THE ANATOMICAL RECORD 231218-224 (1991)

I m m u nochem ical Localization of Extracel I u lar Materials in Bone Marrow of Rats

REBECCA HAMILTON AND FERRELL R. CAMPBELL Department of Anatomical Sciences and Neurobiology, Health Sciences Center,

University of Louisville, Louisville, Kentucky

ABSTRACT The distribution of type I collagen, fibronectin, laminin, and heparan sulfate was studied in marrow of rats by indirect immunof luorescence. Most of the type I collagen of marrow is associated with large blood vessels and connective tissue trabeculae, but type I collagen was also localized in a delicate meshwork throughout the marrow and in the basement membrane of the sinu- soidal endothelium. Fibronectin is partially co-distributed with type I collagen, but is much more widely distributed. Sheets or septa of fibronectin-rich material divide the marrow into small compartments that contain and appear to separate clusters of developing blood cells. These septa may serve as a substrate for anchorage and migration of blood cells. Labeling of laminin was observed in the basement mem- branes of blood vessels, of fat cells, and of the sinusoidal wall, but only scattered labeling was seen in other extracellular materials. Heparan sulfate proteoglycan was poorly labeled in the extracellular matrix of marrow.

Bone marrow, the primary organ of hematopoiesis in adult mammals, is a site of continuous proliferation, maturation, and release of blood cells. This ongoing production of cells is made possible by a unique mi- croenvironment thought to be established and main- tained by stromal cells. A crucial part of the microen- vironment is the extracellular matrix elaborated by the stromal cells. Thus information about the distribu- tion and chemical composition of this matrix is of prime importance in the study of hematopoiesis.

The composition of the extracellular matrix of mar- row is readily studied in long-term culture systems. In these systems, collagen types I, 111, and IV, fibronectin, laminin, and heparan sulfate (Zuckerman and Wicha, 1983; Zuckerman et al., 1985; Gallagher et al., 1983; Gordon et al., 1988) have been identified as important components of the extracellular matrix. According to Roberts et al. (19881, heparan sulfate may be a key component of the extracellular material because it is capable of binding the growth factors necessary for he- matopoiesis.

Numerous studies have described the extracellular materials of marrow in situ. Electron microscopic stud- ies have shown that collagen is distributed widely throughout marrow (see Campbell, 1987) in a pattern similar to that of the reticular fibers shown by classical techniques. In the case of fibronectin, Reilly et al. (1985) reported that in human bone marrow fibronec- tin is found only in the wall of arteries and in extra- cellular material adjacent to megakaryocytes, but the studies of Sorrell (1988) indicated that fibronectin is widely distributed in the marrow of chick embryos. Laminin is present in the wall of large blood vessels and a discontinuous layer is seen adjacent to marrow sinusoids (Apaja-Sarkkinin et al., 1986). However, laminin has not been reported to be a component of the general extracellular matrix of intact marrow. Hep- 0 1991 WILEY-LISS, INC.

aran sulfate, an important proteoglycan in many tis- sues, has been labeled in the extracellular material of embryonic chick marrow (Sorrell et al., 1987), but the distribution of this proteoglycan in mammalian mar- row has not been studied.

The present study was undertaken to determine, with indirect immunof luorescent techniques, the dis- tribution of type I collagen, fibronectin, laminin, and heparan sulfate in intact marrow of rats.

MATERIALS AND METHODS Tissue Preparation

Bone marrow was obtained from male Wistar rats weighing about 150 g. The rats were anesthetized, the thoracic aorta was cannulated, and the vascular sys- tem was flushed with isotonic Krebs’ buffer, pH 7.2. After the vascular system cleared, 100 ml of fixative (3% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate) was infused into the vascular bed. Mar- row was removed from the femora, cut into blocks, and stored in fresh fixative for 2 hours a t 4°C. Blocks of marrow were rinsed in 0.1 M cacodylate overnight, in- filtrated with sucrose (30 minutes each in 0.5 M, 1.0 M, and 1.5 M), and stored in 2.0 M sucrose at 4°C. Blocks were frozen, mounted on cryostat chucks, and sectioned with a Reichert-Jung Frigocut 2800 N (-28°C) at a thickness of 8 pm. Sections were mounted on glass slides coated with TES (3-aminopropyltriethoxysilane).

Received February 7, 1991; accepted March 26, 1991 Address reprint requests to Ferrell R. Campbell, Department of

Anatomical Sciences and Neurobiology, Health Sciences Center, Uni- versity of Louisville, Louisville, KY 40292.

EXTRACELLULARMATRIXOFMARROW 219

lmmunochemistry Sections were wetted and blocked for 10 minutes

with 1.0% ovalbumin (Sigma Chemical Co., St. Louis, MO) in phosphate buffered saline (PBS). Excess oval- bumin was removed and sections were incubated over- night a t 4°C in appropriate antiserum. The next morn- ing sections were rinsed with PBS for 10 minutes, blocked again for 10 minutes with ovalbumin, and in- cubated at 42°C for 1 hour in rhodamine-conjugated secondary antibody. The sections were rinsed exten- sively with PBS (3 x 10 minutes) and mounted in glyc- erol-glycine medium (3 parts 0.1 M glycine and 7 parts glycerol, pH 8.6). Sections were examined with a Nikon Optiphot epif luorescence microscope using appropriate filters and photographed with 35 mm Kodak Tri-X 400 pan film.

Antisera All of the primary antisera were purchased from

Chemicon International (Temecula, CA) and diluted 1 5 0 with PBS immediately before use. They were rab- bit anti-rat type I collagen polyclonal antiserum, rabbit anti-rat fibronectin polyclonal antiserum, rabbit anti- rat laminin polyclonal antiserum, and mouse anti- heparan sulfate proteoglycan monoclonal antiserum. The secondary antisera were rhodamine-conjugated anti-rabbit IgG (also from Chemicon), diluted 1:20 with PBS, and rhodamine-conjugated anti-mouse IgG (Cap- pel Products, Malvern, PA), diluted 1:15 with PBS.

Controls Sections were processed routinely except 1) 1% oval-

bumin was substituted for the primary antiserum, 2) the primary antibody was absorbed with excess anti- gen shortly before use, and 3) pre-immune serum was used in place of primary antiserum. In all three in- stances no labeling occurred.

OBSERVATIONS Much of the type I collagen in bone marrow is located

in or near the wall of large blood vessels. In arteries of marrow (Fig. 1) some labeling is seen in the intima and media but the adventitia is most intensely labeled. From the adventitia a meshwork of collagen radiates among the cellular elements contributing as it does to the extracellular stroma of the marrow (arrows, Fig. 1). This meshwork is most extensive near the large blood vessels but offshoots often can be followed to collagen- rich trabeculae scattered about in the marrow (T, Fig. 2). A layer of collagen also is associated with the base- ment membrane of marrow sinusoids (S, Fig. 2). The endothelial cells are poorly preserved and difficult to see in these sections, but collagen seems to be localized along the basal surface of the endothelium and at times in the endothelial cells. Delicate strands of collagen (arrows, Fig. 2) often pass from the sinusoidal wall to adjacent trabeculae, to adjacent blood vessels, or to nearby sinusoids, interconnecting various regions of the marrow stoma.

Intense labeling of fibronectin is also seen in the wall of the large arteries and central veins of marrow (Fig. 3). Strands of fibronectin-rich material radiate from these large vessels into the surrounding marrow stroma. Fibronectin also is heavily labeled in the tra-

beculae and in the basement membrane of the sinu- soids (S, Fig. 4). Much of the fibronectin appears to be co-distributed with type I collagen since the patterns of distribution are so similar, but fibronectin is much more abundant than type I collagen particularly adja- cent to the developing blood cells (compare Figs. 2, 4). Sheets or septa of fibronectin-rich material (arrows, Fig. 4) are numerous among the developing blood cells. These septa divide the marrow into small compart- ments that appear to separate, a t least in part, clusters of blood cells. The septa are continuous with other ac- cumulations of fibronectin in marrow, interconnecting the fibronectin of large blood vessels, of the trabeculae, and of the sinusoids.

Intense labeling of laminin in bone marrow (Fig. 5) is observed in the wall of arteries (A) and central veins (CV). Heavy labeling of laminin is also observed around fat cells (not shown) where it appears to be a major component of the basement membrane of these cells. Also, the sinusoids of marrow have a thin, dis- continuous layer of laminin associated with them (ar- rows, Fig. 5). Thin strands of laminin were sometimes observed throughout the extracellular material of the marrow cords and were sometimes noted to be contin- uous with the laminin of fat cells, of the large blood vessels, or of the sinusoid wall. However, labeling of laminin in the extracellular matrix of the marrow stroma is scattered and weak as compared with type I collagen or fibronectin.

Heavy labeling of heparan sulfate proteoglycan in marrow was noted in the cytoplasm of some marrow cells (Fig. 6). Judging from the shape and size of the labeled cells, they appear to be granulocytes in various stages of development. The techniques used make it difficult to distinguish granulocytic cell types, but all seem to be labeled. In contrast,the scattered islands of erythroblastic cells (arrowheads) are largely unla- beled.

Labeling of heparan sulfate proteoglycan within the extracellular material of bone marrow was weak. A band of heparan sulfate-positive material is observed in the adventitia of the arteries (arrow, Fig. 6), but no heparan sulfate labeling was seen near central veins or sinusoids. In regions containing many granulocytes, the intense labeling of the cytoplasm of these cells ob- scures any possible extracellular labeling, but a small amount of heparan sulfate is observed that outlines the individual erythroblasts (arrowheads). It is not appar- ent whether this labeling is associated with extracel- lular material or with the surface of the erythroblasts.

DISCUSSION Contradictory findings have appeared concerning

the predominate collagen type of marrow. According to Apaja-Sarkkinen et al. (1986) the distribution of type I11 collagen in human marrow resembles the reticulum seen after silver staining. Also, Bainton et al. (1986) found that cultured marrow fibroblasts synthesize about three times as much type I11 collagen as type I. However, according to Bentley (1982) and Gordon (19881, type I collagen is the predominant type synthe- sized by marrow stromal cells. The present study shows that type I collagen has a wide distribution in marrow of rats, being particularly concentrated near the large blood vessels and in the trabeculae. A delicate layer of

220 R. HAMILTON AND F.R. CAMPBELL

Fig. 1. Immunofluorescence of type I collagen in rat bone marrow. A section of marrow with several arteries (A) and a megakaryocyte (m) is shown. Labeling is observed in the wall of the arteries and within a connective tissue meshwork (arrows) that extends from the arteries into the surrounding stroma. x 270.

type I collagen was observed along the basal surface of the sinusoidal endothelium. Also, type I collagen was localized in thin strands of extracellular material that radiate throughout the marrow supporting the stroma. Type 111 collagen was not labeled in the present study because of problems with available antisera.

Calvo (19681, using silver-impregnation, reported small nerves of the marrow stroma that were closely

Fig. 2. Immunofluorescence of type I collagen in rat bone marrow. Two small arteries (A), sinusoids ( S ) , and megakaryocytes (m) are shown. Heavy labeling of type I collagen is seen in the arterial wall, near the sinusoidal wall, and in a trabecula (TI. Thin strands of col- lagen (arrows) extend from the arteries, from the sinusoidal wall, and from the trabeculae to interconnect different regions of marrow. X 540.

associated with blood cells and the sinusoidal wall. However, DePace and Webber (1975) believed that many of these “nerves” were silver-impregnated strands of connective tissue. The present study shows delicate strands of extracellular material containing type I collagen that well might be mistaken for nerves.

Labeling of fibronectin in marrow of rats by the tech- niques of the present study was widespread and rather

EXTRACELLULAR MATRIX OF MARROW 22 1

Fig. 3. Immunofluorescence of fibronectin in rat bone marrow. A central vein (CV), adjacent arteries (A), and sinusoids (S) are shown. Labeling of fibronectin is intense in the wall of the central vein, in the wall of the arteries, near the wall of the sinusoids, and throughout the marrow stroma where it outlines small compartments. x 270.

Fig. 4. Immunofluorescence of fibronectin in rat bone marrow. A higher power view showing two sinusoids (S), a megakaryocyte (m), and the surrounding marrow stroma. Not the intense labeling of fi- bronectin along the sinusoidal wall and in septa farrows) that divide the marrow into small compartments. x 540.

intense. In many regions the distribution of fibronectin is similar to that of type I collagen; both are found in the wall and adventitia of the large blood vessels. In organs other than marrow fibronectin and collagen are co-distributed within the wall and adventitia of blood vessels (see Voss and Rauterberg, 1986). Since the fi- bronectin molecule has a collagen-binding domain, it seems likely that collagen forms a supporting frame- work and that fibronectin molecules are attached to

this framework. However, in the present study fibro- nectin is more widely distributed than collagen indi- cating that some of the extracellular material contains fibronectin but not type I collagen. Such a wide distri- bution of fibronectin in bone marrow was unexpected since the studies of Reilly et al. (1985) and Sorrel1 (1988) reported little fibronectin in bone marrow.

In the present study fibronectin of the extracellular material forms septa among the developing blood cells,

222 R. HAMILTON AND F.R. CAMPBELL

Fig. 5. Immunofluorescence of laminin in rat bone marrow. This section shows a central vein (CV), two arteries (A), and several sinu- soids (S). Labeling of laminin is seen in the wall of the arteries, the wall of the central vein, and the wall of the sinusoids (arrows). x 540.

dividing the marrow into numerous, small compart- ments. It seems likely that these compartments con- tain closely related clones of cells and that this segre- gation plays a significant role in differentiation of blood cells. The studies of Gordon et al. (1985), Pate1 et al. (1985), and Campbell et al. (1987) indicate that cer- tain lineage and stage-specific progenitor cells may re- quire different microenvironments.

Fig. 6. Immunofluorescence of heparan sulfate proteoglycan in rat bone marrow. A central vein (CV), an adjacent artery (A), sinusoids (S), and numerous blood cells are shown. Heparan sulfate is labeled in the wall of the artery (arrow) and in the cytoplasm of many of the blood cells, but only weak labeling is observed in the extracellular material (arrowheads). x 540.

Fibronectin also may play a key role in the anchor- ing of developing blood cells to the extracellular ma- trix. Tsai et al. (1987), Campbell et al. (19871, and Cou- lombel et al. (1988) demonstrated that immature blood cells adhere to fibronectin. According to Coulombel et al. (1988), receptors on marrow cells bind to fibronectin molecules anchored within the extracellular matrix. Furthermore, the studies of Weinstein et al. (1989) in-

EXTRACELLULAR MATRIX OF MARROW 223

dicate that fibronectin anchors immature blood cells and simultaneously stimulates their proliferation.

With many different cell types, fibronectin may be an important guidance molecule for migration of cells (Duband et al., 1986; McDonald, 1988; Tucker et al., 1988). In bone marrow it seems likely that the septa of fibronectin could serve as convenient “tracks” for cel- lular migration.

Laminin, an important component of the extracellu- lar material in embryonic systems (Little et al., 1989) where cellular division is rapid, has been demon- strated in the extracellular matrix of bone marrow cultures (Zuckerman and Wicha, 1983; Campbell et al., 1987; Gordon, 1988). Thus, we anticipated that laminin would be labeled in extracellular materials of bone marrow. As expected intense labeling of laminin was obsrved around fat cells and in the wall of arteries, sites where laminin has been localized in several tissues other than marrow (Bosman et al., 1985). Also, a discontinuous layer of laminin was observed along the wall of marrow sinusoids similar to that previously reported by Apaja-Sarkkinen et al. (1986). However, labeling of laminin throughout the marrow stroma was observed only in small scattered patches indicating that laminin is not a major com- ponent of the extracellular material of marrow. Any role it may play in the hematopoietic microenviron- ment is unclear. Sorrell found no laminin in bone marrow of chicks (1988) and the studies of Giancotti et al. (1986) showed that immature blood cells do not bind to laminin.

Heparan sulfate proteoglycans are important regu- lators of cell to cell and cell to matrix interactions (see Hedman et al., 1982; Gallagher et al., 1986). Also, heparan sulfate is an important component of long- term murine marrow cultures (Gallagher et al., 1983) and of human marrow cultures (Wight et al., 1986). Sorrell et al. (1987) reported that granulopoiesis in chick bone marrow occurs in an environment rich in heparan sulfate. According to Roberts et al. (1988) heparan sulfate may be of particular importance in he- matopoiesis because it binds growth factors and pre- sents them to the blood cell surface.

In view of these data, one might expect heparan sul- fate to be a major component of the extracellular ma- terial of marrow. However, in the present study heparan sulfate was poorly labeled in the extracellular material. The scant labeling we noted may have oc- curred on the cell surface rather than within the ex- tracellular matrix. Intense labeling was observed only in the cytoplasm of marrow granulocytes where a high concentration of heparan sulfate has been reported (Parmley et al., 1980, 1983).

In conclusion the present study shows that type I collagen forms the structural framework of the marrow stroma with other extracellular materials apparently attached to it. Fibronectin seems to be involved in the anchorage, segregation, and migration of blood cells within the marrow. Laminin is present in extracellular materials of marrow but its role in the hematopoietic environment is unclear. Little extracellular heparan sulfate proteoglycan appears to be present in marrow of rats.

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