Immunofluorescence and Confocal Laser Scanning Microscopy of Chronic Myeloproliferative Disorders on Archival Formaldehyde-fixed Bone Marrow
Maurice E. Mueller Institute at Biozentrum, Basle, Switzerland (RS,WB), and Department of Pathology, Kantonsspital, Aarau, Switzerland (RHL)
Correspondence to: R. Hubert Laeng, MD, Dept. of Pathology, Kantonsspital, CH-5001 Aarau, Switzerland. E-mail: laeng{at}ksa.ch
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Summary |
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Key Words: immunofluorescence confocal laser scanning microscopy chronic myeloproliferative disorders bone marrow fibrosis formaldehyde-fixed paraffin sections archival tissue
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Introduction |
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Materials and Methods |
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The slides removed from the MHB storage buffer were treated for 30 min at 37C with hyaluronidase (1 mg/ml testicular hyaluronidase; Sigma, St Louis, MO), conditioned for 20 min in 5075 µl of MHB containing 10% swine serum (Jackson Immunoresearch; West Grove, PA) and immersed for 60 min in the primary antibody diluted in MHB. Sections were washed three or four times for 5 min with 100200 µl of MHB, incubated for 60 min with the secondary antibody diluted in 75 µl MHB, and washed as above. The primary antibodies chosen for the study of bone marrow stromal cells were directed against tenascin (1:50, polyclonal rabbit anti-chicken antibody recognizing all three isoforms of the tenascin molecule; a kind gift from M. Chiquet, ME Muller Institute, Berne, Switzerland) (Chiquet 1999; and personal communication), smooth muscle
-actin (SMA) (1:500, clone 1A4; Sigma), fibronectin (1:300, polyclonal anti-rabbit antibody; Calbiochem, La Jolla, CA), myeloperoxidase (MPO, 1:1000, polyclonal; DAKO, Glostrup, Denmark) and CD68 (1:50, clone PG-M1; DAKO). Secondary antibodies were Alexa 488-nm goat anti-mouse or Alexa 488-nm goat anti-rabbit (both 1:800, 488-nm; Molecular Probes, Eugene, OR) and Cy3 donkey anti-mouse or Cy3 goat anti-rabbit (both 1:3000, 568-nm; Jackson Immunoresearch). After fluorescence labeling, the slides were immersed for 30 min in 70% ethanol supplemented with 0.1% Sudan Black B (Merck; Darmstadt, Gremany), washed with MHB and transferred into MHB. All sections were mounted with Mowiol-1188 (Hoechst; Frankfurt, Germany) containing 0.75% of the anti-fading agent N-propyl-gallate (Baschong et al. 1999
), dried overnight, and stored in the dark until microscopic analysis.
For removal of autofluorescence, immersion for 60 min in 70% ethanol supplemented with 0.25% NH3 while rehydrating deparaffinized sections in graded ethanol (see above) proved optimal if combined with Sudan Black B on fluorescence labeling (Baschong et al. 2001). After this step, the rehydration procedure was resumed by immersion in 50% ethanol for 10 min and transfer to MHB. The removal of excess Sudan Black from the slides proved to be decisive. It had to be wiped off manually from the back and along the edges of the slides with a soft paper, while the front required a jet wash with MHB (Baschong et al. 2001
). This procedure was followed by the transfer of the slides into MHB for 10 min before mounting.
The sections were viewed in a Leica TCS 4-D CLSM. Fluorescence image stacks were registered as 0.30.5-µm optical sections in parallel in the 488-nm (green) and 568-nm (red) channels. The series of differential interference contrast (DIC) was registered in a subsequent scan (Baschong et al. 2001).
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Results |
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Discussion |
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Our study, focused on the cytoskeletal SMA fibers and the two extracellular matrix proteins tenascin and fibronectin, provides further proof of a close interplay between neoplastic myeloid cells and polyclonal non-neoplastic stromal cells resembling myofibroblasts (SchmittGräff et al. 1994; Hauser et al. 1995
; Schürch et al. 1998
; Chiquet 1999
). Our results suggest that tenascin at a given stage of stromal fibrosis in the progress of CMPD may be co-expressed by both neoplastic cells of the myeloid lineage and reactive stromal cells of myofibroblastic phenotype. In stages of severe medullary fibrosis observed in IMF and occasionally in CML, however, tenascin is predominantly expressed by stromal cells and is released into the interstitial compartment, as confirmed by our results.
Tenascin has been localized in vivo and in vitro in the cytoplasm of a wide array of both mesenchymal and epithelial cells, including myofibroblasts involved in wound healing, normal and neoplastic mammary gland epithelia, oral squamous cell carcinomas, fat-storing cells of the liver, bone marrow stromal cells, and glioma cells (Van Eyken et al. 1992; Klein et al. 1993
; Lightner et al. 1994
; Redick and Schwarzbauer 1995
; Mori et al. 1996
; Seiffert et al. 1998
). Proof of tenascin formation by mRNA demonstration in neoplastic myeloid cells is a subject of further study. On the other hand, phagocytosed tenascin in stromal macrophages has not been detected in our material by CD68 immunofluorescence, nor has it been reported in the literature to the best of our knowledge.
SMA and fibronectin have not been detected in myeloid cells by immunofluorescence. They reflect cytoskeletal and extracellular matrix constituents of stromal cells. Differences in the distribution pattern of tenascin and fibronectin are shared with differences in the kinetics of molecule formation. Whereas the tenascin molecule is a hexamer (termed hexabrachion) and rapidly assembled with ongoing translation before transfer to the Golgi apparatus and secretion, fibronectin molecules are gradually assembled into disulfide-bonded dimers (Redick and Schwarzbauer 1995). The rate-limiting step in the release of tenascin into the extracellular space, however, is found in the posttranslational transport from the endoplasmic reticulum to the Golgi complex, which may be less effective for slow assemblage of fibronectin dimers (Redick and Schwarzbauer 1995
). Both cytoskeletal and extracellular matrix proteins may communicate across the cell membrane, as observed in various cultured cells; this connection is receptor-mediated (Alberts et al. 1989
).
The stromal bone marrow compartment includes various types of differentiated mesenchymal cells such as adipocytes, vascular smooth muscle cells, fibroblasts, and myofibroblasts. The latter are believed to derive from a common ancestor cell and may represent various isoforms evolving on stimulation by environmental factors (SchmittGräff et al. 1994; Schürch et al. 1997
,1998
). The cytokine microclimate reflects the functional demands in reactive processes, may mediate differentiation in cells displaying corresponding receptors, and stimulates extracellular matrix synthesis (Schürch et al. 1997
,1998
). Furthermore, an example of a crosstalk between the stromal and myeloid compartments may be recognized in the adhesive and mitogenic effects of tenascin on normal hematopoietic cells (Seiffert et al. 1998
). Similar mechanisms appear to be effective also in neoplastic conditions (Schürch et al. 1997
,1998
). The documented heterogeneity in the cytoskeletal phenotype of the versatile mesenchymal stromal cell compartment is a reflection of this issue (SchmittGräff et al. 1994
; Schürch et al. 1997
,1998
).
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Acknowledgments |
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We acknowledge Dr L. Landmann, Institute of Anatomy, University of Basle, for the complimentary access to CLSM facilities.
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Footnotes |
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Literature Cited |
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