ARTICLE |
Correspondence to: Anthony A. Capehart, Dept. of Cell Biology and Anatomy, Medical U. of South Carolina, 171 Ashley Ave., Charleston, SC 29425.
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Summary |
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We report the production of a monoclonal antibody (d1C4) by in vitro immunization that has immunoreactivity with a native chondroitin sulfate epitope in embryonic chick limb and heart. Murine lymphocytes were stimulated by direct exposure to unfixed, unsolubilized precartilage mesenchymal aggregates in high-density micromass culture derived from Stage 22-23 chick limb buds. Specificity of d1C4 reactivity was demonstrated by sensitivity of immunohistochemical staining to pretreatment with chondroitinase ABC or AC, preferential immunoreactivity with chondroitin-6-sulfate glycosaminoglycan (CS-C GAG) in ELISA, and competition of immunohistochemical staining with CS-C GAG. Immunohistochemical analysis of the expression of the d1C4 epitope revealed a striking localization of immunoreactivity in the extracellular matrix (ECM) of precartilage aggregates of chick limb mesenchyme in high-density micromass culture by 16 hr and the prechondrogenic limb core at Stage 23 in vivo. Immunoreactivity in both cultured limb mesenchyme and the embryonic limb continued through differentiation of prechondrogenic condensations into cartilage tissue. In the developing chick heart, d1C4 staining was found throughout the ECM of atrioventricular cushion tissue by Stage 25, but was localized to mesenchyme adjacent to the myocardium in the outflow tract cushions. There was an abrupt demarcation between d1C4-reactive intracardiac mesenchyme and unreactive extracardiac mesenchyme of the dorsal mesocardium in the Stage 22 embryo. This study demonstrates the efficacy of in vitro immunization of lymphocytes for the production of MAbs to native ECM constituents, such as CS-GAGs. Immunohistochemical data utilizing d1C4 suggest that CS-GAGs bearing this epitope may be important in early morphogenetic events leading to cartilage differentiation in the limb and valvuloseptal morphogenesis in the heart. (J Histochem Cytochem 45:1567-1581, 1997)
Key Words: in vitro immunization, monoclonal antibodies, extracellular matrix, chondroitin sulfate, proteoglycan, chondrogenesis, limb development, heart development
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Introduction |
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Proteoglycans (PGs) are integral constituents of the extracellular matrix (ECM) and are of critical importance during many morphological processes, including proliferation, migration, tissue remodeling, and differentiation. Many of the roles played by PGs during development are attributed to their associated glycosaminoglycan (GAG) moieties (
To study the intricate processes involved in these critical morphological events, it is often advantageous to utilize immunological probes to native ECM components. Such reagents are useful in the evaluation of ECM interactions by avoiding artifactual alterations in the organization of the matrix caused by the enzymatic creation or unmasking of epitopes for immunohistochemical analyses. One problem with the identification of ECM components such as GAGs, however, has been the difficulty in producing antibodies to the native structure. This difficulty is due in large part to the poor immunogenicity of many ECM molecules, particularly carbohydrate sequences of GAG chains (
In this article we report the preparation of a monoclonal antibody (d1C4) by in vitro stimulation of isolated lymphocytes with cultured limb mesenchyme. Characterization of MAb d1C4 demonstrates that it preferentially recognizes a native epitope on GAG chains enriched in chondroitin-6-sulfate. Immunofluorescence microscopy localizes the d1C4 epitope in prechondrogenic aggregates of chick limb mesenchyme in vitro and in the prechondrogenic limb core and cardiac cushion tissue in vivo.
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Materials and Methods |
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Cell Cultures
High-density micromass cultures were prepared using Stage 22-23 (
Production of Monoclonal Antibodies In Vitro
Four 0.1-ml micromass cultures of chick limb mesenchyme (prepared as described above) were established in separate wells of a 24-well tissue culture plate (Corning; Ithaca, NY). After 24 hr of incubation, cultures were washed three times with sterile PBS and air-dried under laminar flow following the method of
Thymocyte conditioned medium was prepared by a modification of the method of
Macrophages were obtained from a nonimmunized 7-week female Balb/C mouse by peritoneal lavage with Hank's balanced saline solution (
Each of four air-dried prechondrogenic micromass cultures of chick limb mesenchyme received 1 x 107 splenocytes and 1.8 x 104 peritoneal macrophages in 1 ml SFRPMI containing 30 µg/ml N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) as adjuvant (Calbiochem; San Diego, CA) (
Stimulated splenocytes were harvested by centrifugation and fused with the myeloma cell line SP2/0 at a ratio of 3:1 with 50% polyethylene glycol (MW 1500; Boehringer Mannheim, Indianapolis, IN). After fusion, cells were seeded in a single 24-well tissue culture plate (Corning) at a density equivalent to 2 x 105 myeloma cells/ml (pre-fusion count) in RPMI containing 20% HIFCS and 150 µg/ml oxaloacetate, 50 µg/ml sodium pyruvate, 5 µg/ml insulin (modified from
Antibody Purification
Antibodies were purified from tissue culture supernatants using anti-mouse IgM-agarose (Sigma). Bound antibodies were eluted with 0.1 M glycine, pH 2.6, containing 150 mM NaCl. Fractions were neutralized immediately with 1 M Tris, pH 8, and the aliquots were dot-blotted onto nitrocellulose (Bio-Rad Laboratories; Hercules, CA) and stained with 0.1% amido black (Sigma) to detect protein. Fractions containing eluted antibodies were concentrated and exchanged into PBS using Centricon-10 ultrafiltration devices (Amicon; Beverly, MA). Protein content was determined using the Micro BCA protein assay kit (Pierce; Rockford, IL).
Immunohistochemistry
Micromass cultures of chick limb mesenchyme were fixed in 80% methanol-20% DMSO (Dent's fixative) at room temperature (RT) and processed for indirect immunofluorescence microscopy in the tissue culture dishes. Cultures were rehydrated in PBS and blocked for 30 min at RT with PBS containing 2% bovine serum albumin (BSA; Sigma) and 1% normal goat serum (NGS; Cappel Research Products, Durham, NC). Fixed specimens were incubated for 1 hr with 10 µg/ml d1C4 or undiluted tissue culture supernatants, washed three times with PBS, then incubated for 1 hr with a 1:100 dilution of fluorescein-conjugated goat anti-mouse IgM or IgG (Cappel) in BSA-PBS. For double labeling with rhodamine-conjugated peanut agglutinin (RhPNA; Vector Laboratories, Burlingame, CA), selected cultures that had been reacted with antibodies as described above were washed with PBS and incubated with 0.1 mg/ml RhPNA in PBS for 30 min. All specimens were washed four times with PBS and mounted in 10% PBS-90% glycerol containing 25 mg/ml 1,4-diazabicyclo(2,2,2)octane (DABCO; Sigma) (
Chick embryos were fixed in 80% methanol-20% DMSO at -20C, embedded in paraffin, and sectioned at 5 µm. Alternatively, embryonic chick thoracic regions with attached hearts were cryopreserved by freeze-substitution into ethanol (
Controls for immunohistochemistry included omission of the primary antibody or use of an irrelevant IgM. Specificity of d1C4 immunoreactivity was also tested by inclusion of a 100-fold (w/w) excess of chondroitin sulfate Type A (CS-A, sturgeon notochord; Seikagaku America, Rockville, MD) or chondroitin sulfate type C GAGs (CS-C, shark cartilage; Sigma) with the primary antibody incubation. II6B3, an MAb to Type II collagen, was obtained through the Developmental Studies Hybridoma Bank maintained by the Department of Biological Sciences, University of Iowa. Specimens were examined with a Zeiss Axioskop equipped with epifluorescence optics and were photographed using Kodak TMAX 400 film.
Characterization of the d1C4 Epitope
Fixed micromass cultures were treated for 2 hr at 37C with either 0.1 U/ml chondroitinase ABC (Sigma) in 0.1 M Tris, pH 8, or 0.2 U/ml chondroitinase AC (Sigma) in PBS, pH 7.2, before immunostaining. For hyaluronidase digestion, micromass cultures and deparaffinized tissue sections were incubated for 30 min at RT in 0.1% testicular hyaluronidase (Sigma) in 50 mM sodium acetate, pH 6, with 0.9% NaCl, 1 mM phenylmethylsulfonylfluoride (PMSF), 1 µM leupeptin, 1 µM pepstatin A, and 1 mM EDTA before blocking and immunofluorescence staining. Periodate oxidation of fixed micromass cultures was performed using 10 mM periodic acid in 50 mM sodium acetate, pH 4.5, for 1 hr in the dark as described previously (
Microtiter plates (Immulon 4; Dynatech Laboratories, Chantilly, VA) were treated with 1 µg/ml poly-L-lysine (MW >300,000; Sigma) in PBS overnight at 4C, washed four times with PBS, then incubated for 2 hr at RT with CS-A (sturgeon notochord; Seikagaku) or CS-C GAGs (shark cartilage; Sigma), either alone or at various ratios of CS-A:CS-C. In other experiments, poly-L-lysine-coated plates were treated as above with 60 µg/ml CS-C (shark cartilage; Sigma and Seikagaku), chondroitin (from CS-A; Seikagaku), heparan sulfate (bovine kidney; Seikagaku), keratan sulfate (bovine cornea; Seikagaku), or dermatan sulfate GAGs (hog skin; Seikagaku). Wells were washed three times with PBS and treated for 1 hr with 1% BSA-PBS. Plates were washed with PBS and incubated for 1 hr at RT with 5 µg/ml d1C4 IgM in BSA-PBS or an irrelevant monoclonal IgM. After four washes with PBS, plates were incubated with a 1:5000 dilution of alkaline phosphatase-conjugated goat anti-mouse IgM (Sigma) in BSA-PBS for 1 hr at RT. Wells were washed five times with PBS before visualization of bound antibodies with 1 mg/ml p-nitrophenyl phosphate (Sigma) in 10 mM diethanolamine, pH 9.5, containing 0.5 mM MgCl2. Absorbance was measured at 405 nm using a Bio-Tek Microplate Autoreader (Bio-Tek Instruments; Winooski, VT).
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Results |
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Mouse lymphocytes were immunized in vitro with unfixed, air-dried micromass cultures of chick limb mesenchyme to produce MAbs reactive with native ECM molecules expressed during the critical process of cellular aggregation preceding overt limb chondrogenesis. At the time of screening, macroscopic hybridoma colonies were found in 83% of wells (20/24 wells), with 40% of these (8/20) yielding MAbs reactive by indirect immunofluorescence microscopy on frozen chick limb sections. Several antibodies recognized ECM components in both embryonic limb buds and high-density cultures of limb mesenchyme, one of which, d1C4, is the subject of this report.
The MAb d1C4 exhibited particularly strong immunoreactivity with mesenchymal condensations in 24-hr micromass cultures (Figure 1A). Digestion of micromass cultures or tissue sections by testicular hyaluronidase pretreatment in the presence of protease inhibitors before antibody staining abolished reactivity with d1C4 (data not shown). This result suggested that the antigen was either hyaluronic acid or a chondroitin sulfate GAG, because both are digested by testicular hyaluronidase. Pretreatment of 24-hr micromass cultures with either chondroitinase ABC or chondroitinase AC also revealed sensitivity of the d1C4 epitope (Figure 1B), suggesting that the antigen was a chondroitin sulfate. Periodate treatment of micromass cultures of limb mesenchyme to oxidize nonreducing termini of the GAG chains did not inhibit or diminish immunoreactivity (Figure 1C), which is consistent with an internal carbohydrate epitope.
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To assess the type of chondroitin sulfate recognized by d1C4, reactivity with chondroitin-4-sulfate (CS-A; sturgeon notochord) and chondroitin-6-sulfate (CS-C; shark cartilage) GAGs was tested by ELISA. The results indicated a preferential reactivity of d1C4 in a concentration-dependent manner with this CS-C GAG preparation and little or no reactivity with the CS-A GAG (Figure 2A). Little or no concentration-dependent reactivity of d1C4 was observed with CS-A GAGs from another tissue source as well (bovine trachea, Sigma and Calbiochem; data not shown). The preferential reactivity of d1C4 with CS-C GAG was corroborated with various ratios of CS-A:CS-C at a constant total concentration of GAG (Figure 2B). The presence of the d1C4 epitope in other GAGs was also tested by ELISA. Little or no reactivity of d1C4 was seen with chondroitin, heparan sulfate, keratan sulfate, or dermatan sulfate (Figure 2C). In addition, immunoreactivity of d1C4 with micromass cultures could be competed by preincubation of the antibody with excess CS-C GAG (Figure 3), whereas preincubation of antibody with the same concentration of CS-A GAG was ineffective. Taken together, these data suggest that the d1C4 epitope is preferentially found in GAGs enriched in CS-C type GAGs.
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High-density micromass culture of prechondrogenic chick limb mesenchyme duplicates processes that occur during chondrogenesis in vivo (
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Immunolocalization of d1C4 epitopes in paraffin sections of developing chick limb buds paralleled the micromass data and revealed other interesting patterns of reactivity (Figure 5). At Stage 20, a low level of pericellular immunoreactivity with d1C4 was distributed widely in limb mesenchyme. Limb ectoderm, the ectodermal basement membrane, and the immediately subjacent mesenchyme were strongly immunoreactive (Figure 5A). As shown at higher magnification in Figure 5B, there was an abrupt loss of reactivity associated with the apical ectodermal ridge (AER) and subridge basement membrane of the Stage 20 limb bud. In contrast, PNA binding molecules were localized throughout the ridge, nonridge ectoderm, and associated basement membrane (Figure 5C). The staining pattern found in the body of the Stage 20 embryo showed d1C4 immunoreactivity in and around the neural tube and notochord, and also associated with basement membranes of the dorsal ectoderm, gut, and mesonephros (Figure 5A).
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By Stage 22, d1C4 immunoreactivity was prominent in the ECM of the proximal limb, associated primarily with mesenchyme extending from the proximal limb core to the ventral sulcus (Figure 5D). At Stage 23, increased immunopositive material localized to the prechondrogenic core of the developing limb (Figure 5E). It was also noted at these latter stages that the nonridge limb ectodermal basement membrane remained immunoreactive, whereas staining was absent in the basement membrane underlying the AER and the mesenchyme of the distal limb. At Stage 23, immunoreactivity extended into the mesenchyme surrounding the gut epithelium. By Stage 28, d1C4 reactivity was associated predominantly with nascent cartilage tissues (Figure 5F). Immunostaining was also seen surrounding larger blood vessels of the limb.
In addition to the limb, interesting patterns of d1C4 immunoreactivity were found elsewhere in extracellular matrices of the developing chick, including the posterior portion of the somites, surrounding the dorsal aorta, in the eye, and in lung mesenchyme (data not shown). A particularly striking distribution of d1C4 reactivity was found in the developing heart (Figure 6). As shown in Figure 6A, the d1C4 epitope was expressed primarily by mesenchyme within the atrioventricular canal (AV) in the Stage 19 heart. At higher magnification it was apparent that those mesenchyme closest to the AV myocardium were only weakly positive relative to regions nearer the endothelium (Figure 6B and Figure 6C). The AV endothelium was unreactive, as were the premigratory cardiac jelly and the AV myocardium (Figure 6B and Figure 6C). Staining was also observed along the subendothelial myocardium in the atrium and at scattered foci throughout the myocardium of the atrial/AV junction (Figure 6A).
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By Stage 25, the entire inferior AV cushion ECM and all but the distal aspect of the superior AV cushion stained with d1C4 (Figure 7B and Figure 7C). The acellular matrix of the atrium adjacent to the AV cushions was also positive. In contrast, mesenchyme in the conal ridges of the outflow region differentially expressed the d1C4 epitope. Only those mesenchyme in the outflow tract near the myocardium were stained. Interestingly, the dense layer of myocardium (asterisks in Figure 7B and Figure 7C) in the outflow tract and AV region, as well as the epicardium (arrowheads), were unreactive, whereas the intervening mesenchyme and matrix were positive for d1C4. There was also an abrupt lack of immunoreactivity in the extracardiac mesenchyme of the dorsal mesocardium at the level of the spina vestibuli (Figure 8) that demarcates intra- from extracardiac mesenchyme.
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Discussion |
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The use of unfixed, air-dried micromass cultures as an in vitro immunogen proved to be a successful method for obtaining MAbs to precartilage aggregate matrix components in their native state, without modification by solubilization or other typical methods of immunogen preparation. The procedure utilized was an adaptation of that described by
Successful production of hybridomas utilizing the in vitro immunization of mouse lymphocytes in the present study was dependent on several other factors. Micromass cultures fixed lightly with either ethanol or glutaraldehyde (
Several lines of evidence indicate that the epitope recognized by d1C4 resides on GAG chains enriched in chondroitin sulfate type C: (a) d1C4 immunohistochemical reactivity was sensitive to pretreatment with chondroitinase ABC, chondroitinase AC, and testicular hyaluronidase; (b) ELISA results showed a concentration-dependent reactivity with CS-C but not with CS-A GAG; and (c) immunohistochemical reactivity was abolished by preadsorption of d1C4 with CS-C but not with CS-A GAG. In addition, ELISA results showed little or no reactivity of d1C4 with chondroitin, heparan sulfate, keratan sulfate, or dermatan sulfate. The sensitivity of d1C4 reactivity towards preparations digested with chondroitinase AC suggests that it is not crossreactive with dermatan sulfate, nor is the d1C4 epitope a heparan sulfate or keratan sulfate, because these GAGs are not digested by chondroitinases ABC or AC (
Although the data to date are consistent with the d1C4 epitope being found on GAGs enriched in CS-C, the precise structure of the reactive carbohydrate moiety recognized by d1C4 has yet to be identified. However, mild periodate oxidation (
Immunostaining of d1C4 in the developing limb is similar but not identical to that reported for other antibodies to CS-GAG epitopes. Using several MAbs,
The CS-GAG epitope recognized by d1C4 is likely to be represented on several PGs. PGs are composed of GAG chains linked covalently to a core protein, exhibit a wide tissue distribution, and represent a major component of the ECM (
Of particular interest in the present study was the spatial and temporal localization of d1C4 reactivity in prechondrogenic aggregates of chick limb mesenchyme that enabled us to monitor the process of mesenchymal condensation in culture. In the limb, chondrogenesis is preceded by the aggregation of mesenchymal cells into cellular condensations (
Many of the ECM molecules expressed in precartilage aggregates of the developing limb are also expressed in the developing endocardial cushions, including Type I collagen (
Functions attributed to PGs are numerous and range from organization of the ECM (
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Footnotes |
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1 Present address: Pediatric Cardiology, Cook Children's Hospital, Fort Worth, TX.
2 We wish to dedicate this paper to the memory of Professor Michael Solursh, in whose laboratory this work was initiated and who died while this study was in progress.
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Acknowledgments |
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Supported by grants from the NIH to EK (HL52813), MS (HD05505), and GK (HL42266) and from the AHA to MW (94004580).
We thank Sue Tjepkema-Burrows and John Busey for photographic assistance, Dr Tom Trusk and Josh Spruill for help in preparing the figures, and Jan King, Sandra Kolker, Tanya Rittmann, and Alison Chilton for preparation of tissue sections. Thanks also to Dr Roger Markwald for insightful discussion and critical comments and to Dr Corey Mjaatvedt for suggesting poly-L-lysine treatment of microtiter plates.
Received for publication January 31, 1997; accepted May 28, 1997.
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