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The Journal of Immunology, 1999, 162: 3549-3558.
Copyright © 1999 by The American Association of Immunologists

The T Cell-Specific CXC Chemokines IP-10, Mig, and I-TAC Are Expressed by Activated Human Bronchial Epithelial Cells1

Alain Sauty*, Michelle Dziejman*, Rame A. Taha{dagger}, Albert S. Iarossi*, Kuldeep Neote{ddagger}, Eduardo A. Garcia-Zepeda*, Qutayba Hamid{dagger} and Andrew D. Luster2,*

* Infectious Disease Unit, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129; {dagger} Meakins Christie Laboratories, Montreal Chest Institute Research Centre, McGill University, Montreal, Quebec, Canada; and {ddagger} Department of Molecular Sciences, Pfizer Inc., Groton, CT 06340


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recruitment of activated T cells to mucosal surfaces, such as the airway epithelium, is important in host defense and for the development of inflammatory diseases at these sites. We therefore asked whether the CXC chemokines IFN-induced protein of 10 kDa (IP-10), monokine induced by IFN-{gamma} (Mig), and IFN-inducible T-cell {alpha}-chemoattractant (I-TAC), which specifically chemoattract activated T cells by signaling through the chemokine receptor CXCR3, were inducible in respiratory epithelial cells. The effects of proinflammatory cytokines, including IFN-{gamma} (Th1-type cytokine), Th2-type cytokines (IL-4, IL-10, and IL-13), and dexamethasone were studied in normal human bronchial epithelial cells (NHBEC) and in two human respiratory epithelial cell lines, A549 and BEAS-2B. We found that IFN-{gamma}, but not TNF-{alpha} or IL-1ß, strongly induced IP-10, Mig, and I-TAC mRNA accumulation mainly in NHBEC and that TNF-{alpha} and IL-1ß synergized with IFN-{gamma} induction in all three cell types. High levels of IP-10 protein (>800 ng/ml) were detected in supernatants of IFN-{gamma}/TNF-{alpha}-stimulated NHBEC. Neither dexamethasone nor Th2 cytokines modulated IP-10, Mig, or I-TAC expression. Since IFN-{gamma} is up-regulated in tuberculosis (TB), using in situ hybridization we studied the expression of IP-10 in the airways of TB patients and found that IP-10 mRNA was expressed in the bronchial epithelium. In addition, IP-10-positive cells obtained by bronchoalveolar lavage were significantly increased in TB patients compared with normal controls. These results show that activated bronchial epithelium is an important source of IP-10, Mig, and I-TAC, which may, in pulmonary diseases such as TB (in which IFN-{gamma} is highly expressed) play an important role in the recruitment of activated T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tcells play a central role in host defense and mucosal immunity, particularly in the respiratory tract. Activated T cells are recruited into the lungs, where they contribute to the cell-mediated immune response following Th1-type differentiation or participate in the humoral and allergic responses following Th2-type differentiation 1 . Th1 cells mainly secrete IFN-{gamma}, while Th2 cells principally produce IL-4, IL-5, IL-10, and IL-13 1 . Th1 cells and IFN-{gamma} expression are increased in several pulmonary diseases, including pulmonary tuberculosis (TB)3, 2 . In TB, CD4+ T cells are required to provide a state of delayed-type hypersensitivity to Mycobacterium tuberculosis 3 , and lack of CD4+ T cells, as found in AIDS patients, favors the development of TB as well as other mycobacteria diseases 4, 5 .

Recruitment of T cells into sites of airway inflammation is a multistep process involving adherence to and Migration across the pulmonary endothelium, trafficking through the interstitium, and finally moving into and through the airway epithelium 6, 7 . Airway epithelial cells are likely to play an important role in this process 8, 9 . They can express adhesion molecules, such as ICAM-1 and VCAM-1 10 , facilitating the adherence of lymphocytes to the epithelial surface. Airway epithelial cells can also be induced to express HLA-DR 11 and therefore may present Ag to T cells. In addition, activated bronchial epithelial cells secrete a variety of mediators, including prostaglandins, platelet-activating factor, and proinflammatory cytokines, as well as chemokines important for the process of inflammatory cell recruitment into the lung interstitium and alveolar spaces 8 .

Chemokines are a superfamily of 8- to 10-kDa secreted proteins that direct the recruitment of leukocytes to sites of inflammation. All but two of the chemokines belong to the CC (ß-chemokine) or CXC ({alpha}-chemokine) family, defined by the primary sequence of the first two of four invariant cysteine residues 12 . In general, CC chemokines chemoattract monocytes, eosinophils, basophils, and T cells and signal through the chemokine receptors CCR1 to CCR9 (reviewed in 12 . The CXC chemokine family can be divided in two classes based on the presence or absence of an NH2-terminal ELR sequence (Glu-Leu-Arg). The ELR-containing CXC chemokines (e.g., IL-8) chemoattract neutrophils 13 , while the non-ELR CXC chemokines chemoattract lymphocytes. For example, IFN-inducible protein of 10 kDa (IP-10), monokine induced by {gamma}-IFN (Mig), and IFN-inducible T-cell {alpha}-chemoattractant (I-TAC) are potent chemoattractants for activated T lymphocytes 14, 15, 16, 17, 18 , while stromal cell-derived factor-1 is active on resting T 19 and immature B cells 20 , and B cell-attracting chemokine-1 is specific for mature B cells 21 . The CXC chemokines signal through the chemokine receptors CXCR1 to CXCR5.

Several members of the CC chemokine family, including monocyte chemoattractant protein (MCP)-1 22 , MCP-3 23 , MCP-4 24, 25 , RANTES 23 , and eotaxin 26, 27 are expressed by activated bronchial epithelial cells. Likewise, the ELR-containing CXC chemokines IL-8 28 and epithelial cell-derived neutrophil-activating peptide-78 29 have been shown to be inducible in bronchial epithelium. In contrast, non-ELR chemokines have not been demonstrated to be expressed in bronchial epithelium. Since bronchial epithelium is likely to regulate T cell trafficking into the lung, we asked whether human bronchial epithelial cells could express IP-10, Mig, and I-TAC.

IP-10 was initially identified as an abundantly induced mRNA in U937 cells upon IFN-{gamma} stimulation 30 , and its expression is predominantly induced by IFN-{gamma} in endothelial cells, monocytes, fibroblasts 30 , astrocytes 31 , keratinocytes 32 , and neutrophils 33 . Human Mig, which is 37% identical to IP-10 at the amino acid level, is expressed in IFN-{gamma}-induced THP-1 cells, PBMCs, endothelial cells, keratinocytes, and fibroblasts 17, 34 . I-TAC, a newly identified non-ELR chemokine, is ~40% identical at the amino acid level to IP-10 and Mig and is expressed by activated monocytes and astrocytes 18 . Both IP-10, Mig, and I-TAC chemoattract activated T cells and NK cells, but not resting T cells, B cells, or neutrophils 14, 15, 16, 17 . IP-10 expression has been found in various clinical conditions such as psoriasis 35 , tuberculoid leprosy 36 , sarcoidosis 37 , and viral meningitis 38 , as well as in experimental animal models of autoimmune encephalomyelitis 31 and nephrosis 39 . Mig was also found in psoriatic lesions by in situ hybridization 17 . All of these diseases are associated with an increased expression of IFN-{gamma} (Th1-type diseases) 1 , which may induce IP-10 Mig and I-TAC expression in involved tissues.

IP-10, Mig, and I-TAC share a common receptor, CXCR3, which is specific for these three chemokines 15 . CXCR3 is expressed on peripheral blood T cells activated in vitro and on a significant fraction of circulating CD4+ and CD8+ T cells, B cells, and NK cells 40 , but not on monocytes or neutrophils. In addition, most peripheral CXCR3+ T cells expressed CD45RO+ (memory T cells) and ß1 integrins 40 , which are implicated in the binding of lymphocytes to endothelial cells, epithelial cells, and extracellular matrix 6 . Recent studies revealed that CXCR3+ T cell clones were predominantly of the Th1 type 41, 42, 43 . In addition, it was recently reported that virtually all synovial fluid T cells isolated from patients with rheumatoid arthritis expressed CXCR3 40, 42 . Furthermore, the vast majority of T lymphocytes found in colonic tissue obtained from patients with ulcerative colitis were CXCR3+ 40 . These data suggest that in Th1-type diseases, in which IFN-{gamma} is up-regulated, IP-10, Mig, and I-TAC play an important role in the recruitment of activated CXCR3+ T cells into inflamed tissue.

In this study, we demonstrate that human bronchial epithelial cells can be induced to express high levels of IP-10, Mig, and I-TAC mRNA and secrete high levels of IP-10 protein in response to IFN-{gamma} stimulation. In addition, we show that IP-10 is expressed in bronchial epithelial cells and in cells recovered from bronchoalveolar lavage (BAL) in patients with active pulmonary TB.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

BEAS-2B cells and A549 cells were obtained from the American Type Culture Collection (Manassas, VA) and cell culture reagents from Cellgro (Mediatech, Herndon, VA). Recombinant human IFN-{gamma}, TNF-{alpha}, IL-4, IL-10, IL-13, IP-10, and Mig were obtained from PeproTech (Rocky Hill, NJ), and IL-1ß from R&D Systems (Minneapolis, MN). Normal human bronchial epithelial cells (NHBEC) and their specific medium were purchased from Clonetics (Walkersville, MD). Chemicals were obtained from Sigma (St. Louis, MO).

Cell culture

BEAS-2B, an SV-40-transformed human bronchial epithelial cell line, was cultured in F12/DMEM supplemented with 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, and 10% heat-inactivated FCS (Mediatech) (referred to as complete F12/DMEM). A549, a human lung adenocarcinoma cell line with the alveolar type II phenotype, was maintained in complete F12K in a 5% CO2 atmosphere at 37°C. NHBEC were maintained in serum-free bronchial epithelial cell basal medium (Clonetics) supplemented with 50 µg/ml bovine pituitary extract, 50 ng/ml human epidermal growth factor, 0.5 µg/ml hydrocortisone, 0.5 µg/ml epinephrine, 10 µg/ml transferrin, 5 µg/ml insulin, 0.1 ng/ml retinoic acid, 6.5 ng/ml triiodothyronine, 50 µg/ml gentamicin, and 50 ng/ml amphotericin-B. BEAS-2B and A549 cells were grown to confluence in 100-mm cell culture petri dishes, and 24 h before activation they were serum starved in their respective media without FCS. Twelve to 16 h before activation, medium from 90% confluent NHBEC was replaced by BEBM alone. Cells were then stimulated with IFN-{gamma}, TNF-{alpha}, or IL-1ß alone or in combination at indicated doses and times. In experiments involving cycloheximide (10 µg/ml) 26 ; dexamethasone; and IL-4, IL-13, and IL-10 (20 ng/ml), the agents were added 1 h before cell stimulation by cytokines.

RNA analysis

Total RNA was extracted from samples using Stat-60 (Tel-Test, Friendswood, TX). For Northern analysis, 20 µg of total RNA was electrophoresed on a 1.2% agarose-formaldehyde gel and then capillary transferred to a GeneScreen membrane (NEN Life Science Products, Boston, MA). Following overnight prehybridization (50% formamide, 1% SDS, 4x SSC, 4x Denhardt’s solution, 0.8% glycine, and 0.17 mg/ml denatured salmon sperm DNA) at 42°C, blots were hybridized at 42°C in 50% formamide, 10% dextran, 1% SDS, 5x SSC, 1x Denhardt’s solution, and 0.17 mg/ml denatured salmon sperm DNA with 1 x 106 cpm/ml [{alpha}-32P]dCTP-radiolabeled cDNA probe prepared by nick translation. The following fragments were used as probes: a 1-kb PstI fragment from IP-10 cDNA, a 3-kb NotI fragment from human Mig cDNA (kindly provided by J. Farber, National Institutes of Health, Bethesda, MD), a 300-bp BamHI/AvaI fragment from human I-TAC cDNA, and a 346-bp EcoRI fragment from human IL-8 cDNA (a kind gift from H. Oettgen, Children’s Hospital, Boston, MA). A glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe was used as a control for RNA loading and to normalize signals obtained with the different probes. Signal quantitation was determined using a PhosphorImager (Molecular Imager System, Bio-Rad, Hercules, Ca). The data shown in all of the figures are representative of at least two independent experiments.

Generation of anti-human IP-10 (hIP-10) mAbs

Fifty micrograms of histidine-tagged hIP-10 44 in CFA was injected i.p. into 6-wk-old female BALB/c mice. Mice were injected i.p. with 25 µg of His-tagged hIP-10 in IFA on days 14 and 21. On day 25, spleen cells were harvested and fused with P3X63 myeloma cells using a standard PEG 1500 protocol. Hybridoma cells were cloned by two rounds of single-cell cloning using medium supplemented with 10% IL-6-conditioned supernatant from Sp2/IL-6 cells. Direct ELISA, using recombinant non-histidine-tagged hIP-10 (PeproTech) containing only amino acids found in the mature IP-10 protein, was used to screen 750 colonies. Ten of 18 initial positive clones survived two rounds of single-cell cloning. All clones were found to be specific for rhIP-10 when tested by ELISA and dot blot analysis using the following CXC chemokines: human growth-regulated oncogene {alpha} (GRO{alpha}), IL-8, Mig, neutrophil-activating peptide-2(NAP-2), platelet factor 4 (PF4), stromal cell-derived factor-1, and murine IP-10. One clone, D1D2, was chosen for use in Western blots and ELISA. D1D2 mAb was purified from serum-free supernatants using protein G chromatography (Pharmacia, Piscataway, NJ).

Western blot analysis of cell supernatants

Unconcentrated cell supernatants and different amounts of rhIP-10 were separated by SDS-PAGE using 12.5% Tris-tricine gel. Proteins were then transferred to a polyvinylidene difluoride membrane (NEN Life Science Products). The membrane was blocked 1 h in 5% dry milk diluted in PBS-0.1% Tween (PBST) at room temperature and incubated with D1D2 mAb (2 µg/ml in 1% BSA) overnight at 4°C. After the membrane was washed in TBST, 1/10,000 dilution of peroxidase-conjugated goat anti-mouse Ig Ab (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added for 1 h, followed by additional washes and detection solution for chemiluminescence (Amersham Life Science, Arlington Heights, IL).

Sandwich ELISA

D1D2 mAb (100 µl) at 10 µg/ml in PBS was added to wells of a high binding-efficiency 96-well ELISA plate (Costar, Cambridge, MA) and incubated overnight at 4°C. The plate was then incubated with blocking buffer (3% BSA and 3% goat serum in PBS) overnight at 4°C. After the plate was washed with PBS, 100 µl of standards or unconcentrated cell supernatants (in triplicates) were added to each well and incubated for 2 h at 37°C. The plate was washed again three times with PBST and three times with PBS and incubated with 15 ng/well of affinity-purified rabbit anti-hIP-10 44 for 1 h at 37°C. Following similar washes, 100 µl of peroxidase-conjugated goat anti-rabbit Ig Ab (Kirkegaard & Perry Laboratories) diluted 1/10,000 in blocking buffer was added for 1 h at 37°C, followed by detection solution (Kirkegaard & Perry Laboratories). The plate was then read at 650 nm for 15 min. The limit of detection of this assay was ~500 pg/ml.

BALs and bronchial biopsies

Seven control individuals (nonsmokers, nonasthmatics, and nonatopics) and seven patients with active TB (prior antituberculous chemotherapy) underwent bronchofibroscopy and BAL as previously described 2 . Bronchial biopsies of the lower bronchus were performed during bronchoscopy.

In situ hybridization

BAL cells were processed as described 2 . Labeled riboprobe was prepared from IP-10 cDNA using 35S-labeled UTP or digoxigenin-UTP. The prehybridization, hybridization, and posthybridization protocols were as previously described 45 . Hybridization signal was visualized using autoradiography or antidigoxigenin. For negative controls, preparations were hybridized with sense probes. The specificity of the hybridization signal was confirmed by treating preparations with ribonuclease (RNase) before hybridization.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dose-response studies

Preliminary experiments using A549 and BEAS-2B cells showed that IP-10 mRNA was not found in unstimulated cells, but was detectable 2 h after and peaked 8 h after stimulation with IFN-{gamma} (100 ng/ml) or TNF-{alpha} (10 ng/ml) (data not shown). We therefore used the 8-h time point for studying the effect of different doses and combinations of IFN-{gamma}, TNF-{alpha}, and IL-1ß on IP-10, Mig, and I-TAC mRNA accumulation (Fig. 1Go, A and B) and compared it with the expression of the ELR-containing CXC chemokine IL-8. We found that IFN-{gamma} alone induced IP-10 in BEAS-2B but not in A549 cells and that the maximal effect was observed at 100 ng/ml. IL-1ß weakly induced IP-10 in A549 but not in BEAS-2B cells. Both cell lines expressed IP-10 in response to TNF-{alpha} in a dose-dependent manner. Mig and I-TAC expression was barely detected in BEAS-2B cells treated with IFN-{gamma} and was not seen in cells treated with TNF-{alpha} or IL-1ß. TNF-{alpha} and IL-1ß strongly synergized in a dose-dependent manner with IFN-{gamma} (100 ng/ml) in inducing the accumulation of IP-10, Mig, and I-TAC mRNA. IP-10 was induced to higher levels than Mig and I-TAC in both cell lines; this can be appreciated in Fig. 1GoA, which shows blots for IP-10 and Mig that were exposed for similar times (25 and 26 h, respectively) and a blot for I-TAC exposed for 7 days. In contrast, IL-8 was strongly induced in A549 cells by TNF-{alpha} or IL-1ß, with IFN-{gamma} being only weakly synergistic. We also studied the effect of the proinflammatory cytokines IL-6 and granulocyte-macrophage CSF, alone or combined with IFN-{gamma}. These two cytokines had no effect on IP-10 or Mig expression (data not shown).



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  		FIGURE 1. Dose response of proinflammatory cytokine-induced IP-10, Mig, I-TAC, and IL-8 mRNA accumulation. Northern blot analysis of 20 µg of total RNA obtained from A549 and BEAS-2B cells (A) and NHBEC (B) stimulated for 8 h with increasing doses (ng) of IFN-{gamma}, TNF-{alpha}, IL-1ß, or a combination of IFN-{gamma} and TNF-{alpha} or IL-1ß. A GAPDH cDNA probe was used as a control for RNA loading.

 
The induction of IP-10, Mig, and I-TAC in NHBEC in response to cytokine stimulation was different from that observed in the two cell lines (Fig. 1GoB). IFN-{gamma} induced IP-10, Mig, and I-TAC expression to higher levels than those seen in A549 and BEAS-2B cells. In addition, only 10 ng/ml of IFN-{gamma} induced maximal IP-10, Mig, and I-TAC expression, indicating that NHBEC were more sensitive to IFN-{gamma} than the two cell lines. TNF-{alpha} and IL-1ß, alone or together, did not induce IP-10, Mig, and I-TAC expression in NHBEC, while they were potent inducers of IL-8 expression in these cells. Moreover, TNF-{alpha} and IL-1ß only slightly synergized with IFN-{gamma} in the induction of IP-10, Mig, and I-TAC, suggesting that IFN-{gamma} alone was maximally inducing IP-10, Mig, and I-TAC in NHBEC. To determine whether TNF-{alpha} could be induced in response to IFN-{gamma} and therefore have an autocrine effect, NHBEC were treated with IFN-{gamma} in the presence of a neutralizing anti-hTNF-{alpha} goat polyclonal Ab (5 µg/ml) or goat IgG control (5 µg/ml). We found that these treatments had no effect on IP-10 or I-TAC mRNA expression (data not shown). However, as a control, we found that this concentration of anti-TNF-{alpha} Ab completely inhibited the effects of 10 ng/ml TNF-{alpha} (data not shown). These data suggest that TNF-{alpha} did not contribute to the effect of IFN-{gamma} on IP-10 or I-TAC induction in NHBEC. When the level of expression of the four chemokines was compared (Fig. 1GoB) according to their respective times of Northern blot exposure (IP-10, 2 h; Mig, 2 h; I-TAC, 10 h; and IL-8, 3 days), the data show that IP-10 was induced to significantly higher levels than Mig, I-TAC, or IL-8 in NHBEC.

Kinetic studies

The kinetic response of IP-10, Mig, I-TAC, and IL-8 mRNA accumulation after stimulation with combinations of IFN-{gamma}, TNF-{alpha}, and IL-1ß was also examined. Northern blot analysis of NHBEC showed that the pattern of IP-10, Mig, and I-TAC expression was very similar (Fig. 2GoA). IP-10, Mig, and I-TAC mRNA accumulation was detected between 1 and 2 h after cytokines were added, peaked at 8 h, and slowly decreased thereafter. In contrast, IL-8 expression was bimodal with an early peak at 1 h followed by a second peak at 16 h (Fig. 2GoA).



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  		FIGURE 2. Time course of cytokine-induced IP-10, Mig, I-TAC, and IL-8 mRNA accumulation. Northern blot analysis of 20 µg of total RNA obtained from NHBEC, A549 cells, and BEAS-2B cells stimulated for the indicated times with IFN-{gamma} (100 ng/ml) and TNF-{alpha} (10 ng/ml), IFN-{gamma} (100 ng/ml) and IL-1ß (0.1 ng/ml), or TNF-{alpha} (10 ng/ml) and IL-1ß (0.1 ng/ml). A GAPDH cDNA probe was used as a control for RNA loading. A, Northern blot analysis and PhosphorImager quantitation analysis of NHBEC. B, PhosphorImager quantitation analysis obtained from BEAS-2B and A549 cells.

 
Comparable data were obtained with BEAS-2B and A549 cells, and only the PhosphorImager quantitation of Northern blots are shown in Fig. 2GoB. These data revealed minor differences in IP-10 and Mig expression in BEAS-2B and A549 cells compared with NHBEC. There was a suggestion of a bimodal expression of IL-8 in BEAS-2B cells; however, this was not seen in A549 cells.

Effect of protein synthesis inhibition

To examine the effect of protein synthesis inhibition on chemokine mRNA accumulation, cells were pretreated 1 h before cytokine stimulation with cycloheximide at a concentration (10 µg/ml) previously shown to inhibit >90% of protein synthesis in A549 cells 26 . As shown in Fig. 3GoA, in NHBEC, Mig and I-TAC expression induced by IFN-{gamma}, IFN-{gamma}/TNF-{alpha}, and IFN-{gamma}/IL-1ß was significantly inhibited by cycloheximide. The effect of cycloheximide on IP-10 expression was weak compared with the effects of Mig and I-TAC and was even absent in IFN-{gamma}/TNF-{alpha}-stimulated NHBEC. IP-10, Mig, and I-TAC mRNA was not detected in untreated or in TNF-{alpha}- and in IL-1ß-stimulated cells and was not affected by cycloheximide treatment. The effect of cycloheximide on IP-10, Mig, and I-TAC induction in BEAS-2B cells was less pronounced than in NHBEC. However, in A549 cells we observed a superinduction of IP-10, Mig, and I-TAC mRNA accumulation in response to IFN-{gamma}/TNF-{alpha} stimulation (Fig. 3GoB). IL-8 expression was strongly superinduced by cycloheximide in the three cell lines and in all situations studied (Fig. 3Go, A and B). As an example, in A549 cells treated with TNF-{alpha}, cycloheximide increased IL-8 expression 6.6-fold.



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  		FIGURE 3. Effect of protein synthesis inhibition on IP-10, Mig, I-TAC, and IL-8 mRNA accumulation. Northern blot analysis of 20 µg of total RNA obtained from NHBEC, A549 cells, and BEAS-2B cells stimulated for 8 h with indicated cytokines (100 ng/ml IFN-{gamma}, 10 ng/ml TNF-{alpha} or 100 ng/ml IFN-{gamma}, and 10 ng/ml TNF-{alpha}), with or without preincubation with 10 µg/ml cycloheximide. GAPDH cDNA probe was used as a control for RNA loading. A, Northern blot analysis and PhosphorImager quantitation analysis of NHBEC. B, PhosphorImager quantitation analysis obtained from BEAS-2B and A549 cells.

 
Effect of dexamethasone

Glucocorticoids are potent inhibitors of chemokine expression. However, treatment of NHBEC stimulated by IFN-{gamma}, IFN-{gamma}/TNF-{alpha}, and IFN-{gamma}/IL-1ß with 10 µM dexamethasone (Fig. 4Go) was associated with no or only a weak inhibition of IP-10, Mig, and I-TAC expression. This is in contrast to the clear inhibition of IL-8 mRNA expression (Fig. 4Go). Data obtained with A549 and BEAS-2B cells were similar to those obtained with NHBEC (data not shown), although the effect of dexamethasone on IL-8 expression by these cells was significantly less marked.



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  		FIGURE 4. Effect of dexamethasone on IP-10, Mig, I-TAC, and IL-8 mRNA accumulation. Northern blot analysis of 20 µg of total RNA obtained from NHBEC stimulated for 8 h with IFN-{gamma} (100 ng/ml), TNF-{alpha} (10 ng/ml), or IFN-{gamma} (100 ng/ml) and TNF-{alpha} (10 ng/ml) with or without preincubation with 10 µM dexamethasone. GAPDH cDNA probe was used as a control for RNA loading.

 
Effect of Th2 type cytokines

Since IL-4 has been reported to down-regulate IP-10 in monocytes 46 and macrophages 47 , we asked whether IL-4, IL-13, and IL-10 (three Th2-type cytokines) had the capacity to down-regulate IP-10, Mig, and I-TAC expression in epithelial cells. We found that IP-10, Mig, I-TAC, and IL-8 expression by NHBEC treated with IFN-{gamma} or IFN-{gamma}/TNF-{alpha} was not affected by pretreatment with IL-4, IL-13, or IL-10 (all at 20 ng/ml) (Fig. 5Go). However, NHBEC were responsive to IL-4 and IL-13, since IL-8 expression increased by 156% and 93% in TNF-{alpha}-stimulated cells pretreated with IL-4 and IL-13, respectively. Experiments performed on IFN-{gamma}- or IFN-{gamma}/TNF-{alpha}-stimulated A549 and BEAS-2B cells pretreated with IL-4, IL-13, and IL-10 gave results comparable with those described for NHBEC (data not shown).



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  		FIGURE 5. Effect of Th2 cytokines on IP-10, Mig, I-TAC, and IL-8 mRNA accumulation. Northern blot analysis of 20 µg of total RNA obtained from NHBEC stimulated for 8 h with indicated cytokines (100 ng/ml IFN-{gamma}, 10 ng/ml TNF-{alpha}, or 100 ng/ml IFN-{gamma} and 10 ng/ml TNF-{alpha}), with or without preincubation with 20 µg/ml IL-4, IL-13, or IL-10. GAPDH cDNA probe was used as a control for RNA loading.

 
Expression of IP-10 protein in bronchial epithelial cell supernatants

To assess whether IP-10 protein was secreted by human bronchial epithelial cells, we performed Western blot analysis on unconcentrated supernatants from A549 cells, BEAS-2B cells, and NHBEC using a newly generated murine mAb (D1D2) specific for IP-10. We found a significant amount of IP-10 secreted in cell supernatants, as demonstrated by the unique band of ~10 kDa that coMigrated with rIP-10 (Fig. 6GoA). Using a sandwich ELISA developed in our laboratory, we detected high levels of IP-10 in unconcentrated supernatants. IP-10 levels were detected between 4 and 8 h after activation with IFN-{gamma}/TNF-{alpha} (Fig. 6GoB). At 32 h, the amount of IP-10 in supernatants of A549 cells, BEAS-2B cells, and NHBEC was 253.1 ± 36.6 (SEM), 558.7 ± 62.9, and 843.8 ± 73.2 ng/ml, respectively. IP-10 levels at 8, 16, and 32 h were significantly higher than at 4 h in all three cell type supernatants. Moreover, IP-10 levels detected by ELISA were positively correlated with mRNA levels in experiments in which stimulated cells were treated with dexamethasone or with IL-4, IL-13, and IL-10 (data not shown).



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  		FIGURE 6. Expression of IP-10 protein in BEAS-2B cell supernatants. A, Western blot analysis using murine anti-hIP-10 mAb (D1D2) on unconcentrated cell supernatants from BEAS-2B cells treated for indicated times with 100 ng/ml IFN-{gamma} and 10 ng/ml TNF-{alpha}. Forty microliters of supernatant were loaded per lane. Three concentrations of rIP-10 were used as controls. B, Quantitation of IP-10 protein in unconcentrated supernatants from NHBEC, BEAS-2B cells, and A549 cells. Sandwich ELISA was performed on cell supernatants from NHBEC, A549 cells, and BEAS-2B cells treated for the indicated times (h) with IFN-{gamma} (100 ng/ml) and TNF-{alpha} (10 ng/ml). Threshold of detection was 0.250 ng/ml. ND signifies not detected; Ctrl, control. *, p < 0.002; **, p < 0.0001; and ***, p < 0.001, for comparison with supernatants at 4 h.

 
In situ hybridization

To determine whether IP-10 was expressed in the airways of patients with pulmonary TB, a Th1-type disease in which IFN-{gamma} has been shown to be up-regulated, we compared IP-10 mRNA expression by in situ hybridization using an IP-10 cRNA probe on BAL cells obtained from seven patients with active TB and seven normal controls. Quantitation of BAL data is shown in Table IGo and demonstrates a statistically significant increase in the percentage of IP-10 mRNA-positive cells in BAL from TB patients compared with controls (p < 0.001). Fig. 7Go, A and B, shows a representative in situ hybridization of BAL cells from one TB patient and one control. Although the type of the positive cells was not determined, morphologically they appeared to be macrophages. In situ hybridization was also performed on bronchial biopsies obtained from patients with active TB and normal controls. The bronchial epithelium was strongly positive for IP-10 mRNA (Fig. 7GoD). No signal was detected using a sense probe as a control (Fig. 7GoC), confirming the specificity of the hybridization. Bronchial epithelium obtained from normal volunteers had little or no IP-10 signal (data not shown).


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  		Table I. IP-10 mRNA expression in BAL cells1

 


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  		FIGURE 7. IP-10 in situ hybridization of BAL cells and bronchial biopsies of TB patients. BAL cells recovered from a control subject (A) and a TB patient (B) were analyzed by in situ hybridization for IP-10 mRNA expression. C and D, In situ hybridization of a bronchial biopsy specimen from a TB patient using an IP-10 sense probe (C) (negative control) and an antisense probe (D). IP-10 mRNA was primarily seen in the epithelium (thick arrow) and in few subepithelial cells (thin arrow).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Bronchial epithelium is an important source of mediators of lung inflammation, such as chemokines 8, 22, 23, 24, 25, 26, 27, 28, 29, 48 . Chemokines play an important role in the recruitment of leukocytes into sites of tissue inflammation. They increase leukocyte adhesion by activating specific integrins 7 , and they provide a directional signal (chemical gradient) for the movement of these leukocytes into inflamed tissue 12 . Among the CXC family of chemokines, IP-10, Mig, and the newly described I-TAC specifically chemoattract activated T cells through the unique chemokine receptor CXCR3 15 .

In the present study, we demonstrate that primary human bronchial epithelial cells highly express IP-10, and to a lesser extent Mig and I-TAC, in response to IFN-{gamma}, but not in response to TNF-{alpha} or IL-1ß. In addition, as has been seen in other cell types 33, 49 , IL-1ß and TNF-{alpha} synergized with IFN-{gamma} in inducing IP-10 and Mig mRNA expression. Similar synergism was found with I-TAC expression. However, using the human epithelial cell lines A549 (alveolar type II phenotype) and BEAS-2B (bronchial phenotype), we found that IFN-{gamma}, as well as TNF-{alpha} and IL-1ß alone, weakly induced IP-10 but not Mig or I-TAC. This finding may reflect differences between transformed or tumor cell lines and primary cells.

We also found that significant amounts of IP-10 protein were secreted from activated bronchial epithelial cells. Greater than 800 and 500 ng/ml of IP-10 protein was detected in unconcentrated supernatants collected from NHBEC and BEAS-2B cells, respectively, activated for 32 h with IFN-{gamma} and TNF-{alpha}. These are the highest levels reported to date for chemokine secretion from bronchial epithelial cells. In comparison, in a 24-h period, BEAS-2B treated with IFN-{gamma} and TNF-{alpha} released 20-fold less RANTES 48 , and A549 cells maximally stimulated with IL-1ß secreted 10-fold less IL-8 28 and 10,000-fold less eotaxin 26 . IP-10 mRNA accumulation in bronchial epithelial cells closely reflected levels of secreted IP-10 protein, as previously shown in endothelial cells 32 , suggesting that in these cells the IP-10 protein is not stored.

In contrast with IP-10, Mig, and I-TAC, IL-8 expression was weakly induced by IFN-{gamma} but was strongly induced by TNF-{alpha} or IL-1ß, confirming a previous study 28 . These results show that in bronchial epithelial cells, IP-10, Mig, and I-TAC are regulated differently from IL-8. This pattern of differential regulation was also seen when we explored the effects of cycloheximide, glucocorticoids, and Th2 cytokines on the expression of these four chemokines in bronchial epithelial cells.

In NHBEC, cycloheximide reduced IP-10, Mig, and I-TAC mRNA accumulation; however, in BEAS-2B and A549 cells, the effect on IP-10, Mig, and I-TAC expression was less marked. It was previously found that cycloheximide did not affect IP-10 expression on IFN-{gamma}-treated U937 cells 30 but increased its expression in IFN-{gamma}-induced keratinocytes 49 . Therefore, the effect of cycloheximide on IP-10 expression appears to differ according to cell type. Interestingly, the effect of cycloheximide was more pronounced on I-TAC mRNA accumulation in NHBEC, suggesting that different pathways Might be involved in IP-10, Mig, and I-TAC expression, even though they are all induced by IFN-{gamma}. In contrast, cycloheximide increased IL-8 mRNA accumulation in all three cell types, suggesting again that IP-10, Mig, and I-TAC are regulated differently from IL-8.

Glucocorticoids are used in many inflammatory processes involving the lung, such as asthma and sarcoidosis. Steroids modulate cytokine gene expression and lead to an impairment of T cell function in vivo (reviewed in 50 . They also down-regulate the expression of chemokines, including IL-8 51 , MCP-1 52 , RANTES 48 , eotaxin 26 , and MCP-4 25 . In contrast to IL-8, we found that IP-10, Mig, and I-TAC expression was not diminished by pretreatment of epithelial cells with dexamethasone.

Th2 cells secrete IL-4, IL-5, IL-10, and IL-13 and are associated with allergic and humoral-type immune responses 1 . Since IL-4 down-regulates IP-10 expression in human monocytes 46 and murine macrophages 47 , we asked whether IL-4, IL-13, and IL-10 would similarly modulate IP-10 and Mig expression in bronchial epithelial cells. We found that none of these cytokines affected IP-10, Mig, or I-TAC expression induced by IFN-{gamma} or IFN-{gamma}/TNF-{alpha}. Again in contrast, IL-8 expression was increased by IL-4 and IL-13 in TNF-{alpha}-treated NHBEC.

In addition to their differences in leukocyte selectivity and cytokine regulation, IP-10/Mig and IL-8 have opposing effects on endothelial cells. IP-10 and Mig are antiangiogenic 44, 53, 54, 55, 56 , whereas IL-8 is proangiogenic 57 . This has led to speculation that the relative levels of IP-10/Mig and IL-8 may affect neovascularization in the lung following a given inflammatory process 58 . For example, in idiopathic pulmonary fibrosis in which neovascularization is prevalent, pulmonary levels of IP-10 were decreased, while those of IL-8 increased compared with the levels in normal lung 58 . It is interesting to note that high levels of TNF-{alpha} have been demonstrated in the lungs of patients with idiopathic pulmonary fibrosis 59, 60, 61 . Our finding in bronchial epithelial cells that TNF-{alpha} was a much better inducer of IL-8 than IP-10 or Mig may explain the up-regulation of IL-8 compared with IP-10 and resultant angiogenic environment found in idiopathic pulmonary fibrosis.

Activated epithelial cells secrete high levels of IP-10 in vitro and appear to be a major source of IP-10 in vivo. IFN-{gamma}-treated human keratinocytes secreted more IP-10 than IFN-{gamma}-treated endothelial cells, monocytes, or fibroblasts 32 . Furthermore, IP-10 was expressed in keratinocytes in psoriatic lesions, while it was not detected in normal keratinocytes 35 . Similar observations were reported in fixed drug eruptions 62 and in cutaneous T-cell lymphomas 63 . IFN-{gamma} is up-regulated in these skin disorders 1, 64 and Might be responsible for keratinocyte activation and IP-10 expression. Our findings extend these observations to bronchial epithelial cells, which secreted very high levels of IP-10 following IFN-{gamma} stimulation. Furthermore, IP-10 was expressed in the bronchial epithelium in patients with active TB, in which IFN-{gamma} and IL-12 have been shown to be up-regulated 2 . IP-10 secretion from epithelial cells may play an important role in recruiting activated T cells into the epithelium, which is necessary for protective immunity at these surfaces in contact with the environment.

IP-10 is expressed in inflamed tissues in which the Th1-type cytokine IFN-{gamma} is up-regulated. This has been demonstrated for psoriasis 35 , tuberculoid leprosy 36 , ulcerative colitis 65 , sarcoidosis 37 , and atherosclerosis (A. Sauty and F. Mach, unpublished observations), and we now present data for active TB. Interestingly, CXCR3 is found expressed predominantly on Th1 clones and on a significant fraction of memory circulating T cells 41, 42 . Recent studies have implicated CXCR3+ T cells in the pathogenesis of rheumatoid arthritis 42 , ulcerative colitis 40 , and atherosclerosis (A. Sauty and F. Mach, unpublished observations), since the vast majority of infiltrating T cells were CXCR3+. In addition, it has recently been shown that IP-10 selectively activated and enhanced Ag and mitogen-driven IFN-{gamma} (but not IL-4) cytokine gene expression in T cells 66 . Therefore, it is tempting to speculate that in Th1-type disease, IP-10, Mig, and I-TAC are induced by IFN-{gamma} and chemoattract CXCR3+ T cells, which in turn are induced to express more IFN-{gamma}, establishing a mechanism for amplifying a Th1-type immune response.


   Acknowledgments
 
We thank Drs. Joshua Farber and Hans Oettgen for cDNA probes and Dr. R. Menzies (Montreal Chest Institute Research Centre) for performing bronchial biopsies and BAL.


   Footnotes
 
1 A.S. was supported by a grant of the Société Suisse de Pneumologie and Association Suisse contre la Tuberculose et les Maladies Pulmonaires. M.D. was supported by a National Institutes of Health postdoctoral fellowship. R.A.T. was supported by the Medical Research Council of Canada. A.D.L. was supported by a Cancer Research Institute/Benjamin Jacobson Family award, a Culpeper Medical Scholars award, a grant from the Defense Advanced Research Planning Agency, and National Institutes of Health Grants CA69212 and AI40618. Back

2 Address correspondence and reprint requests to Dr. Andrew D. Luster, Infectious Disease Unit, Massachusetts General Hospital East, Building 149, 13th Street, Charlestown, MA 02129. E-mail address: Back

3 Abbreviations used in this paper: TB, tuberculosis; IP-10, IFN-induced protein of 10 kDa; Mig, monokine induced by IFN-{gamma}; I-TAC, IFN-inducible T-cell {alpha} chemoattractant; MCP, monocyte chemoattractant protein; BAL, bronchoalveolar lavage; NHBEC, normal human bronchial epithelial cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; h, human. Back

Received for publication June 17, 1998. Accepted for publication December 7, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mosmann, T. R., S. Sad. 1996. The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol. Today 17:138.[Medline]
  2. Taha, R. A., T. C. Kotsimbos, Y. L. Song, D. Menzies, Q. Hamid. 1997. IFN-{gamma} and IL-12 are increased in active compared with inactive tuberculosis. Am. J. Respir. Crit. Care Med. 155:1135.[Abstract]
  3. Edwards, D., C. H. Kirkpatrick. 1986. The immunology of mycobacterial diseases. Am. Rev. Respir. Dis. 134:1062.[Medline]
  4. Barnes, P. F., A. B. Bloch, P. T. Davidson, J. D. D. Snider. 1991. Tuberculosis in patients with human immunodeficiency virus infection. N. Engl. J. Med. 324:1644.[Medline]
  5. Horsburgh, C. R.. 1991. Mycobacterium avium complex infection in the acquired immunodeficiency syndrome. N. Engl. J. Med. 324:1332.[Medline]
  6. Springer, T. A.. 1994. Traffic signals for lymphocyte recirculation and leukocyte eMigration: the multiple paradigm. Cell 76:301.[Medline]
  7. Butcher, E. C., L. J. Picker. 1996. Lymphocyte homing and homeostasis. Science 272:60.[Abstract]
  8. Thompson, A. B., R. A. Robbins, D. J. Romberger, J. H. Sisson, J. R. Spurzem, H. Teschler, S. I. Rennard. 1995. Immunological functions of the pulmonary epithelium. Eur. Respir. J. 8:127.[Abstract]
  9. Bloemen, P. G. M., M. C. V. D. Tweel, P. A. J. Henricks, F. Engels, S. S. Wagenaar, A. A. J. J. L. Rutten, F. P. Nijkamp. 1993. Expression and modulation of adhesion molecules on human bronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 9:586.
  10. Atsuta, J., S. A. Sterbinsky, J. Plitt, L. M. Schwiebert, B. S. Bochner, R. P. Schleimer. 1997. Phenotyping and cytokine regulation of the BEAS-2B human bronchial epithelial cell: demonstration of inducible expression of the adhesion molecules VCAM-1 and ICAM-1. Am. J. Respir. Cell Mol. Biol. 17:571.[Abstract/Free Full Text]
  11. Rossi, G. A., O. Sacco, B. Balbi, S. Oddera, T. Mattioni, G. Corte, C. Ravazzoni, L. Allegra. 1990. Human ciliated bronchial epithelial cells: expression of the HLA-DR antigens and of the HLA-DR {alpha} gene, modulation of HLA-DR antigens by {gamma}-interferon and antigen-presenting function in the mixed leukocyte reaction. Am. J. Respir. Cell Mol. Biol. 3:431.
  12. Luster, A. D.. 1998. Chemokines: chemotactic cytokines that mediate inflammation. N. Engl. J. Med. 338:436.[Free Full Text]
  13. Clark-Lewis, I., B. Dewald, T. Geiser, B. Moser, M. Baggiolini. 1993. Platelet factor 4 binds to interleukin-8 receptors and activates neutrophils when its N terminus is modified with Glu-Leu-Arg. Proc. Natl. Acad. Sci. USA 90:3574.[Abstract/Free Full Text]
  14. Taub, D. D., A. R. Lloyd, K. Conlon, J. M. Wang, J. R. Ortaldo, A. Harada, K. Matsushima, D. J. Kelvin, J. J. Oppenheim. 1993. Recombinant human interferon-inducible protein 10 is a chemoattractant for human monocytes and T lymphocytes and promotes T cell adhesion to endothelial cells. J. Exp. Med. 177:1809.[Abstract/Free Full Text]
  15. Loetscher, M., B. Gerber, P. Loestcher, S. A. Jones, L. Piali, I. Clark-Lewis, M. Baggiolini, B. Moser. 1996. Chemokine receptor specific for IP-10 and Mig: structure, function, and expression in activated T-lymphocytes. J. Exp. Med. 184:963.[Abstract/Free Full Text]
  16. Liao, F., R. L. Rabin, J. R. Yannelli, L. G. Koniaris, P. Vanguri, J. M. Farber. 1995. Human Mig chemokine: biochemical and functional characterization. J. Exp. Med. 182:1301.[Abstract/Free Full Text]
  17. Farber, J. M.. 1997. Mig and IP-10: CXC chemokines that target lymphocytes. J. Leukocyte Biol. 61:246.[Abstract]
  18. Cole, K. E., C. A. Strick, M. Loetscher, T. J. Paradis, K. T. Ogborne, R. P. Gladue, W. Lin, J. G. Boyd, B. Moser, D. E. Wood, B. G. Sahagan, K. Neote. 1998. Interferon inducible T-cell {alpha} chemoattractant (I-TAC): a novel non-ELR CXC chemokine with potent activity on activated T-cells through selective high affinity binding to CXCR3. J. Exp. Med. 187:2009.[Abstract/Free Full Text]
  19. Bleul, C. C., R. C. Fuhlbrigge, J. M. Casasnovas, A. Aiuti, T. A. Springer. 1996. A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1). J. Exp. Med. 184:1101.[Abstract/Free Full Text]
  20. D’Apuzzo, M., A. Rolink, M. Loetscher, J. A. Hoxie, I. Clark-Lewis, F. Melchers, M. Baggiolini, B. Moser. 1997. The chemokine SDF-1, stromal cell-derived factor 1, attracts early stage B cell precursors via the chemokine receptor CXCR4. Eur. J. Immunol. 27:1788.[Medline]
  21. Legler, D. F., M. Loetscher, R. S. Roos, I. Clark-Lewis, B. Moser. 1998. B cell-attracting chemokine 1, a human CXC chemokine expressed in lymphoid tissues, selectively attracts B lymphocytes via BLR1/CXCR5. J. Exp. Med. 187:655.[Abstract/Free Full Text]
  22. Sousa, A. R., S. J. Lane, J. A. Nakhosteen, T. Yoshimura, T. H. Lee, R. N. Poston. 1994. Increased expression of the monocyte chemoattractant protein-1 in bronchial tissue from asthmatic subjects. Am. J. Respir. Cell Mol. Biol. 10:142.[Abstract]
  23. Humbert, M., S. Ying, C. Corrigan, G. Menz, J. Barkans, R. Pfister, Q. Meng, J. V. Damme, G. Opdenakker, S. R. Durham, A. B. Kay. 1997. Bronchial mucosal expression of the genes encoding chemokines RANTES and MCP-3 in symptomatic atopic and nonatopic asthmatics: relationship to the eosinophil-active cytokines interleukin (IL)-5, granulocyte macrophage-colony-stimulating factor, and IL-3. Am. J. Respir. Cell Mol. Biol. 16:1.[Abstract]
  24. Garcia-Zepeda, E. A., C. Combadiere, M. E. Rothenberg, M. N. Sarafi, F. Lavigne, Q. Hamid, P. M. Murphy, A. D. Luster. 1996. Human monocyte chemoattractant protein (MCP)-4 is a novel CC chemokine with activities on monocytes, eosinophils, and basophils induced in allergic and nonallergic inflammation that signals through the CC chemokine receptors (CCR)-2 and -3. J. Immunol. 157:5613.[Abstract]
  25. Stellato, C., P. Collins, P. D. Ponath, D. Soler, W. Newman, G. L. Rosa, H. Li, J. White, L. M. Schwiebert, C. Bickel, et al 1997. Production of the novel C-C chemokine MCP-4 by airway cells and comparison of its biological activity to other C-C chemokines. J. Clin. Invest. 99:926.[Medline]
  26. Lilly, C. M., H. Nakamura, H. Kesselman, C. Nagler-Anderson, K. Asano, M. E. Rothenberg, E. A. Garcia-Zepeda, J. M. Drazen, A. D. Luster. 1997. Expression of eotaxin by human lung epithelial cells: induction by cytokines and inhibition by glucocorticoids. J. Clin. Invest. 99:1767.[Medline]
  27. Garcia-Zepeda, E. A., M. E. Rothenberg, R. T. Ownbey, J. Celestin, P. Leder, A. D. Luster. 1996. Human eotaxin is a specific chemoattractant for eosinophil cells and provides a new mechanism to explain tissue eosinophilia. Nat. Med. 2:449.[Medline]
  28. Standiford, T. J., S. L. Kunkel, M. A. Basha, S. W. Chensue, III J. P. Lynch, G. B. Toews, J. Westwick, R. M. Strieter. 1990. Interleukin-8 gene expression by pulmonary epithelial cell line: a model for cytokine networks in the lung. J. Clin. Invest. 86:1945.
  29. Walz, A., R. Burgener, B. Car, M. Baggiolini, S. L. Kunkel, R. M. Streiter. 1991. Structure and neutrophil-activating properties of a novel inflammatory peptide (ENA-78) with homology to interleukin 8. J. Exp. Med. 174:1355.[Abstract/Free Full Text]
  30. Luster, A. D., J. C. Unkeless, J. V. Ravetch. 1985. {gamma}-Interferon transcriptionally regulates an early-response gene containing homology to platelet proteins. Nature 315:672.[Medline]
  31. Ransohoff, R. M., T. A. Hamilton, M. Tani, M. H. Stoler, H. E. Shick, J. A. Major, M. L. Estes, D. M. Thomas, V. K. Tuohy. 1993. Astrocyte expression of mRNA encoding cytokines IP-10 and JE/MCP-1 in experimental autoimmune encephalomyelitis. FASEB J. 7:592.[Abstract]
  32. Luster, A. D., J. V. Ravetch. 1987. Biochemical characterization of a {gamma} interferon-inducible cytokine (IP-10). J. Exp. Med. 166:1084.[Abstract/Free Full Text]
  33. Cassatella, M. A., S. Gasperini, F. Calzetti, A. Bertagnin, A. D. Luster, P. P. McDonald. 1997. Regulated production of the interferon-{gamma}-inducible protein-10 (IP-10) chemokine by human neutrophils. Eur. J. Immunol. 27:111.[Medline]
  34. Farber, J. M.. 1990. A macrophage mRNA selectively induced by {gamma}-interferon encodes a member of the platelet factor 4 family of cytokines. Proc. Natl. Acad. Sci. USA 87:5238.[Abstract/Free Full Text]
  35. Gottlieb, A. B., A. D. Luster, D. N. Posnett, D. M. Carter. 1988. Detection of a {gamma} interferon-induced protein IP-10 in psoriatic plaques. J. Exp. Med. 168:941.[Abstract/Free Full Text]
  36. Kaplan, G., A. D. Luster, G. Hancock, Z. A. Cohn. 1987. The expression of a {gamma} interferon-induced protein (IP-10) in delayed immune responses in human skin. J. Exp. Med. 166:1098.[Abstract/Free Full Text]
  37. Agostini, C., M. Cassatella, R. Sancetta, R. Zambello, L. Trentin, S. Gasperini, A. Perin, F. Piazza, M. Siviero, A. D. Luster, et al 1998. Involvement of the IP-10 chemokine in sarcoid granulomatous reactions. J. Immunol. 161:6413.[Abstract/Free Full Text]
  38. Lahrtz, F., L. Piali, D. Nadal, H. W. Pfister, K. S. Spanaus, M. Baggiolini, A. Fontana. 1998. Chemotactic activity on mononuclear cells in the cerebrospinal fluid of patients with viral meningitis is mediated by interferon-{gamma}-inducible protein-10 and monocyte chemotactic protein-1. Eur. J. Immunol. 10:2484.
  39. Gómez-Chiarri, M., A. Ortiz, S. González-Cuadrado, D. Seron, S. N. Emancipator, T. A. Hamilton, A. Barat, J. J. Plaza, E. Gonzáles, J. Egido. 1996. Interferon-inducible protein-10 is highly expressed in rats with experimental nephrosis. Am. J. Pathol. 148:301.[Abstract]
  40. Qin, S., J. B. Rottman, P. Myers, N. Kassam, M. Weinblatt, M. Loetscher, A. E. Koch, B. Moser, C. R. Mackay. 1998. The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions. J. Clin. Invest. 101:746.[Medline]
  41. Sallusto, F., D. Lenig, C. R. Mackay, A. Lanzavecchia. 1998. Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes. J. Exp. Med. 187:875.[Abstract/Free Full Text]
  42. Loetscher, P., M. Uguccioni, L. Bordoli, M. Baggiolini, B. Moser, C. Chizzolini, J. M. Dayer. 1998. CCR5 is characteristic of Th1 lymphocytes. Nature 391:344.[Medline]
  43. Bonecchi, R., G. Bianchi, P. Panina-Bordignon, D. D’Ambrosio, R. Lang, A. Borsatti, S. Sozzani, P. Allavena, P. A. Gray, A. Mantovani, and F. Sinigaglia. 1998. Differential expression of chemokine receptors and chemotactic responsiveness of Th1 and Th2 cells. J. Exp. Med.
  44. Luster, A. D., S. M. Greenberg, P. Leder. 1995. The IP-10 chemokine binds to a specific cell surface heparan sulfate site shared with platelet factor 4 and inhibits endothelial cell proliferation. J. Exp. Med. 182:219.[Abstract/Free Full Text]
  45. Hamid, Q., M. Azzawi, S. Ying, R. Moqbel, A. J. Wardlaw, C. J. Corrigan, B. Bradley, S. R. Durham, J. V. Collins, P. K. Jeffrey, et al 1991. Expression of mRNA for IL-5 in mucosal bronchial biopsies from asthma. J. Clin. Invest. 87:1541.
  46. Larner, A. C., E. F. Petricoin, Y. Nakagawa, D. S. Finbloom. 1993. IL-4 attenuates the transcriptional activation of both IFN-{alpha}- and IFN-{gamma}-induced cellular gene expression in monocytes and monocytic cell lines. J. Immunol. 150:1944.[Abstract]
  47. Deng, W., Y. Ohmori, T. A. Hamilton. 1994. Mechanisms of IL-4-mediated suppression of IP-10 gene expression in murine macrophages. J. Immunol. 153:2130.[Abstract]
  48. Stellato, C., L. A. Beck, G. A. Gorgone, D. Proud, T. J. Schall, S. J. Ono, L. M. Lichtenstein, R. P. Schleimer. 1995. Expression of the chemokine RANTES by a human bronchial epithelial cell line. J. Immunol. 155:410.[Abstract]
  49. Boorsma, D. M., P. D. Haan, R. Willemze, T. J. Stoof. 1994. Human growth factor (huGRO), interleukin-8 (IL-8) and interferon-{gamma}-inducible protein ({gamma}-IP-10) gene expression in cultured normal human keratinocytes. Arch. Dermatol. Res. 286:471.[Medline]
  50. Bloom, J. W.. 1997. Molecular pharmacology of glucocorticoids. Clin. Asthma Rev. 1:99.
  51. Kwon, O. J., B. T. Au, P. D. Collins, J. N. Baraniuk, I. M. Adcock, K. F. Chung, P. J. Barnes. 1994. Inhibition of interleukin-8 expression by dexamethasone in human cultured airway epithelial cells. Immunology 81:389.[Medline]
  52. Mukaida, N., C. C. Zachariae, G. L. Gusella. 1991. Dexamethasone inhibits the induction of monocyte chemotactic-activating factor production by IL-1 or tumor necrosis factor. J. Immunol. 146:1212.[Abstract]
  53. Strieter, R. M., S. L. Kunkel, D. A. Arenberg, M. D. Burdick, P. J. Polverini. 1995. Interferon {gamma}-inducible protein 10 (IP-10), a member of the C-X-C chemokine family, is an inhibitor of angiogenesis. Biochem. Biophys. Res. Commun. 210:51.[Medline]
  54. Angiolillo, A. L., C. Sgadari, D. D. Taub, F. Liao, J. M. Farber, S. Maheshwari, H. K. Kleinmann, G. H. Reaman, G. Tosato. 1995. Human interferon-inducible protein 10 is a potent inhibitor of angiogenesis in vivo. J. Exp. Med. 182:155.[Abstract/Free Full Text]
  55. Sgadari, C., A. L. Angiolillo, G. Tosato. 1996. Inhibition of angiogenesis by interleukin-12 is mediated by the interferon-inducible protein 10. Blood 87:3877.[Abstract/Free Full Text]
  56. Sgadari, C., J. M. Farber, A. L. Angiolillo, F. Liao, J. Teruya-Feldstein, P. R. Burd, L. Yao, G. Gupta, C. Kanegane, G. Tosato. 1997. Mig, the monokine induced by interferon-{gamma}, promotes tumor necrosis in vivo. Blood 1997:2635.
  57. Koch, A. E., P. J. Polverini, S. L. Kunkel, L. A. Harlow, L. A. DiPietro, V. M. Elner, S. G. Elner, R. M. Strieter. 1992. Interleukin-8 as a macrophage-derived mediator of angiogenesis. Science 258:1798.[Abstract/Free Full Text]
  58. Keane, M. P., D. A. Arenberg, III J. P. Lynch, R. I. Whyte, M. D. Iannettoni, M. D. Burdick, C. A. Wilke, S. B. Morris, M. C. Glaas, B. DiGiovine, et al 1997. The CXC chemokines, IL-8 and IP-10, regulate angiogenetic activity in idiopathic pulmonary fibrosis. J. Immunol. 159:1437.[Abstract]
  59. Piguet, P. F., C. Ribaux, V. Karpuz, G. E. Grau, Y. Kapanci. 1993. Expression and localization of tumor necrosis factor-{alpha} and its mRNA in idiopathic pulmonary fibrosis. Am. J. Pathol. 143:651.[Abstract]
  60. Kapanci, Y., A. Desmouliere, J. C. Pache, M. Redard, G. Gabbiani. 1995. Cytoskeletal protein modulation in pulmonary alveolar myofibroblasts during idiopathic pulmonary fibrosis: possible role of transforming growth factor ß and tumor necrosis factor {alpha}. Am. J. Respir. Cell Mol. Biol. 152:2163.
  61. Zhang, Y., T. C. Lee, B. Guillemin, M. C. Yu, W. N. Rom. 1993. Enhanced IL-1ß and tumor necrosis factor-{alpha} release and messenger RNA expression in macrophages from idiopathic pulmonary fibrosis or after asbestos exposure. J. Immunol. 150:4188.[Abstract]
  62. Smoller, B. R., A. D. Luster, J. F. Krane, J. Krueger, M. H. Gray, N. S. McNutt, A. Hsu, A. B. Gottlieb. 1991. Fixed drug eruptions: evidence for a cytokine-mediated process. J. Cutan. Pathol. 18:13.[Medline]
  63. Sarris, A. H., T. Esgleyes-Ribot, M. Crow, H. E. Broxmeyer, N. Karasavvas, W. Pugh, D. Grossman, A. Deisseroth, M. Duvic. 1995. Cytokine loops involving interferon-{gamma} and IP-10, a cytokine chemotactic for CD4+ lymphocytes: an explanation for the epidermotropism of cutaneous T-cell lymphoma?. Blood 86:651.[Abstract/Free Full Text]
  64. Saed, G., D. P. Fiveson, Y. Naidu, B. J. Nickoloff. 1994. Mycosis fungoides exhibits a Th1-type cell-mediated cytokine profile, whereas Sezary syndrome expresses a Th2 profile. J. Invest. Dermatol. 103:29.[Medline]
  65. Grimm, M. C., W. F. Doe. 1996. Chemokines in inflammatory bowel disease mucosa: expression of RANTES, macrophage inflammatory protein (MIP)-1{alpha}, MIP-1ß, and {gamma}-interferon-inducible protein-10 by macrophages, lymphocytes, endothelial cells, and granulomas. Inflammatory Bowel Dis. 2:88.
  66. Gangur, V., F. E. R. Simons, K. T. Hayglass. 1998. Human IP-10 selectively promotes dominance of polyclonally activated and environmental antigen-driven IFN{gamma} over IL-4 responses. FASEB J. 12:705.[Abstract/Free Full Text]



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