Bronchial Branching Correlates with Specific Glycosidase Activity,Extracellular Glycosaminoglycan Accumulation,TGFβ 2,and IL-1 Localization During Chick Embryo Lung Development

Abstract

During organ differentiation, cell-extracellular matrix (ECM) interactions are required. The components of the ECM, such as glycosaminoglycans, fibronectin, laminin, and collagens, change in relation to cytokine and enzyme activity. Moreover, glycosaminoglycans (GAGs) are components of the ECM that play an important role in both cytokine regulation and cell activities. In this work we studied the accumulation of hyaluronic acid and chondroitin sulfate and heparan sulfate proteoglycans (PGs), β-N-acetyl-d-glucosaminidase activity, the presence of transforming growth factor β₂ (TGFβ₂), and interleukin-1 (IL-1), and the localization of fibronectin, laminin, and collagen I and IV during the early stages of chick embryo lung development. We also determined the levels of hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate GAGs and the activity of β-N-acetyl-d-glucosaminidase with biochemical methods. Our data show that β-N-acetyl-d-glucosaminidase activity increases in each cell, especially in the epithelial growth front at the emergence of each bronchial bud, where hyaluronic acid and IL-1 are located in the surrounding mesenchymal areas. Chondroitin sulfate and heparan sulfate PGs, fibronectin, laminin, and collagen I and IV are evident in the area near the basal membrane along the sides where the forming structures are stabilized. Biochemical data show that β-N-acetyl-d-glucosaminidase activity increases in cells during lung development and is related to GAG decrease and to modifications of the nonsulfated/sulfated GAG ratio. These modifications could change cytokine activity and play an important role in bronchial branching development.

Lung rudiments at 6-day development are formed by epithelial tubules and surrounding mesenchyme. The mesobronchus is covered by a pluriseriate epithelium from which entobronchi and ectobronchi, all lined by simple cylindrical epithelium, spring out. It is well known that the epithelial component of the lung during development goes through a complex orderly process of branching, following strict patterns of space and time. Growth factors and glycosaminoglycans (GAGs), which are fundamental components of the extracellular matrix (ECM), perform a key role in morphogenetic and histodifferentiative processes. In particular, there is a specific GAG composition at different levels of the anlagen and at different embryonic stages, revealing heterogeneous distribution of the ECM (Becchetti et al. 1988; McGowan 1992). Many growth factors, such as epidermal growth factor (Gross et al. 1986), basic fibroblast growth factor (Sannes et al. 1992), transforming growth factor β (TGFβ), and interleukin-1 (IL-1) (Locci et al. 1993; Bodo et al. 1994) contribute to controlling the synthesis of ECM components such as hyaluronic acid (HA), chondroitin sulfates-proteoglycans (CS-PGs), laminin, and fibronectin (Schuger et al. 1990; Sannes et al. 1993). Remodeling of the ECM is a necessary condition for both cell adhesion and morphogenetic movements during organ development (Perris and Perissinotto 2000). The variation in hyaluronic acid (HA) distribution plays a basic role in directing myogenetic migrations (Brand-Saberi and Krenn 1991, Krenn et al. 1991), whereas CS impedes them (Landolt et al. 1995; Canning et al. 2000). Furthermore, TGFβ and IL-1 stimulate HA and CS synthesis in the ECM and, in turn, the GAGs modulate the availability and accessibility of cytokines with respect to cell proliferation (Locci et al. 1995). The continuous quali/quantitative variations in GAGs of the ECM involve the processes of their production and degradation. Lysosomal enzymes (exo- and endoglycosidases) are linked to these processes. The activity of these enzymes is quite different depending on the various GAG classes. Exo-glycosidase, such as β-N-acetyl-d-glucosaminidase (β-NAG) acts on HA and keratan sulfate (KS). Through histochemical and biochemical methods it has been demonstrated that, during the development of chick embryo lung, the mesenchyme shows a different GAG composition along the craniocaudal axis. This is correlated both to a different concentration of enzymes such as β-NAG and to bronchial branching development (Becchetti et al. 1988; Stabellini et al. 2002). Because TGFβ₁ and TGFβ₂ expression increases (Adab et al., 2002) and is developmentally regulated during organ morphogenesis (Schaeffer et al. 2000), and moreover stimulates production of various TGFβ isoforms (When et al. 2003), it is extremely interesting to investigate the relationship between growth factors and ECM composition in the various phases of bronchial branching at different development stages.

For this purpose, we have studied the changes in ECM composition, β-NAG enzyme activity, and TGFβ₂ and IL-1 distribution in lung buds during bronchial branching at different stages of development. Moreover, we have analyzed the total GAG and single classes of GAG in the whole lung bud at the same stages.

Materials and Methods

Hubbard fertilized eggs provided by Selice Incubator (Bubano, Imola, Italy) were incubated at 38C and 60% relative humidity. Chick embryo lungs at 5, 7, 9, 11, 14, and 18 developmental days (20 eggs per stage), according to the Hamburger-Hamilton tables (Hamilton 1965), were removed, fixed in 10% formalin, cut in serial sections at 4–7 μm, and stained for histochemical and immunohistochemical data. At the same lung development time, GAG and β-N-acetyl-d-glucosaminidase activity was determined by biochemical methods.

Histochemical Technique

Alcian Blue Staining. The histochemical study was performed on homologous sections of intraclavicular air sac, ectobronchi, parabronchi, and entobronchi on the basis of previous indications of the spatial GAG distribution and tridimensional reconstruction of epithelial lung branching in vivo (Becchetti et al. 1988; Stabellini et al. 2002). GAGs were identified by critical electrolyte concentrations at which the polyanions changed from binding Alcian to Mg⁺⁺ (Scott and Dorling 1965). Alcian stained polyanions with increasing selectivity as the MgCl₂ concentration in the staining solution increased: at MgCl₂, all GAG as well as nucleic acid and sulfate glycoproteins; at 0.3 M MgCl₂, the only macro-molecules stained positively were the GAGs chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), and heparan sulfate (HS). The Alcian blue technique, used to distinguish different GAGs has been described previously (Becchetti et al. 1988). Briefly we used 1% Alcian blue 8GX staining (AB) (Sigma-Aldrich; St Louis, MO) in 0.1 M acetate buffer, pH 5.8, in the presence of 0.025 M or 0.3 M or 0.65 M MgCl₂ solution (Sigma-Aldrich) for 2 hr. For enzymatic digestion, the sections were incubated with testicular hyaluronidase (Merck, Darmstadt, Germany; 1 mg/ml in 0.1 M phosphate buffer, pH 7.6, 6 hr at 37C). Control sections were incubated in buffer alone. The action of specific enzymes on the section, followed by Alcian blue staining, allowed us to determine the distribution of individual gly-cosaminoglycans. Digestion with testicular hyaluronidase, in particular, selectively removed HA and CS. GAG values were obtained by connecting a Zeiss Axioplane Microscope to a Kontron Electronic Scanner using Vidas Software with a specific canal that converted the blue color into gray scale (arrangement: black = 0, white = 1). We prepared three slides of whole lung sections for samples. The values are expressed as relative optical density and were the mean ± SD of five determinations per slide. The sections were stained with hematoxylin-eosin for morphological examination.

β-NAG Localization

For histochemical localization the samples were fixed in 3.7% formaldehyde in 0.1 M PBS, pH 7.25. They were frozen in liquid nitrogen and sectioned with cryostat in 7-μm sections. The β-NAG staining was assayed according to the method previously described (Stabellini et al. 2002). Briefly, sections were immersed in 0.1 M Na-citrate buffer, pH 5.2, containing 0.25 mg/ml Fast-Garnet GBC (Sigma-Aldrich; F-8761) and 0.2 mg/ml naphthol AS-BI N-acetyl-β-d-glucosaminide (Sigma; N-4006) for 1 hr at room temperature (RT). Relative control sections were determined in 0.1 M Na-citrate buffer, pH 5.2.

Immunohistochemistry

CS and HA Localization. For CS analysis, a monoclonal mouse antibody (Bio Makor, Rehovot, Israel; code 6505) diluted 1:100 was used. The secondary antibody against mouse IgG was conjugated with alkaline phosphatase (Sigma, A-3688). For HA analysis, we used a probe constituted of hyaluronectin, a glycoprotein extracted from brain by Delpech et al. (1991), which is able to bind to HA and not to any other GAG. The hyaluronectin conjugated with alkaline phosphatase was provided by Girard Nicole (Centre Henry Becquerel-Roven) and utilized as described by Marret et al. (1994). For HS analysis, a primary polyclonal rabbit anti-HS antibody diluted 1:250 (kindly supplied by Patricia Simonn-Assmann; Iserm, Strassburg) was used.

Cytokine Localization. For TGFβ₂ localization, we used an Rb34 antibody obtained from rabbits immunized against a synthetic peptide consisting of the first 29 amino acids of TGFβ, supplied by Celtrix Laboratories (Palo Alto, CA), diluted 1:40. IL-1 was localized by a rabbit anti-IL-1 antibody (Genzyme; Milan, Italy), diluted 1:40. The secondary antibody against the primary rabbit antibody was a biotinylated goat anti-rabbit IgG (Bio-Division, Milan, Italy, R001–63), diluted 1:250. The third molecule conjugated with peroxidase was streptavidin (Bio-Division, G014–63). For peroxidase visualization, diaminobenzidine was used (DAKO; Carpinteria, CA; S 3000). For alkaline phosphate, Fast Red (Sigma; F-5146) and phosphate naphthol AS-TR (Sigma; N-8518) were used.

Sequential Staining for HA or CS and Glycosidase Reaction. According to previous studies on the spatial distribution of GAGs in chick embryo lung (Becchetti et al. 1988), we also performed a sequential reaction for β-NAG and for HA and CS in 9-day-old lung sections. Control sections for each reaction were also performed without primary antibody.

Biochemical Procedures

GAG Analysis. For biochemical determination of GAG, lung rudiments were removed at 4C from 5-, 7-, 9-, 11-, 14-, and 18-day-old embryos, pooled (30 for each experiment), sonicated for 1 min at 21 Hz (MSE instrument, Model 44), lyophilized, dissolved in distilled water, and further sonicated for 30 sec. This was done for maximum solubility. The GAGs were isolated according to a method previously described (Evangelisti et al. 1984). Individual GAGs were separated by two-dimensional electrophoresis on cellulose acetate plates and identified by comparing them with standard GAG (HA, CS, DS, HS; Sigma) and by their specific ennzymatic susceptibility. Lyophilized samples were digested with bovine hyaluronidase (EC 3.2.1.35) (Miles Italiana; Milan, Italy) and with Streptomyces hyaluronidase lyase (EC 4.2.2.1) (Streptomycetes hyaluroniticus; Seikagaku Kogyo, Tokyo, Japan) at 37C for 24 hr with 30 and 5 enzyme units, respectively, or with chondroitin AC-II lyase (EC 4.2.2.5) (Arthrobacter aurescens; Seikagaku Kogyo) for 24 hr with 0.5 and 0.01 enzyme units (Conrad et al. 1977). The single classes of GAG were quantified using the microcolorimetric methods of Bertold and Page (1985).

β-N-acetyl-d-glucosaminidase Activity. For enzymatic activity, lung rudiments were homogenized in 2 ml of 0.1 M citrate/0.2 M phosphate buffer, pH 4.5, then centrifuged at 25,000 rpm for 10 min, after which the supernatants were used. In preliminary experiments, no enzymatic activity was detected in the sediments. β-N-acetyl-d-glucosaminidase (EC 3.2.1.30) was assayed as previously described by Orlacchio et al. (1984) using as substance 4-methyl-umbelliferyl-β-N-acetyl glucosaminidase in 0.1 M citrate/0.2 M phosphate buffer, pH 4.5 (50 μl of enzyme preparation). One enzyme unit is defined as the amount of the enzyme that converts 1 nmol/hr substrate into 4-methyl-umbelliferone at 37C. Specific activity is expressed as u/mg protein. Proteins were determined according to the Bradford method (1976), using crystalline calf gamma globulin (Merck) as standard.

Statistical Analysis

The statistical analysis was performed using the Student's t-test for paired and unpaired data. The differences were considered significant at p = 0.05.

Results

Histochemistry

Alcian Blue. Developing lung rudiments show a progressive increase of second-order tubules emerging from the bronchus, such as ento, ecto, laterobronchi, and then of third-order tubules, the parabronchi (P), always surrounded by mesenchyme. The mesobronchus (Ms) is covered by a pluriseriate epithelium, the second- and third-order bronchi instead by a simple cylindrical or cubic epithelium. After AB reaction at 0.025, 0.3, and 0.65 M MgCl₂ concentrations (Figure 1), 5-day-old lung rudiments show alcianophilia around the Ms, which is completely removed by testicular hyaluronidase and chondroitinase ABC, corresponding to the presence of HA and CS, respectively. The mesenchyme areas where the entobronchi emerge show greater alcianophilia at 0.025 and 0.3 M MgCl₂, totally removed by testicular hyaluronidase and chondroitinase (HA and CS). In lungs incubated for 14 days (Figure 1) at the same molarity, alcianophilia significantly increases till day 9 of incubation (appearance of laterobronchi), and then decreases (p<0.01) from day 11 (appearance of parabronchi), reaching the lowest point at day 14 of chick embryo lung development (complete bronchial branching). The Alcian blue reaction is lower (p<0.01) around the ectobronchi, laterobronchi, and parabronchi than the entobronchi and very low around the air sac. The alcianophilia at 0.025 and 0.3 M MgCl₂ completely disappears after hyaluronidase and chondroitinase digestion (HA and CS).

β-NAG Reactivity. We examined the localization of β-NAG in lungs at developmental days 5, 7, 9, 11, 14, and 18. The enzyme is generally more active in cell compartments than in the extracellular component. The highest degree of β-NAG reactivity was observed in the more recently developed areas, especially on the growth front of epithelial lung branching and in the basal lysosomal compartment within the membrane surrounding the mesenchyme, where the epithelium of second order bronchi grew first, at 5 (Figure 2A) and 7 (Figure 2C) days, followed by that of parabronchi at 9 (Figure 2E) and 11 days (Figure 3A). Histochemical controls of β-NAG reactions were negative (Figures 2B, 2D, 2E, 3A, and 3C).

Figure 1

Alcian blue staining at different MgCl₂ concentrations. Reactivity of mesenchyme surrounding the mesobronchus, ecto-, latero-, endo-, and parabronchi of bronchial branching in chick embryo lungs at 5, 7, 9, 11, and 14 days of incubation.

Immunohistochemistry

Glycosaminoglycans and β-NAG Localization. The 9-day-old lungs showed a positive reaction for HA and CS PGs in the mesenchyme and basal membrane. The CS PGs were particularly evident along the lateral portion of the bronchial branching (Figure 4A), whereas HA was located either in the mesenchyme or in the most external areas of the expanding lung bud (Figure 4C). HS PG appeared near the basal membrane except in the epithelial growth front (Figure 4E).

Localization of β-NAG/CS or β-NAG/HA reactions in the mesenchyme around the bronchial branching at 9 days showed that HA and CS were poorly reactive in the area where the enzyme was more concentrated (Figures 4B and 4D). Immunohistochemical controls were negative (Figure 4F). The lungs at 11 days showed that fibronectin (Figure 5A), laminin (Figure 5B), collagen type I (Figure 5C) and type IV (Figures 5D) had a strong positive reaction along the lateral part of the bronchus, whereas they were nearly negative in the growth front.

Cytokine, Chondroitin 4–6 Sulfate and Hyaluronic Acid Immunolocalization. TGFβ₂ was located in the basal membrane and particularly in the lateral subepithelial regions of second- and third-order bronchi (Figure 6B). Immunostaining of IL-1 showed the same localization, but with greater reaction in the growth front with respect to the lateral regions of the epithelial tubulus (Figure 6A). The localization of cytokines and GAGs showed that TGFβ₂ and CS were expressed in the same regions (particularly in the lateral region of branching) (Figures 4A and 6B), while IL-1 was correlated with HA reaction (particularly in the growth front of branching) (Figures 4C and 6A).

Biochemical Data

GAG Evaluations. Figure 7 shows the trend of non-sulfated (HA) and sulfated GAGs (CS, DS, HS) in lungs at different incubation days. GAGs increased from 5 days and reached their greatest concentration at 11 days, then decreased to their lowest point at 18 days. In addition, single classes (HA, CS, DS, and HS) displayed the same trend, with a peak of greater concentration at the 11 day. The HA/sulfated GAG ratio showed a significant and continuous decrease from 5 to 18 days (Figure 8).

Figure 2

β-NAG reaction in day 5, 7, 9, 11, and 14 chick embryo lungs. Strong positivity in the epithelial growth front (asterisks) where first secondary bronchi, then parabronchi emerge. Five-day-old chick embryo lung (A) Me, mesobronchus; En, Entobronchus. Seven-day-old chick embryo lung (C). B1, B2, B3 ectobronchi, nine-day-old chick embryo lung (E) P, parabronchi.

β-N-acetyl-d-glucosaminidase Biochemistry Data. Figure 7 also shows the β-NAG activity in whole embryonic chick lungs at the considered developmental days. β-NAG showed constant specific activity from 5 to 7 days, increased till 14 days (p≤0.01), and then decreased (p≤0.01) until day 18.

Figure 3

Eleven-day-old chick embryo lung (A). B2, ectobronchus; P, parabronchus. (C) A 14-day-old chick embryo lung. P, parabronchi; a.c. = air capillaries. The controls are negative (B,D). See also Figures 2B, 2D, and 2F.

Discussion

During chick embryo lung development, bronchial branching shows progressive complexity of ramifications from the day 6 to day 14 of incubation (Hamilton 1965). The gradual development of ento-bronchus, ectobronchi, laterobronchi, parabronchi, and air sacs is related to mesenchymal GAG changes and to cytokine and glycosidase activity. Our data show that during lung branching, when a new bronchial branch emerges, the involved mesenchyme accumulates HA. Instead, when the new branch gradually lengthens, HS PG and CS PG accumulate in the lateral regions. In the same lateral regions of bronchial tubuli, at the epithelial-mesenchymal interface, fibronectin, laminin, and collagen IV are evidenced, whereas they are strongly reduced in the growth front. Collagen I can be detected when the entobronchi have reached complete differentiation, while the underlying mesenchyme shows a decrease in HA and sulfated GAG (day 11 development). During lung development, the decreased HA/sulfated GAG ratio shows that ECM remodeling processes play an important morphogenetic role (Becchetti et al. 1988; Caniggia et al. 1992; Stabellini et al. 2002). At days 6, 9,11, and 14 of lung development, the accumulations of HA in the growth front and of CS in the lateral regions of epithelial branching are in agreement with β-NAG reactivity. The different distribution of degradative enzyme remodels the GAG of the ECM. In fact, a high β-NAG concentration correlates with a decrease in HA and CS. In the epithelial-mesenchymal interface of tubuli, HA co-localizes with IL-1 and CS with TGFβ₂. Therefore, during bronchial branching development, GAGs are able to continuously modulate cytokine activity. In the lung the TGFβ isoforms may stimulate production of various TGFβ isoforms (Jakowlew et al. 1994; When et al. 2003). IL-1 shows greater concentration in the growth front of bronchial branching, co-localizing with HA and laminin, whereas TGFβ₂ is localized mainly in the lateral regions of bronchial branching, together with CS PG and HS PG, fibronectin, laminin, and collagen IV. Because IL-1 increases synthesis of HA (Bodo et al. 1994), TGFβ₁ that of CS (Locci et al. 1993), and HA blocks the effects of TGFβ₁ (Locci et al. 1995), these linkages could prevent the inhibiting action of growth factor on epithelial cell proliferation.

Figure 4

Day 11 chick embryo lungs. (A,C,E) Immunohistochemical localization of CS, HA, and HS. CS (A), parabronchus (P), and ectobronchus (B2) show strong positivity for CS in lateral region underlying the epithelial wall (arrows) and poor positivity in growth front (asterisks). HA reaction (C). Positivity predominates around the emergence of parabronchi (P) (arrows), and is scarce in lateral regions (asterisks). HS immunohistochemistry (E). Positivity is evident in lateral regions of bronchial wall (arrows) and poor in growth front (asterisks). (B,D) β-NAG + HA or β-NAG + CS double reaction. β-NAG reactivity is prevalently located in epithelium of parabronchial growth front (asterisks), and the underlying mesenchyme shows less positivity for HA (arrow) (D). In epithelial lateral region there is less β-NAG reaction (dot) and strong CS positivity in underlying mesenchyme (double arrows) (B). All controls are negative (F).

Figure 5

(A-D) Day 11 chick embryo lungs. Extracellular matrix component. Fibronectin (A), laminin (B), collagen I (C), and IV (D) immunohistochemistry. Fibronectin, laminin, and collagens I and IV are mainly located in lateral regions of bronchial branching (arrows). Less reaction is seen in the growth front (asterisks).

During development of bronchial branching (6–14 days) in the lung, mesenchyme, GAG classes, fibronectin, laminin, and collagen I and IV accumulate along the lateral region of the growing tubules. The stabilization of branching, HS proteoglycan, fibronectin, laminin, and collagen IV therefore play an important role because they intervene in the processes of cell-matrix adhesion and in the regulation of growth factor activities (Miao et al. 1997). Fibronectin is able to link both HS and collagen networks during basal membrane formation. It is present in the lateral regions, where the membranes stabilize the structures, whereas it is absent in the growth front, where HA supports cell proliferation and migration phenomena. The absence of fibronectin, laminin, and collagen IV prevents the formation of basal membrane. It is therefore impossible for them to bind to the cadherin of the cell surface and to form cell-matrix adhesions according to Sakai et al. (2003) in developing structures. Recent studies show that some components of the basal membrane, such as laminin and collagen IV, are able to modulate gene expression of in vitro hepatocytes (Srebrow et al. 2002). It is therefore feasible to outline a sequence of events in which β-NAG activity plays a role in ECM remodeling alongside other enzymes such as N-acetyl-β-d-glucosaminidase and metalloprotein-ases 2 and 9, whose activity is stimulated by the cytokines (Edwards et al. 1987; Mauviel 1993; Paoletti et al. 2001; Wormstone et al. 2002). β-NAG digests HA and the decrease of HA could be able to carry out its action in stimulating CS PG synthesis. In turn, CS PG could activate genes related to the synthesis of fi-bronectin, laminin, and collagen IV and stabilize bronchial branching through the formation of basal membrane. These hypotheses are in agreement with literature data, which show that CS PG destruction compromises branching morphogenesis (Spooner et al. 1993) and that TGFβ induces ECM deposition (Akimov and Belkin 2001).

Figure 6

Day 11 chick embryo lungs. IL-1 and TGFβ₁ immunostaining. IL-1 is mainly located in underlying basal membrane in the growth front (A, asterisk), while TGFβ₂ is seen along lateral regions of bronchial branching (B, arrows).

In conclusion, GAG distribution changes during lung development and permits or prevents the stimulation of epithelial cells through IL-1 and TGFβ₂ in basal membrane formation or in cell proliferation.

Figure 7

β-NAG, HA, and sulfated GAGs (CS, DS, HS) trends during chick embryo lung development.

Figure 8

Trend of HA/sulfated GAG ratio in chick embryo lung development.