Journal of Histochemistry and Cytochemistry, Vol. 45, 1069-1082, Copyright © 1997 by The Histochemical Society, Inc.


ARTICLE

Fibrillin-1 in Human Cartilage: Developmental Expression and Formation of Special Banded Fibers

Douglas R. Keenea, C. Diana Jordanc, Dieter P. Reinhardta, Catherine C. Ridgwaya, Robert N. Onoa, Glen M. Corsona, Margaret Fairhursta, Michael D. Sussmana, Vincent A. Memolic, and Lynn Y. Sakaia,b
a Shriners Hospital for Children, Portland, Oregon
b Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon
c Department of Pathology, Dartmouth Medical Center, Lebanon, New Hampshire

Correspondence to: Lynn Y. Sakai, Shriners Hospital for Children, 3101 SW Sam Jackson Park Rd., Portland, OR 97201.


  Summary
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The molecular basis for Marfan's syndrome (MS), a heritable disorder of connective tissue, is now known to reside in mutations in FBN1, the gene for fibrillin-1. Classic phenotypic manifestations of MS include several skeletal abnormalities associated primarily with overgrowth of long bones. As a first step towards understanding how mutations in FBN1 result in skeletal abnormalities, the developmental expression of fibrillin-1 (Fib-1) in human skeletal tissues is documented using immunohistochemistry and monoclonal antibodies demonstrated here to be specific for Fib-1. At around 10-11 weeks of fetal gestation, Fib-1 is limited in tissue distribution to the loose connective tissue surrounding skeletal muscle and tendon in developing limbs. By 16 weeks, Fib-1 is widely expressed in developing limbs and digits, especially in the perichondrium, but it is apparently absent within cartilage matrix. Fib-1 appears as a loose meshwork of fibers within cartilage matrix by 20 weeks of fetal gestation. Until early adolescence, Fib-1 forms loose bundles of microfibrils within cartilage. However, by late adolescence, broad banded fibers composed of Fib-1 are found accumulated pericellularly within cartilage. Because these fibers can be extracted from cartilage using dissociative conditions, we postulate that they are laterally packed and crosslinked microfibrils. On the basis of these findings, we suggest that the growth-regulating function of Fib-1 may reside persistently within the perichondrium. In addition, the accumulation of special laterally crosslinked Fib-1 microfibrils around chondrocytes during late adolescence suggests that growth-regulating activities may also be performed by Fib-1 at these sites. (J Histochem Cytochem 45:1069-1082, 1997)

Key Words: fibrillin, cartilage, microfibrils, Marfan's syndrome, special banded fibers


  Introduction
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Introduction
Materials and Methods
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Discussion
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Microfibrils are recognizable structural elements found throughout the connective tissue. They were first defined as small-diameter fibrils present near basement membranes (Low 1962 ). Unlike collagen fibers, microfibrils do not demonstrate any well-defined banding pattern. Characteristic features of microfibrils include a "hollow" appearance in cross-sections and occurrence as loose bundles of individual microfibrils. As a component of elastic fibers, microfibrils are aggregated apparently through association with amorphous material composed primarily of elastin.

The structural backbone of all microfibrils is believed to be composed of end-to-end polymers of fibrillin (Fib) molecules (Reinhardt et al. 1996a ; Sakai et al. 1986 , Sakai et al. 1991 ). Cloning and sequencing of fibrillin has resulted in the discovery of two highly homologous molecules, Fib-1 (Corson et al. 1993 ; Pereira et al. 1993 ; Lee et al. 1991 ; Maslen et al. 1991 ) and Fib-2 (Zhang et al. 1994 ; Lee et al. 1991 ). Fib-2 has been immunolocalized to microfibrillar bundles similar to those that contain Fib-1 (Mariencheck et al. 1995 ; Zhang et al. 1994 ), suggesting that microfibrillar bundles could be heteropolymers of both fibrillins. However, in situ hybridization data demonstrated differential expression of fibrillins: in mouse, fbn2 expression is turned on early in fetal development and appears to decline as development proceeds, whereas fbn1 expression appears later but remains high during fetal development (Zhang et al. 1995 ). Additional evidence suggesting differential expression of fibrillins comes from the heritable disorders of connective tissue. When mutated, FBN1 results in Marfan's syndrome (MS), whose cardinal manifestations are found in the cardiovascular, ocular, and skeletal systems (Dietz and Pyeritz 1995 ). In contrast to the multiple manifestations present in MS, mutations in FBN2 result in a more limited phenotype, congenital contractural arachnodactyly (Putnam et al. 1995 ).

Because the fibrillins are highly homologous molecules, monoclonal antibodies (MAbs) that can distinguish between Fib-1 and Fib-2 are required to clarify the tissue distribution of the two fibrillins and to determine whether microfibrils are homopolymers or heteropolymers of the fibrillins. Here we report the characterization of two MAbs specific for Fib-1 and use them to demonstrate the distribution of Fib-1 in hyaline cartilage, the surrounding perichondrium and attachment sites for tendon, and in joint capsules. In addition, we identify a novel banded fiber unique to cartilage. This novel banded fiber contains Fib-1 and represents a special type of microfibril that appears to be laterally crosslinked.


  Materials and Methods
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Characterization of Monoclonal Antibodies
MAbs 26, 69, and 201 have been previously described and demonstrated to bind specifically to epitopes present in fibrillin-1 (Reinhardt et al. 1996a ; Maddox et al. 1989 ; Sakai et al. 1986 ). To exclude crossreactivity with Fib-2, we have constructed recombinant peptides of Fib-2 and tested these for reactivity with MAb 26 and MAb 201.

Construction and Expression of Recombinant Fibrillin Subdomains. To assemble expression vectors for recombinant production of Fib-1 and Fib-2 subdomains, FBN1 or FBN2 clones were PCR-amplified using Vent polymerase (New England Biolabs; Beverly, MA). To subclone the PCR products, the primers were designed to introduce an additional NheI restriction site at the 5' end and either a NotI or XhoI restriction site at the 3' end. In addition, the sequences for 6 histidine residues were introduced into the PCR product to facilitate purification of the recombinant peptide using chelating chromatography. Ligation of the PCR products into the episomal expression vector pCEP4/{gamma}2III4, which contains the sequence for the BM40/SPARC signal peptide (Mayer et al. 1995 ), results in secretion of recombinant peptides with four additional N-terminal amino acid residues (APLA) preceding the authentic fibrillin sequence. Human embryonic kidney cells, 293/EBNA (Invitrogen; San Diego, CA), were transfected and selected with Hygromycin B (Sigma; St Louis, MO). These methodologies and protein purification methods have been described in more detail in a previous publication (Reinhardt et al. 1996a ).

Fib-1 Recombinant Subdomains. Expression vectors rF30 (coding for D330 to I489) and rF31 (coding for D288 to I489) were constructed from human FBN1 clone HFBN29 (Corson et al. 1993 ) by PCR amplification using sense primers DR 64 (5'-CGTAGCTAGCGGATGTTCGCCCAGGATACTG-3') (rF30) or DR 65 (5'-CGTAGCTAGCGGATATTGATGAATGCAGCACC-3') (rF31) and anti-sense primer DR 51 (5'-ATAGTTTAGCGGCCGCTAGTGATGGTGATGGTGATGAATACACTCCCCACGGAGG-3'). A 508-BP (rF30) or a 634-BP (rF31) NheI-NotI fragment was then ligated with the NheI-NotI restricted pCEP4/{gamma}2III4 to yield the plasmids pCEPSP-rF30 and pCEPSP-rF31. The construction, expression, and purification of rF18 and rF20 have been previously described in detail (Reinhardt et al. 1996a ).

Fib-2 Recombinant Peptides. Expression vectors rF33 (coding for D317 to I533) and rF37 (coding for S145 to L995) were constructed from human FBN2 cDNA clones obtained by screening a {lambda}gt11 unamplified placenta library (Clontech; Palo Alto, CA) with FBN1-specific PCR products, as described previously (Corson et al. 1993 ). One clone, UP{psi}22-3, was used to amplify sequences for rF33 and rF37. For rF33, primers DR 67 (5'-CGTAGCTAGCAGACATTGATGAGTGCAGCATC-3') and DR 70 (5'-ATAGTTTAGCGGCCGCTAGTGATGGTGATGGTGATGTATACAATC-TCCATTTGCATCCTGC-3') were used. A 679-BP NheI-NotI fragment was then subcloned into pCEP4/{gamma}2III4 and designated pCEPSP-rF33. For rF37, primers DR 77 (5'-CCCGGTGTATTGACGCAGATTCC-3') and DR 86 (5'-ATA- AGAATGCGGCCGCGCTAGCATCAATTCAGCAGTGCA- GTGTGAG-3') were used, which introduced an additional NheI and EagI restriction site 5' to the FBN2 sequence (nucleotides 432-3404). A 2893-BP EagI-XbaI fragment was then subcloned into EagI-XbaI restricted pBluescript II SK (+) (Stratagene; La Jolla, CA). The resulting plasmid was designated pBS-UP{psi}22-3. To modify the 3' end, pBS-UP{psi}22-3 was PCR-amplified with primer F5.306S (5'-TGAATGTGAAAGCAACCCATG-3') and DR 89 (5'-ACCGCTC- GAGCTAGTGATGGTGATGGTGATGCAAACATACAC-GGCCAGTCCC-3'). The MunI-XhoI fragment from pBS-UP{psi}22-3 was then replaced by the 391-BP MunI-XhoI PCR fragment to yield plasmid pBS-rF37. The 2581-BP NheI-XhoI fragment from pBS-rF37 was subcloned into pCEP4/{gamma}2III4 and designated pCEPSP-rF37.

The complete sequences of subcloned PCR amplified products were confirmed by automated DNA sequencing (Applied Biosystems; Foster City, CA). The sequences obtained from pCEPSP-rF30, pCEPSP-rF31, and pCEPSP-rF33 corresponded with the expected sequences. Some differences were observed for FBN2 sequences from pBS-UP{psi}22-3 and pBS-rF37: 574insGA and 582insC leading to an amino acid exchange from residues 192-194 (AQP to GPNR) [a sequencing error also previously reported (Zhang et al. 1995 )]; C725T resulting in amino acid exchange T242I; T1777A resulting in S593T. These differences may reflect sequencing errors in the original published FBN2 sequence, because the amino acid exchanges result in sequences homologous to FBN1. Two silent nucleotide exchanges were observed (C1560T and A3147G), as well as two nucleotide exchanges (G2890A and A3138T) resulting in conservative exchanges (V964I and E1046D, respectively). The silent nucleotide exchange C1560T was introduced into rF37 by PCR amplification, because this exchange was not observed in rF33, which was amplified from the same template. It is not clear whether the other differences reflect sequencing errors in the published sequence, polymorphisms, or mismatches introduced by PCR amplification.

Immunoblotting
Recombinant subdomains of Fib-1 (rF18, rF20 (Reinhardt et al. 1996a ) and rF31) and Fib-2 (rF33 and rF37) were run on SDS-PAGE and transferred onto nitrocellulose membranes (BioRad; Hercules, CA) in 10 mM borate, pH 9.2, at 0.4 A for 30 min. To confirm sufficient transfer, the proteins were stained with Ponceau S. Free binding sites were blocked with 5% (w/v) dry milk powder in 50 mM Tris-HCl, pH 7.4, 0.15 M NaCl (TBS) for 1 hr at 25C. The nitrocellulose membranes were then incubated for 1.5 hr at 25C with MAb 26 or MAb 201 [10 µg/ml in TBS including 2% (w/v) dry milk powder (incubation buffer)]. The membranes were washed three times for 10 min in TBS-0.05% (v/v) Tween-20 and then were placed in goat anti-mouse IgG conjugated with horseradish peroxidase (BioRad), diluted 1:800 in incubation buffer, for 1.5 hr at 25C. After washing again as above, membranes were developed in 100 ml TBS-0.02% (v/v) H2O2 and 3 mg/ml 4-chloro-1-naphthol in 20 ml methanol.

Other Analytical Methods
Molecular masses of recombinant proteins were determined by comparison of mobilities in SDS-PAGE with globular marker proteins.

N-terminal sequence analyses were performed using a gas phase sequencer (Applied Biosystems 475). Amino acid compositions and protein concentrations were determined after hydrolysis of peptides in 6 M HCl (24 hr at 110C) on an amino acid analyzer (Beckman 6300; Palo Alto, CA).

Light Microscopy
AMEX immunostaining was performed on fetal human tissues (10-11 and 16 weeks' gestation) fixed in acetone (Sato et al. 1986 ; Tanaka et al. 1984 ). Fresh tissue, obtained according to institutional human use guidelines, was placed in room temperature (RT) acetone for a minimum of 4 hr and then was stored frozen at -20C. Before processing, fixed frozen tissues were transferred to 4C acetone for 15 min, followed by RT acetone for 15 min and RT methyl benzoate (Fisher; Pittsburgh, PA) for 30 min, and then were cleared with xylene (Chempure; Curtin Matheson, Houston, TX) for 30 min. Tissues were infiltrated with paraffin (Paraplast, melting point 56C; Curtin Matheson) in a Tissue Tek vacuum infiltrator for 2 hr and embedded with the same paraffin. Sections were cut at 4-5 µm and placed on silane-treated slides or Biotek (Santa Barbara, CA) positively charged slides. Slides were air-dried and then dried at 55-60C for 1 hr before immunohistochemical staining or staining with hematoxylin and eosin. Stock solutions (1 mg/ml) of MAbs specific for Fib-1 (MAb 26 and MAb 201) were utilized at dilutions of 1:20-1:100 for MAb 26 and 1:200-1:500 for MAb 201. A standard avidin-biotin-horseradish peroxidase technique utilizing 3,3'-diaminobenzidine tetrahydrochloride (DAB) as the chromogen (Biotek Chemate detection kit, peroxidase/DAB) was employed. Sections stained using a Biotek 1000 automatic stainer demonstrated equivalent reactions to those stained manually. Photographs were taken with an Olympus Microscope Model AHBT.

Immunofluorescence was performed on acetone-fixed frozen sections as previously described (Sakai and Keene 1994 ).

Confocal Microscopy
A specimen of 14-year-old human rib cartilage was frozen in hexanes and sectioned to a thickness of 40 µm using a Leitz cryostat. Sections were collected on poly-L-lysine-coated slides, dried, then fixed in ice-cold acetone for 10 min. Sections were then removed from the slides by immersion in PBS and submersed in chondroitinase ABC (Sigma) (290 U/ml PBS) for 90 min at 37C. The sections were rinsed in PBS, immersed overnight at 4C in MAb 69 diluted 1:50 in PBS, washed, then immersed in goat anti-mouse TRITC conjugate (Jackson ImmunoResearch Laboratories; West Grove, PA) diluted 1:50 in PBS for 30 min at RT. The sections were rinsed, mounted on slides using Aquamount (Lerner Laboratories; Pittsburgh, PA), and viewed with a Leica confocal microscope equipped with an argon/krypton laser. Images from 2-µm-thick optical sections were collected and recorded.

Electron Microscopy
An overview of the methods used in this study has been described previously (Sakai and Keene 1994 ). Ultrathin sections of cartilage, obtained from 44 individuals according to approved institutional human use protocols, were prepared and examined in a Philips (Santa Clara, CA) 410-LS TEM. Examples included sections of 1.5-year-old normal articular cartilage from the thumb, 9-year-old annulus fibrosis, 11-, 18-, and 71-year-old rib, 43-year-old normal trachea, and 43- and 33-year-old normal elastic cartilage from the ear. Cornea from a normal 80-year-old and skin from a 74-year-old individual were also examined. All tissues were immersed in 0.1 M cacodylate buffered 1.5% glutaraldehyde-1.5% paraformaldehyde containing 0.1% (w/v) tannic acid for 60 min, followed by 1% OsO4 for 120 min, then dehydrated and embedded in Spurr's epoxy. Individuals were scored for the presence or absence of the special banded fibrillin fibers after a period of observation lasting 10 min.

Extracts of 14-year-old rib cartilage were prepared by first trimming away all exterior surfaces of the rib (eliminating perichondrium), mincing the remaining tissue with a sharp blade, then homogenizing the tissue in ice-cold 0.2 M ammonium bicarbonate, pH 7.4, using the 1/4'' blade of a polytron homogenizer until no visible tissue fragments remained. The resulting homogenate was then either exposed to MAb 69 overnight at 4C, followed by washing and an additional overnight exposure to a 1:1 mixture of 5- and 10-nm gold particulates conjugated to goat anti-mouse IgG (Amersham; Arlington Heights, IL) or to 20 U/ml collagenase (Worthington; Freehold, NJ) in 0.05 M Tris-HCl, pH 7.5, 5 mM CaCl2 for 4 hr at RT, followed by exposure to 6 M guanidine-HCl (Amresco; Solon, OH) in water. All homogenates were allowed to settle onto the surface of carbon-coated grids made hydrophilic by glow-discharge, then stained in 3% phosphotungstic acid, pH 7.0.

Immunolocalization of fibrillin within 13-year-old humeral head articular cartilage and also in 13-, 14-, and 31-year-old rib was accomplished by first digesting the tissue with chondroitinase ABC (Sigma) (290 U/ml PBS) for a minimum of 2 hr at RT, followed by en bloc incubation overnight at 4C in either MAb 69, MAb 26, or MAb 201, followed by washing and immersion overnight at 4C in goat anti-mouse IgG 5-nm colloidal gold conjugate (Amersham; Arlington Heights, IL). Control antibodies included those specific for collagen Types II, VI, IX, XI, and XII. Immunolabeled tissues were then fixed, dehydrated, and embedded as described above.

In addition, one sample of 20-year-old cartilage was fixed in ice-cold 4.0% paraformaldehyde-0.1% glutaraldehyde, rinsed in cacodylate buffer and then in 0.15 M Tris-HCl overnight, then dehydrated in a graded alcohol series at progressively lower tempertures (30% EtOH -10C; 50% EtOH -20C; 70% EtOH -20C), infiltrated in LR White embedding Resin (Electron Microscopy Sciences; Fort Washington, PA), and polymerized at 60C for several days. Ultrathin sections were mounted on formvar-coated single-hole slot grids, which were floated section side down at RT in the following solutions: Tris-HCl with 0.1% Tween-20 for 15 min; Tris-HCl containing 0.05M glycine for 60 min; Tris-HCl with 0.1% Tween-20, 0.5% ovalbumin, 0.5% fish gelatin, and 2% nonfat dry milk for 15 min; Tris-HCl containing 0.1% Tween-20 for 5 min; MAb 26 diluted 1:10 in Tris-HCl with 0.1% Tween-20, 0.5% ovalbumin, 0.5% fish gelatin for 120 min; rinsed for 30 min in several changes of Tris-HCl with 0.1% Tween-20; goat anti-mouse 10-nm gold conjugate (Amersham) diluted 1:15 in Tris-HCl with 0.1% Tween, 0.05% ovalbumin, 0.05% fish gelatin; rinsed in Tris-HCl with 0.1% Tween-20; and finally rinsed in distilled water for 30 min.


  Results
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Materials and Methods
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Characterization of Fib-1-specific Antibodies
Previously published results positioned the epitopes recognized by MAb 26, MAb 201, and MAb 69 to distinct regions in Fib-1 (Reinhardt et al. 1996a ). The epitope for MAb 26 was mapped between amino acid residues 45 and 450; the epitope for MAb 201 was found between residues 451 and 909; and the epitope for MAb 69 was between residues 2093 and 2871. To further specify the location of the epitope for MAb 26, we have produced and characterized additional recombinant Fib-1 peptides (rF30 and rF31). A comparable recombinant Fib-2 peptide (rF33), as well as a large recombinant Fib-2 peptide (rF37), spanning approximately one third of the N-terminal end of Fib-2, have also been produced and characterized.

The apparent molecular masses of rF30 (27 kD), rF31 (32 kD), rF33 (30 kD), and rF37 (110 kD) corresponded well with their calculated masses, and Edman degradation resulted in the expected N-terminal sequences (Figure 1). Amino acid compositions also agreed with calculated values. Rotary shadowing of the large Fib-2 peptide rF37 (not shown) demonstrated an extended thread-like shape 38.1 ± 3.6 nm long, similar to shapes of recombinant Fib-1 peptides (Reinhardt et al. 1996a ) and to authentic Fib molecules from cell cultures (Sakai et al. 1991 ). Together with previously published data demonstrating that similarly produced and characterized recombinant Fib peptides are properly folded and functional (Reinhardt et al. 1996a , Reinhardt et al. 1996b ), these data indicate that these new recombinant peptides can be used to further determine the epitopes of MAb 26 and MAb 201.



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Figure 1. Design and properties of recombinant subdomains of fibrillin-1 and fibrillin-2. The major domains of recombinant fibrillin peptides and the position of the peptides within fibrillin-1 and fibrillin-2 is illustrated.

MAb 26 reacted positively with both rF30 (not shown) and rF31 (Figure 2A), locating the epitope to a position between amino acid residues 330 and 489 of Fib-1. rF20, which begins with the EGF-like motif (residues 450-489) adjacent to the proline-rich region, does not react with MAb 26 (Reinhardt et al. 1996a ). Therefore, it is likely that the first "8-cysteine" motif contains the epitope for MAb 26, because this epitope is dependent on disulfide bonds, and there are no cysteine residues in the proline-rich region. MAb 26 did not bind to rF33, a recombinant Fib-2 peptide comparable to rF31 (Figure 2A), or to rF37, the large N-terminal Fib-2 peptide (not shown). MAb 201, which binds to rF20 but not to rF18, also failed to bind to rF37 (Figure 2B). These data demonstrate that MAb 26 and MAb 201 bind to specific regions in Fib-1 but not to similar regions in Fib-2.



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Figure 2. Analysis of recombinant Fib subdomains by SDS gel electrophoresis and immunoblotting with MAbs. Purified recombinant subdomains of Fib-1 (rF18, rF20, and rF31) and of Fib-2 (rF33 and rF37) were analyzed under nonreducing conditions by Coomassie Blue staining (A,B, left panels) and by immunoblotting with MAb 26 (A, right panel) and with MAb 201 (B, right panel). The molecular masses of globular marker proteins are indicated at left in kD.

Fib-1 Expression in Fetal Tissues
Immunohistochemical staining of tissues from a developing human fetus (10-11 weeks' gestation) showed a limited distribution of Fib-1. In Figure 3, MAb 26 (Figure 3B and Figure 3E) and MAb 201 (Figure 3C and Figure 3F) demonstrate similar staining patterns, which are restricted to the loose connective tissue surrounding skeletal muscle and tendon. Endomysium and perimysium, as well as dermis, vasculature, nerves, perichondrium, joint capsules, and cartilage, are not stained. A positive control antibody specific for tenascin-C, which stains the perichondrium, is shown in Figure 3D. Other control antibodies specific to components known to be present in fetal cartilage, trabecular bone, and skin were also positive in sections similar to these (not shown).



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Figure 3. Immunohistochemical localization of Fib-1 in 10-11 week fetal human arm. Sections were stained with MAb 26 (B,E), MAb 201 (C,F), and an MAb specific for tenascin C (D). The section in A was not incubated in primary antibody. Muscle (m), tendon (t), cartilage (c), and perichondrium (p) are designated. Bars: A-C = 500 µm; D-F = 50 µm.

By 16 weeks' gestation, Fib-1 is widely expressed in the fetus. Figure 4 shows cross-sections of a finger (Figure 4A and Figure 4D) demonstrating intense Fib-1 staining throughout the connective tissue (Figure 4D), except within the matrix of cartilage. Increased staining is apparent in the loose connective tissue, around nerves as well as muscle (Figure 4B). By this developmental stage, there is abundant and classical staining of Fib-1 in both skin (Figure 4E) and perichondrium (Figure 4C and Figure 4F). High-magnification views suggested that Fib-1 staining entered the periphery of the cartilage matrix but was largely absent from the interior of the cartilage (Figure 4F). The abundant Fib-1 staining of the perichondrium seen at this time persists through maturity (data not shown).



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Figure 4. Immunohistochemical localization of Fib-1 in 16-week fetal human finger. Compared with control section (A), cross-sections stained with MAb 201 (D) demonstrated staining throughout the connective tissue, except in the cartilage matrix. MAb 26 was used for B; MAb 201 was used to stain sections in C-F. Cartilage (c) and nerve (n) are designated. Bars: A,D = 500 µm; B,C = 50 µm; E,F = 50 µm.

Figure 5. Immunofluorescence of 20-week fetal human humeral cartilage. MAb 201 demonstrated a loose Fib-1 network of fibrils within the cartilage matrix. Bar = 50 µm.

Figure 6. Confocal immunofluorescence of the same 14-year-old rib used in Figure 10A-C. Using MAb 69, fibrillin was localized to the periphery of a human chondrocyte. The approximate periphery of the chondrocyte is delineated with lines. Bar = 29 µm.

Fib-1 staining was found within the matrix of cartilage from the arm after 20 weeks of fetal gestation (Figure 5). A large mesh network of fine fibrils was present. This network of fibrils became more flattened closer to the peripheries of the cartilage (not shown).

Special Banded Fib-1 Fibers in Aging Human Cartilage
Immunofluorescence confocal microscopy of cartilage from adolescents showed Fib staining primarily pooled around chondrocytes within resting and proliferative zones (data not shown). Serial immunofluorescence images demonstrated bright baskets around chondrocytes. Figure 6 is one of a series of sections of Fib staining around a single chondrocyte. Yellow immunofluorescence indicates the Fib network encapsulating the cell. Hypertrophic zones were relatively negative for Fib-1 (data not shown).

Electron microscopy revealed typical microfibrils (Figure 7A and Figure 7B), fibrils that were more densely packed (Figure 7C), as well as very densely packed banded fibers (Figure 8). All these fibrils labeled with Fib-1-specific antibodies (Figure 7A, Figure 8E) but did not label with antibodies specific for collagens II, VI, IX, XI, and XII. These banded Fib-1 fibers most commonly demonstrated a periodicity of approximately 50 nm (with a range up to 65 nm) and could be distinguished ultrastructurally from thin collagen fibers as well as from thick "amianthoid" fibers (Mallinger and Stockinger 1988 ), also occasionally present in cartilage matrix (Figure 8A). These unique banded Fib-1 fibers were found primarily around chondrocytes (Figure 8B, Figure 8F, and Figure 9A). Immunofluorescence shown in Figure 6 corresponds to the location of the banded Fib-1 fibers primarily around cells.



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Figure 7. Immunolocalization of microfibrils in cartilage from a 13-year-old rib (A), humeral head cartilage from a 13-year-old (B), and rib cartilage from a 14-year-old (C). Microfibrils were labeled with MAb 201 (A) and MAb 69 (B,C). Thin bundles or individual microfibrils, although not heavily concentrated, were found within the matrix of human cartilage from children and young adolescent individuals. Although rarely seen together, a banded Fib fiber was found in association with individual microfibrils (C). Bars = 100 nm.



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Figure 8. Electron micrographs of 1.5-year-old articular cartilage (A), 9-year-old annulus fibrosis (B), 18-year-old rib cartilage (C), 13-year-old humeral head cartilage (D), 31-year-old rib cartilage (E), and 11-year-old rib cartilage (F). At low magnification, amianthoid fibers (A) were easily distinguished from banded fibrillin fibers (B-F). Amianthoid fibers are usually much thicker than banded Fib fibers, and they are not distributed in a pericellular capsule as are the banded Fib fibers (arrowheads, B). At higher magnification (C), banded Fib fibers demonstrated a periodicity of 40-50 nm and the ends were often tapered. Immunolabeling with both MAb 69 (D) and MAb 26 (E) demonstrated specific labeling of the banded fibers, often matching the periodicity of the fibers. Banded Fib fibers were found most often very close to chondrocytes (F). Bars: A,B = 1 µm; C-F = 100 nm.



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Figure 9. Electron micrographs of human 71-year-old rib cartilage (A), 74-year-old skin (B), and 80-year-old cornea (C). Banded Fib fibers were abundant in cartilage from elderly individuals (arrows, A). No banded Fib fibers were found in noncartilaginous tissues from comparably aged individuals, although classical bundles of individual microfibrils were found and immunolabeled with MAb 201 (B). Banded fibers present in corneal stroma (C) appeared similar to banded Fib fibers but demonstrated approximately a twofold greater periodicity and did not label with antibodies to Fib. Bars: A = 1 µm; B,C = 100 nm.

Of 44 cartilage samples scored for the presence or absence of the banded Fib-1 fibers, 19 came from individuals under the age of 8, and 17 of these samples did not appear to contain the banded fibers. Of 25 samples from individuals ranging in age from 9 to 71 years, all but one (a sample from an 18-year-old) contained banded fibers. These data are shown in Table 1. Athough most of these samples were rib cartilage and articular cartilage, samples of elastic cartilage, fibrous cartilage, and vertebral cartilage were also included. Samples were not scored for their apparent abundance.


 
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Table 1. Occurrence of special banded fibers related to agea

Because banded Fib-1 fibers were always detected in the matrix of aged cartilage (Figure 9A), other comparably aged tissues were also examined. Laterally packed and banded Fib-1 fibers were not found in any other tissue. In skin, Fib-1 microfibrils appeared typically loosely bundled (Figure 9B). In cornea, laterally packed and banded fibers were seen (Figure 9C). However, the periodicity of the banding pattern of the corneal fiber was was much longer (~115 nm compared to ~50 nm), and these corneal fibrils were not recognized by Fib-specific antibodies (data not shown).

To test whether the banded Fib-1 fibers might be a peculiar artifact of preparing cartilage for microscopy, cartilage homogenates were extracted with collagenase and guanidine, and were stained with phosphotungstic acid. Even after exposure to these dissociative conditions, large banded Fib-1 fibers were visualized (Figure 10A and Figure 10B) and could be immunolabeled with some periodicity using an antibody specific to fibrillin (Figure 10C). The variable thickness, tapered ends (Figure 10A), and the banding pattern (Figure 10B) of the extracted fibers probably represent their authentic morphologies because these features were comparable to those noted in fixed, dehydrated, and embedded tissues (e.g., Figure 8C).



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Figure 10. Negatively-stained images of banded Fib fibers isolated from 14-year-old rib cartilage after digestion with collagenase and extraction with 6 M guanidine. Banded Fib fibers were of variable lengths with tapered ends (A). Bar = 200 nm. Higher magnification revealed a periodicity of approximately 50 nm (B). Fine filaments within the fiber appeared to be oriented parallel with the axis of the fiber (B). Examination of the homogenate after exposure to MAb 69 followed by a mixture of 5- and 10-nm gold secondary antibody conjugates resulted in labeling of the banded fibrillin fibers (C). Bars = 100 nm.

Because en bloc incubation with antibodies results in labeling of fiber surfaces but not interiors, techniques for labeling section surfaces were employed to investigate the presence of Fib-1 within the dense banded fibers. After exposure of fiber interiors to MAbs 69 (not shown), 15 (not shown), and 26 (Figure 11), gold labeling was found throughout the interior of the fiber rather than only at the surface.



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Figure 11. Electron micrograph of 20-year-old human rib cartilage labeled with MAb 26 after exposure of fiber interiors via sectioning. Antibody labeling is specific to banded Fib fibers and is seen throughout the fiber interior. Bar = 200 nm.


  Discussion
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Summary
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Materials and Methods
Results
Discussion
Literature Cited

Using MAbs (26 and 201) that bind to specific epitopes in Fib-1 but do not recognize similar domains present in Fib-2, we have documented the developmental expression of Fib-1 in human fetal limbs and digits. Early in fetal gestation (10-11 weeks), Fib-1 is first expressed in the loose connective tissue around skeletal muscle and tendon. However, by 16 weeks' gestation, abundant and classical immunohistochemical staining is found in all tissues, with the exception of cartilage matrix. At this time, and continuing through adulthood, perichondrium contains abundant Fib-1 microfibrils, which are also endowed with elastin. By 20 weeks of fetal gestation, a loose meshwork of immunofluorescent Fib-1 fibers is detected within cartilage matrix. These results are consistent with the general pattern of expression of fbn1 in fetal mouse tissues examined by in situ hybridization techniques (Zhang et al. 1995 ), although the endochondral ossification process appears to occur relatively much earlier in gestation in human than in mouse.

Between these fetal times and adolescence, immunoelectron microscopy reveals sparse but otherwise classic loose bundles of Fib-1 microfibrils within cartilage matrix. The thin meshwork of Fib-1 immunofluorescence in fetal cartilage matrix corresponds to these loose bundles of microfibrils. By late adolescence, densely packed and laterally cross-striated fibers containing Fib-1 accumulate around the periphery of resting chondrocytes, but hypertrophic chondrocytes appear not to be encapsulated by these fibers. Because fetal expression of Fib-1 by hypertrophic chondrocytes has been reported (Zhang et al. 1994 , Zhang et al. 1995 ), loss of Fib-1 from the hypertrophic zone may also play a role in bone growth.

Fib-1 broad banded fibers can be distinguished morphologically and immunochemically. They have not been described previously and appear to be unique to cartilage. To investigate whether the appearance of these special banded fibers might be due to artifacts associated with dehydration and preparation of cartilage for microscopic examination, cartilage was extracted using dissociative conditions and extracts were examined by electron microscopy. Identical banded fibers were found in these extracts, suggesting that these fibers represent densely packed and laterally crosslinked Fib-1 microfibrils. Additional immunolabeling of the interiors of these fibers by Fib-1-specific antibodies further supports this suggestion. How Fib-1 microfibrils become packed and crosslinked in cartilage and determination of other molecules that might be present in these banded fibers require further investigation. Collagens II, VI, IX, XI, and XII, however, do not appear to be components of these fibers.

Mutations in FBN1 result in Marfan's syndrome, a heritable disorder of connective tissue affecting 1:10,000 individuals. A single mutation in FBN1 can cause multiple and variable phenotypic manifestations (Dietz and Pyeritz 1995 ). Common cardinal manifestations associated with FBN1 mutations include skeletal abnormalities associated with long bone overgrowth, dolichostenomelia, arachnodactyly, scoliosis, and pectus deformities. To elucidate how mutations in FBN1 may result in these skeletal abnormalities, we have documented the developmental expression of Fib-1 in human skeletal tissues.

McKusick 1972 suggested that in Marfan's syndrome "the factor that is missing during morphogenesis and growth of bone ... is a binding force which placed a rein on longitudinal growth." Furthermore, referencing the experience of orthopedic surgeons, McKusick suggested that the location of a molecular defect might be in the periosteum, because cutting the periosteal cuff can stimulate long bone growth. The periosteum, which is loosely attached to the bone, is quite strongly attached to the growth plate at the perichondrial ring. During long-bone growth, stretching of the periosteum is believed to exert a mechanical restraint on elongation of the bone at the perichondrial ring; cutting the periosteum releases this restraint and stimulates long-bone growth (Houghton and Dekel 1979 ).

In the perichondrium/periosteum, Fib-1 microfibrils are highly organized into long elastic fibers uniformly aligned parallel to the long axis of the bone (data not shown) (Wlodarski 1989 ). At the light microscopic level, these well-oriented fibers resemble the circumferential lamellar organization of elastic fibers in large blood vessels such as the aorta. It may be that Fib-1 serves a mechanical function in the perichondrium/periosteum to restrain growth of long bones. A similar mechanical function may be performed by Fib-1 in large blood vessels to resist the deforming effects of blood flow. In the perichondrium and the aorta, continuous pressure against defective, more compliant Fib-1 microfibrils may, over time, result in overgrowth of long bones as well as aortic dilatation and rupture.

Alternatively, or in addition, growth-regulating activities of Fib-1 may be performed specifically at the time when growth ceases. We have documented the presence of special banded Fib-1 fibers that accumulate around chondrocytes at the time when growth ceases (late adolescence) and which are abundant in adult cartilage. This new finding suggests the possibility that Fib-1 fibers may exert a special influence on chondrocytes to cease proliferation and differentiation. Because fibrillins are highly homologous to the family of latent transforming growth factor-ß-binding proteins (Saharinen et al. 1996 ; Yin et al. 1995 ; Moren et al. 1994 ; Corson et al. 1993 ), Fib-1 may be capable of binding to, sequestering, and presenting members of the transforming growth factor-ß family (which include the bone morphogenetic proteins and growth and differentiation factors) to chondrocytes. The special laterally aggregated form of Fib-1 fibers around chondrocytes may indicate a supramolecular structure that is required for Fib-1 to properly perform these activities around chondrocytes.


  Acknowledgments

Supported by grants from the Shriners Hospitals for Children (DRK, LYS) and by the Deutsche Forschungsgemeinschaft (DPR).

We thank Maudine Waterman (Dartmouth Medical Center) and Noe Charbonneau (Shriners) for excellent technical assistance. Expertise provided by Kenneth Fish at the Laboratory for Confocal Microscopy, Oregon Health Sciences University, is sincerely appreciated. Electron microscopy facilities were provided in part by the Fred Meyer and R. Blaine Bramble Charitable Trust Foundations.

Received for publication October 3, 1996; accepted January 21, 1997.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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