CCR2 and CXCR3 agonistic chemokines are differently expressed and regulated in human alveolar epithelial cells type II
© Pechkovsky et al. 2005
Received: 16 February 2005
Accepted: 20 July 2005
Published: 20 July 2005
The attraction of leukocytes from circulation to inflamed lungs depends on the activation of both the leukocytes and the resident cells within the lung. In this study we determined gene expression and secretion patterns for monocyte chemoattractant protein-1 (MCP-1/CCL2) and T-cell specific CXCR3 agonistic chemokines (Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11) in TNF-α-, IFN-γ-, and IL-1β-stimulated human alveolar epithelial cells type II (AEC-II). AEC-II constitutively expressed high level of CCL2 mRNA in vitro and in situ , and released CCL2 protein in vitro . Treatment of AEC-II with proinflammatory cytokines up-regulated both CCL2 mRNA expression and release of immunoreactive CCL2, whereas IFN-γ had no effect on CCL2 release. In contrast, CXCR3 agonistic chemokines were not detected in freshly isolated AEC-II or in non-stimulated epithelial like cell line A549. IFN-γ, alone or in combination with IL-1β and TNF-α resulted in an increase in CXCL10, CXCL11, and CXCL9 mRNA expression and generation of CXCL10 protein by AEC-II or A549 cells. CXCL10 gene expression and secretion were induced in dose-dependent manner after cytokine-stimulation of AEC-II with an order of potency IFN-γ>>IL-1β ≥ TNF-α. Additionally, we localized the CCL2 and CXCL10 mRNAs in human lung tissue explants by in situ hybridization, and demonstrated the selective effects of cytokines and dexamethasone on CCL2 and CXCL10 expression. These data suggest that the regulation of the CCL2 and CXCL10 expression exhibit significant differences in their mechanisms, and also demonstrate that the alveolar epithelium contributes to the cytokine milieu of the lung, with the ability to respond to locally generated cytokines and to produce potent mediators of the local inflammatory response.
Many pulmonary disorders are characterized by accumulation and activation of inflammatory cells within the lung, followed by the release of regulatory mediators, resulting in macrophage/lymphocyte alveolitis. Sarcoidosis, tuberculosis, hypersensitivity pneumonitis, eosinophilic pneumonia, and usual interstitial pneumonia represent such lung diseases that have in common the selective recruitment and activation of different types of leukocytes, and therefore, exhibit distinct forms of alveolitis [1–5]. The inflammatory phase of alveolitis is initiated by epithelial and/or endothelial injury involving the structures of the alveolar wall. The alveolar surface area of the lung is covered with a layer of alveolar epithelial cells type I and type II. Type I cells function as a physical barrier, whereas type II cells produce surfactant and act as progenitors to replace injured alveolar epithelial cells type I . Thus, located at the boundary between the alveolar airspace and the interstitium, alveolar epithelial cells type II (AEC-II) are ideally situated to regulate the recruitment and activation of different types of leukocytes through the production of chemokines/cytokines in response to inflammatory stimulation from the alveolar space. Recent studies have suggested that AEC-II secrete a variety of mediators, including proinflammatory cytokines and chemokines important for the recruitment of monocytes / macrophages and T cells into the lung interstitium and alveolar space [7–10].
Although leukocyte recruitment is a complex and multistep process with involvement of different types of cells, cell-surface adhesion molecules, and soluble inflammatory mediators, the prominent role of the attractant molecules such as chemokines has widely been appreciated [11, 12]. Chemokines are a superfamily of small, secreted proteins that direct the recruitment of leukocytes to the sites of inflammation. They are classified into four subfamilies on the basis of the primary sequence of the first two of four invariant cysteine residues, and named according to the recommendation for new systematic nomenclature for human chemokines . CC chemokines/CCL attract monocytes, eosinophils, basophils, dendritic and T cells and signal through chemokine receptors CCR1 to CCR10. In contrast to CC chemokines, the CXC chemokines (CXCL) are divided into two classes depending on the presence of the glutamate-leucine-arginine motif (ELR) in the NH2-terminal domain. The CXC chemokines signal through the chemokine receptors CXCR1 to CXCR5 (reviewed in ). The CC chemokine, monocyte chemoattractant protein-1/CCL2 (CCL2), has been shown in vitro and in vivo to target preferentially monocytes and memory T cells through the CCR2 [13–16]. Monokine induced by IFN-γ (Mig/CXCL9), IFN-induced protein of 10 kDa (IP-10/CXCL10), and IFN-inducible T-cell α-chemoattractant (I-TAC/CXCL11) are all members of the non-ELR CXCL class and target preferentially memory T cells and natural killer cells through the single and shared receptor CXCR3 [17, 18]. Recently, it has been reported that some chemokine receptors are associated with human Th1 or Th2 cells, and therefore the respective agonists can selectively attract the respective Th cell subset into inflammatory sites (reviewed in ).
In this context, we hypothesized that AEC-II are an important source of CCL2 and the CXCR3 agonistic chemokines in the lung, and through expression of these mediators involved in the homing of immune effector cells during lung inflammatory processes. As a model we investigated the gene expression and production of chemokines, important for the recruitment of CCR2 and CXCR3 bearing mononuclear leukocytes, by human primary AEC-II and airway epithelial like cell line A549 after exposure of the cells to the proinflammatory cytokines TNF-α, IFN-γ, and IL-1β. A striking result was the difference between spontaneous and cytokine-induced CCL2, CXCL9, CXCL10, and CXCL11 mRNA expression and/or protein production in both human AEC-II and A549 cell cultures. Finally, we provide evidence of selective CCL2 and CXCL10 mRNA expression of human AEC-II in vivo .
Materials and Methods
The following materials were purchased from GIBCO BRL (Paisley, Scotland): PBS, RPMI 1640 medium with 2 mM L-glutamine, FCS, HEPES, TRIZOL Reagent, SuperScript™ RNase H- reverse transcriptase (RT), oligo (dT)12–18 primer and agarose; penicillin/streptomycin solution and sodium pyruvate from Biochrom (Berlin, FRG); trypsin/EDTA solution from Boehringer-Mannheim (Mannheim, FRG); collagen R from Serva (Heidelberg, FRG); chloroform and isopropanol from Merck (Darmstadt, FRG); recombinant human IFN-γ (specific activity 3 × 107 U/mg) and recombinant human IL-1β (specific activity 2 × 108 U/mg) from Biotrend (Cologne, FRG); recombinant human TNF-α was a courtesy of Dr. E. Schlick (Knoll AG, Ludwigshafen, FRG); dexamethasone from Sigma (St. Louis, MO); 100 mm plastic dishes, 75 cm2 tissue culture flask and 24-well cell culture plates from NUNC (Wiesbaden, FRG). All reagents used were of the highest available grade and were dissolved in pyrogen-free water.
Human Lung Tissue
Lung tissue samples were obtained from subjects with lung cancer undergoing lobectomy or pneumectomy. Twelve patients with bronchogenic carcinoma, without any other systemic or pulmonary diseases, were enrolled in this study. All subjects were smokers and have had no respiratory tract infection within the last month. None of them was taking immunosuppressants within one month before surgery. In addition, lung tissue samples were obtained from 3 patients with pulmonary sarcoidosis who had undergone diagnostic wedge biopsies and from 3 patients with pulmonary tuberculosis who had undergone upper lobectomy due to destructive tuberculoma. Informed consents were obtained from all subjects. The study was approved by the medical ethics committees of the involved institutions.
Primary Human Alveolar Epithelial Cells Type II
Samples from macroscopically tumor-free lung tissue were cut from the surgical specimens and used for cell isolation procedure as described previously . In brief, the lung tissue was first sliced and slices were washed three times at 4°C in PBS. The washed slices were incubated in sterile dispase solution at 37°C for 45 min. After dispase digestion the lung tissue slices were cut into small, pipetable pieces, and thoroughly pipetted for several min. Crude tissue and cell suspensions were filtered through nylon gauze with meshes of 100 μm, 50 μm, and 20 μm. The resulting single cell suspension was placed on Ficoll separating solution and centrifuged at 800 × g for 20 min. The AEC-II-enriched cells from the interphase were incubated in 100 mm plastic dishes at 37°C in humidified air containing 5% CO2 for 15, 20 and 30 min with seeding of non-adherent cells on fresh dishes for each time interval to remove adherent cells (alveolar macrophages, monocytes, fibroblasts, and endothelial cells). To remove remaining monocytes/macrophages and lymphocytes, antibodies against CD3 (OKT3, ECACC 86022706) and CD14 (HB-246 ATCC) were added and the antibody-binding cells were removed by anti-mouse IgG coated magnetic beads and Magnetic Activated Cell Sorting (MACS) system (Miltenyi Biotec, Bergisch Gladbach, FRG) as suggested by the supplier. Identity of type II alveolar epithelial cells was confirmed by a modified Papanicolaou staining, their alkaline phosphatase activity, and SP-A mRNA expression in RT-PCR (see below). Cell purity was assessed by immunoperoxidase staining with monoclonal antibodies directed against CD3 and CD14 (Immunotech, Marseille, France) as previously described . Viability of the AEC-II after isolation was > 97% as determined by trypan blue exclusion. After the final step of MACS purification, the AEC-II preparations included in this report were free of CD14+ and CD3+ cells as determined by immunocytochemistry. 98 ± 1.3% of cells were identified as AEC-II by the presence of dark blue inclusions as revealed by modified Papanicolaou staining and 93 ± 2.1% of cells were positive for alkaline phosphatase (data not shown). All RNA samples isolated from these AEC-II preparations contained SP-A mRNA, and CD3 and CD14 mRNA were found in four of twelve samples by RT-PCR (data not shown). In order to avoid false positive results from contaminated cells, these four AEC-II preparations were excluded from further experimental data analysis.
A549 Cell Line
A549 cells were used as the positive control for CCL2, CXCL9, CXCL10, and CXCL11 mRNA expression and protein production upon stimulation with proinflammatory cytokines. Experiments were performed with cells after 7, 8 and 9 passages after thawing and inoculation in culture. Cells were grown on 75 cm2 tissue culture flask in culture medium (CM) (RPMI1640 medium, 10% heat inactivated FCS, 1% penicillin/streptomycin solution, 1% sodium pyruvate solution and 20 mM HEPES) in a humidified atmosphere containing 5% CO2 at 37°C for 5 days. After this culture period, cells were removed from plastic surfaces by treatment with trypsin/EDTA solution (0.05/0.02% in PBS) for 10 min at 37°C, washed twice in PBS and suspended in CM.
Immediately after purification, AEC-II were suspended in CM (1 × 106 cells/ml) and treated with TNF-α (1 – 10 ng/ml), IFN-γ (10 – 100 U/ml) or IL-1β (10 – 100 U/ml) in collagen R-coated 24-well plates at 37°C, 5% CO2 atmosphere. A549 cells were plated at 1 × 106 /ml in 24-well plates in the same culture condition as for AEC-II and stimulated with TNF-α (1 – 10 ng/ml), IFN-γ (50 – 500 U/ml) or IL-1β (50 – 500 U/ml) in different combinations as indicated in the Results section. At the indicated time, cell-free supernatants were harvested and stored at -70°C, and cell pellets were extracted for total RNA. The cell viability after culture always exceeded 95% in both AEC-II and A549 cells as determined by trypan blue exclusion. For samples of RNA from freshly isolated AEC-II or harvested A549 cells, they were subjected to RNA isolation procedures before cultures, henceforth referred to as non-cultured controls.
Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
Primers used in RT-PCR analysis
Product Size (bp)
F† : 5'-CAA ACT GAA GCT CGC ACT CTC GCC-3'
R† : 5'-ATT CTT GGG TTG TGG AGT GAG TGT TCA-3'
F: 5'-CGT GGT AAA ACA CTT GCG GAT ATT-3'
R: 5'-CAA TCA TGC TTC CAC TAA CCG ACT-3'
F: 5'-CCA TGA ATC AAA CTG CGA TTC TG-3'
R: 5'-CTT GGA AGC ACT GCA TCG ATT T-3'
F: 5'-AAA GGC TGG TTA CCA TCG GAG T-3'
R: 5'-RTGT TGC CAG TAT CCC ATA GCG T-3'
F: 5'-GGC TGT CCT CAT CCT GGC TAT CAT-3'
R: 5'-ACT GGT TTC CTT GAA GGT GGC TGT-3'
F: 5'-ACT CCC TCA ATC TGT CGT TCG CTG-3'
R: 5'-CTG AAG CCA AGG CAG TTT GAG TCC-3'
F: 5'-TCT TTG GAT GCC AAC TCA GC-3'
R: 5'-CTT TAT TCA GCT CAG GGG TG-3'
F: 5'-AGC GGG AAA TCG TGC GTG-3'
R: 5'-CAG GGT ACA TGG TGG TGCC-3'
Measurement of CCL2 and CXCL10 Concentrations
Chemokines concentrations in A549 cell and primary cultured AEC-II supernatants were measured in duplicate by commercial available ELISA kits. Human CCL2 and CXCL10 ELISA kits were from HyCult biotechnology (Uden, the Netherlands). The assays were performed as suggested by the suppliers. Optical density readings were obtained with a MRX Microplate Reader and analyzed with Revelation 2.0 software (both from Dynex Technologies, FRG). The lower detection limit of the assays was 10 pg/ml for CCL2 and 20 pg/ml for CXCL10. For duplicate samples an intra assay coefficient of variation (CV) of < 10% and interassay CV of < 20% was accepted.
In Situ Hybridization (ISH)
Paraffin embedded lung tissue samples were prepared from the same surgical specimens as described above and used for ISH. These tissue samples showed normal architecture with few intra-alveolar macrophages and edema. Some lung tissue explants were placed in CM alone or with IFN-γ (500 U/ml) and IL-1β (500 U/ml), and/or 10-4 M dexamethasone and incubated at 37°C in humidified air containing 5% CO2 for 24 h. After incubation, these lung tissue explants were further used for ISH. The cDNA probes corresponding to CCL2 and CXCL10 mRNAs were produced by PCR as described before, filtered through Centri-Sep spin columns (Applied Biosystems, Foster City, CA), and labeled with digoxigenin (DIG) following the manufacturer's instructions (Dig-High-Prime, Roche, FRG). After deparaffinization, in situ hybridization was carried out overnight and, after washing at high stringency, detection was performed by application of Anti-Dig/alkaline-phosphatase-conjugate and new-fuchsin as substrate for alkaline phosphatase . Slides were counterstained with Mayers hemalum and mounted with Kayser's glyceringelatine. For negative control, sections were hybridized with hybridization buffer in the absence of labeled cDNA probes. Hybridization of a probe targeting the mRNA of SP-A, a specific product of AEC-II, served as an additional positive control.
Data are expressed as means ± SEM. Statistical comparisons were made by ANOVA with post hoc Fisher's protected least significant difference (PLSD) for each agent separately. Probability values were considered significant if they were less than 0.05. All testing was done using StatView 5.0 program (SAS Institute Inc., Cary, NC) for Macintosh computers.
Chemokine mRNA expression by A549 cells
Chemokine mRNA Expression by AEC-II in Primary Culture
Production of CCL2 and CXCL10 by AEC-II in Primary Culture
CCL2 and CXCL10 mRNA expression by AEC-II in vivo
To increase our knowledge in mechanisms controlling the recruitment and activation of inflammatory cells in the alveolar space and the role of alveolar epithelial cells type II in the cytokine network of the lung, we investigated the effects of proinflammatory cytokines on chemokine gene expression and production by human primary AEC-II. We examined CCL2, a CC chemokine that attracts predominantly monocytes/macrophages and activated T cells by binding to CCR2, and CXCL9, CXCL10, and CXCL11, T cell-specific chemokines binding to CXCR3. In this work, we demonstrate that CCL2 mRNA is present in freshly isolated AEC-II and its level is significantly up-regulated during culture. The proinflammatory cytokines IL-1β, TNF-α, and IFN-γ increased the accumulation of CCL2 mRNA in 24 h cultured AEC-II in a dose-dependent manner, however these effects were not statistically significant compared with non-stimulated cells. CCL2 mRNA patterns of resting and cytokine-stimulated A549 cells, which were used as control, disclosed the same expression profiles as observed in primary AEC-II. The highest CCL2 mRNA level was detected in A549 cells stimulated with a combination of IL-1β, TNF-α, and IFN-γ, and this effect was time-dependent.
In agreement with mRNA data, we also found that significant amounts of CCL2 protein were spontaneously secreted from primary cultured AEC-II, and IL-1β or TNF-α, but not IFN-γ, up-regulated CCL2 production, confirming a previous study . In contrast to studies of IFN-γ effects on CCL2 release by human bronchial and endothelial cells and fibroblasts we could not demonstrate that IFN-γ up-regulates the CCL2 protein production by AEC-II and A549 cells [23–25]. Although, IFN-γ modulates CCL2 mRNA expression in AEC-II, time course experiments showed that IFN-γ does not significantly influence CCL2 release. These results are in line with other observations of IFN-γ being rather an inhibitor than promoter of spontaneous and LPS-induced tissue-specific CCL2 releases [26, 27]. Interestingly, IFN-γ also selectively inhibits the CCR2 expression on human monocytes . It seems that differences in IFN-γ regulation of CCL2 may result from differential responses of target cells to pro- and anti-inflammatory stimuli and to cell type-specific patterns of stimulus sensitivity. In contrast to IFN-γ, IL-1β was the most potent stimulus on CCL2 release by AEC-II. More than 25 ng/ml/106 cells of CCL2 were detected in supernatants collected from AEC-II, activated with IL-1β for 24 h. This is the highest level reported to date for cytokine-stimulated CCL2 secretion from airway epithelial cells. In comparison, in a 24-h period, human bronchial epithelial cells treated with IL-1β released 25-fold less CCL2 , and in our experiments, A549 cells maximally stimulated with TNF-α secreted 10-fold less protein. Furthermore, Sadek and associates demonstrated that human AM retrieved from BAL generate the same levels of CCL2 after 72-h stimulation with LPS in culture . Thus, spontaneous CCL2 level produced by AEC-II is of biological importance since on a per cell basis it is three-fold higher than baseline CCL2 level generated by human AM for 72 h in vitro . Although we did not determine the capacity of IL-1β-stimulated AEC-II to attract mononuclear cells, previous studies have shown that AEC-II-derived CCL2 is strongly chemotactic for CD14+ and CD3+ cells in vitro and in vivo [8, 10, 15].
Under normal condition the alveolar space contains a low number of leukocytes with AM forming about 95% of total cell population. As AEC-II are uniquely positioned in the borders between the microvascular compartment and the alveolar space, and constitutively generate considerable amounts of CCL2, one may hypothesize that this AEC-II-derived chemokine is responsible, at least partially, for basal recruitment of monocytes and their differentiation into AM, in healthy humans. In this respect Gunn and colleagues have demonstrated that CCL2 overexpression in the lung of transgenic mice leads to a marked increase in the number of BAL monocytes, but does not cause inflammatory activation of cells . According with our in vitro and in vivo results, it is necessary to have additional inflammatory agonists such as macrophage-derived IL-1β and/or TNF-α for increasing and amplifying CCL2 expression by AEC-II, which in turn might be important factors for further development and manifestation of lung inflammation.
In contrast to CCL2, we could demonstrate that IFN-γ induces the expression of CXCL9, CXCL10, and CXCL11 by AEC-II. In fact, IFN-γ significantly up-regulated CXCL9, CXCL10, and CXCL11 mRNA accumulation and CXCL10 production of AEC-II and A549 cells. Albeit at low levels, CXCL10, CXCL11, and CXCL9 were also induced directly by IL-1β and TNF-α in primary cultured AEC-II. Interestingly, the kinetics of CXCL11 mRNA expression in IFN-γ- or IFN-γ plus TNF-α and IL-1β-treated A549 cells were faster than those of CXCL9 or CXCL10 mRNA expression. Furthermore, the effect of TNF-α and IL-1β was more pronounced on CXCL11 mRNA accumulation in A549 cells compared with CXCL10 or CXCL9, suggesting that different pathways might be involved in CXCL9 and CXCL10 expression at one side, and CXCL11 on the other side, even though they are all induced by IFN-γ. The significance of a sequential regulated expression of CXCL10, CXCL11, and CXCL9 by AEC-II is only a matter of speculation, but a similar pattern has been found in other cell types, for instance, in endothelial cell, bronchial epithelial cells, and neutrophils [30–32]. CXCL10 mRNA accumulation in primary cultured AEC-II and in the control A549 cells closely reflected levels of secreted CXCL10 protein, as previously shown in human bronchial epithelial cells , suggesting that the CXCL10 production is strongly regulated and dependent on the transcriptional mechanisms.
To date, a number of cellular sources of CCL2 and CXCL10 in the inflamed lung have been identified, including macrophages, fibroblasts, airway epithelium, and endothelial cells. In the present study, we have evaluated the ability of human AEC-II to express and produce CCL2 and CXCL10 in vitro , and express CCL2 and CXCL10 mRNA in situ . ISH studies revealed intense positive signals for CXCL10 mRNA in AEC-II, as well as in interstitial and alveolar macrophages in lung tissue explants after in vitro treatment with IL-1β and/or IFN-γ. In contrast, no positive signal was detected in non-cultured or cultured lung tissue explants with medium alone or with IFN-γ/IL-1β and 10-4 M dexamethasone. On the other hand, CCL2 mRNA expression was detected in AEC-II and AM in all lung tissue explants, and IL-1β, alone or in combination with IFN-γ, markedly up-regulated CCL2 mRNA expression of AEC-II and AM in situ . Treatment with dexamethasone attenuated the signal intensities in cytokine-stimulated, but not in non-stimulated preparations. These results from in vitro stimulation and dexamethasone inhibition experiments demonstrated that AEC-II were capable of expressing significant quantities of CXCL10 mRNA in situ only under local IFN-γ or IL-1β activation, in contrast to CCL2, which is expressed constitutively, and proinflammatory cytokines only up-modulated the steady state mRNA levels of CCL2. These data corroborate well observations from van der Velden et al. and Witowski et al., which have shown that dexamethasone and actinomycin D strongly inhibit cytokine-driven but not constitutive CCL2 release in human bronchial epithelium and peritoneal fibroblasts [23, 33].
Our in situ hybridization data are consistent with observations from Jaffe and associates, and Martin et al., which have shown that only after local, but not after systemic administration of recombinant human IFN-γ in healthy volunteers, CXCL10 mRNA was induced in AM [34, 35]. Moreover, production of CXCL10 and CXCL9 in the injured lung was not completely suppressed in mice deficient for IFN-γ or the IFN-γ receptor , and CXCL10, but not CCL2, failed in the human T cell transendothelial migration model . All these data suggest that in contrast to CCL2, CXCL10 is directly induced by IFN-γ released from cells within the lung rather than by IFN-γ derived from distant sites, and that alternate agonists are present in the alveolar compartment, which may together with IFN-γ or separately amplify CXCL10 expression, and subsequently promote local accumulation of CXCR3+ cells in the alveolar space. In addition, ISH data strongly suggest that effects of the proinflammatory cytokines on CCL2 and CXCL10 mRNA accumulation and protein generation in primary cultured AEC-II were not due to artifacts elicited by cell isolation procedure or culture conditions.
The quantitative differences, time and cytokine-inducing profile dependencies in CCL2, CXCL9, CXCL10, and CXCL11 expression of AEC-II suggest that there are several cytokine/chemokine cascades within the injured lung, which in turn, determine the flexible programs of recruitment and activation of inflammatory cells into the alveolar space. For instance, alveolar macrophage-derived pro-inflammatory cytokines such as IL-1 and TNF-α, but not IFN-γ, directly up-regulate expression by AEC-II of certain chemokines including CCL2 and CXCL8 [8, 10, 19, 38]. In our previous report we demonstrated that in close similarity with CCL2, TNF-α and IL-1β, but not IFN-γ, up-modulated constitutive interleukin-8/IL-8 (CXCL8) release in primary cultured AEC-II, and cytokine-derived increase of CXCL8 basal level was only two-fold lower than LPS-induced CXCL8 release in AM . Our results demonstrating high levels of CCL2, CXCL8 and CXCL10 production by AEC-II in vitro indicate that AEC-II have the potential to participate in physiologic and pathologic macrophage, neutrophil and T cell responses within the alveolar space. By chemoattracting IL-1-producing monocytes/macrophages, TNF-α-producing neutrophils and IFN-γ-producing CD4+ or CD8+ cells in close proximity to the epithelium, AEC-II-derived CCL2, CXCL8, CXCL9, CXCL10, and CXCL11, which themselves are up-regulated by IL-1β and/or TNF-α, and IFN-γ, may activate several positive feedback loops. In addition, it has been reported that CXCL10 selectively activated and enhanced antigen-driven IFN-γ gene expression in T cells . Thus, it is tempting to speculate that locally produced CCL2 and CXCL10 by AEC-II attract activated memory T cells into alveoli and further amplify antigen-driven IFN-γ response of Th1 cells. The strong confirmation of this hypothesis results from animal models of the lung inflammation and some clinical observations in pulmonary diseases. It has been demonstrated that bronchoalveolar lavage (BAL) cells disclosed the Th1-dominated pattern in transgenic rats that express CXCL10 in the lung, and BAL lymphocytes of HIV-infected patients with T-cell alveolitis were CD8+ T cells expressing high levels of CXCR3 and IFN-γ, which exhibited a high migratory capability in response to CXCL10 and CXCL9 [40, 41]. Similarly, over-expression of human CCL2 in transgenic AEC-II or intra-tracheal administration of murine MCP-1 caused a substantial accumulation of activated monocytes within the bronchoalveolar space of mice [15, 16]. Additionally, Miotto et al. reported that CXCL10 expression was strongly up-regulated in Th1-mediated lung diseases, whereas increased CCL2 expression was not specifically associated with Th1 or Th2 patterns. Our in situ hybridization data confirm that in contrast with normal lung tissue preparations a strong expression of CXCL10 can be detected in sarcoidosis and tuberculosis – diseases in which the Th1-type cytokine IFN-γ is up-regulated.
Beside the pro-inflammatory effects of CXCL9, CXCL10, and CXCL11 they also exhibit down-regulation e.g. on the migration of eosinophils  or on angiogenesis . This is of interest, because it could be shown in a mouse model that CXCL11 attenuates fibrosis by inhibition of vascular remodeling . In addition, it could be demonstrated that CXCL9 down-regulates IL-4 expression but up-regulates IFNγ expression by T cells. This illustrates that AEC-II not only induce the migration of Th1 cells by the release of CXCR3-ligands but they also participate in T cell activation  and Th1 polarization of lung T cells.
Since it is known that AEC-II also express CXCR3A and CXCR3B  it is tempting to speculate about a possible activity of CXCL9, CXCL10, and CXCL11 on AEC-II in an autocrine fashion, e.g. in re-epithelialization after lung injury. Therefore, induction of CXCR3 agonists may in some respects of benefit and may represent a possible therapeutic mechanism of IFNγ therapy of IPF patients .
List of abbreviations
alveolar epithelial cells type II
CC-ligand 2 (= MCP-1 (monocyte chemoattractant protein-1))
CXC-ligand 9 (= MIG (monokine induced by interferon-γ))
CXC-ligand 10 (= IP-10 (Interferon-inducible protein-10))
- CXCL11 CXC-:
ligand 11(= I-TAC (Interferon-inducible T-cell alpha chemoattractant))
in situ hybridization
surfactant protein A
tumor necrosis factor α
The authors would like to thank Dr. E. Richter, National Reference Center for Mycobacteria, for sequencing of the PCR-products, S. Adam, D. Bubritzki, H. Kühl, and N. Husmann for technical assistance.
This work was supported by grant from the Deutsche Forschungsgemeinschaft (No. Mu 692/5-5). D.V.P. is the recipient of a Research Fellowship from the Alexander von Humboldt Foundation.
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