Bronchial Branching Correlates with Specific Glycosidase Activity, Extracellular Glycosaminoglycan Accumulation, TGFß2, and IL-1 Localization During Chick Embryo Lung Development
Experimental Medicine and Biochemistry Science Department, University of Perugia (MC,TB,CL,EB); Morphology Embriology Department, Section of Histology and Embryology, University of Ferrara (CC); Histology and Embryology Institute and Center of Molecular Genetics, University of Bologna (EC,PC); Human Anatomy DepartmentPolo of Vialba, University of Milano (LV); and Human Anatomy DepartmentL.I.T.A of Segrate, State University of Milano (GS), Milan, Italy
Correspondence to: Giordano Stabellini, Dipartimento di Anatomia Umana, Via Mangiagalli 31, 20133 Milano, Italy. E-mail: giordano.stabellini{at}unimi.it
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Summary |
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Key Words: bronchial branching chondroitin sulfate proteoglycans heparan sulfate proteoglycans interleukin-1 transforming growth factor ß2 laminin fibronectin collagen I collagen IV
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Introduction |
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For this purpose, we have studied the changes in ECM composition, ß-NAG enzyme activity, and TGFß2 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.
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Materials and Methods |
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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 macromolecules 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) (SigmaAldrich; 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 (SigmaAldrich) 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 glycosaminoglycans. 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 hematoxylineosin for morphological examination.
ß-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 ß-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 (SigmaAldrich; F-8761) and 0.2 mg/ml naphthol AS-BI N-acetyl-ß-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 SimonnAssmann; Iserm, Strassburg) was used.
Cytokine Localization
For TGFß2 localization, we used an Rb34 antibody obtained from rabbits immunized against a synthetic peptide consisting of the first 29 amino acids of TGFß, 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 (BioDivision, Milan, Italy, R001-63), diluted 1:250. The third molecule conjugated with peroxidase was streptavidin (BioDivision, 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 ß-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)
.
ß-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. ß-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-ß-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.
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Results |
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and 18. The enzyme is generally more active in cell compartments than in the extracellular component. The highest degree of ß-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 ß-NAG reactions were negative (Figures 2B, 2D, 2E, 3A, and 3C).
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Discussion |
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During development of bronchial branching (614 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 cellmatrix 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 cellmatrix 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 ß-NAG activity plays a role in ECM remodeling alongside other enzymes such as N-acetyl-ß-D-glucosaminidase and metalloproteinases 2 and 9, whose activity is stimulated by the cytokines (Edwards et al. 1987
; Mauviel 1993
; Paoletti et al. 2001
; Wormstone et al. 2002
). ß-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 fibronectin, 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 TGFß induces ECM deposition (Akimov and Belkin 2001
).
In conclusion, GAG distribution changes during lung development and permits or prevents the stimulation of epithelial cells through IL-1 and TGFß2 in basal membrane formation or in cell proliferation.
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Acknowledgments |
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Footnotes |
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Received for publication June 23, 2003; accepted October 23, 2003
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