Regulation of extracellular matrix synthesis by TNF-alpha and TGF-beta 1 in type II cells exposed to coal dust

Yu-Chen Lee1 and D. Eugene Rannels1,2

Departments of 1 Cellular and Molecular Physiology and 2 Anesthesia, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Type II pulmonary epithelial cells respond to anthracite coal dust PSOC 867 with increased synthesis of extracellular matrix (ECM) components. Alveolar macrophages modulate this response by pathways that may involve soluble mediators, including tumor necrosis factor-alpha (TNF-alpha ) or transforming growth factor-beta 1 (TGF-beta 1). The effects of TNF-alpha (10 ng/ml) and/or TGF-beta 1 (2 ng/ml) were thus investigated in dust-exposed primary type II cell cultures. In control day 1 or day 3 cultures, TNF-alpha and/or TGF-beta 1 had little or no effect on the synthesis of type II cellular proteins, independent of whether the cells were exposed to dust. With PSOC 867 exposure, where ECM protein synthesis is elevated, TNF-alpha and TGF-beta 1 further increased both the absolute and relative rates of ECM synthesis on day 3 but had little effect on day 1. Each mediator increased expression of fibronectin mRNA, as well as of ECM fibronectin content, in a manner qualitatively similar to their effects on synthesis. Thus TNF-alpha and TGF-beta 1 modulate both ECM synthesis and fibronectin content in coal dust-exposed type II cell cultures.

tumor necrosis factor-alpha ; transforming growth factor-beta 1; alveolar epithelium; anthracite coal dust; fibronectin; lung injury; protein synthesis

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

CHRONIC INHALATION of exogenous mineral or coal dust leads to progressive changes in lung structure and function, including development of pulmonary fibrosis and consequent reductions in lung compliance, characteristic of chronic interstitial lung disease (18). These clinically significant effects of particle injury are thought to involve activated inflammatory cells, including alveolar macrophages, in the gas-exchange region of the lung (10, 36). Numerous studies (2, 4, 9, 20, 21) support the premise that soluble products of both resident and recruited macrophages and inflammatory cells can initiate both acute and long-term lung injury through release of soluble mediators such as eicosanoids, cytokines, and free radicals. The response to silica dioxide, for example, is associated with an increased release of cytokines such as tumor necrosis factor-alpha (TNF-alpha ) and interleukin-1 by alveolar macrophages (12), along with structural changes in the alveolar region. The extent of this response is dependent on dose, time, and dust composition.

Previous reports from this laboratory demonstrated that type II pulmonary alveolar epithelial cells exposed in primary culture to a generic anthracite coal dust, PSOC 867, exhibit a dose-dependent increase in expression and accumulation of extracellular matrix (ECM) proteins (22) including fibronectin (23). Effects of the dust on the synthesis of ECM proteins, expression of fibronectin mRNA, and ECM fibronectin content are enhanced if the epithelial cells are cocultured with alveolar macrophages (23). The observation that coculture has little effect on the same parameters when dust is absent suggests that dust-mediated activation of the macrophage population in vitro may potentiate the epithelial cell response to dust exposure. Soluble mediators appeared to make a minimal contribution to those results. Macrophage-conditioned culture medium did not mimic the effects of coculture nor were similar effects evident when type II cells and macrophages were separated by a permeable membrane (23). The latter observations suggested that the effects of coculture require that epithelial cells and macrophages be in close proximity and thus indicated a role for direct cell-cell interactions in potentiation of the dust effects.

The present experiments were initiated to investigate further the possibility that the above interactions of epithelial cells and macrophages in vitro may involve soluble mediators. Specifically, TNF-alpha and transforming growth factor-beta 1 (TGF-beta 1) were selected for further study on the basis of preliminary experiments that suggested relevant activity of both agents (24; unpublished observations). The results demonstrate that either or both of these mediators increase synthesis of ECM proteins, expression of fibronectin mRNA, and fibronectin content of the ECM in coal dust-exposed type II cell cultures. These results reproduce the response pattern observed in dust-treated cocultures of alveolar epithelial cells and macrophages.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Isolation and primary culture of type II pulmonary epithelial cells. Type II pulmonary epithelial cells were isolated from the lungs of anesthetized (60 mg pentobarbital sodium/kg body weight) male Sprague-Dawley rats (200-250 g) obtained from Charles River Laboratories. Type II cell isolation was according to published procedures (28). Briefly, cells dispersed by intratracheal elastase were purified by density gradient centrifugation and differential adherence. For radioisotopic labeling studies, the final cell preparation was dispersed in six-well culture plates (Falcon) in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) at 1.5 × 106 cells/well. Cells not attached to the plate were removed with a change of medium after 16-18 h. The day of cell isolation was designated day 0.

Preparation of coal dust samples. Anthracite coal dust PSOC 867 (1.65-µm median particle size; Primrose Mine) was supplied by Dr. Richard Hogg (Mineral Engineering Laboratory, The Pennsylvania State University) (13). The dust was freshly ground in a zirconium oxide ball mill (International Equipment model P7) for 10 min, weighed, and suspended (20 mg/ml) in DMEM deficient (DMEM-D) containing 80 µM leucine but without FBS. The concentrated dust suspension was sonicated and diluted to 750 µg/ml in cell culture medium. Cell toxicity due to dust exposure was minimal (22, 23).

Preparation of TNF-alpha and TGF-beta 1. TNF-alpha was purchased from Sigma. The human recombinant product expressed in yeast is supplied by the manufacturer at 10 µg/ml in PBS containing 0.1% bovine serum albumin (BSA). This stock solution was diluted to a final concentration of 10 ng/ml with DMEM-D containing 10% FBS immediately before the experiment. TGF-beta 1 from human platelets was purchased from Sigma as an aseptically lyophilized product containing 1 µg of TGF-beta 1 and 50 µg of human serum albumin. The powder was first dissolved in 1 ml of 4 mM HCl containing 1 mg/ml of BSA; the final concentration of TGF-beta 1 was 2 ng/ml culture medium. Each mediator was tested in preliminary dose-response experiments to establish a maximally effective concentration (data not shown).

Radiolabeling of cell and ECM proteins. DMEM containing 80 µM leucine was prepared by a 1:10 dilution of DMEM (GIBCO) with leucine-free DMEM-D (Sigma); concentrations of additional amino acids deficient in DMEM-D were adjusted as described (27). L-[4,5-3H]leucine (10 µCi/well; 2 × 106 dpm/µmol) was added to each well for radiolabeling, which was continued for 6 h. Rates of protein synthesis, calculated as described elsewhere (27), were expressed on the basis of cellular DNA content (22) before comparison of experimental values with those from control cultures. This calculation ensured normalization of data from independent cell isolations.

Preparation of cell and ECM fractions. Preparation of cell and ECM fractions was according to well-defined procedures (22, 27). After the fractions were radiolabeled, the medium was sampled to determine radioactivity. The remaining medium was aspirated from the culture well, and the monolayer was washed once with cold PBS containing 1 mM EDTA before separation of the cell and ECM fractions by differential extraction of the cell monolayer with ammonium hydroxide and high salt in the presence of a protease inhibitor. The "cell fraction" was precipitated in 10% trichloroacetic acid (TCA), washed two times by resuspension in 10% TCA containing 1 mg/ml of leucine, and then solubilized for scintillation counting in biodegradable counting scintillant (Amersham) with a Beckman LS-3801 instrument.

Material remaining on the culture surface after extraction of the cell monolayer, the "ECM fraction" (15), was solubilized in 1 N NaOH and then precipitated in TCA with BSA as the carrier. The resulting TCA-insoluble fraction was washed and prepared for scintillation counting as above.

Measurements of ECM fibronectin content. After resolution of the proteins by SDS-PAGE (33), the gels were equilibrated in transfer buffer containing 192 mM glycine and 20% methanol in 25 mM Tris (pH 8.3). The proteins were transferred to polyvinyl difluoride membranes for 1 h at room temperature in transfer buffer containing 0.1% SDS. Before Western blots were immunostained, nonspecific binding sites on polyvinyl difluoride membranes were blocked by incubation in BLOTTO (33). The membranes were washed in 500 mM NaCl in 20 mM Tris (Tris-buffered saline), pH 7.5, and 0.5% Tween 20 (TBS-T) before incubation with rabbit anti-rat fibronectin (Calbiochem). The membranes were then washed two times in TBS-T and incubated with goat anti-rabbit IgG conjugated to alkaline phosphatase. Before color development, the membranes were washed in TBS-T and TBS (pH 7.5). Immunoreactive fibronectin was visualized by incubating the membranes with substrate provided in an alkaline phosphatase development kit (Bio-Rad).

Fibronectin was quantified in Western blots with a laser densitometer (Molecular Dynamics). The optical density of fibronectin was compared with a standard curve prepared from samples containing 1, 5, 20, and 50 ng of rat fibronectin. Each Western blot contained both a standard curve and the experimental samples to control for interblot variability of color development.

Northern blot analysis. For isolation of RNA, type II epithelial cells (9 × 106) were cultured on 100-mm plates (Falcon). After a specified interval, the medium was aspirated, and the plates were stored immediately at -70°C. The cells were lysed in TRI REAGENT (Molecular Research Center, Cincinnati, OH); after the addition of chloroform and centrifugation, RNA was collected and precipitated by the addition of isopropanol. The pellet was washed with ethanol and solubilized in formazol. RNA concentration was estimated spectrophotometrically. RNA samples of 20 µg were applied to 1.2% agarose gels (containing 0.4 M formaldehyde) and electrophoresed at 55 V for 4 h in 20 mM MOPS buffer (pH 7.0) containing 1 mM EDTA. Corrections for uneven loading of the lanes were made based on data from a control probe against the rat homologue of prokaryotic elongation factor EFTu (25; data not shown).

RNA was transferred overnight to a nylon membrane by capillary action with 10× saline-sodium phosphate-EDTA (SSPE) buffer (1.5 M NaCl, 100 mM NaH2PO4, and 12.5 mM EDTA, pH 7.4) treated with 0.1% diethyl pyrocarbonate. The blots were baked for 2 h at 80°C and cross-linked by ultraviolet light. cDNA probes for fibronectin (30) were labeled with [alpha -32P]dCTP (NEN) by random-primer procedures as previously described (23). The Northern blots were prehybridized for 2 h at 42°C in 50% formamide, 5× Denhardt's solution, 5× SSPE, 0.2% SDS, 10% dextran sulfate, and 100 µg/ml of denatured salmon sperm DNA. Hybridization with the [32P]cDNA probe was continued overnight. After a thorough washing, the membrane was exposed to Fuji X-ray film at -70°C in the presence of one intensifying screen. [32P]cDNA hybridized to specific mRNAs was quantified by using a Betagen instrument (Betagen, Mountain View, CA).

Analysis of data. Data are expressed as means ± SE. In cases where SE does not appear, the value did not extend beyond the symbol. Statistical analysis was performed with one-way analysis of variance (ANOVA) followed by a modified t-test as described in Fig. 1. Values of P < 0.05 were considered to be of significance. All observations were made using at least two independent cell preparations.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Effects of anthracite coal dust on synthesis of cell and ECM proteins in vitro. A previous study (22) suggested that the synthesis of proteins in the ECM of type II pulmonary epithelial cells is increased in vitro in a dose-dependent manner by specific anthracite and bituminous coal dusts. Data in Table 1 extend those observations to the context of the present experiments. Samples of a well-characterized anthracite coal dust, PSOC 867 (13), were freshly ground, sonicated into cell culture medium, and applied to primary cultures of type II alveolar epithelial cells (22).

                              
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Table 1.   Effect of anthracite coal dust PSOC 867 on synthesis of cell and ECM proteins

In untreated control type II cells, synthesis of the proteins recovered in the cell fraction (27) increased more than ninefold between day 1 and day 3 of primary culture (Table 1). Exposure of the cells to anthracite dust PSOC 867 in vitro had no significant effect on the synthesis of cellular proteins on either day 1 or day 3. Rates of synthesis of ECM proteins were elevated somewhat (17%) on day 1 in PSOC 867-treated cultures (P < 0.05), whereas the magnitude of the dust effect increased threefold by day 3 (P < 0.01). Differential effects of dust exposure on the synthesis of ECM proteins were more evident when the ECM data are expressed relative to the synthesis of cellular protein data (ECM/cell). Exposure to PSOC 867 increased the relative rate of synthesis of ECM proteins by 28 and 48% on day 1 and day 3, respectively (Table 1). The latter values confirm that the effects of dust on the synthesis of ECM components do not simply reflect global changes in cellular protein metabolism. These results demonstrate culture time-dependent effects of PSOC 867 to increase both the absolute and relative rates of synthesis of ECM proteins by alveolar epithelial cells.

TNF-alpha and TGF-beta 1 modulate the type II cell response to coal dust. A previous study (24) showed that the effect of PSOC 867 on the synthesis of ECM proteins by type II cells is enhanced significantly when the pneumocytes are cocultured with alveolar macrophages. It is well known that particle exposure stimulates macrophages to release a spectrum of soluble mediators (17, 32, 34). These mediators, which include TNF-alpha and TGF-beta 1, could account for the observation of macrophage-dependent stimulatory effects. To investigate this possibility, the effects of TNF-alpha and TGF-beta 1 on the synthesis of cell and ECM proteins were examined in day 1 and day 3 epithelial cell cultures. TNF-alpha and TGF-beta 1, alone or in combination, had only small effects on the synthesis of cellular proteins in the presence and absence of PSOC 867 (Fig. 1). TNF-alpha alone caused a small inhibition in the synthesis of cell proteins in day 1 control cultures but was without effect on day 3 or in combination with TNF-alpha (Fig. 1A). TGF-beta 1 had no significant effects under the same conditions (Fig. 1A). In PSOC 867-treated cells (Fig. 1B), TNF-alpha again had a small inhibitory effect on day 1; TGF-beta 1, alone or in combination with TNF-alpha , was slightly inhibitory to the synthesis of cellular proteins on day 3.


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Fig. 1.   Effects of tumor necrosis factor-alpha (TNF) and transforming growth factor-beta 1 (TGF) on synthesis of cell proteins by control (A) and coal dust (PSOC 867)-treated (B) type II cells. Experimental conditions were as described in Table 1. As indicated, control and PSOC 867-treated cultures were exposed to either TNF (10 ng/ml), TGF (2 ng/ml), or both (TNF+TGF) at the beginning of experimental interval on day 1. Data from day 1 and day 3 control cultures (without cytokine treatment) were normalized to 100% (A, left bars). Data from PSOC 867-treated cultures without cytokines are expressed relative to normalized values from untreated control cells (B, left bars). Actual data (means ± SE) from both control groups are shown in Table 1, as well as within the corresponding bars in Figs. 1-5. Normalized data are expressed as means ± SE and represent 6 observations derived from 2 independent experiments. In all cases, statistical analysis of cytokine effects compares values from cytokine-treated cultures with those of the corresponding control without cytokine (Control or Control+867). Significantly different from corresponding control: a P < 0.05; b P < 0.01.

The effects of TNF-alpha and TGF-beta 1 on the synthesis of ECM proteins are shown in Fig. 2. Again, there was little effect of either mediator in control cultures (without dust; Fig. 2A). As noted above, the synthesis of ECM proteins was increased by dust on both day 1 and day 3 (Fig. 2B, control cells+867). Although the magnitude of the dust effect was not changed by TNF-alpha or TGF-beta 1 on day 1, both mediators had substantially stimulated ECM protein synthesis by day 3 in dust-treated cells (Fig. 2B). Differential stimulation of ECM protein synthesis by TNF-alpha and TGF-beta 1 in dust-treated cultures is emphasized in Fig. 3, where the effects on cell and matrix components are expressed on a relative basis. These differences are especially evident on day 3 when TNF-alpha and TGF-beta 1, alone or in combination, had no effect in control cultures (Fig. 3A) but increased the synthesis of ECM proteins in dust-exposed cells more than twofold (Fig. 3B).


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Fig. 2.   Effects of TNF-alpha and TGF-beta 1 on synthesis of extracellular matrix proteins by control (A) and coal dust-treated (B) type II cells. Both experimental conditions and format are described in Fig. 1. Normalized data are expressed as means ± SE and represent 6 observations derived from 2 independent experiments. Significantly different from corresponding control: a P < 0.05; b P < 0.01; c P < 0.001.


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Fig. 3.   Effects of TNF-alpha and TGF-beta 1 on relative rates of synthesis of extracellular matrix proteins by control (A) and coal dust-treated (B) type II cells. Both experimental conditions and format are described in Fig. 1. Normalized data are expressed as means ± SE and represent 6 observations derived from 2 independent experiments. Significantly different from corresponding control: b P < 0.01; c P < 0.001.

TNF-alpha and TGF-beta 1 modulate fibronectin content of the type II cell matrix. In control type II cell cultures, matrix fibronectin content, measured by Western blot analysis, increased nearly 30-fold (4.9 ± 0.2 vs. 141 ± 4 ng/well; P < 0.001) between day 1 and day 3 of primary culture (Fig. 4A). This result is in qualitative agreement with recent observations from independent experiments (33). Although matrix fibronectin was unaffected (5.2 ± 0.1 ng/well) in PSOC 867-treated cultures on day 1 (Fig. 4B), matrix levels of the glycoprotein increased 60% by the third day of dust exposure (224 ± 7 vs. 141 ± 4 ng/well; P < 0.001).


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Fig. 4.   Effects of TNF-alpha and TGF-beta 1 on extracellular matrix fibronectin in control (A) and coal dust-treated (B) type II cell cultures. Both experimental conditions and format are described in Fig. 1. Normalized data are expressed as means ± SE and represent 6 observations derived from 2 independent experiments. Significantly different from corresponding control: a P < 0.05; b P < 0.01; c P < 0.001.

As shown above, TNF-alpha and TGF-beta 1 had only small, if any, effects on the absolute synthesis of ECM proteins in day 1 control cell cultures (Fig. 2) and caused no significant changes in the relative rates of ECM protein synthesis (Fig. 3). Similarly, TNF-alpha and TGF-beta 1 were without effect on matrix fibronectin content on day 1 (Fig. 4B). In contrast, both TNF-alpha and TGF-beta 1 significantly increased matrix fibronectin levels on day 3 by 25 and 50%, respectively (Fig. 4A). The latter effects were not additive.

In PSOC 867-treated cultures, matrix fibronectin content was unaffected on day 1, independent of the treatment condition (Fig. 4B). In contrast, matrix fibronectin increased 50% in response to dust by day 3; the magnitude of this effect was comparable to both the absolute and relative rates of matrix protein synthesis (compare with Figs. 2 and 3, respectively). Furthermore, the increase in matrix fibronectin in response to TNF-alpha and/or TGF-beta 1 was qualitatively similar to, but somewhat smaller in magnitude than, that in matrix protein synthesis under each treatment condition (Fig. 4B).

Effects of TNF-alpha and TGF-beta 1 on relative abundance of fibronectin mRNA. In parallel with measurements of ECM fibronectin content, steady-state levels of fibronectin mRNA were determined by Northern analysis (Fig. 5). Data are expressed relative to those for a control cDNA probe against the rat homologue of prokaryotic protein synthesis elongation factor EFTu (data not shown). In day 1 control cultures, expression of fibronectin mRNA was not affected by TNF-alpha or TGF-beta 1 alone but increased to a small extent in the presence of the combined mediators (Fig. 5A). Fibronectin message doubled by day 3 (P < 0.05; Fig. 5A, numbers in left bars) but increased further only on treatment with TNF-alpha (P < 0.05).


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Fig. 5.   Effects of TNF-alpha and TGF-beta 1 on expression of fibronectin mRNA by control (A) and coal dust-treated (B) type II cells. Both experimental conditions and format are described in Fig. 1. Normalized data are expressed as means ± SE and represent 6 observations derived from 2 independent experiments. Significantly different from corresponding control: a P < 0.05; c P < 0.001.

Again, more substantial changes in fibronectin mRNA expression were evident in PSOC 867-treated cultures (Fig. 5B). In control cells, PSOC 867 significantly reduced fibronectin mRNA on day 1, whereas the mRNA was increased fourfold by day 3 (Fig. 5B, numbers in left bars) to levels significantly above those in control cultures (P < 0.05). Similarly, both TNF-alpha and TGF-beta 1, alone or in combination, reduced fibronectin mRNA expression on day 1 but elevated the message on day 3. In general, dust- and treatment-associated changes in fibronectin mRNA expression on day 3 are in qualitative agreement with those in matrix protein synthesis under the same conditions (compare with Fig. 3).

Parallel changes in ECM synthesis and ECM fibronectin content. Examination of the data in Figs. 3 and 4 suggests that time in culture, dust exposure, and treatment with TNF-alpha and/or TGF-beta 1 have parallel effects both on the synthesis of ECM proteins and on ECM fibronectin content. The apparent correlation between these parameters was examined further by direct comparison of these parameters with data from the 16 experimental conditions of the present study (Fig. 6). In all cases, both synthesis of ECM proteins and ECM fibronectin content were measured in experimental samples from the same culture well. The data demonstrate a direct correlation between these parameters, described by the equation y = 8.39x + 5.49 (R2 = 0.91). This analysis suggests a strong correlation between the experimental data sets, with a slope > 1.0. The result is consistent with the premise that accumulation of fibronectin in the type II cell ECM cannot be accounted for by de novo synthesis of the glycoprotein (33).


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Fig. 6.   Relative changes in extracellular matrix protein synthesis and matrix fibronectin content in type II cell cultures. Corresponding values for rates of extracellular matrix protein synthesis and matrix fibronectin content, taken from experiments in Figs. 2 and 4, are shown as means ± SE. Data for both parameters represent 16 groups of 6 corresponding observations each, derived from day 1 and day 3 cultures. The curve, drawn by least squares linear regression, is described by the equation y = 8.393x + 5.486 (correlation coefficient = 0.911 for 96 observations).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Results of the present experiments supplement previous observations (23) in vitro that demonstrate a significant role for the interactions of alveolar epithelial cells and pulmonary macrophages to determine the epithelial cell response to coal dust exposure. A substantial literature (1, 11, 20) implicates soluble products of the macrophage population in the pathophysiological consequences of particle injury to the lung. The acute response involves phagocytosis-initiated activation of macrophages (21, 26) to release cytokines and other mediators (2-4, 29) that promote fibroblast recruitment and growth, ultimately leading to fibrotic changes in the alveolar region (32).

TNF-alpha and TGF-beta 1 were selected for the present experiments based on observations that demonstrated their release by macrophages (5, 6, 18) and suggested that they may regulate activation and metabolic activity, including synthesis of ECM components (24), in type II cells. The rationale for conditions of coculture and the basis of measurements made in these experiments has been discussed in detail elsewhere (23). Coculture has been applied frequently to address the role of both direct and indirect cell-to-cell interactions in a variety of physiological and pathophysiological responses including cellular differentiation (8), wound healing (31), and expression of ECM components (23, 35).

A previous study (22) established that type II cells in primary culture respond to anthracite coal dust PSOC 867 with a time- and dose-dependent increase in the relative rates of synthesis of ECM proteins. This response, which does not involve cytotoxicity, is qualitatively similar to that observed in the presence of bituminous coal dust PSOC 1451 or a mine dust, MIT-3 (24). The fact that a second bituminous coal dust (PSOC 1361) was not active in the same cell preparations mitigates against these observations being due to a nonspecific response to particles. Effects of the three active dust samples were not correlated with SiO2 content nor were they associated with cell detachment or overt toxicity (22). The observation that alveolar macrophages further augment the response of type II cells to PSOC 867, but have little or no effect in the absence of dust, suggests involvement of activated macrophages.

The observations outlined above indicated that the effect of coculture on the type II cell matrix likely involved direct cell-to-cell interaction rather than soluble mediators (23). Nevertheless, the possibility remained that relevant mediators were not stable in conditioned medium or that they were released in sufficient concentrations to act locally but not after dilution in several milliliters of conditioned medium. Thus the present results supplement previous data and emphasize the complexity of a cellular response to particles or other injurious stimuli. Clearly, the situation in vivo is even more complex because it involves both additional cell types and their products.

In the present experiments, TNF-alpha and TGF-beta 1 had only small effects on control cultures by day 1. These effects, although significant in some cases, remained modest on day 3. In dust-treated cultures, synthesis of matrix proteins, as well as both ECM fibronectin content and expression of fibronectin mRNA, were elevated on day 3. Specific cytokine effects were enhanced in the latter cultures. In independent studies where changes in matrix characteristics of similar magnitude were observed, cell-free ECM was tested for biological activity against freshly isolated type II cells. Results demonstrated an increased biological response to the latter matrix (data not shown). Although these observations require more detailed study, they suggest that changes in matrix composition on the order of those presently observed are biologically relevant.

Several investigators (16) reported that ECM synthesized by alveolar epithelial cells is rich in fibronectin and exerts biological activity, supporting loss of the differentiated type II cell phenotype. The present data suggest a strong positive correlation between synthesis of total ECM proteins and accumulation of fibronectin in the type II cell matrix. The slope of the regression curve that describes these variables over a sevenfold range of synthesis rates is >8. This comparison suggests that de novo protein synthesis cannot account for accumulation of fibronectin in the type II cell matrix. The result thus extends previous observations that type II cells assemble ECM using both newly synthesized fibronectin and fibronectin derived from the culture medium (33), with the exogenous glycoprotein being predominant. The data also support results from independent injury models wherein serum fibronectin is utilized in the pathway of ECM assembly (7, 19).

It is important to recognize that these interpretations of the results in Fig. 6 incorporate the effects of culture time on changes in matrix protein synthesis and fibronectin content. The data thus extend beyond the major focus of the present work on cytokine effects, which are of more modest magnitude. The slope of the data largely reflects the range of individual values measured on day 3, along with increasing rates of synthesis between day 1 and day 3. About 40% of fibronectin newly synthesized by type II cells is released to the medium, whereas 10-20% distributes to the ECM (14). Nevertheless, molecules of endogenous origin appear to constitute only a small proportion of the total fibronectin recovered in the type II cell matrix (33). The observation that the slope of the data in Fig. 6 is >1.0 is thus consistent with the conclusion that exogenous fibronectin contributes to the pathway of ECM assembly in type II cell cultures.

In summary, exposure of type II pulmonary epithelial cells in primary culture to anthracite coal dust has little effect on the overall synthesis of cellular proteins. In contrast, both the absolute and relative rates of synthesis of ECM proteins are increased significantly. The latter response to dust, along with parallel increases in fibronectin mRNA expression and in matrix fibronectin content, increases with culture time. Treatment of type II cells with either TNF-alpha , TGF-beta 1, or both causes only minimal changes in the same parameters under control culture conditions or in dust-exposed cells on culture day 1. In contrast, on day 3, TNF-alpha and TGF-beta 1 stimulate matrix synthesis and assembly in dust-treated cultures, thus demonstrating a pattern of activity that parallels that observed in epithelial cells cocultured with alveolar macrophages (23). These results suggest that TNF-alpha and/or TGF-beta 1 may play a role in the effect of cocultured alveolar macrophages to modulate the type II cell response to coal dust.

    ACKNOWLEDGEMENTS

We thank Dr. John W. Swisher for helpful discussions. We also thank Drs. Jean Schwarzbauer and Roy Levine for the generous gifts of complementary deoxyribonucleic acid probes against rat fibronectin and elongation factor Tu, respectively.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant R37-HL-31560 and US Bureau of Mines Generic Mineral Technology Center for Respirable Dust Grant G1145242-42A8.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: D. E. Rannels, Dept. of Cellular and Molecular Physiology, The Pennsylvania State Univ. College of Medicine H166, 500 University Dr., Hershey, PA 17033.

Received 11 February 1998; accepted in final form 22 May 1998.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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