Monoclonal antibodies with selective reactivity against osteoblasts and osteocytes in human bone

.IOURNAL OF BONE AND MINERAL RESEARCH Volume 9, Number 11, 1994 Mary Ann Liehert, Inc., Puhlishers

Monoclonal Antibodies with Selective Reactivity Against Osteoblasts and Osteocytes in Human Bone

SUSAN WALSH.] ROBERT A. DODDS.'.* I A N E. JAMES,'.* JEREMY N . BRADBEER,'.* and MAXlNE GOWEN'.*

ABSTRACT

Monoclonal antibodies (MAb) may provide valuable tools for studying osteoblast differentiation. We therefore raised a panel of MAb reactive with cells of this phenotype using 1,25(OH),D,-treated human trabecular osteoblast-like cells (HOBS) as the immunogen. lmmunohistochemical studies on various tissues, including undecalcified cryostat sections of fetal and adult human bone, identified 1 I bone cell-reactive MAb. Of these, 2 demonstrated particularly selective reactivities against osteocytes (OB/M) and osteoblasts (OB/L). These reactivities were also seen in developing bone from rat, rabbit, and marmoset. OB/L and OB/M demonstrated limited reactivity against a small number of human tissues from the extensive panel of substrates tested. Both MAb exhibited reactivity against discrete populations of cells in the large and small intestine. In addition, OB/L reacted with cells in the basal epidermis of skin and OB/M with cells in blood vessel walls. Both antibodies demonstrated reactivity against a variety of cultured osteoblast-like cell lines and other cultured cell types. These MAb may therefore provide a valuable means of studying osteoblast ontogeny.

INTRODUCTION

t i t i ) i t t . i ~ . ~ E N r i m o N I'.vftiWAY of osteohlasts (OB) has not T k e n fully elucidated. To date, the best available markers o f cells of the osteoblast lineage are the components prtxluccd by these cells. Unfortunately . these biochemical markers do not clearly identify specilic stages in the maturation o f osteoblasts. To address this problem. a number of groups have raised montdonal antihxlies reactive with cells of this lineage." ' ' I However. the majority of this work has k e n carried out using cells derived from nonhuman species and/or cells derived from cultured cell lines for hoth inimunogen and screening. This study describes the production ol' iiiontxlonal antihxlies (MAh) using human cells as the inimunogen and the subsequent screening prottxol. which eni- ployed in situ techniques. This allowed the identification of specific h m e cell types and their relationship to the surrounding histology.

MATERIALS AND METHODS

Pri.pirrritioti of' rititigiw t o r itntnirriizutions

Confluent primary cultures o f human osteoblasts were treated with I .2S-dihydroxyvitamin D,. 1 1 ,2S(OH)2D,. 10 MI for 3 days before the immunizations. The cells from one to three 1 0 cm petri dishes were washed with phosphate-buffered saline (PBS). harvested using a cell scraper. and pelleted by centrifugation at 100 X ,q for 5 minutes. Following two further washes. the cells were resuspended in PBS and used to immunize intraperitoneally two 6-week-old female BalbiC mice. A total of five immunizations were performed. followed by a booster injection 3 days before fusion. Terminal bleeds were taken from the mice and the sera used as positive controls in thc hybridoma screening step.

'Bath Institute lor Rheumatic Disease\, and University o f Bath, Bath. England. -'Bone I h e a s c Rewarch Group. MRC Clinical Research Centre. Harrow. England. *Prc\ent addrcss. Department o f Cellular Biochemistry. SrnlthKline Hecchani Pharmaceuticals. King of Prussia. Pennsylvania.

1688 WALSH ET AL.

Production of monoclonul antibodies osteoid lined with alkaline phosphatase-positive osteoblasts. the

Spleen cells from the immunized mice were fused with NS- I -Ag4- I murine myeloma cells at a ratio of 10: I using SO% polyethylene glycol IS00 (Boehringer Mannheim, Sussex, UK) in RPMI medium. The hybridomas were selected in hypoxanthine-aminopterin-thy midineesupplemented RPMI medium for 3 weeks' ") and were subsequently maintained in hypoxanthine- thymidine (HT) supplemented RPMI medium containing 100 U/ml of interleukin-6 (Boehringer Mannheim. Sussex, UK) .

Screening of hybridomus

Hybridomas were selected initially for their reactivity against acetone-fixed cryostat sections of human fetal rib (aged 8-1 1 weeks). Cross sections of the ribs (three donors) revealed a cartilage core lined by osteoblasts laying down woven bone (Fig. la). The antiosteoblastic reactivity of the MAb was verified on undecalcified cryostat sections of developing osteophytic bone' obtained from osteoarthritic femoral heads (mean age 65 years; range 50-82 years; Fig. 2a) and 1.25(OH),D,-treated (10 M) human osteoblaMike cells (HOBS).

Reactivity with osteoblasts in normal adult human bone was tested on cryostat sections of transiliac bone biopsies (7.5 mm diameter) taken from each of three osteologically normal patients (aged 27, 33, and 38 years) before treatment with a luteinizing hormone releasing hormone analog for endometrio- sis. Informed consent was obtained as required by the hospital ethical committee. Patients were double labeled with demethyl- chlortetracycline (300 mg twice per day) using a 2, 12,2, and 4 schedule. MAb OB/L and OB/M were tested on consecutive sections. Sections adjacent to those used for testing MAb were either stained using the von Kossa method and counterstained with eosin to visualize osteoid or reacted for alkaline phosphatase activity, as previously described."" In each biopsy, 30-40 bone formation sites were identified by the presence of

state of tetracycline labeling (absent, single. or double) being used as an indication of the relative degree of advancement through the formation phase.""

Todetermine furtherthe selectivityofthe MAbreactivities. they were also screened on a panel of human soft tissues. The species specificity of the MAb was tested against developing metatarsal bone derived from I-day-old marmosets, rats. and rabbits.

Cryostat sections (8 p,m) of the tissues were prepared and acetone fixed as previously described, and a standard indirect immunofluorescence method was performed.'

Control experiments consisted of tissue sections incubated with unrelated MAb supernatant of the same isotype, culture supernatant. or second antibody alone.

Churucterizution of MAb on cultured cells und humun peripheral blood mononuclear cells

The reactivity of the MAb was tested against cultured normal HOBS and a number of immortalized osteoblast-like cell lines. This latter group included a selection of human bath osteoblast prototype (BOP) cell lines that were previously shown to demonstrate a number of osteoblastic characteristics.' "' Each cell type was cultured for 72 h in the presence or absence of I ,25(OH),D, ( lo- ' M).

The MAb reactivities were further characterized against a range of human cell lines of fibroblastic (HFI9and MCS/SV2). endothelial (HUVEC), epithelial (HEp2 and Chang liver), and hematopoietic origin (KS62 and HL-60). The reactivity against normal keratinocytes was also studied

Normal peripheral blood mononuclear cells were harvested by density gradient centrifugation, as previously described.' 17' The isolated cells were adjusted to a density of 1 x 10h/ml in medium; 40 p1 aliquots of the suspension were settled overnight at 37°C onto poly-L-lysine-coated wells (0. I mg/ml in PBS) of 10-well multispot slides. The medium was removed, and the

TABLE 1, REACTIVITY OF THE MARS WITH HUMAN BONE BY INDIRECT IMMUNOFLUORESCENC"1

MAh Other reuctivitic~s isotvpe Osteoblasr Osteocytr Osiiwid o.stl'ol~lo.st rn hone

OB/A lgGl

OB/C IgM OB/H IgM

OB/J IgM

OB/K IgM

OB/L IgM

OB/M IgM

OB/N IgM

OB/P IgM

++ Cytoplasm

+ + Cytoplasm

+ + * Cytoplasm

+* Cytoplasm

+++ Cytoplasm

+* Cytoplasm

+ + Cytoplasm

+ Cytoplasm

+* -

+*

+++ +* -

+++ -

+*

Calcifying cartilage, blood

None Fibrocartilage cells,

preosteoblastsh None

vessels

None

Bone marrow stromal cells near bone surface, preosteoblastsh

Preosteoblasts.h blood vessels

Bone marrow cells

Preosteoblastsh

~

"The reactivities of the MAbs with osteogenic cells were assessed using human osteophyte and fetal bone tissues as the substrates. hReactivity observed only in fetal bone. Intensity of staining: - negative; +/- weak staining, + + + strong staining, * very

occasional expression.

OSTEOBLAST- AND OSTEOCLAST-REACTIVE MAb 1689

adherent cells were fixed in acetone for I0 minutes. All tissue and blood cell substrates were subsequently screened by indirect itiiniunotluorescence.

f lu t i l (.olot.tili~[itioti stirdics

The reactivity of the MAh OBiL and OB/M with HOBS cultured o n poly-L-lysine-coated slides was compared to the expression of alkaline phosphatase. as described previously.' ")

The expression ofthc antigens recognized by MAb OBiL and OB/M was assessed quantitatively using a method optimized for measuring intracellular antigens.' I") MG-63 o r HOBS were treated with either S )LM moncnsin and ethanol o r ethanol alone l o r 12 h at 37°C. The cells were harvested by trypsinization. washed once in cold PBS, and their density adjusted to 4 X

I0.5/ml. All subsequent incubation steps were performed on ice; IOU FI volumes o f the cells were fixed for 5 minutes in 2% paraformaldehyde and PHS (pH 7.4) and washed once by centrifugation at 800 X ,q in 2 ml PBS supplemented with I % bovine seruiii alhumin (PBSB). Background fluorescence was nssesscd using culture medium alone and an unreactive IgM MAb supernatant. The control IgM antihody used in these experiments was raised against an antigen that should not demonstrate reactivity in these expcriments (I>ro.uphi/tr arylsul- fatase). The purpose o f including this was to detect any nonspecific hinding of the IgM antihodies. The cells were permeabi- l i d hy incubation for 5 minutes with 0. I % saponin in PBSB (washing buffer). pellcted hy centrifugation. and incubated with thc MAb for 30 minutes. Optimally diluted negative (culture medium and irrelevant antibody) and positive (mouse IgM antivimentin asciteh) control\ were run in par;rllel. The cells wcrc washed twice hy centrifugation and incubated for 30 minutes with optimally diluted p a t antinlouse IgM FITC con.jugatc. Finally. the cells were washed twice by centrifuga- t i o n in washing bufler and once in PBSB alone. A total of 5 X

10' ccllsisamplc wcrc studied by fluorescence-activated cell sorting (FACS) analysis (FACStar Plus: Becton Dickinson). All samples were run in triplicate. and the data were analyzed using geometric analysis.

Wt~stcwi blotting

In an attempt to determine the molecular weights of the antigens recognized hy MAb OB/L and OB/M. a standard western blotting technique was used.'"'.."' I .2S(OH),D, (10 ' M) and/or nionensin-treatetl(5 p M ) MG-63 cells were boiled in

sodium dodecyl sulfate sample buffer and used as the antigen source. Optimally diluted aliquots of the antigens were loaded onto a 10% polyacrylamide gel and separated electrophoreti- cally. The proteins were transferred to a nitrocellulose sheet; the sheet was blocked for 2 h in 5% milk powder in PBS (pH 7.3) and cut into representative strips. These were probed with the MAbs and any reactivity detected as a colored band by the addition of a peroxidase-labeled secondary antibody and a chromogenic substrate. Molecular weight markers run in paral- lel with the lysates provided a standard curve from which the weights of the unknown proteins could be determined.

/sotyping of MAb

The isotype of the MAb was assessed using reversed passive hemagglutination performed according to the manufacturer's instructions (Serotec. Oxford, U K ) . Agglutination of the cells after 1 h incubation indicated a positive reaction.

RESULTS

Monctc~lonut rintihorly production und initiul screening

A total of I590 supernatant samples were tested for reactivity against human fetal bone, and 65 of these were selected for screening on cryostat sections of osteophyte and I .25(OH),D,- treated HOBS. Based o n the reactivity observed against these substrates. nine MAb were chosen and successfully cloned twice by limiting dilution. Of these. seven MAb were found tobe reactive with cytoplasmic antigens and two MAb recognixd predominantly cxtracellularantigens(Tab1e I ). Subsequently the MAb weretested on a panel of human soft tissue substrates. Based o n the findings, we report here the charactenzition oftwo MAb, OB/Land OB/M, 0 1 IgM subclass. that showed distinctive reactivity with cells at certain stages of osteoblast differentiation.

Rctictivity of MAA OBIL und OBIM with human hone

The reactivity of the MAb was assessed by indirect immunofluorescence using cryostat sections of human fetal bone, developing osteophyte. and normal adult human iliac crest biopsy. Both OB/L and OB/M were isotyped as IgM antibodies that reacted with cytoplasmic antigens (Table I ) .

In the fetal rib (Fig. la), OB/L demonstrated strong reactivity with cuboidal osteoblasts apposed to newly secreted osteoid (Fig. Ib). Weaker reactivity was observed in the osteoblasts associated with areas o f mineralization and in the preosteoblasts within the peripheral stacked cell layer. N o expression was detected in chrondrocytes.

~ ~ - O R I L + + - + + + + Cells within basal Cells within Cells within the

layer of dermis villi tips tips of the lamina propria

- OH/M + + + - + -

Blood ves5eI Blood vessel Cells along the Cells along the cells cells lamina propria lamina propria

1690 WALSH ET AL.

Cuboidal osteoblasts forming trabecular bone in the developing osteophyte (Fig. 2a) demonstrated strong reactivity with OB/L (Fig. 2b). In addition, a minor population of bone marrow cells adjacent to areas of bone formation demonstrated weak reactivity with this antibody. A gradient of OB/L expression was detected at sites of intramembranous ossification similar to that observed in the fetal bone; osteoblasts differentiating from the surrounding connective tissue demonstrated moderate reactivity that intensified in osteoblasts secreting osteoid (not shown). Connective tissue cells and chondrocytes were unreactive with this antibody.

In sections of fetal rib, OB/M demonstrated reactivity with recently encased osteocytes (Fig. 3a). Weak reactivity was observed with the osteoprogenitor stacked cell layer and osteoblasts. Similarly, in sections of osteophyte. the major character- istic of OB/M was the intense staining of recently encased osteocytes within both woven and fibrolamellar bone (Fig. 3b and c). This expression was lost in the majority of osteocytes in mineralized bone (Fig. 3b). There was occasional OB/M reactivity in osteoblasts (Fig. 3b and c) and. in some instances. with their basolateral surfaces. including the processes extending into the osteoid. No reactivity was detected in bone marrow cells with OBIM.

In contrast with fetal and obteophytic bone, only I in 10 formation sites in each iliac crest biopsy was stained with MAb OB/L and OB/M. lmmunoreactivity was not restricted to any particular stage of the formation period; OBLreactive cells were found lining new osteoid (not shown) that had yet to commence mineralization, as well as at single- and double- tetracycline-labeled sites (Fig. 4a). At those sites bearing OB/L-reactive cells, all osteoblasts were positive. Furthermore. when OB/M reactivity was examined in the adjacent section, positive osteoid osteocytes tended to be located within the osteoid lined with OB/L-reactive osteoblasts. An example of an OB/M-positive osteocyte is shown in Fig. 4b. No cells were stained at sites judged to have ceased formation. OB/L, but not OBIM, also stained a minor population of bone marrow cells that were dispersed throughout the marrow.

Reactivity of MAh OBIL and OBIM with murmoset, ruhhit, und rut hone

Moderate reactivity against osteoblasts within the periosteal layer was observed with OB/L and OB/M in sections of metatarsal bone derived from l-day-old marmosets (Fig. 5a and b). Similar results were observed in the metatarsals derived from rabbit and rat (not shown). Within this modeling bone, in areas of both periosteal and metaphyseal bone formation, OB/L reacted intensely with the osteoblasts lining the bone surfaces (Fig. 5a); at the same sites OB/M reacted predominantly with osteocytes (Fig. 5b and c). However, the number of osteoblasts and osteocytes reactive with OB/L and OB/M, respectively, was significantly reduced in bone closer to the endosteal surface and trdbeculae distant from the growth plate, where the onset of fibrolamellar bone formation was apparent (Fig. 5a). This distinct pattern of reactivity was difficult to discern in the developing human bone that was available for study.

Further charucterization of OBIL und OBIM

The specificity of OB/L and OB/M for osteoblast reactivity was assessed against a panel of human tissue substrates (Table 2) . In addition. their reactivities were checked against a large panel of cultured osteoblast-like. nonosteoblastic cell lines (Table 3). and human blood mononuclear cells

In the tissue substrates tested, OB/L reacted with basal cells in the epidermis of skin (Fig. 6a) and a select population of cells resident in the apex of villi within the lamina propria of the small intestine (Fig. 6b). These intestinal cells were located on the underside of the matrix immediately beneath the basal lamina of the surface epithelium. Similarly. in the crypts of the large intestine, OB/L demonstrated reactivity against cells within the tips of the lamina propria (mucosal surface). In contrast, OB/M showed reactivity against discrete populations of cells along the entire length of the lamina propria in both small and large intestine (not shown). In addition, OB/M exhibited reactivity against cells in blood vessel walls (Fig. 6c).

OB/L and OB/M demonstrated reactivity against HOBS, MG-63, and HOS cells, although OB/M demonstrated reactivity only against a minor population of cells in each of these cultures (Table 3 and Fig. 7a). The level of reactivity in each of these cell populations was not affected by the presence of I .25(OH),D,

b FIG. 1. Reactivity of MAb OB/L within sections of human fetal rib. (a) A histologic section reacted for alkaline phosphatase activity (red) and counterstained with methylene blue. The stacked cell layer of preostoblasts (SC), the bone (B), and the surface osteoblasts rich in alkaline phosphatase activity (arrowheads), and the underlying cartilage (C) are indicated. Original magnification. x40. (b) Indirect immunoiluorescence using OB/L demonstrates strong reactivity with osteoblasts (large arrowheads) apposed to osteoid. Reactivity in osteoblasts is reduced or lost at sites of mineralization (small arrowhead). Fluoroscein isothiocyanate (FITC). Original magnification. X 20. (c) The negative control (irrelevant. isotype-matched antibody) demonstrates no specific reactivity. The nuclei were counterstained with 0.002% ethidium bromide (red stain). FITC/ethidium bromide. Original magnification. ~ 4 0 . FIG. 2. OB/L reactivity against sections of human osteophyte. (a) Histology shows osteoblasts (large arrowhead) secreting osteoid (0) on trabecular bone (B). Associated positive marrow cells are also indicated (small arrowheads). Wright stain. Original magnification. X 20. (b) OB/L demonstrates strong reactivity against cuboidal osteobl and weaker staining was observed against the neighboring bone marrow cells (small arrowheads) in the adjacent section (higher magnification). FITC/ethidium bromide. Original magnification. x40. (c) The negative control demonstrates no reactivity (c). FITC/ethidium bromide. Original magnification. X 20. FIG. 3. Reactivity of MAb OB/M against sections of human osteophyte. (a) Strong reactivity of OB/M is demonstrated against recently encased osteocytes (arrowheads) in developing fetal rib (B). FITC. Original magnification. x20. (b) Developing osteophyte demonstrates reactivity of OB/M against surface osteoblasts (large arrowhead) and recently encased osteocytes (arrows). Osteocytes in mineralized ( M ) bone (small arrowheads) show no reactivity. FITC/ethidium bromide. Original magnification. X40. (c) Reactivity against the basolateral processes of osteoblasts (arrowheads) lining osteophyte trabecular bone and adjacent osteocytes (arrows). FITC. Original magnification: X40.

OSTEOBLAST- AND OSTEOCLAST-KEACTIVE MAb 1691

1692 WALSH ET AL.

FIG. 4. Reactivity of MAb OB/L and OB/M against sections of human iliac crest biopsy. (a) Both a single- (sl) and double- (dl) tetracyline-labeled site. Each site is lined with a continuous pallisade of OB/L-reactive osteoblasts (arrows; green fluorescence). Original magnification: X 20. (b) Double-tetracycline-labeled site showing the first ( L I ) and second (L2) labels and an OB/M-reactive osteocyte (arrow) embedded in the osteoid. Original magnification: X40.

(results not shown). Of the BOP cell lines tested, both antibodies reacted against lines 12, 26, and 3 I , which were previously shown to demonstrate the most osteoblastic phenotype of these cell lines."" OB/L alone demonstrated reactivity with two other BOP cell lines (7 and 37; Table 3). Neither OB/L nor OBiM reacted with human peripheral blood mononuclear cells.

Studies were performed in which we attempted to colocalize alkaline phosphatase activity and the immunoreactivity of OB/L and OB/M (Fig. 7b) in the same osteoblast-like cell populations (MG-63 and HOBS). There was no apparent correlation of alkaline phosphatase activity with either antibody. That is, cells were observed that expressed only alkaline phosphatase or MAb reactivity, neither. or both reactivities. Thus the expression o f the antigens recognized by the MAb was not coincident with alkaline phosphatase expression.

In the non4steoblast-like cell lines tested. OB/L reacted with fibroblast (HF 19) and endothelial (HUVEC) cells. OB/M stained minor populations of cells in endothelial (HUVEC) and epithelial (Hep2 and Chang liver) cell lines (Table 3).

FACS unulwis

OB/M demonstrated reactivity with only a minority of cells within the osteoblast-like cell populations. This appeared to be increased in the presence of monensin, an ionophore that prevents the secretion of proteins from the Golgi apparatuh. To verify this, we cultured MG-63 and HOB cells in the presence or absence of monensin and quantified the reactivity of OB/M and OB/L by FACS analysis.

b FIG. 5. Reactivity of MAb OB/L and OB/M against sections of developing marmoset bone. (a) Reactivity of OB/L against periosteal cells (P) and osteoblasts (arrows) lining the diaphyseal surfaces (D). The reactivity is lost in osteoblasts closer to the endosteal surface (arrowheads). Original magnification: X 10. (b) In a serial section. OB/M denionstrates reactivity against periosteal cells and osteocytes (arrows); reactivity was lost in osteocytes closer to the endosteal surface (arrowheads). FITC/ ethidium bromide. Original magnification: X 10. (c) Reactivity of OB/M is demonstrated in osteocytes (arrows) and occasional osteoblasts (basolateral services: arrowheads) within metaphyseal trabeculae (T). Osteoblasts apposed to trabeculae distant from the growth plate area are negative (large white arrowheads). Marrow cells are negative (M). FITC/ethidium bromide. Original magnification: x20 . FIG. 6. OB/L and OB/M reactivity against a panel of human tissues. (a) OB/L reactivity with basal cells in the epidermis of skin. (b) The reactivity of this antibody against cells resident in the apex of villi within the lamina propria of the small intestine (arrows). (c) Weak OB/M reactivity against cells in blood vessel walls (arrows). FITC/ethidium bromide. Origi- nal magnifications: X40. FIG. 7. Reactivity of OBlM against cultured osteoblast- like cells. (a) Reactivity of OB/M against occasional MG-63 cells (arrowhead). FlTCiethidium bromide. Original magnification: ~ 4 0 . (b) The reactivity of this antibody (shown as pink staining) is colocalized (arrow) with endogenous alkaline phosphatase reactivity (blue staining) in a primary culture of human osteoblast-like cells (HOBS). Many of the cells in these cultures did not show this colocalization. Avidin/biotin alkaline phosphatase and Fast Blue B. Original magnifications: X40.


1694

TABLE 3. POSITIVE IMMUNOREACTIVITY OF OB/L AND OB/M WITH CULTURED CELLS BY IMMUNOFLUORESCENCE

WALSH ET AL.

OB/L HOB" MG-63 HOS BOP 7, 12, 26, 31. 37

OBiM HOB"

HOS" BOP 12, 26, 31"

MG-63"

HF 19 fibroblast HUVEC endothelial cells

HUVEC endothelial cells" HEp2 epithelial cells" Chang liver epithelial cells"

"MAb stained only a few cells within the population studied.

Treatment ofthe cells with saponin did not appreciably affect their scatter characteristics (Fig. 8a and b). Treatment of MG-63 and HOB cells with monensin produced a significant increase in the mean fluorescence intensity (MFI) of OB/M staining in both cell types (Fig. 8c; p < 0.05). However, the MFI values recorded for the HOBS cells were lower (Fig. 8d). OB/L exhibited reactivity against the permeabilized cells, but the level of this reactivity was not significantly affected by monensin treatment. The culture medium and conjugate controls did not show nonspecific binding.

Western blotting results

denatured MG-63 proteins. Neither OB/L or OB/M demonstrated any reactivity with the

DISCUSSION

Within developing bone, areas of intramembranous bone formation exhibit the differentiation of osteoblasts in an orga- nized sequence of histologically observable stages. During endochondral bone formation and the normal turnover of adult trabecular bone. however, it is difficult to discern the sequence of events that occur in the differentiation of preosteoblastic populations into the mature osteoid-secreting osteoblast.'"' Reagents, such as monoclonal antibodies, would therefore be valuable in the study of the events that occur during osteoblast ontogeny. In this paper we describe the production of MAb that have been characterized in situ on bone sections, which allowed the identification of osteoblasts at various stages of differentiation and function.

Two monoclonal antibodies, designated OB/L and OB/M. have been characterized that demonstrate selective reactivity against cells of the human osteoblast lineage. This reactivity was localized to specific cell populations within developing bone by indirect immunofluorescence. Using this technique. we were able further to characterize the reactivitive of OBiM and OB/L against a variety of tissue and cell types.

The reactivities of OBlL and OBiM in developing human bone tissues suggest that they both recognize osteoblasts. How- ever, whereas OBlL recognizes the majority of mature osteoblasts, OB/M reacts with a distinctly smaller population. Fur-

thermore. although OB/L and OBiM also react weakly with immature osteoblastic cells. OB/M predominantly recognizes the majority of the terminally differentiated osteocytes in newly formed osteoid. There was occasional OB/M reactivity in osteoblasts and. in some instances, with their basolateral surfaces. including the processes extending into the osteoid. These cytoplasmic processes are thought to derive mainly from the osteocytes and preosteocytes.'"' This pattern of overlapping reactivities is supportive of the differentiation of osteogenic cells from bone marrow stromal cells to cells apposed to forming matrix surfaces and, finally. into osteocytes. In the developing animal bone, osteoblasts and osteocytes exhibited a more defined reactivity with OB/L and OB/M. respectively. Thus reactivity was observed only in newly modeling bone. The osteoblasts and osteocytes in remodeling bone (woven and fibrolarnellar) were, in general, negative.

In contrast to the widespread reactivity seen in human fetal and osteophytic bone, only a minority of active formation sites in iliac crest biopsies of normal bone contained cells reactive with the MAb. Expression of immunoreactivity appeared not to be related to any particular stage of the formation phase because it could be found at sites where bone formation had just commenced as well as at more advanced sites. At those sites where OBLpositive osteoblasts were found, all cells in the cohort were positive. This pattern of all or nothing staining would be expected if immunoreactivity were expressed intermit- tently throughout the formation phase. Bone lamellae may differ principally in their content of noncollagenous matrix proteins,'"' which may require that osteoblasts undergo periodic synchronous changes in behavior depending on the composition of the lamella under construction. The differences in distribution of MAb staining between woven and lamellar bone may reflect differences in the composition of the matrix being synthesized. OB/M stained osteoid osteocytes, which were usually at the same sites bearing OB/L-positive osteoblasts. This suggests coordinated expression of OB/L and OB/M antigens and sup- ports the view that the MAb distinguish consecutive maturational stages in the life span of osteogenic cells.

OB/L appeared to react with the majority of cells present in the transformed osteoblast-like cell lines. However, in the primary cultures of human osteoblast-like cells from trabecular explants (HOBS), only a minority of the cells demonstrated reactivity. This may reflect the heterogeneity of these cultures


in terms of both osteoblast differentiation state and cell lineage. However. the reactivity in all these cultures was unaffected by the presence of I .2S(OH)2D,. Thus, i f either antibody detects an antigen that is affected by differentiation, as appears to be the case, I .2S(OH),D, had no effect on this differentiation process.

In contrast, OBiM reacted with only select populations of cells in all the cell lines tested in the presence or absence of I ,2S(OH)$,. The number of OB/M-positive cells in both the MG-63 and HOB cultures was increased in the presence of monensin. These observations suggested that the antigen re- sponsible for OB/M reactivity was secreted. This is particularly interesting because n o immune reactivity was detected in the hone matrix in situ, which may indicate either that the antigen is not laid down (i.e., is soluble) or that it is modified in the matrix. If the former is true. this may indicate that the antigen plays a role in cellular communication. This is further supported by the observation in situ that OBiM was expressed in the osteoblast cell processes extending into the matrix.

The lack of correlation of reactivity of these antibodies with alkaline phosphatase expression in cultured osteoblasts suggests that the differentiation state of a cell is unlikely to be related to the expression of a single marker. The correlation of antibody reactivity with the synthesis of other osteoblast products may clarify this issue. This is of particular interest in view of the studies demonstrating temporal variations in osteoblast products in rat osteoblast-like cell

Further characterization of OB/L and OBiM showed them to be reactive with a number oftissue substrates. In both the small and large intestine, the reactivities of these antibodies demon-

a lo00 I--

b lo00 I--

600 7 C d -

100

80

60

40

20

CIM U OEM. O m VIM Treatment Treatment

FIG. 8. FACS analysis of OB/L and OB/M reactivities against monensin-treated and untreated MG-63 and HOB cells. The scatter characteristics of the cells were not affected by the saponin permeabilization step, as demonstrated by a (MG-63 cells before saponin treatment) and b (after saponin treatment). (c and d) The reactivity of the antibodies against MG-63 and HOBS. respectively, as expressed by their mean fluorescence intensity (MFI). The solid columns represent the results for untreated cells and the hatched columns after saponin treatment. The antibody reactivities were compared with negative control antibody (U). culture medium (C/M), and an antivimentin

strated distinct patterns. Thus, OBiL was reactive against a population of cells solely within the apex of the lamina propria. OBiM, however, was reactive with cells throughout the length of the lamina propria. This pattern of expression possibly reflects the upward migration of fibroblastic cells that participate in the synthesis of the lamina propria, a supportive matrix rich in collagen and proteoglycans. OB/L additionally reacted with the germinal layer of the epidermis of skin, where the basal cells undergo regular mitotic division, giving rise to a succession of cells that are rapidly pushed up toward the free surface.

OB/M also showed immunoreactivity with cells surrounding small blood vessel walls in bone, kidney, and skin. Although there is very little understanding of the relationship of perivas- cular cells (pericytes, fibroblasts, and endothelial cells of vessel walls) to other cells of the stromal cell system, this reactivity is intriguing because it is thought that pericytes represent a primi- tive pluripotent stromal cell population capable of differentiating into other cell types in the adult."" Significantly, pericytes are considered to represent an osteoprogenitor cell population. They can differentiate along the osteogenic pathway in vitro and are capable of calcifying their extracellular mat r i~ . '~ ' .*~ ' The relationship of pericytes to other blood vessel endothelial cells is unclear, and the precise reactivity of MAb OB/M with these cell types will be part of our future studies. This pattern of reactivity (observed also in osteoblasts) may imply that the OB/M antigen is expressed at both early and late stages of differentiation. This feature of reexpression of antigens in cell differentiation has also been noted for the SB-S antigen and N-CAM.'h.2y'

Unlike our observations in bone and the intestine, there was no overlap of reactivity with the antibodies detected in other tissues. Interestingly, osteogenic reactive MAb raised by other workers show reactivity with extraskeletal tissues. Some of the most highly characterized are those reported by Bruder and Caplan, which show a different pattern of reactivity with soft tissues from those observed OB/L and OB/M but predominantly react with epithelial and endothelial cells in different tissue compartments.'h-x' In conjunction with our findings, these reactivities support the idea that the stromal fibroblast system of bone and marrow may form part of a wider stromal cell system o f the body, each organ containing pluripotent and restricted stem cells that generate the cell lines of the particular organ con- cerned.'30' The immunoreactivity of MAb with tissues other than bone may therefore indicate reactivity with inducible osteogenic precursor cells that form part of the pluripotent stem cell compartment, which under the correct stimulus may differentiate into osteogenic cells.

In agreement with other workers, our findings support the hypothesis that during osteogenic differentiation, specific alter- ations in cell-associated antigens reflect the position of cells within the osteogenic lineage. The differentiation of osteogenic cells assumes that a discrete series of steps or transitions exists between the osteoprogenitor cell, the fully expressive osteoblast, and the terminally differentiated osteocyte. The pattern of sequential reactivity observed with OBiL and M in the sections of developing osteophyte is supportive of the differentiation of osteogenic cells from bone marrow stromal cells from cells apposed to forming matrix surfaces and, finally, into osteocytes. Furthermore, osteocytes recently encased in new matrix are distinct from "older" osteocytes. Bianco and coworkers recently

antibody (Vim, positive control). defined two distinct maturational compartments (proliferative

1696 WALSH ET AL.

and secretory) for osteoblasts as defined by bone sialoprotein (BSP) production, alkaline phosphatase activity, proliferative capacity, and cell shape."" The relationship of the reactivity of MAb OB/L with regard to these two osteogenic compartments and the expression of alkaline phosphatase remains to be explored.

In conclusion, although we do not know the precise identity of the antigens recognized by MAb OB/L and M , these osteoblast- selective monoclonal antibodies may have a number of impor- tant applications in the study of osteoblast differentiation in both human and animal bone.

ACKNOWLEDGMENTS

We thank Mr. A. Ross ofthe Royal United Hospital, Bath and the orthopaedic surgeons of Northwick Park Hospital, for providing the bone samples. This work was supported by a Program Grant from Glaxo Group Research, U K .

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Address reprint requests to: Marine Gowen. P h . 0 .

Department of Cellular Biochemistry SmithKline Beecham Pharmaceuticals

709 Swedeland Road P . O . Box 1539

King ($ Prussia. PA 19406-0939

Received in original form July 26. 1993; in revised form March 8, 1994; accepted April 7 , 1994.

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Monoclonal antibodies with selective reactivity against osteoblasts and osteocytes in human bone