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Am. J. Respir. Cell Mol. Biol., Volume 17, Number 4, October 1997 501-507

KL-6, a Human MUC1 Mucin, Is Chemotactic for Human Fibroblasts

Yutaka Hirasawa, Nobuoki Kohno, Akihito Yokoyama, Yoshikazu Inoue, Masahiro Abe, and Kunio Hiwada

Second Department of Internal Medicine, Ehime University School of Medicine, Onsen-gun, Ehime, Japan


    Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

KL-6 in serum and bronchoalveolar lavage fluid has been reported to be a sensitive marker indicating the activity of fibrosing lung diseases. The molecule is clustered in MUC1 mucin according to the findings of immunohistochemical and cytometric studies. To elucidate the pathogenic role of KL-6 in fibrosing lung disease, we characterized its biochemical properties and examined whether purified KL-6 is chemotactic for human fibroblasts in vitro using modified Boyden chambers. Biochemical properties of purified KL-6 were similar to those of other MUC1 mucins previously reported. KL-6 promoted the migration of 5 of 5 human lung fibroblasts and 3 of 4 human skin fibroblasts. Checkerboard analysis revealed that KL-6 was chemotactic as well as chemokinetic. Though platelet-derived growth factor, fibroblast growth factor, or fibronectin were also chemotactic for fibroblasts in the experimental system, only fibronectin augmented KL-6-induced chemotaxis. These observations indicate that KL-6 is one of the chemotactic factors for most fibroblasts and that the increased KL-6 in the epithelial lining fluid in small airways may cause the intra-alveolar fibrosis in fibrosing lung diseases.


    Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Fibrosing lung diseases (FLD) such as idiopathic pulmonary fibrosis (IPF) (1, 2) and histiocytosis X (3) are characterized by alveolitis and intra-alveolar fibrosis. Normally, the alveolar epithelium consists of two types of cells: the type I and the type II pneumocytes. In alveolitis, the type I pneumocytes are damaged and desquamated. The alveolar lining is then repaired by the proliferation of regenerating type II pneumocytes when the basement membrane is intact (4). Where the basement membrane is injured, fibroblasts migrate into the alveolar space from the alveolar interstitium through the crevices in the injured basement membrane. The intra-alveolar fibroblasts produce collagens resulting in intra-alveolar fibrosis (2).

KL-6 is a high-molecular-weight glycoprotein, and is classified as "Cluster 9 (MUC1)" of lung tumor and differentiation antigens according to the findings of immunohistochemical and flow cytometry studies (5). The molecule consists of multiple heterogeneous submolecules (6). KL-6 is detected by a murine monoclonal antibody (mAb), KL-6 antibody (IgG1), which recognizes a sialylated sugar chain on the molecule (9). Type II pneumocytes express KL-6 in the normal lung. KL-6 expression by regenerating type II pneumocytes is stronger in the FLD. Circulating KL-6 has been shown to be a sensitive marker indicating disease activity of interstitial pneumonitis such as IPF, hypersensitivity pneumonia, radiation pneumonia, and sarcoidosis (10). Furthermore, it is a marker indicating the degree and extent of pulmonary fibroproductive lesions in pulmonary tuberculosis (13). In such diseases, the levels of KL-6 are also markedly increased in bronchoalveolar lavage (BAL) fluid (14).

It has been reported that BAL fluids from patients with fibrosing alveolitis are chemotactic for human fibroblasts (15). Since a large quantity of KL-6 exists in BAL fluid, we attempted to determine whether or not KL-6 is chemotactic for human fibroblasts.

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

Materials

The KL-6 mAb (IgG1) was purified from ascites of mice bearing KL-6 antibody-producing hybridoma using a protein A affinity column (Affi Gel Protein A MAPS II Kit; Bio-Rad Lab, Hercules, CA) (16).

Preparation of Immunoaffinity Column

The KL-6 mAb was coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia Fine Chem., Uppsala, Sweden) according to the method reported previously (17). An affinity column (10 × 60 mm) was prepared using the gel.

Purification of KL-6

KL-6 was purified from ascites obtained from a patient (age 64 yr, male) with metastatic liver tumor derived from differentiated adenocarcinoma of the lung. KL-6 levels in his serum and ascites were 880 and 63,000 U/ml, respectively.

All procedures described below were performed at 4°C. The ascites were centrifuged at 420 × g for 15 min, and the supernatant was dialyzed overnight against 0.1 M Tris-HCl buffer (pH 8.0) containing 0.05% NaN3. The dialyzed sample was centrifuged at 17,000 × g for 30 min, and the supernatant was applied to the KL-6 immunoaffinity column. The column was washed with 10 column volumes of the starting buffer. The molecules retained on the column were eluted with 0.1 M Tris-HCl buffer (pH 8.0) containing 3 M MgCl2 and dialyzed against 0.1 M Tris-HCl buffer (pH 8.0). The eluate was concentrated by ultrafiltration (Model YM-10; Amicon Corp., Danvers, MA) and lyophilized. The lyophilized sample was resolved in 2 ml of distilled water and was gel filtrated on the Superose 6 (Pharmacia) column (15 × 320 mm) in 0.1 M Tris-HCl buffer (pH 8.0) containing 0.05% NaN3. The concentrations of protein and KL-6 in the fractions were measured, and the fractions showing high KL-6 levels were collected and lyophilized for experiments.

SDS-PAGE, Western Blotting, and Immunostaining

Vertical slab gel electrophoresis was carried out using sodium dodecyl sulfate (SDS)-4% polyacrylamide gel. Western blotting and immunostaining were performed according to the method of Towbin and colleagues (18). The gel was stained by periodic acid Schiff reaction (Schiff's Reagent; Wako Pure Chem., Osaka, Japan) for sugar (19) and by silver stain (Silver Stain Kit Wako; Wako Pure Chem.) for protein (20). DF3 mAb (kindly provided by Toray-Fuji Bionics Inc., Tokyo, Japan), as well as KL-6 mAb, were used for immunostaining of nitrocellulose membranes (21).

Determination of KL-6 Concentration

The concentration of KL-6 was measured with a sandwich-type enzyme-linked immunosorbent assay using immobilized KL-6 antibody and horseradish peroxidase-labeled KL-6 antibody, as described previously (9).

Amino Acid Analysis

The protein concentration was determined by the method of Bradford (22) using bovine serum albumin (BSA; Sigma, St. Louis, MO) as a standard. The purified KL-6 (100 µg) was prepared by hydrolysis with 6 M HCl at 135°C for 3 h. Amino acid analysis of purified KL-6 was performed using an automatic amino acid analyzer (Hitachi L-8500; Hitachi Ind., Tokyo, Japan) (23).

Sugar Analysis

The sugar content was measured by the method of Dubois and associates (24) using sucrose (Sigma) as a standard. For the analysis of neutral sugar, the purified KL-6 was hydrolyzed in 4 M trifluoroacetic acid at 100°C for 4 h and analyzed using TSK-gel Sugar AXG (4.6 × 150 mm) (Tosoh Co., Tokyo, Japan). For analysis of amino sugar, the purified KL-6 was hydrolyzed in 4 M HCl at 100°C for 6 h and analyzed using TSK-gel SCX (6.0 × 150 mm) (Tosoh). For analysis of sialic acid, the purified KL-6 was hydrolyzed in 0.05 M H2SO4 at 80°C for 1 h and analyzed using Gelpack c-620-10 (H+) (10.7 × 300 mm) (Hitachi-Kasei Ind., Tokyo, Japan) (25).

Uronic Acid Analysis

For the analysis of uronic acid, the purified KL-6 was hydrolyzed in 2 M trifluoroacetic acid at 100°C for 3 h and analyzed using SHIM-pack ISA 07/2504 (4.0 × 250 mm) (Shimadzu Co., Kyoto, Japan) (26). In this assay, the column was standardized with galacturonic acid (Kishida Chemical Co., Osaka, Japan), glucuronic acid (Kishida), and iduronic acid prepared by hydrolysis of calcium isopropylidine iduronate (Sigma). The minimal detection level was 50 ng.

Cell Culture

CCD-11Lu, CCD-13Lu, CCD-16Lu, and CCD-18Lu cells were nontransformed human lung fibroblast lines received from the American Type Culture Collection (Rockville, MD). Strain WI-38 was a nontransformed human fibroblast line from the lung, and RB16KY, AT2KY, CS2AW, and CS2OS were nontransformed human fibroblast lines from the skin, all of which were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan). Cells were seeded into 100-mm-diameter culture dishes and were cultured until confluent at 37°C in 5% CO2 in DME (Nikken BioMed. Lab., Kyoto, Japan) with 10% fetal calf serum (FCS) (Gibco, Grand Island, NY) supplemented with 100 U/ml of penicillin and 100 µg/ml of streptomycin.

Evaluation of Fibroblast Chemoattractant Activity

Chemoattractant activities of recombinant human platelet-derived growth factor-BB (PDGF-BB) (Becton Dickinson Lab., Bedford, MA), recombinant human basic-fibroblast growth factor (b-FGF) (Becton Dickinson), human plasma fibronectin (Funakoshi Co. Ltd., Tokyo, Japan), and the purified KL-6 were determined using 24-well microchemotaxis assemblies (Transwell; Costar, Cambridge, MA), as reported previously (27, 28). Briefly, 600 µl of the test sample were placed, in triplicate, in the lower compartment of the chemotaxis assembly. Polycarbonate filters (Costar), 8-µm pore size, which were pretreated with 0.01% type I collagen solution (Böttger GmbH., Berlin, Germany) to obtain attachment and spreading of the fibroblasts to the membrane, were then applied. Subsequently, 2 × 105 fibroblasts in 100 µl of conditioned medium (DMEM [Dulbecco's modified Eagle's medium] supplemented with 0.1% BSA), which were detached from the culture dishes with 0.25% trypsin (Flow Lab., Irvine, Scotland), were added to the upper compartment of the assembly. Following 4-h incubations, the conditioned medium was discarded and the filters were fixed with 5% glutaraldehyde solution (Wako) and stained with Giemsa's solution (Merck, Darmstadt, Germany). The number of fibroblasts migrating completely through the filter was counted in 10 high-power fields (hpf) at a magnification of ×300 and was shown as in 1 hpf. In each experiment, a set of "negative control" wells, run in parallel, contained conditioned medium alone.

To evaluate whether KL-6 was truly chemotactic, rather than chemokinetic, fibroblasts were exposed to the gradients of concentration of the purified KL-6 in a "checkerboard" arrangement, as reported previously (27). In brief, conditioned medium containing various concentrations of the purified KL-6 (0 to 100 U/ml) was placed in the upper and lower compartments of the chemotaxis assembly to attain appropriate positive and negative gradients of chemoattractant activity.

The effects of anti-KL-6 mAb on the KL-6-induced chemoattractant activity were examined. In brief, the purified KL-6 (100 U/ml) was incubated with mouse anti-KL-6 mAb (0 to 100 µg/ml) at room temperature for 1 h. The chemotactic activity of the mixture was tested. Mouse anti- Eimeria tenella HB8337 mAb (IgG1), which was received from the American Type Culture Collection, was used as a negative control antibody.

Statistics

Results are expressed as mean ± SD for each category examined in triplicate. Differences between two categories were tested by Student's t test. The concentration effects were evaluated by one-way analysis of variance (ANOVA). Significance was accepted when P < 0.05.

    Results
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Purification of KL-6

Figure 1 demonstrates elution patterns of KL-6 and protein in the gel-filtrated fractions. Only the first peak of protein eluates contained KL-6. Starting from 390 ml of ascites (a total of 2.46 × 107 U of KL-6), 1.53 × 106 U of the purified KL-6 was obtained. The final yield of KL-6 was 6.2% and the purified KL-6 was 1.56 × 107 U/mg protein. Sugar content of purified KL-6 was 73.8% per weight.


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  		Figure 1.   Gel filtration patterns on Superose 6 column of a specimen purified with an immunoaffinity column. Closed circles indicate KL-6 concentrations and open circles, protein concentrations. Fraction size: 0.25 ml.

Biochemical Properties of KL-6

When ascites and KL-6 were subjected to SDS-PAGE (Figure 2), the purified KL-6 was not detected by silver stain but was stained by periodic acid Schiff reaction. In the immunostaining of ascites with KL-6 antibody (Figure 2), there were a major high-molecular-weight strong band, showing a polydisperse pattern (more than 200 kD), and a low-molecular band (150 kD). Only the major band was observed in the immunostaining of purified KL-6. DF3 antibody, which recognizes the tandem repeat portion of MUC1 core protein (21), reacted with the purified KL-6 similarly to KL-6 antibody.


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  		Figure 2.   SDS-PAGE, Western blotting, and immunostaining. Lane 1: molecular weight standards. Lane 2: silver stain of ascites. Lane 3: silver stain of purified KL-6. Lane 4: PAS stain of purified KL-6. Lanes 5 and 6: immunostaining with KL-6 antibody of ascites and purified KL-6, respectively. Lane 7: immunostaining of purified KL-6 with DF3 antibody.

The amino acid analysis demonstrated that KL-6 had a high proportion of proline (19.1 mol %), alanine (17.5%), threonine (13.7%), serine (12.3%), and glycine (9.4%), as shown in Table 1. The results of sugar analysis (neutral sugar, amino sugar, and sialic acids) are shown in Table 2. KL-6 had high contents of N-acetylneuraminic acid (30.2% per weight), galactose (18.9%), N-acetylgalactosamine (11.9%), and N-acetylglucosamine (9.7%). Uronic acid was not detected in 1,344 µg/10 ml of the purified KL-6.

                              
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TABLE 1
Amino acid composition of purified KL-6

                              
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TABLE 2
Sugar composition of purified KL-6

Effect of KL-6 on Fibroblast Migration

Conditioned medium containing 100 U/ml of the purified KL-6 significantly enhanced the number of migrated cells in 5 of 5 lung fibroblasts and 3 of 4 skin fibroblasts examined, as shown in Table 3. KL-6 in the concentration of 1 U/ml or more dose-dependently enhanced the migration of CCD-11Lu cells, as shown in Figure 3. The KL-6-induced migration of the fibroblast was dose-dependently inhibited by mouse anti-KL-6 mAb and 100 µg/ml anti-KL-6 mAb completely inhibited the migration enhanced by 100 U/ml of the purified KL-6, as shown in Figure 4.

                              
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TABLE 3
Effects of KL-6 on the migration of fibroblasts*


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  		Figure 3.   Effects of KL-6 on the migration of a human lung fibroblast, CCD-11Lu. The number of migrated fibroblasts was significantly augmented according to the increase of KL-6 concentration (*P < 0.001, one-way ANOVA). When the KL-6 concentration was 1, 10, 100, or 1,000 U/ml, the number of migrated fibroblasts was significantly higher than those without KL-6 (dagger P < 0.001, Student's t test).


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  		Figure 4.   Effects of mouse anti-KL-6 mAb on KL-6-induced fibroblast migration. Anti-KL-6 mAb dose-dependently inhibited the migration of CCD-11Lu fibroblast induced by 100 U/ml of KL-6 (*P < 0.001, one-way ANOVA). The inhibitory effect was obtained with 0.1 µg/ml or more concentration of anti-KL-6 mAb (dagger P < 0.001, Student's t test). Mouse IgG1 control mAb had no effect.

Well-defined chemoattractants such as PDGF-BB, b-FGF, and fibronectin were examined to determine whether they enhance the KL-6-induced migration of the fibroblasts. As shown in Figures 5 and 6, each of them alone showed a significant chemoattractant activity to CCD-11Lu lung fibroblast. Only fibronectin, however, promoted the KL-6-induced migration of the fibroblast, whereas PDGF-BB and b-FGF did not.


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  		Figure 5.   Effects of PDGF and FGF on the migration of a human lung fibroblast, CCD-11Lu. Closed circle indicates the number of migrated fibroblasts, which was dose-dependently increased at the concentrations of 0.3 to 30 ng/ml of PDGF (A; *P < 0.001, one-way ANOVA) and of 16 to 250 ng/ml of FGF (B; *P < 0.001, one-way ANOVA). Open column indicates the number of fibroblasts which migrated to 1,000 U/ml of purified KL-6, and 0-300 ng/ml of PDGF (A) or 0-250 ng/ml of FGF (B). In the presence of 1,000 U/ml of KL-6, no concentrations of PDGF or FGF promoted the KL-6-induced migration.


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  		Figure 6.   Effect of fibronectin on the migration of a human lung fibroblast, CCD-11Lu. Closed circle indicates the number of migrated fibroblasts, which was dose-dependently increased at the concentrations of 1.25 to 20 µg/ml (Dagger P < 0.001, one-way ANOVA). Open column indicates the number of fibroblasts which migrated 0-20 µg/ml of fibronectin and 1,000 U/ml of purified KL-6. Even in the presence of purified KL-6, the number of migrated fibroblasts was significantly increased according to the fibronectin concentration (*P < 0.001, one-way ANOVA).

Checkerboard Analysis

A set of "checkerboard" experiments was performed to determine whether the augmented fibroblast chemoattractant activity induced by purified KL-6 was due to enhancement of true chemotaxis (i.e., induced directed migration), or whether this was merely a reflection of stimulated chemokinesis (i.e., accelerated random migration). The results are shown in Table 4. All samples tested displayed a greater capacity to induce lung fibroblast CCD-11Lu cell migration in positive gradients of KL-6 than in negative gradients (i.e., in each instance, KL-6 was truly chemotactic for a human lung fibroblast).

                              
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TABLE 4
Effect of concentration gradients of KL-6 on CCD-11Lu fibroblast migration*

    Discussion
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

This study clarified that KL-6, a human MUC1 mucin, is chemotactic for human fibroblasts. KL-6 had chemokinetic activities for 8 of 9 human fibroblasts and the activity was shown to be chemotactic by the checkerboard analysis. The chemotactic activity of KL-6 was comparable to those of PDGF, FGF, and fibronectin. In our previous studies (10, 14), clinical evidence has shown that KL-6 levels in serum and BAL fluid reflect disease activity of diffuse lung diseases revealing interstitial pneumonitis and pulmonary fibrosis. The role of KL-6 in the pathogenesis of these diseases, however, has not been elucidated. Since KL-6 is chemotactic for fibroblasts, the molecule might promote alveolar fibrosis by inducing the migration and accumulation of fibroblasts in the damaged alveolar spaces in FLD.

KL-6 molecules were refined using immunoaffinity and gel filtration columns to ascertain whether or not KL-6 is really a MUC1 mucin. Not enough biochemical studies have been performed to confirm that KL-6 really is a MUC1 mucin, although KL-6 has been classified as a MUC1 mucin based on the findings of immunohistochemical and cytometric studies (5). Biochemical studies such as SDS-PAGE and amino acid and sugar analysis revealed that the purified KL-6 is quite similar to MUC1 mucins reported previously (16, 21).

It has been reported that MUC1 mucin has various pathophysiologic roles. First, MUC1 mucin inhibits cell- cell adhesion of epithelial cells (29). Second, it has the immunomodulatory effect of decreasing the susceptibility of malignant cells to cytotoxic T cells through the inhibition of cell-cell adhesion between target and effector cells (30). Third, MUC1 mucin itself could be a target molecule of HLA-unrestricted cytotoxic T cells (31). Cancer cells expressing a large amount of mucin not defined as MUC1 were more invasive and more metastatic compared with cells with a low amount of mucin (32). Moreover, it has been reported that colonic mucin glycoprotein might act in conjunction with trefoil peptides to promote the migration of an intestinal epithelial cell line (33). Our finding is the first evidence disclosing a novel function of a soluble type of MUC1. In conclusion, since it has been stated that the expression of MUC1 mucin in a human rectal adenocarcinoma was enhanced more than 5-fold by treatment with tumor necrosis factor alpha  (34), KL-6 production might be controlled by cell-cell and cytokine networks in FLD.

The main source of KL-6 in alveolar spaces is type II pneumocytes in normal and fibrosing human lungs (10). Previous reports indicate that, other than KL-6, human type II pneumocytes produce chemoattractants for fibroblasts such as fibronectin (35), transforming growth factor beta  (36), and PDGF (37, 38). In FLD, the increased production of PDGF is observed in activated macrophages and endothelial cells (38), and that of transforming growth factor beta  in macrophages and fibroblasts (36). Fibroblast growth factor is also recognized as an important cytokine which is overproduced by fibroblasts and endothelial cells in IPF (39). In the peripheral lung, there could be many other chemoattractants for fibroblasts, such as epidermal growth factor (40, 41), leukotriene B4 (42), magnesium (43), thrombin (44), and collagens (45, 46). Enhanced expression of PDGF receptor on fibroblasts is also recognized as one of the regulatory factors for fibroblast proliferation and chemotaxis (47). The levels of these substances differ in different stages of fibrogenesis.

Since proteoglycan is a high-molecular-weight glycoprotein, there is a possibility that the KL-6 we purified could have been contaminated with proteoglycans. That possibility was ruled out, however, because no detectable levels of uronic acids were found in the purified KL-6. KL-6- induced fibroblast migration was, furthermore, completely inhibited by mouse anti-KL-6 mAb. This observation indicates that the chemotactic activity of KL-6 needs the sialylated carbohydrate chain specifically recognized by the mAb.

KL-6-induced fibroblast migration was enhanced by the addition of fibronectin, but not by that of PDGF or FGF. It is difficult to explain the difference in the promoting effect between fibronectin and PDGF or FGF. In PDGF-stimulated chemotaxis, Ras was reported to work as an intermediate, but it might not be required for fibronectin-stimulated cell motility (48). It has also been reported that FGF exerts some effects on the G-protein-mediated pathways (49). These findings suggest that remarkably different intracellular signaling pathways may exist between fibronectin- and PDGF- or FGF-dependent cell motilities.

Levels of KL-6 are more than 15,000 U/ml in the epithelial lining fluids of the lower respiratory tract (14) and are always much higher in the alveolar spaces than in the interstitium (10). In this study, at the concentrations of 1 to 1,000 U/ml, KL-6 was chemotactic for lung fibroblasts. Locally existing KL-6 might contribute, at least in part, to the fibrogenesis in FLD in cooperation with other functionally active molecules such as extracellular matrix and cytokines. The mechanism by which KL-6 exerts chemotaxis on fibroblasts should be clarified by further studies.

    Footnotes

Address correspondence to: N. Kohno, Second Dept. of Internal Medicine, Ehime University School of Medicine, Onsen-gun, Ehime 791-02, Japan.

(Received in original form May 23, 1995 and in revised form February 25, 1997).

Acknowledgments: This work was supported in part by Grants-in-Aid for Scientific Research (Nos. 06670618 and 08670665) from the Ministry of Education, Science, Sports and Culture, Japan.

Abbreviations BAL, bronchoalveolar lavage; FGF, fibroblast growth factor; FLD, fibrosing lung diseases; IPF, idiopathic pulmonary fibrosis; PDGF, platelet-derived growth factor.

    References
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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