Immunosuppressive Factors Secreted by Human Amniotic Epithelial Cells

Abstract

purpose. Amniotic membrane has been applied to the ocular surface to restore corneal function. The beneficial effect of amniotic membrane transplantation may be due to the immunosuppressive effects of amniotic epithelial cells. The purpose of this study was to determine whether amniotic epithelial cells (AECs) secrete anti-inflammatory and antiproliferative factors that affect the chemotaxis of neutrophils and macrophages and suppress both T- and B-cell proliferation in vitro.

methods. Human amniotic cells were isolated from human amniotic membrane and cultured in vitro. The supernatants from AEC cultures were collected after 48 hours of incubation. Neutrophil and macrophage chemotactic activity was tested in the presence of AEC supernatant, using 24-well migration assay chambers. Lymphocyte proliferation was tested by H³-thymidine incorporation. Apoptosis was examined by caspase-3 and annexin V assays, and expression of cytokines was assessed by RT-PCR.

results. AEC supernatant significantly inhibited the chemotactic activity of neutrophils and macrophages toward macrophage inflammatory protein (MIP)-2 (P < 0.05). The supernatant significantly reduced the proliferation of both T and B cells after mitogenic stimulation (P < 0.05). Caspase-3 assays revealed that the supernatant induced apoptosis of T and B cells, but not of corneal epithelial cells and liver cells. In contrast to lymphocytes, macrophages and neutrophils were resistant to apoptosis induced by AEC supernatant. The AECs expressed message for TNFα, Fas ligand (FasL), TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), TGFβ, and macrophage migration-inhibitory factor (MIF). However, AEC induction of apoptosis was inhibited (50%) by anti-FasL antibody but not by anti-TRAIL or anti-TNFα antibodies. Moreover, AEC supernatant inhibited macrophage migration in vitro.

conclusions. AECs secrete soluble factors that inhibit cells in both the innate and adaptive immune systems.

The amniotic membrane is the inner layer of the fetal membranes, and it is bathed by amniotic fluid. It consists of a single layer of epithelial cells and an avascular stroma. ¹ Amniotic membrane transplantation has been successfully used in patients with persistent epithelial defects who are unresponsive to medical treatment. ^{2 3 4 5} Amniotic membrane transplantation has been reported to suppress inflammation and promote wound healing in acute burns, herpetic infection, and autoimmune diseases and to prevent corneal perforation in acute infectious keratitis. ^{5 6 7 8 9 10} These studies generally noted that ocular surface inflammation was markedly reduced in the area covered by the amniotic membrane. ^{11 12 13 14} The nature and the exact mechanism of the anti-inflammatory effect of the amniotic membrane remain unclear. 

We have reported that cultured human amniotic epithelial cells (AECs) can be successfully transplanted onto a denuded rabbit cornea without any clinical complications or tissue rejection during 10 days of observation. ¹⁵ Human AECs do not express HLA-A, -B, -C or -DR antigens ^{16 17} and presumably are less likely to be rejected by the host’s immune system. It has been shown that the α5 chain of type IV collagen is present in the amniotic membrane, which suggests that amniotic membrane is a useful substrate for growing corneal epithelial cells. ¹⁸ Clinical observation has shown that transplanted amniotic membrane decreases vascularization of the ocular surface and that the presence of pigment epithelium-derived factor (antiangiogenic factor) plays an important role in reducing vascularization of the cornea. ¹⁹ In addition, the amniotic membrane in general and AECs specifically may directly or indirectly modulate the inflammatory process that is involved in tissue rejection. Considering that the AECs are crucial for protecting the fetus from various injuries including infections, ²⁰ it can be speculated that molecules secreted from AECs play an important role in the anti-inflammatory process. 

Because it is not known whether AECs can suppress the host’s immune and inflammatory responses, the purpose of this study was to determine whether AECs secrete anti-inflammatory and antiproliferative factors that affect cells of the adaptive and innate immune systems. 

Materials and Methods

Animals

BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All animals were handled in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. 

Cell Lines

Jurkat cells (human acute lymphoblastic T-cell leukemia, clone E6.1) were grown in RPMI 1640 medium. Human corneal epithelial cells (HCECs) were a generous gift from Sherry Ward (Gillette Medical Evaluation Laboratories, Gaithersburg, MD). Cells were cultured in keratocyte growth medium (KGM; Bullet Kit CC-3111; Clonetics, Walkersville, MD). Human Chang liver cells (ATCC CCL-13) were obtained from the American Type Culture Collection (Rockville, MD) and cultured in MEM. 

AEC Culture

Human placentas were obtained from normal cesarean deliveries, in accordance with the protocol approved by the Human Subjects Committee of University of Texas Southwestern Medical Center at Dallas. Informed consents were obtained from placenta donors. All placentas were seronegative for human immunodeficiency virus (HIV), hepatitis B and C viruses, and syphilis. Isolation of AECs was performed as described previously. ^{15 21} Briefly, amniotic membrane was peeled from the placenta and washed with sterile saline, transferred into a fresh tissue culture dish, and minced with scissors and incubated in MEM-trypsin solution. Cells were collected by gentle centrifugation and resuspended in F12/DMEM. The supernatant was collected 48 hours after incubation and stored at −80°C. 

Isolation of Murine Macrophages and Neutrophils

Peritoneal macrophages were harvested from C57BL/6 mice by the collection of peritoneal exudates, as described before. ^{22 23} Each mouse received 2 mL of 2.5% thioglycollate injected intraperitoneally 4 to 5 days before death. Immediately after death, the peritoneal cavities were washed two times with 10 mL of Hanks’ balanced salt solution (HBSS). Peritoneal exudate cells were centrifuged and plated onto Petri dishes and incubated in 5% CO₂ at 35°C for 1.5 hours. The dishes were then washed with ice-cold HBSS, and the adherent cells were gently scraped off the bottom of the dish with a cell scraper. Cells were washed three times and resuspended in complete RPMI-1640. Peritoneal neutrophils were harvested from mice by the collection of peritoneal exudates, as mentioned earlier. Each mouse received 2 mL of 2.5% thioglycollate injected intraperitoneally 4 hours before death. Immediately after death, neutrophils were isolated from the peritoneal cavity, as mentioned earlier. The peritoneal exudates were collected and layered onto 3 mL gradient (Histopaque; Sigma-Aldrich, St. Louis, MO) and centrifuged at 3000 rpm for 20 minutes. The neutrophils were collected at the interface, washed three times in HBSS, and resuspended in complete RPMI. Viable cells were counted with a hemocytometer and trypan blue exclusion. 

B-Lymphocyte Preparation

Spleen and peripheral lymph nodes (inguinal, axillary, and cervical) were removed aseptically, minced, and passed through a nylon mesh to obtain single-cell suspensions. After being treated with 5 mL of erythrocyte lysis buffer, the cells were resuspended in RPMI 1640. Cell suspensions (3 mL) were added to plates that had been coated with goat antiserum to mouse gamma globulin (ICN, Aurora, OH), which were prepared before each series of experiments. The plates were incubated at 4°C for 20 minutes. The nonadherent cells were removed gently by swirling with cold HBSS. The B lymphocytes were collected by vigorous pipetting, washed, and counted. 

T-Lymphocyte Preparation

Single-lymphocyte suspensions were collected as just described. A stock of nylon wool columns was prepared before each series of experiments. The cells were eluted from the nylon wool, and T lymphocytes were counted and stored on ice. 

Chemotactic Assays

Chemotactic assays were performed with 6.5-mm diameter, 3.0-μm pore size, 24-well migration assay chambers (Corning-Costar, Inc., Corning, NY). 

The bottom chamber was filled with either 0.1 mL complete RPMI or RPMI containing 60 ng of recombinant mouse macrophage inflammatory protein (rMIP)-2 (R&D Systems, Minneapolis, MN). Neutrophils (5 × 10⁵) or macrophages (1 × 10⁵), with or without AEC supernatant in 0.25 mL medium were added to the top chamber. Plates were incubated at 37°C for 4 hours. After 4 hours, the top chambers were removed, and the number of migrated cells were counted by direct light microscopy. 

Proliferation Assay

T and B lymphocytes (1 × 10⁶/mL) were cultured in medium containing 50% AEC supernatant in 24-well plates (Corning-Costar, Inc.). Concanavalin A (1.0 μg), lipopolysaccharide (5.0 μg/mL), or BSA (1.0 μg) was added to each well before incubation at 37°C for 48 hours. Cultures were pulsed with 1 μCi per well of [³H]-thymidine 18 hours before cell harvesting. The cells were harvested onto glass-fiber filters using an automated cell harvester (Skatron, Lier, Norway) and counted in a liquid scintillation counter (Beckman, Irvine, CA). The data are expressed as mean counts per minute ± SE. 

Apoptosis Assays

Murine neutrophils, macrophages, T and B lymphocytes, HCECs, human Chang liver cells, and Jurkat cells were incubated in respective medium containing 50% AEC supernatant for 24 hours. As a positive control of apoptosis, the cells were treated with 3 μg/mL staurosporine (Sigma-Aldrich). Quantification of apoptosis was performed by an annexin V-FITC kit (R&D Systems) or phycoerythrin (PE)-conjugated polyclonal anticaspase-3 antibody (BD PharMingen, San Diego, CA), as described previously. ²⁴ Inhibition of apoptosis was determined using anti-human FasL (MAB126), TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), and TNFα monoclonal antibodies (20 μg/mL; R&D Systems). Mouse IgG (20 μg/mL; Sigma-Aldrich) was used as a negative control. FasL antibody effects the recombinant FasL protein in trimeric form and can bind the extracellular domain on membrane-expressed FasL, according to the manufacturer’s technical support. 

To determine whether caspase-3 is necessary for apoptosis-induced cell death by an AEC supernatant, Jurkat cells were incubated with the caspase-3 inhibitor (Z-DEVD-FMK; BD PharMingen) at a concentration of 20 μM in the presence or absence of AEC supernatant. The cells were stained with annexin V, as described earlier. Control inhibitor (Z-FA-FMK; BD-PharMingen) or cells treated without inhibitors were used as negative controls. 

RT-PCR Analysis of Apoptosis-Inducing Genes

Amniotic epithelial cell mRNA was isolated (Oligotex Direct mRNA Mini Kit; Qiagen Inc., Valencia, CA) as described elsewhere. ²⁵ The PCR products were visualized in 1.0% agarose gels. β-Actin mRNA levels were used as internal controls. The primers for human MIF, TGFβ, TNFα, Fas-L, and β-actin were purchased from R&D Systems, Inc. and included TRAIL: forward, 5′-AGACCTGCGTGCTGATCGTG-3′; reverse, 5′-TTATTTTGCGGCCCAGAGGCC-3′. Primers were synthesized by Integrated DNA Technologies (IDT, Inc., Coralville, IA). 

Inhibition of Macrophage Migration

Macrophage migration assays were performed as described previously. ^{26 27} The average area of migration (square millimeters) was calculated for each group by image analysis. Data are presented as a mean area of macrophage dispersion (in millimeters) after 24 hours culture. 

Statistical Analysis

Data are expressed as the mean ± SEM. Comparisons were made using Student’s t-test, and differences were considered significant if P < 0.05. 

Results

Inhibition of Chemotactic Responses of Murine Neutrophils and Macrophages by AEC Supernatants

Macrophages and neutrophils play an important role in inflammatory processes. ²⁸ It has been shown that transplantation of amniotic membrane in patients with microbial keratitis reduced ocular inflammation. ⁸ Therefore, it is important to determine whether AEC supernatant is capable of affecting the chemotactic response of neutrophils and macrophages in vitro. A significant reduction in the number of neutrophils (P < 0.0 5) exposed to AEC supernatants was observed in the lower chambers of the migratory assay wells containing the neutrophil chemoattractant rMIP-2 (Fig. 1A) . AEC supernatants also significantly inhibited (P < 0.05) macrophage migration toward rMIP-2 (Fig. 1 B) . In the absence of AEC supernatants, the neutrophils and macrophages migrated toward the rMIP-2 (positive control). The difference between the negative control (medium) and experimental group (AEC+MIP-2) was not statistically significant (P < 0.05). 

Effect of AEC Supernatant on Apoptosis of Neutrophils and Macrophages

Because the AEC supernatants inhibited macrophage and neutrophil chemotactic activities in vitro, experiments were performed to determine whether the inhibition of chemotactic response was not simply due to cells being killed by AEC supernatant. Macrophages and neutrophils were incubated in the presence or absence of AEC supernatant. The percentage of apoptosis was determined by antiactive caspase-3 staining. The results of two independent experiments indicated that neither neutrophils nor macrophages were killed by apoptosis (Figs. 2A 2B) . Staurosporine, a known inducer of apoptosis was used as a positive control that induces significant apoptosis in both neutrophils and macrophages. It is possible that macrophages and neutrophils are killed by mechanisms other than apoptosis. To test this, macrophages and neutrophils were incubated with AEC supernatant, and cell viability was tested by trypan blue exclusion. The results show that AEC supernatants were not toxic to neutrophils and macrophages in vitro (data not shown). 

Macrophage Migration Inhibitory Activity of AEC Supernatants

Our RT-PCR and ELISA results demonstrated that migration inhibitory factor (MIF) is expressed in AECs and present in AEC supernatants (Fig. 3) . To determine whether MIF was biologically active, AEC supernatants were tested in a bioassay. MIF was originally named for its ability to inhibit the migration of macrophages, and the standard bioassay for MIF activity remains the most sensitive one for detecting this cytokine. ²⁹ Accordingly, the biological activity of MIF-containing AEC supernatants was tested in a macrophage migration inhibition assay, using a conventional capillary tube assay, as described previously. ³⁰ Concentrated supernatant (15×) from AEC significantly inhibited the macrophage migration from the capillary tubes (P < 0.05) compared with 15× concentrated control medium (Fig. 4) . 

Effect of AEC Supernatant on T and B Lymphocytes

Histologic examination of corneas from patients with persistent corneal epithelial defects after undergoing amniotic membrane transplantation revealed that lymphocytes were trapped in the amniotic membrane and exhibited the characteristics of cells undergoing apoptosis. ³¹ Based on these observations, we hypothesized that the AECs secrete factors that induce apoptosis of lymphocytes. T and B lymphocytes were incubated in the presence or absence of AEC supernatant, and the percentage of apoptosis was determined by anti-active caspase-3 staining. The results indicated that the AEC supernatant produced a significant (6–7-fold) increase in the apoptosis of T and B lymphocytes compared with the untreated control (Figs. 5A 5B) . 

The response of T and B cells to AEC supernatants was examined in vitro with a lymphoproliferative assay. T and B cells from spleens and mesenteric lymph nodes were isolated from naïve mice and tested for blastogenic responses in the presence of AEC supernatant. AEC supernatant significantly (P < 0.05) inhibited T and B cell proliferative responses to ConA and LPS, respectively (Figs. 6A 6B) . However, no significant inhibition of proliferation was observed when AEC supernatant was added to the unstimulated lymphocytes. 

It is possible that lymphocytes exposed to AECs are susceptible to apoptosis, and normal cells are not. This was tested by evaluating the effect of AECs on normal HCECs and Chang liver cells. HCECs and Chang cells were incubated in the presence or absence of AEC supernatant and the percentage of apoptosis was determined by caspase-3 assay. The results indicate that AECs did not induce apoptosis in HCECs or Chang liver cells (Fig. 7) . 

AEC Supernatant–Induced Apoptosis through Caspase-3 Pathways

The above experiments indicate that AEC supernatant is able to kill the lymphocytes by a process that culminates in apoptosis. To determine whether the classic mechanisms of apoptosis are involved in lymphocyte cell death, an activated T-cell line (Jurkat cells) was incubated in the presence or absence of AEC supernatant and the percentage of apoptosis was determined by annexin V assay. The results indicate that treatment of Jurkat cells with caspase-3 inhibitor completely blocked apoptosis induced by AEC supernatant (Fig. 8) . Staurosporine (positive control) induced significant apoptosis in Jurkat cells (data not shown). Caspase 3 control inhibitor (Z-FA-FMK) or cells treated without inhibitors did not inhibit apoptosis induced by AEC supernatant (Fig. 8) . 

Expression of Apoptosis-Inducing Genes by AECs

It is well known that members of the TNF family (TNF, Fas-L, and TRAIL) are involved in the induction of apoptosis. ^{32 33} Because caspase pathways are involved in the initiation of apoptosis after Fas/Fas ligand, TNF/TNF receptor, and TRAIL/TRAIL receptor interactions, ³⁴ we hypothesized that the AECs express one or more of these ligands. Amniotic epithelial cells were examined for message for the aforementioned apoptosis-related genes by RT-PCR analysis. The results indicate that AECs express human TRAIL, TNF-α, and FasL (Fig. 9) . 

Blockade of AEC Supernatant–Induced Apoptosis

To determine whether TNF, TRAIL, or FasL are responsible for the induction of apoptosis of Jurkat cells by AEC supernatants, Jurkat cells were incubated with anti-TNFα, anti-TRAIL, or anti-FasL antibodies in the presence or absence of the AEC supernatant. Apoptosis was determined by anti-active caspase-3 staining. The results indicate that anti-FasL inhibited the apoptosis of Jurkat cells by 50%. In contrast, anti-TRAIL and anti-TNFα antibodies failed to inhibit the apoptosis of Jurkat cells induced by AEC supernatant (Fig. 10) . Normal mouse serum IgG treatment did not significantly reduce the apoptotic activity of the AEC supernatant compared with the untreated control. 

Discussion

It has been shown that amniotic membrane transplantation is an effective surgical procedure that promotes the reduction of chronic inflammation, increases re-epithelialization, decreases recurrent erosion, and causes regression of neovascularization in affected corneas. ^{3 4 35 36} Although the anti-inflammatory and immunosuppressive effect of the amniotic membrane has been observed clinically, the underlying mechanism is not known. 

The present results demonstrate for the first time that AECs secrete factors that inhibit both the innate and adaptive immune systems. AEC-derived factors inhibit migration of neutrophils and macrophages in vitro. mRNA analysis revealed that AEC produced MIF, a potent inhibitor of macrophage migration and coincidentally, a potent inhibitor of NK cell-mediated lytic activity. ²⁷ Additional analysis revealed that biologically active MIF protein was indeed secreted by AECs. Thus, AECs produce factors that can inhibit cells of the innate immune apparatus that are critical players in the inflammatory responses that lead to the clinical decision to perform amniotic membrane transplantation. 

AECs also secrete factors that inhibit cells of the adaptive immune system, as demonstrated by the steep reduction in T- and B-cell proliferative responses to mitogens. Moreover, AEC factors induced apoptosis of activated T cells (Jurkat cells). The mechanism of AEC-mediated induction of apoptosis of lymphocytes remains a mystery. A growing body of evidence indicates that the amniotic membrane possesses a multitude of immunomodulatory factors. ³⁷ More recently, Soloman et al. ³⁸ reported that the anti-inflammatory effect excreted by the amniotic membrane is mediated by the suppression of IL-1 mRNA and the reduction of IL-1 protein in human limbal epithelial cells. More recently, it has been reported that soluble factors present in the amniotic membranes are capable of suppressing alloreactive T cells in vitro. ³⁹  

It is well known that FasL, TNF, and TRAIL are involved in apoptosis in many cell types. ^{32 33} Because caspase pathways are involved in the initiation of apoptosis after Fas/Fas ligand, TNF/TNF-R, and TRAIL/TRAIL receptor interactions, ³⁴ we hypothesized that AECs mediate caspase-dependent killing through these ligand–receptor interactions. 

Our results demonstrated that mRNA for TNFα, TRAIL, and FasL are expressed in AECs. Although treatment of the AEC supernatant with anti-FasL antibody blocked the killing of Jurkat cells by 50%, treatment with anti-TRAIL and anti-TNFα antibodies did not inhibit AEC-induced apoptosis. These results clearly demonstrate that Fas-ligand plays a major role in AEC-mediated apoptosis. Immune-privileged sites, such as the eye, testis, and uterus, express Fas ligand. ^{40 41 42} It has been shown that expression of FasL within the eye plays an important role in the maintenance of the immune privilege by eliminating infiltrating lymphocytes and other inflammatory cells. ⁴¹ Soluble FasL induces apoptosis in a variety of cells, including T cells. ^{43 44} Thus, the presence of FasL on AECs may prevent inflammation by triggering apoptosis of Fas-positive cells after amniotic membrane transplantation. As yet, we have no explanation as to why the anti-FasL antibody did not completely inhibit AEC-induced apoptosis. Our results show that TNF-α and TRAIL are not involved in induction of AEC-induced apoptosis in Jurkat cells. These results also indicate that AECs secrete soluble factor(s) other than FasL that induced 50% apoptosis in Jurkat cells. Although soluble FasL triggers apoptosis in lymphocytes, macrophages and neutrophils were not eliminated through the apoptosis pathways. The most likely explanation to account for the lack of induction of apoptosis in these cells by soluble FasL is that AECs produce known or unknown factors capable of inhibiting apoptosis in neutrophils and macrophages. In support of this view, it has been demonstrated that aqueous humor–derived soluble FasL induces neutrophil cytotoxicity that is inhibited by human TGF-β. However, TGF-β had no effect on FasL-dependent apoptosis in Jurkat cells. ⁴⁵ Whether similar mechanisms are involved in preventing macrophage and neutrophil apoptosis by AECs remains to be clarified. 

In our prior study, human AECs transplanted onto the denuded corneal surfaces of rabbits reconstituted the corneal epithelium and did not undergo immune rejection during 10 days of observation. ¹⁵ Under normal conditions, AECs do not express classic major histocompatibility complex (MHC) class I antigens ¹⁷ and thus enjoy a degree of immune privilege. The results reported herein suggest that in addition to the absence of MHC class I antigens, the AECs secrete immunosuppressive factors that inhibit both innate and adaptive immune responses, thereby reinforcing the immune privilege of AECs. 

Although the absence of MHC class I antigens on the AECs provides a degree of immune privilege against cells of the adaptive immune system, it ironically renders these cells potentially vulnerable to attack by cells of the innate immune apparatus—namely, NK cells. According to the “missing self” hypothesis, NK cells recognize and lyse cells that do not express or that express low levels of MHC class-I molecules. ⁴⁶ However, we also showed in the current study that AECs express MIF, which is a potent inhibitor of NK-cell–mediated cytolysis. ²⁷ The MIF secreted by AECs was biologically active and inhibited macrophage migration in the classic assay for detecting MIF. ²⁹  

The present results demonstrate for the first time that AECs secrete factors that inhibit cells in the innate and adaptive immune systems. We are attracted to the hypothesis that AECs produce a variety of immunoregulatory factors that accumulate in the amniotic membrane and endow it with anti-inflammatory factors and immunosuppressive properties. Fully characterizing the immunoregulatory factors induced by AECs may have potential application in corneal transplantation and in the treatment of corneal inflammatory disorders. 

 Supported in part by National Eye Institute Grant EY09756 and an unrestricted grant from Research to Prevent Blindness.

 Submitted for publication May 4, 2004; revised June 24, 2004; accepted July 4, 2004.

 Disclosure: H. Li, None; J.Y. Niederkorn, None; S. Neelam, None; E. Mayhew, None; R.A. Word, None; J.P. McCulley, None; H. Alizadeh, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Inhibition of chemotactic responses of macrophages and neutrophils by AEC supernatant. Murine neutrophils (A) and macrophages (B) were incubated in 24-well plates with migration assay membrane with complete F12/DMEM or medium with 50% AEC supernatant. rMIP-2 (60 μg) was used to induce neutrophil and macrophage migration. The transmigrated cells were harvested after 4 hours of incubation at 37°C and were counted under the microscope (×200). Data are representative of two independent experiments and are the mean ± SE of triplicate counts. *P < 0.01 compared with the medium group. The difference between the negative control (medium) and experimental group (AEC+MIP-2) was not statistically significant (P > 0.05).

Figure 2.

 

Effect of AEC supernatant on murine neutrophil and macrophage apoptosis. The neutrophils (A) and macrophages (B) were incubated with or without 50% AEC supernatant in medium at 37°C for 4 hours. The cells were collected, stained with anti-caspase-3 antibody, and analyzed by flow cytometry. For each group, 5000 ungated events were counted. The positive cells in the M₂ area are defined as apoptotic. Staurosporine (3 μg/mL) was the positive control.

Figure 3.

 

Expression of TGF-β2 and MIF by AECs. Agarose gel (1%) showing specific PCR product amplified from mRNA isolated from human AECs. Lane 1: molecular weight marker; lane 2: a 380-bp band corresponding to TGF β2; lane 3: a 300-bp band corresponding to MIF, and lane 4: a 528-bp band corresponding to β-actin.

Figure 4.

 

Inhibition of macrophage migration by AEC supernatant. Capillary tubes were filled with macrophages and incubated with either 50% concentrated (15×) AEC supernatant or complete medium (×15). (A) Representative samples of macrophage migration in 15× complete medium (left) or 15× medium containing 50% AEC supernatant (right). (B) The average area of migration was calculated for each group by image analysis. Data are presented as the mean area of macrophage dispersion after 24 hours in culture (n = 3 for each group; **P < 0.01 compared with medium control).

Figure 5.

 

Effect of AEC supernatant on murine T and B lymphocyte apoptosis. T lymphocytes (A) and B lymphocytes (B) were incubated with or without 50% AEC supernatant in medium at 37°C for 4 hours. The cells were collected, stained with anti-active caspase-3 antibody and analyzed by flow cytometry. For each group, 10,000 ungated events were acquired. The positive cells in the M₂ area are defined as apoptotic. Staurosporine (3 μg/mL) was the positive control.

Figure 6.

 

Effect of AEC supernatant on murine T and B lymphocyte proliferation. T lymphocytes (A) and B lymphocytes (B) were incubated with F12/DMEM or medium containing 50% AEC supernatant for 48 hours at 37°C. Concanavalin A (CON A; 1.0 μg/mL) or lipopolysaccharide (LPS; 5.0 μg/mL) was used to stimulate lymphocyte proliferation. Proliferation was measured by uptake of ³H-thymidine. ***P < 0.001 compared with medium+mitogens.

Figure 7.

 

Effect of AEC supernatant on HCEC and Chang cell apoptosis. HCECs and Chang cells were incubated with or without 50% AEC supernatant in medium at 37°C for 48 hours. Cells were collected, stained with anti-active caspase-3 antibody, and analyzed by flow cytometry. From each sample, 5000 ungated events were acquired. The positive cells in the M₂ area are defined as apoptotic. Staurosporine (3 μg/mL) was the positive control.

Figure 8.

 

AEC supernatant induced apoptosis through caspase-3 pathways. Jurkat cells were incubated in medium with and without AEC supernatant (50%). The supernatants were treated with either caspase-3 inhibitor (Z-DEVD-FMK) or control inhibitor (Z-FA-FMK). Apoptosis was determined by annexin V assay and analyzed by flow cytometry. For each sample, 10,000 ungated events were acquired. ***P < 0.001 compared with control inhibitor or without inhibitor.

Figure 9.

 

RT-PCR analysis of apoptosis inducing genes by AECs. Agarose gel (1%) showing specific PCR product amplified from mRNA isolated from human AECs. Lane 1: molecular weight marker; lane 2: a 414-bp band corresponding to TNFα; lane 3: a 482-bp band corresponding to FasL; lane 4: a 513-bp band corresponding to TRAIL; and lane 5: a 528-bp band corresponding to β-actin.

Figure 10.

 

Effect of anti-human Fas antibody on apoptosis of Jurkat cells induced by AEC supernatant. Jurkat cells were incubated with anti-TRAIL, anti-TNFα, or anti-FasL (20 μg/mL) monoclonal antibodies in the presence or absence of AEC supernatant (50%). Apoptosis was determined by anti-active caspase-3 staining and the percentage of apoptosis was determined by flow cytometry. Mouse IgG (20 μg/mL) was the negative control. ***P < 0.001 compared with control antibody.