Impaired synthesis of prostaglandin E2 by lung fibroblasts and alveolar epithelial cells from GM-CSF-/- mice: implications for fibroproliferation

Prostaglandin E(2) (PGE(2)) is a potent suppressor of fibroblast activity. We previously reported that bleomycin-induced pulmonary fibrosis was exaggerated in granulocyte-macrophage colony-stimulating factor knockout (GM-CSF(-/-)) mice compared with wild-type (GM-CSF(+/+)) mice and that increased fibrosis was associated with decreased PGE(2) levels in lung homogenates and alveolar macrophage cultures. Pulmonary fibroblasts and alveolar epithelial cells (AECs) represent additional cellular sources of PGE(2) within the lung. Therefore, we examined fibroblasts and AECs from GM-CSF(-/-) mice, and we found that they elaborated significantly less PGE(2) than did cells from GM-CSF(+/+) mice. This defect was associated with reduced expression of cyclooxygenase-1 and -2 (COX-1 and COX-2), key enzymes in the biosynthesis of PGE(2). Additionally, proliferation of GM-CSF(-/-) fibroblasts was greater than that of GM-CSF(+/+) fibroblasts, and GM-CSF(-/-) AECs were impaired in their ability to inhibit fibroblast proliferation in coculture. The addition of GM-CSF to fibroblasts from GM-CSF(-/-) mice increased PGE(2) production and decreased proliferation. Similarly, AECs isolated from GM-CSF(-/-) mice with transgenic expression of GM-CSF under the surfactant protein C promoter (SpC-GM mice) produced more PGE(2) than did AEC from control mice. Finally, SpC-GM mice were protected from fluorescein isothiocyanate-induced pulmonary fibrosis. In conclusion, these data demonstrate that GM-CSF regulates PGE(2) production in pulmonary fibroblasts and AECs and thus plays an important role in limiting fibroproliferation.     

Expression and functional implications of CCR2 expression on murine alveolar epithelial cells

Acute lung injury results in damage to the alveolar epithelium, leading to leak of proteins into the alveolar space and impaired gas exchange. Lung function can be restored only if the epithelial layer is restored. The process of reepithelialization requires migration of lung epithelial cells to cover denuded basement membranes. The factors that control the migration of lung epithelial cells are incompletely understood. We examined isolated murine type II alveolar epithelial cells (AECs) for expression of CC chemokine receptor 2 (CCR2) and functional consequences of the binding of the main CCR2 ligand monocyte chemoattractant protein-1 (MCP-1). We found that primary AECs bound MCP-1 and expressed CCR2 mRNA. These cells demonstrated functional consequences of CCR2 expression with migration in response to MCP-1 in chemotaxis/haptotaxis assays. Primary AECs cultured from mice lacking CCR2 did not respond to MCP-1. Monolayers of AECs lacking CCR2 demonstrated delayed closure of mechanical wounds compared with AEC monolayers expressing CCR2. Delayed closure of mechanical wounds of wild-type AECs was also demonstrated in the presence of anti-MCP-1 antibody. These data demonstrate for the first time that AECs express CCR2 and are capable of using this receptor for chemotaxis and healing of wounds. CCR2-MCP-1 interactions may be important in the process of reepithelialization after lung injury.     

Bleomycin-induced E prostanoid receptor changes alter fibroblast responses to prostaglandin E2

Although PGE(2) is a potent inhibitor of fibroblast function, PGE(2) levels are paradoxically elevated in murine lungs undergoing fibrotic responses. Pulmonary fibroblasts from untreated mice expressed all four E prostanoid (EP) receptors for PGE(2). However, following challenge with the fibrogenic agent, bleomycin, fibroblasts showed loss of EP2 expression. Lack of EP2 expression correlated with an inability of fibroblasts from bleomycin-treated mice to be inhibited by PGE(2) in assays of proliferation or collagen synthesis and blunted cAMP elevations in response to PGE(2). PGE(2) was similarly unable to suppress proliferation or collagen synthesis in fibroblasts from EP2(-/-) mice despite expression of the other EP receptors. EP2(-/-), but not EP1(-/-) or EP3(-/-) mice, showed exaggerated fibrotic responses to bleomycin administration in vivo as compared with wild-type controls. EP2 loss on fibroblasts was verified in a second model of pulmonary fibrosis using FITC. Our results for the first time link EP2 receptor loss on fibroblasts following fibrotic lung injury to altered suppression by PGE(2) and thus identify a novel fibrogenic mechanism.     

Role of CC chemokine receptor 2 in bone marrow cells in the recruitment of macrophages into obese adipose tissue

The MCP-1 (monocyte chemoattractant protein-1)/CCR2 (CC motif chemokine receptor-2) pathway may play a role in macrophage infiltration into obese adipose tissue. Here we investigated the role of CCR2 in the recruitment of bone marrow-derived macrophages into obese adipose tissue in vitro and in vivo. Using the TAXIScan device, which can measure quantitatively the directionality and velocity of cell migration at time lapse intervals in vitro, we demonstrated that bone marrow cells (BMCs) from wild type mice migrate directly toward MCP-1 or culture medium conditioned by adipose tissue explants of genetically obese ob/ob mice, which are efficiently suppressed by pharmacological blockade of CCR2 signaling. The number of F4/80-positive macrophages was reduced in the adipose tissue from high fat diet-fed obese KKAy or ob/ob mice treated with a CCR2 antagonist propagermanium relative to vehicle-treated groups. We also found that the number of macrophages is reduced in the adipose tissue from ob/ob mice reconstituted with CCR2(-/-) BMCs (ob/ob + CCR2(-/-) BMCs) relative to those with CCR2+/+ BMCs (ob/ob + CCR2+/+ BMCs). Expression of mRNAs for CD11c and TLR4 (Toll-like receptor 4) markers of proinflammatory M1 macrophages was also decreased in the adipose tissue from ob/ob + CCR2(-/-) BMCs relative to ob/ob + CCR2+/+ BMCs, whereas mannose receptor and CD163, markers of anti-inflammatory M2 macrophages, were unchanged. This study provides in vivo and in vitro evidence that CCR2 in bone marrow cells plays an important role in the recruitment of macrophages into obese adipose tissue.     

Differential monocyte chemoattractant protein-1 and chemokine receptor 2 expression by murine lung fibroblasts derived from Th1- and Th2-type pulmonary granuloma models.

Recent studies suggest that monocyte chemoattractant protein-1 (MCP-1) is involved in fibrosis through the regulation of profibrotic cytokine generation and matrix deposition. Changes in MCP-1, C-C chemokine receptor 2 (CCR2), procollagen I and III, and TGF beta were examined in fibroblasts cultured from normal lung and from nonfibrotic (i.e., Th1-type) and fibrotic (i.e., Th2-type) pulmonary granulomas. Th2-type fibroblasts generated 2-fold more MCP-1 than similar numbers of Th1-type or normal fibroblasts after 24 h in culture. Unlike normal and Th1-type fibroblasts, Th2-type fibroblasts displayed CCR2 mRNA at 24 h after IL-4 treatment. By flow cytometry, CCR2 was present on 40% of untreated Th2-type fibroblasts, whereas CCR2 was present on <20% of normal and Th1-type fibroblasts after similar treatment. IL-4 increased the number of normal fibroblasts with cell-surface CCR2 but IFN-gamma-treatment of normal and Th2-type fibroblasts significantly decreased the numbers of CCR2-positive cells in both populations. Western blot analysis showed that total CCR2 protein expression was markedly increased in untreated Th2-type fibroblasts compared with normal and Th1-type fibroblasts. IL-4 treatment enhanced CCR2 protein in Th1- and Th2-type fibroblasts whereas IFN-gamma treatment augmented CCR2 protein in normal and Th1-type fibroblasts. All three fibroblast populations exhibited MCP-1-dependent TGF-beta synthesis, but only normal and Th2-type fibroblasts showed a MCP-1 requirement for procollagen mRNA expression. Taken together, these findings suggest that lung fibroblasts are altered in their expression of MCP-1, TGF-beta, CCR2, and procollagen following their participation in pulmonary inflammatory processes, and these changes may be important during fibrosis.     

CCR2 Regulates the Uptake of Bone Marrow-Derived Fibroblasts in Renal Fibrosis

Recent studies have shown that bone marrow-derived fibroblasts contribute significantly to the pathogenesis of renal fibrosis. However, the molecular mechanisms underlying the recruitment of bone marrow-derived fibroblasts into the kidney are incompletely understood. Bone marrow-derived fibroblasts express the chemokine receptor - CCR2. In this study, we tested the hypothesis that CCR2 participates in the recruitment of fibroblasts into the kidney during the development of renal fibrosis. Bone marrow-derived collagen-expressing GFP⁺ fibroblasts were detected in the obstructed kidneys of chimeric mice transplanted with donor bone marrow from collagen α1(I)-GFP reporter mice. These bone marrow-derived fibroblasts expressed PDGFR-β and CCR2. CCR2 knockout mice accumulated significantly fewer bone marrow-derived fibroblast precursors expressing the hematopoietic marker-CD45 and the mesenchymal markers-PDGFR-β or procollagen I in the obstructed kidneys compared with wild-type mice. Furthermore, CCR2 knockout mice displayed fewer bone marrow-derived myofibroblasts and expressed less α-SMA or FSP-1 in the obstructed kidneys compared with wild-type mice. Consistent with these findings, genetic deletion of CCR2 inhibited total collagen deposition and suppressed expression of collagen I and fibronectin. Moreover, genetic deletion of CCR2 inhibits MCP-1 and CXCL16 gene expression associated with a reduction of inflammatory cytokine expression and macrophage infiltration, suggesting a linear interaction between two chemokines/ligand receptors in tubular epithelial cells. Taken together, our results demonstrate that CCR2 signaling plays an important role in the pathogenesis of renal fibrosis through regulation of bone marrow-derived fibroblasts. These data suggest that inhibition of CCR2 signaling could constitute a novel therapeutic approach for fibrotic kidney disease.     

TGFβ signaling in lung epithelium regulates bleomycin-induced alveolar injury and fibroblast recruitment

The response of alveolar epithelial cells (AECs) to lung injury plays a central role in the pathogenesis of pulmonary fibrosis, but the mechanisms by which AECs regulate fibrotic processes are not well defined. We aimed to elucidate how transforming growth factor-β (TGFβ) signaling in lung epithelium impacts lung fibrosis in the intratracheal bleomycin model. Mice with selective deficiency of TGFβ receptor 2 (TGFβR2) in lung epithelium were generated and crossed to cell fate reporter mice that express β-galactosidase (β-gal) in cells of lung epithelial lineage. Mice were given intratracheal bleomycin (0.08 U), and the following parameters were assessed: AEC death by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling assay, inflammation by total and differential cell counts from bronchoalveolar lavage, fibrosis by scoring of trichrome-stained lung sections, and total lung collagen content. Mice with lung epithelial deficiency of TGFβR2 had improved AEC survival, despite greater lung inflammation, after bleomycin administration. At 3 wk after bleomycin administration, mice with epithelial TGFβR2 deficiency showed a significantly attenuated fibrotic response in the lungs, as determined by semiquantitatve scoring and total collagen content. The reduction in lung fibrosis in these mice was associated with a marked decrease in the lung fibroblast population, both total lung fibroblasts and epithelial-to-mesenchymal transition-derived (S100A4(+)/β-gal(+)) fibroblasts. Attenuation of TGFβ signaling in lung epithelium provides protection from bleomycin-induced fibrosis, indicating a critical role for the epithelium in transducing the profibrotic effects of this cytokine.     

Effect of PGE2 and indomethacin on human fibroblast collagen production

Fibroblasts can synthetize prostaglandins (particularly PGE2) "in vitro" but it still remains unclear what role they play in the regulation of fibroblast proliferation and collagen production. We report here the effect of PGE2 and indomethacin on collagen synthesis by cultured human dermal fibroblasts. PGE2 (range: 1-300 pmoles/ml) and indomethacin (range: 0.0025-1.0 micrograms/ml) did not significantly affect fibroblast collagen production, when added for 24 hours at 37 degrees C to the cultures, in comparison to controls (fibroblasts incubated for 24 hours at 37 degrees C in medium only). Prostaglandins probably modulate collagen synthesis, as described in a previous report, by means their effect on cell proliferation. It appears they do not affect the intracellular mechanism of collagen production.     

The MCP-1/CCR2 axis in podocytes is involved in apoptosis induced by diabetic conditions

Previous studies have demonstrated the importance of monocyte chemoattractant protein-1 (MCP-1) in the pathogenesis of diabetic nephropathy in terms of inflammation, but the direct role of the MCP-1/CCR2 system on podocyte apoptosis under diabetic conditions has never been explored. In vitro, mouse podocytes were exposed to a medium containing 30?mM glucose (HG) with or without CCR2 siRNA or CCR2 inhibitor (RS102895). Podocytes were also treated with MCP-1 or TGF-β1 with or without anti-TGF-β1 antibody, CCR2 siRNA, or CCR2 inhibitor. In vivo, 20?db/m and 20?db/db mice were divided into two groups, and ten mice from each group were treated with RS102895. Western blot and Hoechst 33342 or TUNEL staining were performed to identify apoptosis. HG-induced apoptosis and TGF-β1 levels were significantly abrogated by CCR2 inhibition. In addition, treatment with MCP-1 directly induced apoptosis via CCR2. Moreover, TGF-β1- and MCP-1-induced apoptosis were significantly ameliorated by the inhibition of CCR2 and anti-TGF-β1 antibody, respectively. Glomerular expression of cleaved caspase-3 and apoptotic cells within glomeruli were also significantly increased in db/db mice compared to db/m mice, and these increases were significantly attenuated in db/db?+?RS102895 mice. These results suggest that interactions between the MCP-1/CCR2 system and TGF-β1 may contribute to podocyte apoptosis under diabetic conditions.     

CC-chemokine receptor 2 required for bleomycin-induced pulmonary fibrosis

MCP-1, which signals via the CC chemokine receptor 2 (CCR2), is induced in lung fibrosis that is accompanied by mononuclear cell recruitment and activation of lung fibroblasts. To evaluate the role of CCR2 in lung fibrosis, CCR2 knockout (ko) mice were used in a model of bleomycin-induced lung fibrosis. Wild type (wt) and ko mice were injected endotracheally with bleomycin to induce lung injury and fibrosis, and then analyzed for degree of lung fibrosis and cytokine expression. The results showed significantly reduced fibrosis in ko mice as evidenced by decreased lung type I collagen gene expression and hydroxyproline content relative to those in wt mice. Lung TNF-alpha and TGF-beta1 expression was significantly lower in ko vs. wt mice, while MCP-1 expression was unaffected. Interestingly, lung alpha-smooth muscle actin (alpha-SMA) expression, a marker for myofibroblast differentiation, was also decreased in ko mice, which was confirmed by analysis of isolated lung fibroblasts. Fibroblasts from ko mice exhibited decreased responsiveness to TGF-beta1 induced alpha-SMA expression, which was associated with reduced expression of TGF-beta receptor II (TbetaRII) and Smad3. These findings suggest that CCR2 signaling plays a key role in bleomycin-induced pulmonary fibrosis by regulating fibrogenic cytokine expression and fibroblast responsiveness to TGF-beta.     

Prostaglandin E(2) inhibits fibroblast chemotaxis

Fibroblasts are the major source of extracellular connective tissue matrix, and the recruitment, accumulation, and stimulation of these cells are thought to play important roles in both normal healing and the development of fibrosis. Prostaglandin E(2) (PGE(2)) can inhibit this process by blocking fibroblast proliferation and collagen production. The aim of this study was to investigate the inhibitory effect of PGE(2) on human plasma fibronectin (hFN)- and bovine bronchial epithelial cell-conditioned medium (BBEC-CM)-induced chemotaxis of human fetal lung fibroblasts (HFL1). Using the Boyden blind well chamber technique, PGE(2) (10(-7) M) inhibited chemotaxis to hFN 40.8 +/- 5.3% (P < 0.05) and to BBEC-CM 49.7 +/- 11.7% (P < 0.05). Checkerboard analysis demonstrated inhibition of both chemotaxis and chemokinesis. The effect of PGE(2) was concentration dependent, and the inhibitory effect diminished with time. Other agents that increased fibroblast cAMP levels, including isoproterenol (10(-5) M), dibutyryl cAMP (10(-5) M), and forskolin (3 x 10(-5) M) had similar effects and inhibited chemotaxis 54.1, 95.3, and 87.0%, respectively. The inhibitory effect of PGE(2) on HFL1 cell chemotaxis was inhibited by the cAMP-dependent protein kinase (PKA) inhibitor KT-5720, which suggests a cAMP-dependent effect mediated by PKA. In summary, PGE(2) appears to inhibit fibroblast chemotaxis, perhaps by modulating the rate of fibroblast migration. Such an effect may contribute to regulation of the wound healing response after injury.     

Release of biologically active TGF-beta1 by alveolar epithelial cells results in pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive fatal fibrotic lung disease. Transforming growth factor (TGF)-beta1 is present in a biologically active conformation in the epithelial cells lining lesions with advanced IPF. To determine the role of aberrant expression of biologically active TGF-beta1 by alveolar epithelial cells (AECs), the AECs of explanted normal rat lungs were transfected with the TGF-beta1 gene using the retrovirus pMX-L-s223,225-TGF-beta1. In situ hybridization using a digoxigenin-labeled cDNA of the puromycin resistance gene contained in the pMX demonstrated that pMX-L-s233,225-TGF-beta1 was selectively transfected into AECs of the explants. Conditioned media overlying explants obtained 7 days after being treated with pMX-L-s223,225-TGF-beta1 contained 14.5 +/- 3.15 pg/ml of active TGF-beta1. With the use of Masson's trichrome staining of explant sections obtained 14 days after transfection, there were lesions similar to those in IPF, characterized by type II AEC hyperplasia, interstitial thickening, extensive increase in interstitial and subepithelial collagen, an increase in the number of fibroblasts, and areas resembling fibroblast buds. Collagens I, III, IV, and V and fibronectin were increased in explants treated with pMX-L-s223,225-TGF-beta1. The findings in the current study suggest that IPF may be a disorder of epithelial cells and not inflammatory cells.     

Monocyte chemoattractant protein-1 and CCR2 interactions are required for IFN-alpha/beta-induced inflammatory responses and antiviral defense in liver

IFN-alpha/beta-mediated functions promote production of MIP-1alpha (or CCL3) by mediating the recruitment of MIP-1alpha-producing macrophages to the liver during early infection with murine CMV. These responses are essential for induction of NK cell inflammation and IFN-gamma delivery to support effective control of local infection. Nevertheless, it remains to be established if additional chemokine functions are regulated by IFN-alpha/beta and/or play intermediary roles in supporting macrophage trafficking. The chemokine MCP-1 (or CCL2) plays a distinctive role in the recruitment of macrophages by predominantly stimulating the CCR2 chemokine receptor. Here, we examine the roles of MCP-1 and CCR2 during murine CMV infection in liver. MCP-1 production preceded that of MIP-1alpha during infection and was dependent on IFN-alpha/beta effects for induction. Resident F4/80(+) liver leukocytes were identified as primary IFN-alpha/beta responders and major producers of MCP-1. Moreover, MCP-1 deficiency was associated with a dramatic reduction in the accumulation of macrophages and NK cells, as well as decreased production of MIP-1alpha and IFN-gamma in liver. These responses were also markedly impaired in mice with a targeted disruption of CCR2. Furthermore, MCP-1- and CCR2-deficient mice exhibited increased viral titers and elevated expression of the liver enzyme alanine aminotransferase in serum. These mice also had widespread virus-induced liver pathology and succumbed to infection. Collectively, these results establish MCP-1 and CCR2 interactions as factors promoting early liver inflammatory responses and define a mechanism for innate cytokines in regulation of chemokine functions critical for effective localized antiviral defenses.     

STAT1 activation causes translocation of Bax to the endoplasmic reticulum during the resolution of airway mucous cell hyperplasia by IFN-gamma

Disruption of the normal resolution process of inflammation-induced mucous cell hyperplasia may lead to sustained mucous hypersecretion in chronic diseases. During prolonged exposure of mice to allergen, IFN-gamma reduces mucous cell hyperplasia, but the signaling responsible for the cell death is largely unknown. A brief phosphorylation of STAT1 by IFN-gamma was required for cell death in airway epithelial cells (AEC), and during prolonged exposure to allergen, mucous cell hyperplasia remained elevated in STAT1(-/-) but was resolved in STAT1(+/+) mice. Although IFN-gamma treatment of primary human AECs and other airway cell lines left Bax protein levels unchanged, it caused translocation of Bax from the cytosol to the endoplasmic reticulum (ER) but not to the mitochondria. Localization of Bax to the ER was observed in IFN-gamma-treated primary AECs isolated from STAT1(+/+) mice but not in cells from STAT1(-/-) mice. In addition, ER Bax was detected in mucous cells of STAT1(+/+) but not STAT1(-/-) airways of mice exposed to allergen for prolonged periods. IFN-gamma did not release cytochrome c from mitochondria but reduced ER calcium stores and dilated the ER, confirming that the IFN-gamma-induced cell death is mediated through changes localized in the ER. Collectively, these observations suggest that STAT1-dependent translocation of Bax to the ER is crucial for IFN-gamma-induced cell death of AECs and the resolution of allergen-induced mucous cell hyperplasia.     

Prostaglandin receptor EP2 mediates PGE2 stimulated hypercalcemia in mice in vivo

Prostaglandin E2 (PGE2) can stimulate bone resorption by a cyclic AMP-dependent pathway. Two PGE2 receptors, EP2 and EP4 have been shown to play a role in PGE2 stimulation of osteoclast formation. In primary osteoblastic cell cultures from EP2 wild type (EP2 +/+) mice, PGE2 (0.1 microM) increased cyclic AMP production 3.5-fold, but PGE2 had no effect on cells from mice in which the EP2 receptor had been deleted (EP2 -/-). To examine the role of the EP2 receptor in the resorption response in vivo we injected PGE2 in EP2 -/- mice, and compared them with EP2 +/+ mice. Injection of PGE2 (3 mg/kg, four times daily for three days) in 9- to 12-month-old male mice on a 129 SvEv background increased serum calcium from 9.8 +/- 0.5 to 10.7 +/- 0.3 mg/dl (P < 0.01) in EP2 +/+ mice but not in EP2 -/- mice (10.1 +/- 0.3 vs. 10.2 +/- 0.3 mg/dl). PGE2 injection (6 mg/kg twice a day for three days) in 3-4 month old male mice on a C57 BL/6 X 129 SvEv background increased calcium from 8.2 +/- 0.1 to 9.0 +/- 0.3 mg/dl (P < 0.05) in EP2 +/+ mice but had no effect in EP2-/- mice (8.4 +/- 0.1 vs. 8.3 +/- 0.2 mg/dl). Injection of PGE2 over the calvariae of EP2 +/+ and EP2-/- mice increased the expression of receptor activator of nuclear factor kappaB ligand (RANKL) both locally and in the tibia, but RANKL responses were lower in EP2 -/- mice. We conclude that EP2 receptor plays a role in the hypercalcemic response to PGE2. This impaired response in EP2 -/- mice may be due to decreased ability to stimulate cyclic AMP and in part, to a smaller increase in the expression of RANKL mRNA.     

Additive roles for MCP-1 and MCP-3 in CCR2-mediated recruitment of inflammatory monocytes during Listeria monocytogenes infection

Chemokine receptor-mediated recruitment of inflammatory cells is essential for innate immune defense against microbial infection. Recruitment of Ly6C(high) inflammatory monocytes from bone marrow to sites of microbial infection is dependent on CCR2, a chemokine receptor that responds to MCP-1 and MCP-3. Although CCR2(-/-) mice are markedly more susceptible to Listeria monocytogenes infection than are wild-type mice, MCP-1(-/-) mice have an intermediate phenotype, suggesting that other CCR2 ligands contribute to antimicrobial defense. Herein, we show that L. monocytogenes infection rapidly induces MCP-3 in tissue culture macrophages and in serum, spleen, liver, and kidney following in vivo infection. Only cytosol invasive L. monocytogenes induce MCP-3, suggesting that cytosolic innate immune detection mechanisms trigger chemokine production. MCP-3(-/-) mice clear bacteria less effectively from the spleen than do wild-type mice, a defect that correlates with diminished inflammatory monocyte recruitment. MCP-3(-/-) mice have significantly fewer Ly6C(high) monocytes in the spleen and bloodstream, and increased monocyte numbers in bone marrow. MCP-3(-/-) mice, like MCP-1(-/-) mice, have fewer TNF- and inducible NO synthase-producing dendritic cells (Tip-DCs) in the spleen following L. monocytogenes infection. Our data demonstrate that MCP-3 and MCP-1 provide parallel contributions to CCR2-mediated inflammatory monocyte recruitment and that both chemokines are required for optimal innate immune defense against L. monocytogenes infection.     

Regulation of alveolar epithelial cell survival by the ACE-2/angiotensin 1-7/Mas axis

Earlier work from this laboratory demonstrated that apoptosis of alveolar epithelial cells (AECs) requires autocrine generation of angiotensin (ANG) II. More recent studies showed that angiotensin converting enzyme-2 (ACE-2), which degrades ANGII to form ANG1-7, is protective but severely downregulated in human and experimental lung fibrosis. Here it was theorized that ACE-2 and its product ANG1-7 might therefore regulate AEC apoptosis. To evaluate this hypothesis, the AEC cell line MLE-12 and primary cultures of rat AECs were exposed to the profibrotic apoptosis inducers ANGII or bleomycin (Bleo). Markers of apoptosis (caspase-9 or -3 activation and nuclear fragmentation), steady-state ANGII and ANG1-7, and JNK phosphorylation were measured thereafter. In the absence of Bleo, inhibition of ACE-2 by small interfering RNA or by a competitive inhibitor (DX600 peptide) caused a reciprocal increase in autocrine ANGII and corresponding decrease in ANG1-7 in cell culture media (both P < 0.05) and, moreover, induced AEC apoptosis. At baseline (without inhibitor), ANG1-7 in culture media was 10-fold higher than ANGII (P < 0.01). Addition of purified ANGII or bleomycin-induced caspase activation, nuclear fragmentation, and JNK phosphorylation in cultured AECs. However, preincubation with ANG1-7 (0.1 μM) prevented JNK phosphorylation and apoptosis. Moreover, pretreatment with A779, a specific blocker of the ANG1-7 receptor mas, prevented ANG1-7 blockade of JNK phosphorylation, caspase activation, and nuclear fragmentation. These data demonstrate that ACE-2 regulates AEC survival by balancing the proapoptotic ANGII and its antiapoptotic degradation product ANG1-7. They also suggest that ANG1-7 inhibits AEC apoptosis through the ANG1-7 receptor mas.     

Alveolar type II cells inhibit fibroblast proliferation: role of IL-1alpha

Alveolar type II (ATII) cells inhibit fibroblast proliferation in coculture by releasing or secreting a factor(s) that stimulates fibroblast production of prostaglandin E2 (PGE2). In the present study, we sought to determine the factors released from ATII cells that stimulate PGE2 production in fibroblasts. Exogenous addition of rat IL-1alpha to cultured lung fibroblasts induced PGE2 secretion in a dose-response manner. When fibroblasts were cocultured with rat ATII cells, IL-1alpha protein was detectable in ATII cells and in the coculture medium between days 8 and 12 of culture, correlating with the highest levels of PGE2. Furthermore, under coculture conditions, IL-1alpha gene expression increased in ATII cells (but not fibroblasts) compared with either cell cultured alone. In both mixed species (human fibroblasts-rat ATII cells) and same species cocultures (rat fibroblasts and ATII cells), PGE2 secretion was inhibited by the presence of IL-1 receptor antagonist (IL-1Ra) or selective neutralizing antibody directed against rat IL-1alpha (but not IL-1beta). Conditioned media from cocultures inhibited fibroblast proliferation, and this effect was abrogated by the addition of IL-1Ra. Addition of keratinocyte growth factor (KGF) resulted in an earlier increase in PGE2 secretion and fibroblast inhibition (day 8 of coculture). This effect was inhibited by indomethacin but was not altered by IL-1Ra. We conclude that in this coculture system, IL-1alpha secretion by ATII cells is one factor that stimulates PGE2 production by lung fibroblasts, thereby inhibiting fibroblast proliferation. In addition, these studies demonstrate that KGF enhances ATII cell PGE2 production through an IL-1alpha-independent pathway.     

Secretion of MCP-1/CCL2 by bile duct epithelia induces myofibroblastic transdifferentiation of portal fibroblasts

Portal fibroblasts (PF) are fibrogenic liver cells distinct from hepatic stellate cells (HSC). Recent evidence suggests that PF may be important mediators of biliary fibrosis and cirrhosis. The cytokine monocyte chemoattractant protein-1 (MCP-1)/CCL2 is upregulated in biliary fibrosis by bile duct epithelia (BDE) and induces functional responses in HSC. Thus we hypothesized that release of MCP-1 may mediate biliary fibrosis. We report that PF express functional receptors for MCP-1 that are distinct from the receptor CCR2. MCP-1 induces proliferation, increase and redistribution of alpha-smooth muscle (alpha-SMA) expression, loss of the ectonucleotidase NTPDase2, and upregulation of alpha(1)-procollagen production in PF. BDE secretions induce alpha-SMA levels in PF, and this is inhibited by MCP-1 blocking antibody. Together, these data suggest that BDE regulate PF proliferation and myofibroblastic transdifferentiation in a paracrine fashion via release of MCP-1.     

Accessory cell function of airway epithelial cells

Accessory cell function of airway epithelial cells. We previously demonstrated that airway epithelial cells (AECs) have many features of accessory cells, including expression of class II molecules CD80 and CD86 and functional Fcgamma receptors. We have extended these studies to show that freshly isolated AECs have mRNA for cathepsins S, V, and H [proteases important in antigen (Ag) presentation], invariant chain, human leukocyte antigen (HLA)-DM-alpha and HLA-DM-beta, and CLIP, an invariant chain breakdown product. A physiologically relevant Ag, ragweed, was colocalized with HLA-DR in AECs, and its uptake was increased by granulocyte-macrophage colony-stimulating factor and IFN-gamma treatments, which had no effect on CD80 and CD86 expression. We demonstrate the presence of other costimulatory molecules, including B7h and B7-H1, on AECs and the increased expression of B7-H1 on AECs after treatment with granulocyte-macrophage colony-stimulating factor and IFN-gamma. Finally, we compared T cell proliferation after allostimulation with AECs and dendritic cells (DCs). The precursor frequency of peripheral blood T cells responding to AECs was 0.264% compared with 0.55% for DCs. DCs stimulated CD45RO(+), CD45RA(+), CCR7(+) and CCR7(-)CD4(+), and CD8(+) T cells, whereas AECs stimulated only CD45RO(+), CD45RA(-), CCR7(-), CD4(+), and CD8(+) T cells. There was no difference in cytokine production, type of memory T cells stimulated (effector vs. long-term memory), or apoptosis by T cells cocultured with AECs and DCs. The localization of AECs exposed to the external environment may make them important in the regulation of local immune responses.