymph nodes from wild-type mice, suggesting that MIG expression in the lymph node is dependent on TNF (47). However, TNF-{alpha} and IL-1ß did not induce IP-10 or MIG in human bronchial cells and respiratory epithelial cells in vitro (33). These results indicate that TNF or IL-1 alone may not be sufficient to induce IP-10 and MIG expression in the lung.

IP-10 and MIG were initially identified as {gamma}-interferon–inducible genes (48, 49). We previously reported increased cord blood {gamma}-interferon levels 1 day after IA endotoxin in this model (24). However, we could not detect {gamma}-interferon mRNA in the lung of preterm lambs exposed to IA endotoxin by RNAse protection assay or reverse transcription-polymerase chain reaction at the time points tested (data not shown). Thus, the induction of IP-10 and MIG in the lungs in our study could be a direct effect of endotoxin or a response to systemic {gamma}-interferon.

Systemic Induction of IP-10 and MIG mRNA
Systemic induction of IP-10 and MIG mRNA after IA endotoxin differed in comparison with the induction of proinflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-{alpha} mRNA. For example, the chorioamnion but not the jejunum responded with an early induction of IL-1ß, IL-6, and IL-8 mRNA (18), whereas IP-10 or MIG mRNA induction was detected in the jejunum but not the chorioamnion in this study. The liver, spleen, and small intestinal expression of IP-10 and MIG mRNAs increased in adult mice given intravenous endotoxin (31). Intestinal epithelial expression of IP-10 and MIG was induced by {gamma}-interferon and is postulated to play an important role in the transepithelial trafficking of lymphocytes during inflammation (50). In our study, the fetal liver and spleen showed modest induction of IP-10 and MIG mRNA expression after IA endotoxin indicating a systemic response. The serum levels of IP-10 and MIG proteins were not measured in this study. Our results demonstrate a low-level systemic induction of IP-10 and MIG mRNA in response to IA endotoxin in fetal sheep.

In summary, IP-10 and MIG mRNAs were highly induced in the lung after IA endotoxin. Both IP-10 and MIG mRNAs were expressed focally in the bronchiolar epithelium, the peribronchiolar region, and the vascular endothelium in the preterm lamb lung after IA endotoxin. IP-10 and MIG are potent angiostatic agents (19). We have previously shown that IA endotoxin administered as either a single dose or a continuous infusion in preterm lambs causes pulmonary changes similar to human BPD (12, 13). The expression of IP-10 and MIG after continuous endotoxin infusion was not studied. Several recent reports suggest decreased proangiogenic signaling as a mechanism underlying some of the pathological features of BPD (51–53). Our results support the hypothesis that increased angiostatic activity after inflammation might be another mechanism causing decreased pulmonary vascular development in infants with BPD. Further research is needed to define the biologic roles of the chemokines IP-10 and MIG in the inflamed preterm lung and the mechanisms of impaired vascular and alveolar development in BPD.


   FOOTNOTES
 
Supported by National Institutes of Health grants HD 12714, HL 65397, and K08 HL 70711.

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Received in original form March 19, 2002; accepted in final form October 4, 2002


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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