IL-4 bone therapy

An in vivo assay for selecting a candidate therapeutic for treating osteoporosis. A candidate reagent is administered to an IL-4 transgenic mammal whose cells contain a recombinant IL-4 coding sequence operably lined to a promoter sequence which is transcriptionally active in bone marrow cells. At the time the candidate reagent is first administered the IL-4 transgenic mammal is either symptomatic of, or asymptomatic of, an osteoporotic phenotype. The candidate reagent is selected as a candidate therapeutic for treating osteoporosis if either amelioration of, or delay in the onset of, the osteoporotic phenotype is observed following administration of the candidate reagent to the IL-4 transgenic mammal.

FIELD OF THE INVENTION 
The invention provides animal and in vitro models for the evaluation of 
candidate drugs and therapies for the prevention and treatment of bone 
diseases, particularly osteoporosis, resulting from defective bone 
remodeling. 
BACKGROUND OF THE INVENTION 
Osteoporosis, a disease in which loss of bone mass causes skeletal 
deformities and fractures, affects nearly 20 million people, mostly women, 
in the United States alone. Development of new therapies for this disease 
has suffered from the absence of an appropriate animal model. 
Osteoporosis has been defined as the clinically significant loss of bone 
mass without abnormality in either its composition or the proportion of 
its mineral and organic phases (1; see the appended Citations). 
Involutional (including post-menopausal and senile forms) and 
glucocorticoid therapy-induced osteoporosis are the two most frequent 
etiologies in developed countries. In the U.S. alone, the cost of 
treatment for osteoporosis-related fractures was estimated at 7-10 billion 
dollars in 1987 (2). It has been calculated that a person in the U.S. dies 
every 20 minutes as a result of complications from osteoporosis (3). The 
burden that osteoporosis places on society will inevitably grow as the 
number of elderly increases in coming decades. Treatment with estrogens 
and other agents can slow or prevent bone loss in women, (particularly if 
administered shortly after menopause; 4). However, effective, safe and 
well-tolerated therapy for established involutional osteoporosis has yet 
to be achieved (reviewed in 5,6). The development of new therapeutic 
approaches for osteoporosis would be facilitated by a better understanding 
of the disease pathogenesis, a goal that has been hindered by the lack of 
appropriate animal models. 
Bone remodeling and the pathophysiology of osteoporosis. Bone remodeling is 
the ever-occurring skeletal process that is ultimately responsible for the 
development of acquired osteoporosis (7). Remodeling occurs at discrete 
sites scattered throughout the skeleton (7). The process is characterized 
by functional coupling of the activities of osteoclasts and osteoblasts 
(reviewed in 8). Thus, in skeletal balance, the mount of bone deposited by 
osteoblasts at a remodeling site is mirrored by the amount removed 
previously by osteoclasts. Regardless of cause, systemic osteoporosis 
always reflects circumstances in which osteoclast activity is enhanced 
relative to that of osteoblasts. Since bone deposition is generally 
initiated following bone resorption, one therapeutic strategy for 
osteoporosis has been to attempt a transient activation of osteoclasts in 
the hope that osteoblasts would subsequently deposit more bone than that 
removed by the osteoclasts. It follows conceptually that suppressed bone 
resorption might herald a reduced rate of bone remodeling and thus 
diminished bone formation. In fact, the most common form of involutional 
osteoporosis is characterized by slow remodeling in which bone formation 
is reduced more than is resorption (9). 
Molecular and cellular mechanisms of bone remodeling. Bone resorption by 
osteoclasts occurs at a given bone remodeling site for only about 7-10 
days (10). Murine models of osteoporosis have demonstrated that generation 
of osteoclasts from hematopoietic precursors is critically dependent on 
the production of macrophage-colony stimulating factor (M-CSF; 11), as 
well as the src gene product, (a protein-tyrosine kinase which osteoclasts 
express at high levels; 12,13). Many molecules which increase bone 
resorption by osteoclasts, (including interleukin-1, IL-1; tumor necrosis 
factors, TNF-.alpha. and -.beta.; parathyroid hormone, PTH; and, 
1,25-(OH).sub.2 vitamin D.sub.3 1,25-(OH).sub.2 D.sub.3 !, may act 
indirectly by activating secretion of factors from osteoblasts that, in 
turn, act on osteoclasts (14-16). 
Candidate molecules for coupling bone resorption by osteoclasts to bone 
formation by osteoblasts include transforming growth factor-.beta. 
(TGF-.beta.) and insulin-like growth factor-1 (IGF-1); both of which 
proteins a) are present in significant amounts in bone matrix and b) have 
been reported to enhance osteoblast activity (17,18). Once activated in 
vitro, osteoblasts appear to undergo a stereotypic sequence of 
proliferation, followed by increased production of alkaline phosphatase, 
followed finally by mineralization and production of bone matrix proteins 
such as osteocalcin and osteopontin (19,20). At a particular bone 
remodeling site, osteoblast activity and mineralization may require 
several months to reach completion (8). In addition to osteoblasts and 
osteoclasts, mononuclear phagocytes (M.phi.) may briefly replace 
osteoclasts at a resorptive cavity surface in the remodeling site (21). 
Osteocalcin and other matrix proteins exposed by osteoclasts may act as 
chemoattractants for M.phi. and possibly other cell types (22). Mast cells 
may also be present in increased numbers at sites of bone remodeling, and 
especially in post-menopausal osteoporosis (23,24). Recently, mast 
cell-deficient W/W.sup.v mice have been reported to have alterations in 
the remodeling process (25) even though no skeletal abnormalities are 
evident. The defective gene product in the W/W.sup.v animals, c-kit, is a 
cell surface receptor for a cytokine encoded by the Sl locus (reviewed 
26,27). Since the in vitro effects of the Sl cytokine are not limited to 
promoting mast cell growth, it seems that this cytokine might well exert 
its effects on remodeling by mechanisms that are independent of the mast 
cell-deficiency. In summary, the role that mast cells, M.phi., or other 
marrow cells, e.g., lymphocytes and fibroblasts, play in normal and 
osteoporotic bone remodeling remains uncertain. 
IL-4 and its potential influence on bone remodeling and osteoporosis. IL-4 
is a 15 kD glycoprotein produced by mast cells and T-lineage cells, 
especially mature CD4.sup.+ T cells (28,29). IL-4 has pleiotropic effects 
on a wide variety of hematopoietic and other cell types, including 
abrablasts and endothelial cells (28-31). The intracellular signal 
transduction pathways by which IL-4 acts remain to be defined, but they 
appear to differ significantly from other pathways of cytokine signaling 
(32). To date, IgE production by B-lineage cells is the only normal 
function that has been reported to depend on the production of IL-4 in 
viva (33,34). 
IL-4 transgenic mice. Three groups have reported generating IL-4 transgenic 
mice. 
Tepper et al. (83) fused a genomic IL-4 coding region to immunoglobulin 
promoter and enhancer elements derived from mouse and human immunoglobulin 
heavy chain loci, respectively. The construct (termed Ig.IL4) was 
introduced into fertilized mouse oocytes, and the resultant 
over-expression of IL-4 in the transgenic mice reportedly induced a 
complex inflammatory reaction resembling that observed in certain human 
allergic disease. In addition, Burstein et al. (83a) observed that IL-4 
over-expression also had profound effects on the B cell function of these 
animals. The authors stated that the IL-4 transgenic animals are 
potentially powerful models for studying the initiation and control of 
inflammatory reactions depending upon Ag-specific IgE production. See also 
international patent publication No. WO 91/13979 (Leder et al.). 
Muller et al. (83b) describes insertion of an IL-4-encoding construct into 
the mouse germ line under the control of the immunoglobulin heavy chain 
enhancer/promoter elements. In contrast to the IL-4 transgenic mice 
described by Tepper et al., the Muller et al. mice reportedly did not 
exhibit measurable alterations in the T cell compartment even though the 
level of transgene expression was sufficient to maintain Ia 
hyper-expression in B cells. The latter IL-4 transgenic mice thus 
reportedly provided an experimental model to test the effects of chronic 
Ia hyperexpression on B cells, and whether this is sufficient, per se, to 
lead to the development of autoimmune disease. No autoimmune disease was 
observed in the initial report (83b). 
Lewis et al. (35) disclosed generation of transgenic mice in which 
increased IL-4 expression was selectively targeted to the thymus. The 
present FIG. 1 details the transgene expression construct (lck-IL-4). 
Three transgene-positive mice expressing detectable transgene-derived mRNA 
were obtained after microinjection of the lck-IL-4 construct into oocytes. 
The founder animals, designated #1315, #4453, and #4475, reportedly 
displayed a distinct perturbation in development of T-lineage cells. 
SUMMARY OF THE INVENTION 
We describe a disorder in bone remodeling in mice that inappropriately 
express the cytokine interleukin-4 (IL-4), the histological features of 
which are strikingly similar to those observed in cases of severe human 
involutional osteoporosis. These findings demonstrate that constitutive 
IL-4 expression in hematopoietic cells can have a dramatic negative 
influence on bone remodeling in vivo, and indicate that IL-4, itself, or 
an IL-4-induced pathway may contribute to the development of osteoporosis 
in humans. The exemplary lck-IL-4 mouse should serve as a useful model for 
the in vitro and in vivo evaluation of therapies for the prevention and 
treatment of osteoporosis as well as other defects in bone remodeling. 
The invention provides such an in vivo assay for selecting a candidate 
therapeutic for treating diseases involving defective bone remodeling such 
as osteoporosis. A candidate reagent is administered to an IL-4 transgenic 
mammal, e.g., a mouse, whose cells contain a recombinant IL-4 coding 
sequence operably linked to a promoter sequence which is transcriptionally 
active in bone marrow cells. The promoter sequence is preferably 
transcriptionally active in lymphoid cells, particularly T cells. 
Representative promoters include the lck promoter and IL-2 promoter. At 
the time the candidate reagent is first administered, the IL-4 transgenic 
mammal is either symptomatic of, or asymptomatic of, an osteoporotic 
phenotype. The osteoporotic phenotype may be characterized by one or more 
symptoms selected from among histopathology showing reductions in cortical 
and trabecular bone mass, tetracycline bone labeling showing reduced 
osteoblast activity, and histomorphometric analysis showing a flattened 
appearance of osteoclasts and osteoblasts. The candidate reagent is 
selected as a candidate therapeutic for treating osteoporosis if either 
amelioration of, or delay in the onset of, the osteoporotic phenotype is 
observed following administration of the candidate reagent to the IL-4 
transgenic mammal. In alternative embodiments, the candidate reagent may 
be administered to an animal symptomatic of an osteoporotic phenotype as a 
result of either adoptive transfer of IL-4 expressing cells into the 
animal or administration of IL-4 protein to the animal. The candidate 
reagent is preferably an antagonist of IL-4, such as an antibody against 
IL-4, an antibody against IL-4 receptor, or a soluble IL-4 receptor 
molecule. The antagonist of IL-4 may be directed against cell types, 
particularly T cells, which constitutively expresses IL-4. 
The invention also provides an in vitro assay for selecting a candidate 
therapeutic for defects in bone remodeling. A candidate reagent is 
administered to a population of cells or tissue isolated from an IL-4 
transgenic mammal whose cells contain a recombinant IL-4 coding sequence 
operably linked to a promoter sequence which is transcriptionally active 
in bone marrow cells. The isolated cells or tissue may be selected from 
among bone explants, lymphoid cells, T cells, bone marrow cells, 
osteoblasts, and osteoclasts. The candidate reagent is selected as a 
candidate therapeutic for treating defects in bone remodeling if either 
amelioration of, or delay in the onset of, an osteoporotic cell phenotype 
is observed following administration of the candidate reagent to the 
isolated cells or tissue. The osteoporotic cell phenotype may be 
characterized by one or more properties selected from among decreased 
osteoblast or osteoclast cell growth, decreased osteoblast or osteoclast 
metabolic activity, and histomorphometric analysis showing a flattened 
appearance of osteoblasts or osteoclasts.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
To study the effects of IL-4 in vivo, we generated transgenic mice in which 
an IL-4 cDNA is expressed under the control of the proximal lck promoter 
(35,36). The lck gene encodes a protein-tyrosine kinase principally 
expressed by T-lineage lymphocytes and NK cells (37-39). Our original 
intent in generating lck-IL-4 mice was to determine the effect of 
increased intrathymic expression of IL-4, since IL-4 has been proposed to 
have a possible physiologic role in thymic development (40-42). We chose 
to use the proximal promoter segment of the lck gone to drive IL-4 
expression, since its transcriptional activity in the context of the 
normal lck gone is approximately 50-100-fold higher in thymocytes than in 
peripheral T cells (36,43). This pattern of increased expression in T 
cells was also true for the lck-IL-4 transgene in mice of the initial 
founder #1315 inbred line of animals. The latter mice also consistently 
exhibited multiple perturbations of T-lineage development that we have 
shown are due to increased IL-4 secretion (35). Remarkably, we found that 
lck-IL-4 mice, by 3 to 4 months of age, also develop severe progressive 
osteoporosis that closely resembles the human involutional osteoporosis. 
This previously unpublished observation forms the basis of the present 
disclosure. 
As discussed below in Examples 3-6, the #1315 line of lck-IL-4 transgenic 
mice have a genetic bone disease characterized by bone loss that is 
grossly evident by an abnormal translucent appearance of the skeleton, 
which is especially well seen in the ribs. With aging, many lck-IL-4 mice 
also develop gross kyphosis of the thoracic spine, a feature which is 
conveniently confirmed radiographically. No fractures of the vertebral 
bodies or of other bones have been found. Microradiography invariably 
demonstrates significant losses of cortical and trabecular bone mass 
throughout the skeleton. This bone disease radiographically and 
histopathologically closely resembles severe osteoporosis which occurs in 
humans. The stigmata of other bone diseases, such as osteomalacia, renal 
osteodystrophy, or primary hyperparathyroidism, are absent. Serum 
chemistries also argue against a primary metabolic or endocrine disorder 
mediating bone disease in these mice. Lower serum alkaline phosphatase and 
osteocalcin levels in lck-IL-4 mice compared to controls are consistent 
with the histopathologic studies, which indicate that osteoblast activity 
is markedly reduced in these animals. Histomorphometric studies confirm 
that lck-IL-4 mice have striking reductions in both cortical and 
trabecular bone mass. In vivo tetracycline bone labeling studies confirm 
that reduced osteoblast activity is the major factor leading to 
osteoporosis. In toto, the characteristics of the bone disease in these 
IL-4 transgenic animals are virtually indistinguishable from those in the 
low turnover form (Type II) of human osteoporosis. 
The osteoporotic disease in lck-IL-4 mice is of similar severity in both 
sexes, and has remained phenotypically stable during propagation of one 
lck-IL-4 inbred animal line, from founder #1315, for more than eight 
generations on a C57BL/6 genetic background. Importantly, 
histomorphometric analysis of the bone tissue from an independently 
derived lck-IL-4 founder animal, #4475, in which the transgene has 
presumably integrated at a different site than for #1315, also 
demonstrates severe bone loss and decreased osteoblast-lineage function as 
compared to an age-matched control animal. This indicates that the bone 
disease phenotype observed in lck-IL-4 mice is mediated by 
transgene-derived IL-4, rather than by a perturbation of an endogenous 
gene as a result of transgene integration. Further evidence that 
expression of IL-4 mediates the observed bone disease comes from an 
analysis of other transgenic mice we have generated using a pIL-2/IL-4 
construct. This construct is similar to the lck-IL-4 construct except that 
a human IL-2 promoter segment has been inserted in place of the proximal 
lck promoter segment. Histomorphometric analysis shows that pIL-2/IL-4 
mice consistently have decreased bone mass compared to littermate 
controls, indicative of osteoporosis. The degree of osteoporosis in 
pIL-2/IL-4 mice is less than in lck-IL-4, consistent with the lower 
overall activity of the IL-2 promoter, compared to the proximal lck 
promoter. Taken together, these findings show that expression of IL-4 in 
vivo in certain contexts can induce a form of osteoporotic bone disease. 
Although it remains to be shown that increased IL-4 secretion, particularly 
within the bone microenvironment, is responsible for the osteoporosis 
phenotype of the #1315 line, our preliminary data showing increased IL-4 
mRNA in transgenic bone marrow is consistent with this possibility. 
Bioassays for IL-4 indicate that bone marrow cells from transgenic mice do 
not produce IL-4 in pharmacologic amounts. Taken together, the findings 
indicate that the #1315 line of lck-IL-4 transgenic mice is a useful model 
for studying the pathogenesis of osteoporosis. 
Significance of the lck-IL-4 mouse model. Information potentially relevant 
to the pathogenesis of osteoporosis has been obtained previously in 
studies using tissue explants or cell lines cultured in vitro, but a major 
limitation has been the lack of a convenient model for extrapolating these 
results to the in vivo situation. Reported in vivo models of osteoporosis 
have required hormonal or pharmacologic manipulations to achieve 
osteopenia (64-66). Further, unlike lck-IL-4 mice, which consistently 
develop severe kyphosis associated with generalized osteoporosis, many of 
these models do not yield such osteopenic sequelae. A mouse strain with an 
uncharacterized genetic predisposition for osteopenia has been reported 
(67), but it is not clear that this strain develops significant bone 
disease. Defining the mechanisms that lead to osteoporosis in our model, 
which is histologically and phenotypically remarkably similar to the most 
common form of the human disease, should provide general insights into the 
pathogenesis of osteoporotic disorders. Most importantly, this model may 
afford insights into designing new forms of treatment for osteoporosis and 
may be a useful tool for evaluating candidate drugs. Since trials of 
systemic IL-4 treatment for neoplasms are already underway (68), the 
subject animals and cells should also provide information as to the 
potential effects of IL-4 in vivo, and could have practical implications 
for understanding risks and benefits of cytokine immunotherapy. 
Selection of candidate therapeutics for treating osteoporosis. The 
invention provides an in vivo assay for selecting a candidate therapeutic 
for treating osteoporosis. A candidate reagent is administered to an IL-4 
transgenic mammal whose cells contain a recombinant IL-4 coding sequence 
operably linked to a promoter sequence which is transcriptionally active 
in lymphoid and preferably bone marrow cells. At the time the candidate 
reagent is first administered the IL-4 transgenic mammal may be either 
symptomatic of, or asymptomatic of, an osteoporotic phenotype. The 
candidate reagent is selected as a candidate therapeutic for treating 
osteoporosis if either amelioration of, or delay in the onset of, the 
osteoporotic phenotype is observed following administration of the 
candidate reagent to the IL-4 transgenic mammal. 
The IL-4 transgenic mammal is preferably a mouse but may alternatively be 
any mammal including rats. Transgenic refers to an animal that is 
genetically recombinant in all its cells by virtue of the introduction of 
a nucleic acid into the germ line. The subject nucleic acid can be 
introduced into the germ line by transduction or transfection using known 
procedures, such as described below for the representative lck-IL-4 
transgenic animals. 
The cells of the IL-4 transgenic mammal contain a recombinant IL-4 coding 
sequence operably linked to a promoter sequence which is transcriptionally 
active in lymphoid and preferably bone marrow cells. IL-4 coding sequences 
are known in the art; see, for example, U.S. Pat. No. 5,017,691 (Lee et 
al.), which is incorporated by reference herein. Representative promoter 
sequences which are transcriptionaly active in bone marrow cells include 
the proximal lck promoter, IL-2 promoter, and ost promoter (68a). Proximal 
lck promoter refers to the region from -584 to +37 with respect to the 
transcription start site of the lck gene (which encodes a 
lymphocyte-specific membrane-associated protein tyrosine kinase, 
p56.sup.lck, that is a member of the src gene family and participates in 
T-lymphocyte signaling in mammals). 
Representative candidate reagents for the subject assay include IL-4 
antagonists which are capable of blocking the normal IL-4/IL-4 receptor 
interaction, and include, for example, IL-4 specific antibodies, IL-4 
receptor specific antibodies, IL-4 receptor polypeptides, and binding 
fragments thereof. IL-4 specific antibodies are available from commercial 
sources, such as ICN Biomedicals (Irving, Calif.) and are disclosed in 
U.S. Pat. Nos. 5,031,824 (Abrams et al.) and No. 5,041,381 (Abrams et 
al.), which are incorporated by reference herein. See also international 
patent publications No. WO 91/09059 (Ramanathan et al.) and No. WO 
89/06975 (Coffman et al.). IL-4 receptor specific antibodies are disclosed 
in international patent publication No. WO 89/09621 (Ritter et al.). 
Soluble IL-4 receptors are disclosed in international patent publications 
No. WO 90/05183 (Cosman et al.) and No. WO 90/03555 (Galizzi et al.). 
A second class of representative candidate reagents and procedures are 
targeted to cells, such as T cells, that constitutively express 
inappropriate amounts of IL-4. These include cell-lineage specific 
monoclonal antibodies (or antigen-binding sites thereof, whether made by 
hybridoma or recombinant techniques), such as anti-CD4, anti-CD8, 
anti-Thy-1, and anti-NK1-1 monoclonal antibodies, either unconjugated or 
conjugated to immunotoxins, as well as procedures such as thymectomy and 
bone marrow transplantation. 
The candidate reagent is typically administered by injection, e.g., 
intravenously, intraperitoneally, subcutaneously, intradermally, or 
intraosteously. 
At the time the candidate reagent is first administered, the IL-4 
transgenic mammal is either symptomatic of an osteoporotic phenotype or 
asymptomatic of an osteoporotic phenotype. This permits selection of 
candidate therapeutics for treating osteoporosis in terms of ameliorating 
the osteoporotic phenotype and/or delaying the onset of the osteoporotic 
phenotype, respectively. The osteoporotic phenotype is characterized by 
one or more findings or symptoms selected from among the following Group 
1, or two or more findings selected from Group 2. Group 1 osteoporotic 
findings include: a) histopathology showing reductions in both cortical 
and trabecular bone mass; b) in vivo tetracycline bone labeling showing 
reduced osteoblast activity in deposition of calcium carbonate 
(hydroxyapatite) and mineralization into bone; and c) histomorphometric 
analysis showing a flattened appearance of osteoclasts and osteoblasts, as 
described in Example 4. Group 2 osteoporotic findings include: a) bone 
loss evident as an abnormal translucent appearance of the skeleton; b) 
kyphosis of the thoracic spine confirmed by radiographic examination 
especially in lateral views of the spine; c) loss of trabecular and 
cortical bone mass throughout the skeleton, grossly evident on dissection 
as bones with an abnormal, semitranslucent appearance and a consistency 
similar to cartilage rather than healthy bone; and d) increased general 
bone fragility with the absence of stigmata of other bone diseases (e.g., 
osteomalacia, renal osteodystrophy, and primary hyperthyroidism), and the 
absence in the serum chemistry of a primary metabolic or endocrine 
disease, but with lowered serum alkaline phosphatase and osteocalcin 
levels. 
The candidate reagent is selected as a candidate therapeutic for treating 
osteoporosis if either amelioration of, or delay in the onset of, the 
osteoporotic phenotype is observed following administration of the 
candidate reagent to the IL-4 transgenic mammal, as compared to control 
IL-4 trangenic mammals. 
In vitro assays. The invention also provides in vitro assays for selecting 
therapeutic agents for treating osteoporosis. In this case the candidate 
reagent is added, e.g., at a concentration of approximately 1 pM to 1 mM, 
to a culture of test cells or tissue (e.g., bone explants, lymphoid cells, 
T cells, bone marrow cells, osteoblasts, and/or osteoclasts) isolated from 
the subject IL-4 transgenic mammal. The test cells are isolated when the 
IL-4 transgenic mammal is either symptomatic of, or asymptomatic of, the 
osteoporotic phenotype. 
The test cell population(s) may alternatively be isolated from mammals that 
possess a bone marrow altered by experimental manipulations, e.g., animals 
subjected to lethal irradiation and bone marrow reconstitution with 
enriched populations of IL-4 producing cells. The test cells, themselves, 
may also be genetically engineered with an IL-4 construct. In an 
alternative embodiment, test cells may be isolated from mammals that have 
been genetically engineered to decrease the level of IL-4 expression in 
lymphoid tissues and bone marrow. 
The in vivo assay provides a preliminary screen for candidate reagents 
which ameliorate or delay an osteoporotic cell phenotype, as indicated by: 
a) decreased osteoblast or osteoclast cell growth (e.g., as measured by 
tritiated thymidine incorporation in vitro in cultures containing optimal 
concentrations of growth factors); b) decreased osteoblast or osteoclast 
metabolic activity (e.g., for osteoblasts synthesis of alkaline 
phosphatase, secretion of tetracycline-labeled calcium phosphate, and the 
like; for osteoclasts synthesis of TRAP, and formation of pits in bone 
slices, and the like); and/or c) an abnormal flattened cell shape observed 
in histological cross-sections of cells grown in vitro on an appropriate 
extracellular matrix. 
The invention also provides a method of transferring an osteoporotic 
phenotype from an IL-4 transgenic mammal to a syngenic recipient mammal, 
by transferring cells such as bone marrow, stromal, lymphoid, and/or T 
cells into the recipient. Such cell transfers are conveniently conducted 
by transplantation of isolated cell suspensions, tissue samples, and the 
like. The transfer of cell suspensions may be made by injection, e.g., 
intravenous, intraperitoneal, intradermal, subcutaneous, or intraosseous 
instillation of cells. A suitable number of cells will depend upon the 
size of the mammal, for example, approximately 10.sup.4 to 10.sup.7 cells 
per mouse and larger numbers of cells for larger animals. 
Directed manipulation of bone remodeling. The invention also provides 
assays for selecting candidate therapeutic agents for altering bone 
remodeling in a mammalian host. By alterating bone remodeling is meant 
qualitative or quantitative inhibition or enhancement of either a normal 
or diseased bone remodeling process that is ongoing in an animal or 
patient. The alteration of bone remodeling may include changes in rate, 
extent, or amount of mineralization, percentage of bone mass attributable 
to trabecular versus cortical bone, the osteoblastic amount of collagen, 
and the like. Bone diseases in which altering bone remodeling may be 
useful include osteoporosis, osteopetrosis, non-union fractures, formation 
of bony spurs (e.g., at tendon insertion sites), Paget's disease, and the 
like. In bone disease such as osteoporosis or osteopetrosis it may be 
useful to treat the disease in a stepwise fashion, first with an agent 
selected to interrupt the ongoing remodeling process by stimulating an 
osteoporotic phenotype with an IL-4 bone therapy, and then with an agent 
selected to reverse the IL-4 osteoporotic phenotype. 
In the subject IL-4 bone therapy, an IL-4 agent is introduced into a 
subject in an amount and for a time sufficient to alter bone remodeling. 
The IL-4 agent may be an IL-4 polypeptide (e.g., introduced systemically 
by intravenous injection or infusion, or infused into a bone cavity 
through a surgically implanted bone catheter, or injected locally at the 
bone remodeling site). Alternatively, the IL-4 agent used in the IL-4 bone 
therapy may take the form of genetically engineered cells, e.g., 
fibroblasts or other cell types expressing a recombinant IL-4 polypeptide, 
in which case the IL-4 expressing cells are adoptively transferred into 
the animal or patient. The IL-4 agent used in the IL-4 bone therapy may 
alternatively be a gene transfer vector, e.g., a retroviral vector capable 
of infecting and inducing IL-4 expression in cells at a the treatment 
site. 
In situations where the bone disease involves a non-union fracture, bony 
spur, or a cosmetic bony defect (e.g., resorption of supernumerary digits, 
or facial reconstruction), a similar stepwise approach is advised, i.e., 
IL-4 bone therapy followed by treating with an agent to reverse the IL-4 
osteoporotic phenotype, followed by treating with an agent to induce 
ossification. For example, following surgical intervention to re-establish 
a mechanical union (e.g., by plastic surgery), it may prove highly 
effective to start treatment with an IL-4 bone therapy that stimulates 
altered bone remodeling in the surgical area. In this manner any bone 
defects are corrected and the site is prepared for ossification. As a next 
step, the altered bone remodeling may be stopped (i.e., by treating with 
an agent reversing an IL-4 osteoporotic phenotype or by discontinuing the 
IL-4 administration), and then ossification may be induced by 
administering a bone osteogenic growth factor or factor stimulating 
mineralization and/or cortical bone formation. By ossification is meant 
mineralization of bone (e.g., as measured using .sup.45 Ca incorporation 
or incorporation of tetracycline), formation of an increased mass of 
cortical bone (e.g., the mass of cortical bone compared with trabecular 
bone), and the like. 
A variety of IL-4 treatments are available for stimulating bone remodeling 
with IL-4 bone therapy. For example, controlled release formulations 
containing an IL-4 agent (i.e., IL-4 polypeptide, recombinant cells, or 
vectors, as described above) may be implanted during surgical intervention 
at a bone site. Illustrative examples of such controlled release materials 
include biocompatible sponges (e.g., fibrillar collagen-heparin surgical 
sponges and the like), capsules (e.g., made of alginates, gelatin, and the 
like), gels (e.g., collagen, hyaluronate), and the like. Those skilled in 
the art will recognize that pharmacological properties and biological 
activities of an IL-4 agent in blood may not model the properties and 
activities in bone, and that local modeling may be required to determine 
the optimal conditions for IL-4 bone therapy. 
The assays disclosed herein provide in vitro and in vivo models that are 
useful in determining pharmacological dosage ranges, timing, rates for 
administration, and delivery methods for IL-4 bone therapy. In one 
example, a 1 mm defect is created in the distal region of the femur of a 
mouse and after a period of healing transgenic murine cells expressing 
recombinant IL-4 (e.g., cells from an IL-4 transgenic animal) are injected 
into the defect site. The transferred cells stimulate altered bone 
remodeling at the site, and may promote the rate at which bone reunion 
occurs in the presence (or absence) of bone growth factors (e.g., 
TGF-.beta., IGF-1, IGF-2, bone morphogenetic proteins, osteogenein, and 
osteocalcin). 
Those skilled in the art will recognize that candidate therapeutic agents 
that are selected in the assays of the invention as agonists or 
antagonists of an osteoporotic phenotype may prove useful in their own 
right as therapeutic agents for promoting ossification in bone. 
Alternatively, the candidate therapeutics may prove useful in a stepwise 
bone therapy protocol. For example, after disrupting an ongoing diseased 
bone process with an IL-4 bone therapy, treatment may next be initiated 
with a candidate therapeutic agent designed to reverse an IL-4 
osteoporotic phenotype. In the murine femoral bone model described above, 
a candidate therapeutic agent may be tested for its ability to promote the 
rate of reunion and ossification. 
The invention is further illustrated by the following representative 
examples. 
EXAMPLE 1 
Preparation of the lck-IL-4 construct 
The lck-IL-4 construct (FIG. 1) was produced by inserting a full-length 
0.7-kb murine IL-4 cDNA BamHI fragment (69) into the calf intestinal 
phosphatase-treated BamHI site of an expression vector, p1017 (70), using 
standard subcloning techniques (71). In the final construct, the murine 
IL-4 cDNA segment was located 3' to a 3.2-kb murine proximal lck promoter 
segment at +37 with respect to the transcription start site (36), and 5' 
to a 2.1-kb BamHI-EcoRI fragment of the human growth hormone (hGH) gene 
(72). Referring to FIG. 1, the transcription start site of the proximal 
lck promoter is indicated by an arrow, the IL-4 cDNA segment is indicated 
by the solid rectangle, and hGH exon sequences are indicated by 
crosshatching. The IL-4 segment was embedded within the hGH gene since 
intronic sequences appear to be required for efficient transgene 
expression; the basis for this requirement remains poorly understood (73). 
Of course, one concern would be that hGH is being produced by the 
construct in biologically significant amounts in these mice. However, 
positioning the cDNA 5' to the translation initiation codon of the hGH 
gene results in undetectable serum levels of human growth hormone (&lt;50 
picograms/ml by RIA) in mice bearing the lck-IL-4 construct, or in other 
lines of mice bearing similarly constructed transgenes containing the 3' 
hGH gene segment (43,70; and data not shown). 
EXAMPLE 2 
Generation of lck/IL-4 transgenic mice 
A 6.0-kb NotI fragment containing the lck-IL-4 construct was purified by 
agarose gel electrophoresis onto DEAE-nitrocellulose paper (71) and 
diluted to 2 ng/ml in 10 mM Tris-Cl, 0.1 mM EDTA, pH 7.5. Aliquots of this 
preparation were micro-injected into the pronuclei of C57BL/6J X DBA/2J 
F.sub.2 hybrid mouse zygotes. The micro-injected embryos were transferred 
to oviducts of anesthetized C57BL/6 pseudopregnant females using standard 
techniques (74). Mice born to these pseudopregnant females were analyzed 
for transgene integration at 2-3 weeks of age by hybridizing blotted tail 
DNA with a hGH fragment probe (72). Three lck-IL-4-positive founder mice 
expressed detectable transgene-derived mRNA in thymic tissue (35; and 
unpublished data). These founder animals, designated #1315, #1453, and 
#4475 displayed a distinct perturbation of T-lineage cell development 
which included thymic hypoplasia and the absence of peripheral CD8.sup.+ T 
cells (35). The estimated number of copies of the lck-IL-4 transgene in 
the #1315, #4453, and #4475 founders was 8,5, and 5, respectively, based 
on densitometry of tail DNA blots hybridized with a 0.6-kb IL-4 cDNA probe 
(data not shown). Since the transgene copy number and the degree of 
perturbation of T-lineage cells in these three founders were similar, this 
indicated that the particular integration site of the transgene did not 
have a significant influence on the T-lineage cell phenotype we observed. 
The #1315 founder and its progeny were bred with C57BL/6 mice to maintain 
this line and permit a more detailed characterization of these lymphocytic 
perturbations. Treatment of #1315 line mice with anti-IL-4 monoclonal 
antibody clearly demonstrated that the perturbations of T-lineage cells in 
these mice was dependent on the secretion of IL-4 and was not due to 
disruption of an endogenous gene as a result of transgene integration 
(35). 
EXAMPLE 3 
Radio graphic abnormalities in the #1315 line of lck-IL-4 mice 
The #1315 male founder was sacrificed at 7 weeks of age after it had 
successfully bred with a normal C57BL/6 female. A translucent appearance 
of the founder animal's ribs was noted at this time. By 12 weeks of age, a 
kyphotic posture was evident in all transgenic progeny of this founder. 
Conventional radiographs which included lateral spine views confirmed 
severe kyphosis of the spinal column of the transgenic progeny (FIG. 2, 
upper view), as compared with nontransgenic littermate controls (FIG. 2, 
lower view). No wedging of vertebral bodies suggestive of compression 
fractures was evident. During dissection of six-week-old lck-IL-4 mice 
which had been sacrificed to obtain tissues for analysis, we also observed 
that their bones had a "washed-out" appearance and were much easier to cut 
than bones of littermate controls, suggesting that lck-IL-4 mice had a 
significant generalized reduction in bone mass. These phenotypic 
abnormalities have remained consistent after more than 9 generations of 
backcrossing the #1315 line with normal C57BL/6 mice, and have been of 
similar severity in males and females. The bone disease in lck-IL-4 mice 
did not appear to be due to a nutritional deficiency. The weight of 
transgenic and nontransgenic littermates prior to 12 weeks of age was 
similar even though marked decreases in bone mass were already evident. 
After the onset of severe kyphosis, the weight of transgenic animals 
begins to decline compared to nontransgenic littermates. This probably 
reflects a decreased caloric intake due to their limited mobility. 
Severely kyphotic mice have been euthanized at this point to prevent 
suffering. 
Although lck-IL-4 mice had generalized bone fragility and progressive 
kyphosis, they were otherwise free of dysmorphic features, suggesting 
their major disease process was osteoporosis. To evaluate in detail the 
entire skeleton for evidence of osteoporosis or other abnormalities, 
microradiography was performed. To allow optimal studies, lck-IL-4 and 
littermate control mice were first euthanized and then fixed in 10% 
neutral formalin prior to radiography. Representative results are shown in 
Table 1. Lck-IL-4 mice had a generalized decrease in cortical and 
trabecular bone mass, which was particularly evident in the long bones and 
vertebrae, respectively. 
Table 1. Cortical thickness and percent cortical area bone indices of 
3-month-old lck-IL-4 (n=4) and nontransgenic littermate control (n=4) 
mice. Shown are mean values.+-.standard error of means. Statistical 
significance was calculated using the unpaired two-tailed Student's t test 
comparing the lck-IL-4 and littermate control means. 
______________________________________ 
Littermate 
lck-IL-4 control Statistics 
______________________________________ 
Mid-radius 
Cortical thickness (mm) 
0.20 .+-. 0.01 
0.29 .+-. 0.01 
P &lt; 0.0001 
Percent cortical area 
52.6 .+-. 1.3 
71.3 .+-. 1.3 
P &lt; 0.00002 
Mid-ulna 
Cortical thickness (mm) 
0.35 .+-. 0.02 
0.52 .+-. 0.05 
P &lt; 0.02 
Percent cortical area 
68.0 .+-. 2.3 
83.8 .+-. 1.5 
P &lt; 0.002 
______________________________________ 
Microradiography documented marked and generalized reductions in cortical 
and trabecular bone mass in lck-IL-4 mice compared to littermate controls 
(FIG. 2). Our microradiographic analysis focused on the long bones, since 
these are particularly useful for quantifying bone mass by calculating 
cortical bone indices (74a). By these criteria lck-IL-4 mice consistently 
had markedly reduced total cortical bone thickness, area, and area as a 
percent of total bone volume compared to littermate controls (Table 1 and 
FIG. 3). 
FIGS. 3A and 3B show lateral survey microradiographs, at identical 
magnification, of the forelimb of a three-month-old male lck-IL-4 mouse 
(3A) and a three-month-old nontransgenic male littermate (3B). Marked 
cortical thinning of the radius and ulna are evident in the transgenic 
mouse compared with the littermate control. 
Importantly, no stigmata typical of endocrine or metabolic bone diseases, 
such as osteomalacia, renal osteodystrophy, primary hyperparathyroidism, 
or of congenital dysmorphic syndromes involving the skeleton were 
observed, indicating that these mice had an osteoporotic disorder. 
EXAMPLE 4 
Histopathology and histomorphometry of bone in lck-IL-4 mice 
Since osteoporosis by definition requires the absence of abnormalities in 
the composition or the proportion of the matrix and mineral bone 
components, bone tissue from lck-IL-4 and nontransgenic littermates was 
examined histologically to exclude these perturbations. By light 
microscopy, decreased cortical and trabecular bone mass in tissue from 
transgenic animals was evident in vertebral bodies as well as in long 
bones. In accord with microradiographic results, trabecular bone volumes 
were decreased and the complexity of the trabecular network appeared 
reduced, a typical finding in severe involutional osteoporosis in humans 
(74b). At higher magnification of lck-IL-4 bone tissue, a particularly 
dramatic reduction in the number and cell height of osteoblasts lining 
trabeculae was found, suggesting that these osteoblasts were largely 
inactive. Again, in agreement with the radiographic studies, no signs of 
osteomalacia, hyperparathyroidism, or skeletal dysplasia were evident. 
Although a mast cell-mediated mechanism for osteoporosis in lck-IL-4 mice 
is plausible, given that systemic mastocytosis can result in generalized 
osteoporosis (60,61), and IL-4 is a well-characterized mast cell growth 
factor in vitro (59), there were no significant differences between 
transgenic and nontransgenic animals in the number of mast cells in 
decalcified bone sections stained by toluidine blue. Furthermore, the 
number and appearance of mast cells in bone tissue or in bone marrow of 
lck-IL-4 and littermate control mice was similar after staining with 
either hematoxylin and eosin or with Giemsa, which enhances the detection 
of these cells (data not shown). As reported previously, we also failed to 
find increased numbers of mast cells at any soft tissue sites, including 
the skin and mucosal areas in lck-IL-4 mice (35). Taken together, these 
initial histologic studies suggested that markedly reduced osteoblast 
activity was likely to be an important mechanism for the development of 
osteoporotic bone disease in the #1315 line of lck-IL-4 mice. Although not 
conclusive, these studies also suggested that this bone disease was 
unlikely to be mediated by mast cells and was not accompanied by an 
increased rate of bone turnover. 
To more precisely quantitate differences in bone volume between transgenic 
and control animals, sections from the third caudal vertebrae were 
histomorphometrically analyzed for trabecular bone volume (the percent of 
marrow space occupied by bone matrix expressed as the "percent total 
osteoblastic surface") and mean cortical width using standard light 
microscopic techniques (75). All samples were coded prior to their 
analysis. Histomorphometric analysis revealed significant decreases in 
total bone volume, total surface osteoid, and osteoblast surface area in 
bone tissue of lck-IL-4 mice (Table 3). Importantly, histomorphometric 
analysis of the bone tissue of an independently-derived lck-IL-4 founder 
animal, #4475, in which the transgene had presumably integrated at a 
different site than for the #1315 line, also demonstrated severe bone loss 
and decreased osteoblast-lineage function as compared to an age-matched 
control animal. This strongly indicated that the bone disease phenotype 
observed in lck-IL-4 mice was directly mediated by the transgene, rather 
than reflecting the perturbation of an endogenous gone as a remit of 
transgene integration. 
To further document that osteoblast activity was depressed in lck-IL-4 
mice, osteoblast function was assessed by determining the incorporation of 
tetracycline into osteoid matrix after intraperitoneal injection (75a). 
Bone tissue from transgenic mice clearly had reduced incorporation of this 
label compared to littermate control cells (Table 2) indicating that the 
formation of mineralizing bone was reduced in lck-IL-4 animals. 
Table 2. Histomorphometric analysis of osteoblast activity in bone tissue 
of lck-IL-4 and or wild-type control mice. Mean values.+-.the standard 
error of the mean are shown for the #1315 line and nontransgenic 
littermates. 
______________________________________ 
Total Total 
Osteoblast Tetracycline 
Surface Surface 
Age (%) (%) 
______________________________________ 
lck-IL-4 #1315 line 
4 months 10.6 .+-. 2.4* 
20.9 .+-. 2.2.sup..dagger. 
(n = 3) 
Nontransgenic littermates 
4 months 18.0 .+-. 1.1 
48.4 .+-. 10.2 
(n = 3) 
lck-IL-4 #4475 female 
9 months 5.4 ND 
founder 
Control.sup.# 
9 months 13.6 ND 
______________________________________ 
*p &lt; 0.05 compared with an age and sexmatched C57BL/6 mouse. 
.sup..dagger. p &lt; 0.03 compared with an age and sexmatched C57BL/6 mouse. 
.sup.# An age and sexmatched C57BL/6 mouse was used as a control. 
ND = Not Determined 
Enzymo-histologic staining of bone sections for alkaline phosphatase (AP), 
an enzyme produced by osteoblasts, and for tartrate-resistant acid 
phosphatase (TRAP), an osteoclast-specific enzyme, provided additional 
insight into the bone disorder in lck-IL-4 mice. In transgenic bone 
tissue, AP activity associated with osteocytes or with osteoblasts lining 
the periosteum, endosteum, and trabeculae was markedly reduced as was TRAP 
activity associated with osteoclasts (FIG. 4). Histomorphometric analysis 
of TRAP-stained bone tissue also revealed that lck-IL-4 mice had 
consistent reductions in the numbers of resident osteoclasts compared to 
nontransgenic littermates. Together, these findings indicated reduced 
function of osteoblasts as well as osteoclasts in lck-IL-4 bone tissue. 
For the enzymo-histological staining, bone tissue was fixed in 70% ethanol 
at 5.degree. C. for 8-12 hrs, embedded in OCT freezing medium, and 
snap-frozen in liquid nitrogen-cooled isopentane. Sections were cut with a 
cryostat, collected onto Vectabone-treated glass slides, air-dried, and 
used immediately or stored at -70.degree. C. Staining for alkaline 
phosphatase and tartrate-resistant acid phosphatase (TRAP) activities 
employed standard procedures C. Liu et al., Histochem. 86:559, 1987!. For 
alkaline phosphatase detection, sections were incubated with a mixture of 
naphthol ASTR phosphate and the coupling azo dye, Fast Blue BB, in Tris-Cl 
buffer, pH 9.0 for 30 min at 37.degree. C., and then rinsed thoroughly 
with distilled water. For TRAP activity, sections were incubated with 
naphthol ASTR phosphate with hexazotized parasoaniline in the presence of 
10 mM tartrate in acetate buffer, pH 5.0 at 37.degree. C. for 1 hr and 
then thoroughly rinsed with distilled water. 
FIG. 4 shows enzymo-histologic staining of bone tissue from lck-IL-4 and 
littermate control mice. Alkaline phosphatase activity (blue staining) of 
frozen sections of tibiae (10.times.) from a two-month-old female lck-IL-4 
mouse (FIG. 4A) is compared with a nontransgenic littermate control (FIG. 
4B). In lck-IL-4 tissue there is a dramatic generalized reduction in 
alkaline phosphatase activity associated with osteoblasts lining the 
trabeculae, periosteum, and endosteum. Thinning of the cortical bone is 
also evident in the transgenic sample. Tartrate-resistant acid phosphatase 
(TRAP) activities (red staining) of metaphyseal regions of tibiae 
(40.times.) from the same mice are shown in FIGS. 4C (transgenic) and 4D 
(control), respectively. The intensity of staining is reduced in 
osteoclasts in lck-IL-4 tissue compared to that in the littermate control. 
EXAMPLE 5 
Serum chemistry studies 
Serum chemistry and enzyme studies also argued against hyperparathyroidism, 
vitamin D deficiency, or renal insufficiency as the etiology for the 
generalized bone disease of lck-IL-4 mice (Table 3). 
Table 3. Serum biochemistries in 9-week-old lck-IL-4 (n=5) and littermate 
control (n=3) mice. Transgenic and littermate control sera were analyzed 
in parallel for serum calcium, phosphorus, creatinine, and alkaline 
phosphatase activity (10 .mu.l/test) using a Kodak EktaChem700XR Analyzer. 
Serum osteocalcin was determined using a commercial radioimmunoassay kit 
(Biomedical Technologies, Sloughton, Mass.) following the manufacturers 
instructions. Shown are mean values.+-.the standard error of the mean. 
______________________________________ 
Phos- Alkaline 
Ca.sup.2+ phorus phosphatase 
Creatinine 
osteocalcin 
(mg/dL) (mg/dL) (U/ml) (mg/dL) 
(ng/ml) 
______________________________________ 
lck-IL-4 
10.0 .+-. 0.1 
9.2 .+-. 0.3* 
113 .+-. 8* 
0.1 .+-. 0.0 
84 .+-. 55* 
litter- 
10.2 .+-. 0.2 
8.1 .+-. 0.5 
176 .+-. 8 
0.1 .+-. 0.0 
162 .+-. 12 
mate 
control 
______________________________________ 
*p &lt; 0.05 compared with LM control using the twotailed Student's t test. 
Transgenic and littermate-control animals had similar total serum calcium 
and protein levels (data not shown), indicating that serum free calcium 
levels were probably normal in lck/IL-4 mice. Lck-IL-4 sera exhibited a 
small but significantly increased phosphorus concentration. The normal 
serum creatinine (Table 3) and normal microscopic renal histology in 
lck-IL-4 mice (data not shown) argued against a renal disorder in 
phosphate clearance being responsible for this very modest 
hyperphosphatemia. The high-normal level of phosphorus also essentially 
excluded a primary hyperparathyroid disorder in lck-IL-4 mice, since 
abnormally increased parathyroid hormone secretion typically lowers serum 
phosphate levels by increasing renal phosphate clearance (76). A 
significant decrease in the level of alkaline phosphatase (AP) activity 
was invariably observed, suggesting an overall reduction in osteoblast 
biosynthetic activity. Since serum AP is not solely derived from 
osteoblasts, decreased levels could also reflect reduced production from 
other tissue sources of this enzyme, e.g., liver and intestine, or, 
alternatively, a selectively accelerated clearance of this enzyme from the 
circulation (76a). However, the serum levels of osteocalcin, a protein 
made exclusively by osteoblasts and odontoblasts (76b), were also 
significantly reduced in lck-IL-4 mice compared to nontransgenic 
littermates. Together, these findings strongly suggested that decreased 
osteoblast activity had a significant role in the bone disorder of 
lck-IL-4 mice. 
EXAMPLE 6 
Expression of the lck-IL-4 transgene in lymphoid, bone marrow, and other 
tissues 
The generalized bone disease in lck-IL-4 mice might be due to a systemic or 
a local (bone microenvironment) effect mediated by expression of the 
transgene. Alternatively, the disease might reflect the disruption of an 
endogenous gene critical in bone remodeling as a result of transgene 
integration. To determine if there was detectable expression of the 
lck-IL-4 transgene in bone marrow cells, a finding which would make a 
local IL-4-mediated mechanism for osteoporosis plausible, these cells were 
assayed for spontaneous production of IL-4 using a sensitive bioassay 
lower limit of detectability &lt;10 pg/ml (35,77)!. Conditioned media from 
bone marrow cells (isolated by irrigation of humeri, femurs, and tibias) 
from lck-IL-4 or littermate control mice was prepared by incubating cells 
(5.times.10.sup.6 /ml) in IL-4-free CT.4S medium (77) for 24 hr. 
Conditioned media from thymocytes, a cell type known to express high 
levels of the lck-IL-4 transgene (35), and from splenocytes, a cell type 
predicted to have low levels of transgene expression, were prepared in 
parallel. IL-4 activity was determined by the ability of conditioned media 
to support .sup.3 H!thymidine incorporation by CT.4S cells as previously 
described (77). The concentration of IL-4 in samples was interpolated from 
a standard curve generated using recombinant murine IL-4. To confirm that 
.sup.3 H!thymidine incorporation was IL-4 dependent, an aliquot of each 
sample was preincubated with saturating amounts of purified 11B11 
anti-murine IL-4 mAb (78)! for 45 min at 4.degree. C. prior to the assay. 
Table 4 shows representative results from one of three experiments in 
which cells from 6-wk-old mice were analyzed. Spontaneous IL-4 production 
was detectable by transgenic thymocytes, but not by transgenic splenocyte 
or bone marrow cells. This result was not unexpected since the lck 
proximal promoter is mainly active in immature T-lineage cells. 
Neutralization of this thymocyte-derived activity by 11B11 mAb confirmed 
its identity as IL-4. As expected, none of the supernatants from these 
cell types in littermate controls had detectable activity. 
Table 4. IL-4 secretion (pg/ml) by unstimulated cells from lck-IL-4 and 
littermate control mice. 
______________________________________ 
without 11B11 
with 11B11 
Cell Type mAb mAb 
______________________________________ 
lck-IL-4 thymocytes 
29 &lt;10 
nontransgenic littermate 
&lt;10 ND 
thymocytes 
lck-IL-4 splenocytes 
&lt;10 &lt;10 
nontransgenic littermate 
&lt;10 ND 
splenocytes 
lck-IL-4 bone marrow 
&lt;10 &lt;10 
nontransgenic littermate 
&lt;10 ND 
bone marrow 
______________________________________ 
If bone-marrow derived IL-4 was responsible for the osteoporotic phenotype, 
the above negative results indicated that relatively low, nonpharmacologic 
levels of IL-4 were sufficient for this to occur. This is a reasonable 
possibility, given the potency of IL-4 in mediating many of its biologic 
activities (28,29) and the likelihood that any transgene expression in 
bone marrow would be constitutive. As an alternative to the IL-4 bioassay, 
we analyzed bone marrow from lck-IL-4 mice for IL-4 transcripts using RNA 
blotting. While transgene-derived transcripts were evident in thymus and 
spleen tissue, they were not detectable in bone marrow cells (FIG. 5). It 
should be noted that transgene-encoded IL-4 transcripts, which contain 3' 
hGH sequences (see FIG. 1), were significantly larger and readily 
distinguished from the endogenous 0.7-kb IL-4 transcripts found in EL-4 
cells. 
FIG. 5 (35) shows total RNA from tissues of lck-IL-4 mice hybridized with 
an IL-4 cDNA probe. All lanes were loaded with 10 .mu.g. RNA from 
PMA-stimulated IL-4 cells, which express endogenous IL-4 gone transcripts, 
served as a positive control. 
Given these negative results, we chose to determine the mount of IL-4 mRNA 
transcripts in bone marrow cells from lck-IL-4 mice using a 
highly-sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) 
assay (79). Special precautions were taken to insure specificity and that 
the assay was at least semiquantitative. Two .mu.g of total RNA from all 
samples were reverse-transcribed in parallel using a standard protocol 
(79). Various dilutions of the final RT reaction from each sample were 
amplified by PCR under identical conditions. To prevent amplification of 
any contaminating genomic DNA, murine IL-4 primers from the first and 
fourth exons, which are separated by more than 6 kb of genomic sequence, 
were used (80,81). PCR reaction products were electrophoresed and 
transferred to nylon filters by Southern blotting (71). The filters were 
hybridized with a murine IL-4 probe internal to both primers, to insure 
that detection of IL-4 sequences was specific. Scanning densitometry was 
used to quantitate the intensity of signals on autoradiographs of these 
filters. The portion of the curve for which RT concentration was linear 
with autoradiographic signal was then determined. In comparing 
autoradiographic signals from different samples, only an RT dilution which 
fell in this range of linear response was used. To control for possible 
differences between the efficiency of the reverse transcription reaction, 
the same RT dilution used to detect murine IL-4 mRNA was also used to 
amplify sequences for .beta.-actin (82), an abundant housekeeping gene. 
Using the RT-PCR assay described above, IL-4 mRNA was detectable in bone 
marrow and spleen cells, and thymocytes from lck-IL-4 mice, but was 
undetectable from these cells in nontransgenic littermates. FIG. 6 shows 
RNA levels in tissues from lck-IL-4 (TG) and nontransgenic littermates 
(CTL) assayed by RT-PCR. Spleen and bone marrow from two individual 
transgenic and control mice were assayed. FIG. 6A: PCR reaction products 
using first and fourth exon piers from the murine IL-4 gene, after 
Southern transfer and hybridization with an internal murine IL-4 cDNA 
probe. FIG. 6B: PCR reaction products using primers for .beta.-actin after 
electrophoresis and ethidium bromide staining. All RT and PCR reactions 
shown were performed in parallel. 
The low levels of IL-4 transcripts observed in transgenic kidney and liver 
tissue were presumably the result of blood contamination, since the 
endogenous proximal lck promoter segment is normally transcriptionally 
inactive in these tissues (39). The fact that the levels of IL-4 mRNA in 
bone marrow is markedly higher than kidney or liver argues against this 
merely representing blood contamination. The ready detection of IL-4 
transcripts in spleen and thymic tissue from lck-IL-4 but not control mice 
was expected based on the results using RNA blotting (FIG. 5). The primer 
pair used to detect IL-4 in these assays does not distinguish between 
transgenic and endogenous IL-4 transcripts. However, the lack of 
detectable signals in any of the nontransgenic littermate samples 
indicated that virtually all IL-4 mRNA detected in transgenic samples was 
derived from the transgene. The similar amount of .beta.-actin product in 
all samples suggested that the differences in IL-4 transcript levels were 
not attributable to differences in the overall efficiency of the reverse 
transcription reaction. 
EXAMPLE 7 
Determine whether the development of osteoporosis observed in lck-IL-4 mice 
depends on the production of IL-4 in vivo 
We hypothesize that this will require transgene expression, particularly 
within the bone microenvironment, and will not be due to the disruption by 
transgene integration of a genetic locus crucial for bone metabolism. We 
predict that: a) osteoporosis will occur reproducibly in independently 
generated lines of mice bearing the lck-IL-4 transgene, and that its 
severity will correlate with transgene expression by bone marrow cells; b) 
osteoporosis will depend on the presence of mature T cells, and will occur 
only after these cells appear in the periphery, including the bone marrow, 
during postnatal development; c) treatments which decrease transgene 
expression or neutralize secreted IL-4 will ameliorate osteoporosis; d) 
osteoporosis will be adoptively transferred to wild-type mice by bone 
marrow transplantation or by mature lck-IL-4 T cells; conversely, lck-IL-4 
mice will be cured of osteoporosis by transplantation with wild-type bone 
marrow; and e) transgenic mice in which IL-4 production is targeted to 
osteoblasts using the osteocalcin promoter (ost-IL-4 mice) will also 
develop osteoporosis. If none of these predictions are correct, and the 
development of osteoporosis appears independent of the expression of IL-4 
in vivo, one can, as an alternative approach, clone and characterize any 
endogenous genes which the lck-IL-4 transgene may have disrupted or 
altered by its integration in the initial line. 
Additional lines of lck/IL-4 transgenic mice are generated and examined for 
evidence of osteoporosis or other bone disease using microradiography and 
histomorphometry after in vivo tetracycline labeling (see Materials and 
Methods below). If bone disease similar to that found in the #1315 line is 
observed in these additional lines, this implies that the construct per 
se, rather than a particular integration site, is responsible for this 
phenotype. Of interest on this point is the recent report by Tepper et al. 
(83) of mice with an IL-4 transgene under the control of the 
immunoglobulin heavy (.mu. H) chain enhancer. These mice displayed 
perturbed T-lineage cell development similar to that observed in lck-IL-4 
mice, but it is not known if bone abnormalities were also present in these 
animals. Should .mu. H chain enhancer-IL-4 mice lack bone disease, this 
could reflect differences in the tissue pattern of transgene expression 
because of the promoter system used, and would not be informative as to 
whether insertional mutagenesis from transgene integration was likely to 
be the cause of osteoporosis in the #1315 lck-IL-4 line. For example, the 
lck promoter is active in NK cells (84), a cell type which does not 
normally express immunoglobulin genes. Hence, it is still important to 
generate additional lck-IL-4 mice for mechanistic studies. 
The following studies are pursued for the #1315 line and any other lck-IL-4 
lines manifesting bone disease: 
1. Determine whether them is a positive correlation between the levels of 
transgene expression in the bone microenvironment and the severity of bone 
disease in lck-IL-4 transgenic mice. Transgene expression by bone marrow 
cells is compared to that by other tissues using RNase protection assays 
or reverse transcriptase-polymerase chain reaction (RT-PCR) (see above). 
Both assays are highly sensitive and do not require large amounts of total 
RNA to be performed. Although the RNase protection assay is more 
technically difficult to perform than RT-PCR, it is also a more 
quantitative assay, and is therefore the preferred approach. 
2. To confirm that bone disease is dependent on IL-4 secretion, any lines 
manifesting bone disease are treated with anti-IL-4 monoclonal antibody 
(mAb) beginning at approximately two weeks of age (see section N, below, 
for details). This is a convenient age to begin treatment, since tailblots 
can be readily used to identify transgenic progeny. If we are unable to 
ameliorate or cure bone disease by anti-IL-4 treatment in the #1315 or 
other lines, one possibility would be that there is a critical 
developmental stage in bone, beyond which IL-4-induced abnormalities are 
irreversible. This would be more likely if there were evidence of bone 
disease in early ontogeny (see below). To address this possibility, we 
treat pregnant mice which have been mated with lck-IL-4 males with 
anti-IL-4 starting at day 10 of gestation. This precedes by several days 
the first detectable transcriptional activity of the endogenous proximal 
lck segment within the thymus and, presumably, the activity of the 
lck-IL-4 transgene as well. All progeny continue to be treated with 
anti-IL-4 for several weeks after birth. The anti-IL-4 antibody we use, 
11B11 (78), has previously been shown to effectively cross the mouse 
placenta (Robert Tepper, personal communication). If treatment beginning 
in fetal life is still unsuccessful in partially or completely alleviating 
bone disease, this might reflect an inability to achieve high enough local 
concentrations of antibody at sites of IL-4 secretion. As an alternative 
approach, we use an anti-murine IL-4 receptor mAb (85) in place of, or in 
addition to, mAb 11B11. However, the absence of a blocking effect will 
still not exclude inadequate local concentrations of antibody. Therefore, 
regardless of the outcome of anti-IL-4 and/or anti-IL-4 receptor 
experiments, we proceed to determine the effect on bone disease of the 
transplantation of normal bone marrow as well as decreasing transgene 
expression by reducing or eliminating particular T-lineage or NK 
populations, as described below. 
To determine if the osteoporosis of lck-IL-4 mice is mediated by 
hematopoietically derived cells, lck-IL-4 mice are irradiated and 
reconstituted with bone marrow from wild-type (nontransgenic) mice. 
Experiments in which normal mice are reconstituted with lck-IL-4 bone 
marrow are also performed to determine if this is sufficient to produce 
the osteoporotic phenotype. Since the #1315 line of lck-IL-4 mice has been 
backcrossed with homozygous C57BL/6 mice for more than 9 generations, it 
is essentially congenic on the C57BL/6 background, and should accept bone 
marrow from normal C57BL/6 donors. A failure to cure osteoporosis in 
lck-IL-4 mice by transplantation of normal bone marrow, or, conversely to 
adoptively transfer osteoporosis to wild-type mice by transplantation of 
lck-IL-4 marrow, could have several explanations. One would be that the 
osteoporotic phenotype is mediated by transgene-expressing cells which are 
not hematopoietically derived. This seems remote, given that the lck gene 
is not normally expressed by any nonhematopoietic cell type. A more likely 
explanation would be that the phenotype is due to transgene integration 
altering the function Of nonhematopoietic cells involved in bone 
remodeling. This possibility would be particularly likely if all of the 
predictions of the above study are incorrect. A final possibility is that 
osteoporosis is transgene-dependent, but is irreversible, or cannot be 
induced, after a critical development stage. This possibility cannot 
readily be addressed by transplanting bone marrow into mice younger than 
four to six weeks of age, since this results in an unacceptably high rate 
of mortality. However, other manipulations to reduce transgene expression 
in lck-IL-4 mice can be performed during the fetal or neonatal period of 
development to address this possibility. These are described below. 
To test our hypothesis that the development of bone disease is mediated by 
mature peripheral T cells, we first determine when significant bone 
disease is first evident in post-natal development. Mature T cells do not 
begin to accumulate in significant numbers in the periphery until after 
birth (86) and do not reach peak adult levels until after 6-12 weeks of 
age. Therefore, if osteoporosis is dependent on the presence of mature 
peripheral T cells, it is unlikely that bone disease will occur before 2-3 
weeks of age. We thymectomize neonatal lck-IL-4 and littermate controls to 
limit the accumulation of mature T cells and determine how this impacts on 
the development of osteoporosis. If thymectomy ameliorates or eliminates 
bone disease, we conclude that this phenotype is mediated by T-lineage 
cells which express the transgene. We then determine if elimination of 
mature T-lineage cells is sufficient to completely or partially prevent 
bone abnormalities. Lck-IL-4 mice are treated beginning in the neonatal 
period with monoclonal antibodies, such as anti-Qa-2 (86,87), which 
preferentially deplete mature peripheral T cells but leave the majority of 
thymocytes intact. If the elimination of peripheral T cells ameliorates or 
cures osteoporosis in lck-IL-4 mice, we perform adoptive transfer 
experiments to prove that T cells are sufficient to induce bone disease: 
Peripheral T cells are purified from the spleens and lymph nodes of 
lck-IL-4 mice, and injected intravenously into normal adult mice. If our 
ontogeny studies indicate that osteoporosis is evident relatively early in 
lck-IL-4 mice (i.e., before 2-3 weeks of age), T cells are injected 
intraperitoneally into normal neonatal mice. (The intravenous route is 
technically difficult until mice are about 2-3 weeks of age.) Recipient 
mice are analyzed at various times after injection for the development of 
osteoporosis, as described below. In the event that elimination of mature 
T cells does not ameliorate bone disease but thymectomy is effective, we 
conclude that it is likely that IL-4 is mediating its effect systemically 
on bone rather than by local secretion within the bone microenvironment. 
If, contrary to our prediction, bone disease is evident in lck-IL-4 mice 
before two to three weeks of age, we still perform neonatal thymectomies 
to determine if disease is mediated by T-lineage cells. If thymectomy does 
not ameliorate bone disease, this suggests that transgene-mediated effects 
are likely to be due, at least in part, to expression by non-T lineage 
cells. NK cells and, to a much smaller extent, B cells are the only non-T 
lineage cell types which have been shown to express significant amounts of 
lck (37-39,84). Therefore, to determine if bone disease is mediated by NK 
cells, lck-IL-4 and control mice are treated with antibodies which 
effectively deplete NK cells in vivo, such as asialo-GM.sub.1 antiserum 
(88) or mAb NK1.1 (89). Similar to the anti-IL-4 experiments described 
above, antibody treatments are initiated during pregnancy to attempt to 
deplete NK cells during fetal ontogeny. These NK cell depletion studies 
are also performed in the event that bone disease is not evident until 
after two weeks of age, but neither thymectomy nor the elimination of 
peripheral T cells effectively ameliorates bone disease. 
3. If any of the above studies with lck/IL-4 mice indicate that 
osteoporosis is mediated by transgene expression, we proceed to generate 
new lines of mice with a transgenic construct in which the osteocalcin 
promoter is used to direct transcription of an IL-4 cDNA. Osteocalcin is 
produced exclusively by osteoblasts and odontoblasts (20,76), and the 
osteocalcin promoter has been characterized in some detail (90,91). 
Therefore, this promoter appears to be a reasonable choice by which to 
selectively increase IL-4 production within the bone microenvironment, and 
to determine whether this production is sufficient to cause severe 
osteoporosis. As an alternative approach, less well-characterized 
promoters from a number of other cloned bone matrix proteins (e.g., 
osteopontin) can be employed. If osteoporosis or other bone disease is 
observed in ost-IL-4 mice, we compare the mount of transgene expression in 
bone tissue by several independent lines generated with this construct, 
and determine if this positively correlates with the severity of disease. 
Analogous to the lck-IL-4 experiments outlined above, we also treat 
ost/IL-4 mice which manifest bone disease with anti-IL-4 and/or anti-IL-4 
receptor mAbs to confirm that the disease is IL-4 dependent. 
The fact that the osteoporotic phenotype of the #1315 line of lck-IL-4 mice 
has an autosomal dominant inheritance pattern suggests that disruption of 
an endogenous gene via transgene integration is probably not responsible 
for the bone disease. Most cases of insertional mutagenesis in transgenic 
mice have been expressed in an autosomal recessive pattern (92-97). 
However, if the osteoporosis is limited only to the #1315 line, and is not 
influenced by anti-IL-4 treatment, neonatal thymectomy, or apparently 
mediated by bone marrow-derived cells, we can critically examine the 
hypothesis that this phenotype is the result of transgene integration 
perturbing an endogenous genetic locus which is critical for normal bone 
remodeling. We prepare a genomic DNA library from the #1315 line and then 
clone and sequence the integration site and attempt to determine any gene 
product(s) which may have been perturbed. Although this approach has 
successfully been used by others to identify endogenous genes disrupted by 
transgene integration (92,98,99), there are a number of potential pitfalls 
to be considered. Transgene integration could perturb an endogenous 
genetic locus by a number of different mechanisms: The insertion of 
transgenic DNA within a genetic locus could prevent or reduce its 
expression, or alter the protein produced. This can occur as a result of 
either the deletion and/or rearrangement of flanking genomic DNA (92, 100, 
101). Alternatively, the transgenic promoter could act to enhance 
expression of genes flanking the integration site; if the transgene acted 
as a locus activating region, such enhancement could potentially occur 
over more than 100 kb of genomic DNA (102). Regardless of the mechanism by 
which insertional mutagenesis might act to produce the phenotype, a major 
task would be to identify the affected gene products. This is not a 
trivial task since the size of mammalian genes varies from a few kilobases 
to more than 2 megabases. Although PCR-based techniques have recently been 
described which may identify functional exons within genomic DNA (103); 
see section O below), it remains to be shown that this procedure can 
reliably identify all such segments (103). Therefore, in the event of 
negative results with this technique, probes derived from nonrepetitive 
regions flanking the integration site, or from segments which have been 
deleted as a consequence of integration, are used to identify perturbed 
genes. The probes are used to screen for alterations in transcripts in 
bone marrow, osteoblasts, and other tissues from transgenic and normal 
animals. The chromosomal location of the transgene integration site is 
also determined and, if possible, more finely mapped by linkage analysis 
by crossing lck-IL-4 mice with appropriate mutant strains. This 
comprehensive approach offers the best chance for identifying a genetic 
locus which is critical in bone remodeling. 
If an endogenous gene product which has been perturbed by transgene 
integration is identified, the scope of subsequent studies depends on 
whether this is a novel gene product. Assuming that it is previously 
undescribed, we determine which tissues express the gene's cognate mRNA. 
We particularly focus on characterizing mRNA expression by normal bone 
tissue cells, as well as relevant osteoblast and osteosarcoma cell lines. 
High priority is given to the generation of polyclonal and preferably 
monoclonal antibody reagents against the gene product, to determine 
protein expression by various tissues, particularly bone. These studies 
lay the groundwork for experiments to determine the normal function(s) of 
the cloned gene product. 
EXAMPLE 8 
Define alterations in osteoblast and/or osteoclasts which are responsible 
for osteoporosis in lck-IL-4 mice and, if applicable, ost-IL-4 mice 
We hypothesize that decreased osteoblast activity due to locally produced 
IL-4 will be a major mechanism for both types of transgenic mice, and 
predict that: a) osteoclast activity will be reduced, resulting in 
decreased bone remodeling; b) osteoblast activity will be depressed to a 
greater extent than osteoclast activity, accounting for progressive 
osteoporosis; c) osteoblast and osteoclast abnormalities will be 
ameliorated by manipulations which reduce transgene-mediated IL-4 
production; and d) osteoporosis will not be associated with significant 
increases in mast cell abundance nor with major alterations in systemic 
hormones affecting bone metabolism. 
These studies should substantially enhance our understanding of the 
pathogenesis of osteoporosis, and the role that IL-4 may play in this 
disease process. They may also point out potential pitfalls of systemic 
immunotherapy with cytokines such as IL-4. 
The skeletal status of all lines of lck-IL-4 and ost/IL-4 mice are screened 
microradiographically and by routine histology. The remodeling dynamics of 
those with osteoporosis are examined histomorphometrically using double 
tetracycline labeling and nondecalcified histological sections. This 
approach enables one to qualitatively determine the effects of the 
transgene on the rates of bone formation (mineralization) and the numbers 
of osteoblasts and osteoclasts involved in the remodeling process. These 
examinations include stains to determine if the numbers of mast cells are 
increased in bone tissue from any of our transgenic lines. New lines are 
screened at 12 weeks of age. This should provide sufficient time for the 
expression of bone disease, based on our experience with the lck-IL-4 
line, in which severe osteoporosis is readily apparent at 6 weeks of age. 
However, some mice are allowed to age for up to one year to insure that a 
late-appearing phenotype is not missed. 
We specifically quantitate osteoblasts and osteoclasts in all lines which 
have evidence of bone disease using enzymo- and immuno-histochemical 
techniques. Osteoclasts are identified by staining bone in situ for 
tartrate-resistant acid phosphatase and osteoblasts by staining for 
alkaline phosphatase and osteocalcin using enzymatic and 
immunohistochemical techniques, respectively (104-107). To evaluate 
overall osteoblast activity, serum levels of osteocalcin and alkaline 
phosphatase are determined in affected mice and sex-matched littermate 
controls. Osteocalcin level is the preferred assay, because of its greater 
specificity for osteoblast activity than alkaline phosphatase level 
(20,76,108). Once these more detailed studies are completed, we then 
examine the effect of anti-IL-4 treatment and manipulations performed to 
reduce transgene expression (see above) on the severity of bone disease. 
For these experiments, microradiography, histomorphometry with 
tetracycline labeling, in situ assays of osteoblast and osteoclast 
function, and serum assays of osteoblast activity are employed. 
Sera from lines which manifest bone disease are screened for endocrine and 
metabolic abnormalities, including total calcium, phosphorus, creatinine, 
total protein, PTH, corticosterone the major circulating glucocorticoid 
in mice (109)! and osteocalcin concentrations as well as alkaline 
phosphatase activity. It is particularly important to exclude 
hyperglucocorticoidism since there are recent in vitro data that low 
concentrations of another T-cell derived cytokine, IL-2, can potentiate 
the release of anterior pituitary hormones, including ACTH (110). Further, 
our group has encountered patients with Cushings syndrome in whom 
osteoporosis has been the sole clinical manifestation. 
MATERIALS AND METHODS 
A. Transgene constructs. Purification of DNA plasmids, restriction 
endonuclease digestion, calf intestinal phosphatase-treatment of vectors, 
and ligation of DNA fragments use standard methods (71). Completed 
constructs are purified from vector sequences by restriction endonuclease 
digestion and electrophoresis onto DEAE-nitrocellulose paper (71), to 
result in DNA free of particulate matter which can interfere with 
microinjection. The sequences across ligation sites are verified by using 
standard dideoxynucleotide chain-termination sequencing methods (71). 
For the generation of additional lines of lck-IL-4 mice, a candidate 
alteration from the construct used in the generation of the #1315 line is 
in the hGH segment. As discussed above, it is extremely unlikely that 
biologically significant amounts of hGH protein are produced by the 
lck-IL-4 transgene in vivo. However, this possibility can be completely 
excluded in all future transgene constructs by replacing the hGH segment 
with hGX, which carries a frameshift mutation in the hGH coding sequence 
(111). hGX protein has no significant biologically active in mice, even at 
high levels (111). 
To make the ost-IL-4 transgene construct, the osteocalin promoter is 
subcloned immediately 5' to the hGX segment contained in the pBS/KS 
vector. The osteocalcin segment is either obtained from the laboratories 
in which it has been cloned and characterized (90,91), or is isolated by 
PCR amplification of murine genomic DNA. If PCR is used, primers include 
convenient internal restriction sites to facilitate subcloning. The murine 
IL-4 cDNA clone (69) is then inserted at a BamHI site in the 5' 
untranslated region of the first exon of hGX. The ost-IL-4 transgene 
construct is similar to the lck-IL-4 construct shown in FIG. 1, except 
that the osteocalcin promoter is substituted for the lck promoter segment 
5', and the hGX segment is substituted for the hGH segment 3'. The human 
rather than murine osteocalcin promoter is used in this construct since it 
is readily available and has been well-characterized. The 
tissue-specificity of expression of transgenes driven by human promoters 
has matched that of homologous endogenous murine promoter in most 
instances. (data not shown). Our experience with the lck-IL-4 construct 
has been that its expression in vivo in mice is compatible with viability 
and fertility. However, other IL-4 transgenic mice generated by Tepper and 
colleagues (83), in which the transgene was transcriptionally driven by 
immunoglobulin heavy chain enhancer, uniformly died shortly after birth. 
This presumably was due to a toxic effect of IL-4. To attenuate expression 
of the transgene, these investigators inserted prokaryotic sequences 
between the promoter and IL-4 segments. For unknown reasons, prokaryotic 
sequences appear to generally inhibit transgene expression (112). 
Transgenic mice bearing these attenuated constructs were both viable and 
fertile. If expression of the ost-IL-4 occurs at high enough levels to 
interfere with either viability or fertility, constructs are redesigned to 
include attenuating prokaryotic sequences between the osteocalcin promoter 
and IL-4 cDNA. An alternative approach is to delete the hGX segment of the 
construct, replacing it with a polyadenylation signal segment; the lack of 
intronic sequences in this construct should also lead to lower levels of 
transgene expression. 
B. Generation of transgenic mice and tailblots. Transgene construct DNA 
diluted in Tris-EDTA buffer is microinjected into the pronuclei of 
C57BL/6J X DBA/2J F.sub.2 hybrid mouse zygotes. The injected embryos are 
transferred to the oviduct of female Swiss-Webster previously made 
pseudopregnant by mating with vasectomized SJL male mice. These 
pseudopregnant mice are anesthetized using an intraperitoneal injection of 
0.4-0.5 ml of a solution of ketamine (6.5 mg/ml) and xylazine (0.4 mg/ml) 
in phosphate-buffered saline prior to the exteriorization of the oviduct. 
After embryos are transferred, the oviduct is returned to the pelvic 
cavity and the wound sutured closed. 
Mice born to pseudopregnant mothers are screened at 2-3 weeks of age for 
integration of the transgene by the tailblot technique. To obtain tail 
tissue, mice are anesthetized using ether or a single 0.1 ml 
intraperitoneal injection of ketamine/xylazine solution described above. 
Approximately 1 cm of tail tissue is removed using a sharp scissors; after 
which the tail wound is electro-cauterized to stop bleeding. Mice are also 
ear-tagged at this point for identification purposes. Tail tissue is 
digested with proteinase K (250 .mu.g/ml) in the presence of 1% SDS and 
Tris-EDTA buffer for 4 h at 37.degree. C. Protein is precipitated by the 
addition of NaCl and KCl to final concentrations of 0.4M and 20 mM, 
respectively. The DNA contained in the supernatant is ethanol 
precipitated, dissolved in 0.1M NaOH/2M NaCl, boiled for three min., and 
dotted onto nitrocellulose filters. The filter is neutralized by 
moistening with 2.times. standard saline citrate SSC (71)!, baked at 
80.degree. C. for 1 h, and then hybridized with a 0.6-kb hGH probe (72) in 
Stark's buffer (113) at 42.degree. C. for 12-24 h. Blots are then washed 
with 6.times. SSC, 0.1% SDS for 30 min, 0.1.times. SSC, 0.1% SDS for 30 
min, and then autoradiographed. 
Mice which are positive for transgenic constructs are backcrossed to 
C57BL/6 mice. Nontransgenic littermates are kept with transgenic 
littermates of the same sex to serve as wild-type controls for all 
experiments. 
C. Cell isolation. Murine bone marrow cells are isolated by irrigation of 
femurs and humeri from euthanized mice with phosphate-buffered saline 
(PBS), pH 7.4. To isolate spleen or lymph node mononuclear cells, tissue 
is disrupted with a fine-mesh sieve, and the mononuclear fraction is 
purified by Ficoll-Hypague density gradient centrifugation (114). In cases 
of significant red blood cell contamination, brief hypotonic lysis with 
NH.sub.4 Cl is performed (115). T lymphocytes are purified from splenic or 
lymph node mononuclear cells by passage over nylon wool (116) followed by 
treatment with mAb and complement to deplete B and monocyte-lineage cells 
as described (117). The purity of all final cell preparations are 
determined by immunofluorescent staining with appropriate mAbs analyzed by 
flow cytometry. 
D. RNA isolation. Total cellular RNA is isolated from cells or tissue using 
the guanidinium isothiocyanate/CsCl method (118). The RNA pellet is 
dissolved in KNase-free water, ethanol precipitated in 0.3M Na acetate (pH 
5.3), redissolved in RNase-free water, and quantitated by 
spectrophotometry. A small aliquot is also run on a Tris-borate agarose 
gel after brief heat denaturation in 50% formamide. Only those samples in 
which the 28S and 18S ribosomal RNA bands are intact are used for RNase 
protection or RT-PCR assays. 
E. RNA blotting. Total RNA (5-20 .mu.g/lane) is heat-denatured in 2.2M 
formaldehyde and 50% formamide and electrophoresed in 2.2M formaldehyde 
gels. The RNA is capillary transferred to nylon membranes using 20.times. 
SSC (pH 7.0). RNA is cross-linked to the membrane by brief exposure to 
short-wave UV light and then baked at 80.degree. C. Filters are 
prehybridized with Stark's buffer (113) for 1-2 h at 42.degree. C. and 
then hybridized with .sup.32 P-labeled DNA probes at 1-2.times.10.sup.6 
units/ml in Stark's buffer at 42.degree. C. for 12-24 h. After 
hybridization, the blots are washed and autoradiographed as described 
above for tail blots. 
F. RNase protection assays. Total RNA (0.1-2 .mu.g) is denatured for 5-10 
min at 85.degree. C. in a mixture containing 1-2.times.10.sup.5 cpm/tube 
of an antisense RNA probe, 80% formamide, 40 mM PIPES. 0.4M NACl, 1 mM 
EDTA. After overnight hybridization at 52.degree.-53.degree. C., the 
sample is incubated with RNase A (100 .mu.g/ml) and RNase T1 (20 U/ml) for 
30 min at room temperature, followed by digestion with proteinase K (500 
.mu.g/ml) for 30-45 min at 37.degree. C. Both hybridization temperature 
and concentrations of RNase may be varied as needed depending on the 
particular probe. After phenol/chloroform extraction, the RNA remaining in 
the aqueous phase is ethanol precipitated in the presence of carrier tRNA, 
washed in 80% ethanol, desiccated, and redissolved in 80% formamide/TBE 
buffer. After heat-denaturation at 100.degree. C. for 5 min, the samples 
are loaded onto a5% acrylamide, 8M urea sequencing gel, electrophoresed, 
and then autoradiographed at -80.degree. C. 
G. Reverse-transcriptase-polymerase chain reaction (RT-PCR) analysis. Total 
RNA (1-2 .mu.g) is heat-denatured at 90.degree. C. for 5 minutes, rapidly 
iced, and then allowed to anneal to random hexamers for 5 minutes at room 
temperature. Reverse-transcription using murine Moloney virus reverse 
transcriptase enzyme is performed at 42.degree. C. for 1 hr as described 
(79). The reaction mixture is incubated at 95.degree. C. for 5 minutes to 
denature the RT enzyme activity and is then stored at -80.degree. C. until 
PCR is performed. For PCR, 1/1000th to 1/20th of the total RT reaction is 
amplified using 1 .mu.M of specific 5' and 3' primers, 200 .mu.M of all 
four dNTPs, and 40 U/ml of Taq polymerase in PCR buffer (Amersham Corp., 
Arlington Heights, Ill.). The reaction mixture is overlayered with mineral 
oil and then incubated in a thermal cycler. Standard reaction conditions 
are 30 cycles of denaturation at 95.degree. C. for 1 min., annealing at 
58.degree. C. for 1 min., and extension at 72.degree. C. for 2 min. 
H. Southern transfer and hybridization. Aliquots of PCR reaction mixtures 
or restriction-endonuclease digested DNA are electrophoresed in 
Tris-borate agarose gels. The gel is sequentially soaked in 0.2N HCl for 
10 min., 1.5M NACl, 0.5N NaOH for 30 min, and 1M Tris-Cl (pH 7.4), 1.5M 
NaCl for 30 min. The DNA is then capillary transferred to nylon membrane 
(MSI, Lowell, Mass.) using 20.times. SSC (pH 7.0). The blot is UV shadowed 
while moist, baked at 80.degree. C. for 1 hr, and then hybridized with 
.sup.32 P-labeled DNA probes (1.times.10.sup.6 cpm/ml) in Stark's buffer 
(113) for 6-18 hrs in a rotating oven at 42.degree. C. In the analysis of 
PCR products, to prevent the detection of artifacts all probes should 
consist of sequences which are internal to the primers used for the PCR 
amplification. Blots are sequentially washed in 6.times. SSC, 0.1% SDS for 
30 min. at42.degree. C., and 0.2.times. SSC, 0.1% SDS for 30 min. at 
65.degree. C. After washing, blots are autoradiographed at -80.degree. C. 
in cassettes with intensifying screens. Scanning densitometry is used to 
quantitate the intensity of bands on autoradiographs. 
I. Enzymo- and immunohistochemical analysis of bone tissue. Simultaneous 
determination of bone alkaline phosphatase and acid phosphatase activity 
in situ is performed using the protocol of Liu et al. (105). Tibiae and 
femora are fixed in 70% ethanol overnight and then embedded in 
glycolmethacrylate using an embedding kit (Polysciences) as described 
(105). Sections (5 .mu.m) are cut with a sliding microtome, mounted, and 
air-dried. Sections are first stained for alkaline phosphatase activity by 
incubation with a mixture of naphthol ASTR phosphate and a coupling azo 
dye, Fast Blue BB, at pH 9.0 for 30 min at 37.degree. C. After thorough 
rinsing with distilled water, the sections are stained for acid 
phosphatase activity by incubating with naphthol ASTR phosphate with 
hexazotized parasoaniline in the presence of tartrate (10 mM) at pH 5.0 at 
37.degree. C. for 1 hr. Using this procedure, alkaline phosphatase 
activity stains blue to purple while acid phosphatase activity stains red. 
This technique has been successfully used for murine, rat, as well as 
human bone tissue (105). Alternatively, separate staining procedures can 
be performed for acid phosphatase and alkaline phosphatase activity as 
previously described (104, 106). 
Osteocalcin is detected immunohistochemically using a modification of the 
method of Ohta et al. (107). Bone tissue is rapidly fixed in 
periodate-lysine-paraformaldehyde solution at 4.degree. C. for 6 hr. The 
tissue is rinsed in 20% polyethylene glycol (MW 20,000.+-.5,000) in PBS 
(pH 7.4) and frozen in liquid nitrogen. Sections (5 .mu.m) are cut on a 
cryostat and mounted on albumin-coated slides. Slides are incubated with 
0.6% hydrogen peroxide in methanol for 30 min at room temperature to block 
endogenous peroxidase activity. Sections are then sequentially incubated 
in 5% skim milk, goat anti-mouse osteocalcin antibody (Biomedical 
Technologies, Stoughton, Mass.), peroxidase-conjugated F(ab').sub.2 
fragments of donkey anti-goat IgG (Pel-Freez, Rogers, Ark.), followed by 
rinsing in complete Graham-Karnovsky medium (0.03% 3,3'-diaminobenzidine, 
0.01% H.sub.2 O.sub.2 in 0.05 Tris-HCl, pH 7.6). Sections are mounted on 
glycerin-agar. 
J. Serum endocrine and metabolic studies. Serum osteocalcin and 
corticosterone are determined using commercially available RIA kits 
osteocalcin: Biomedical Technologies, Stoughton, Mass.; corticosterone: 
Analytics, Gaithersburg, Md.). The osteocalcin assay only requires 10 
.mu.l of serum to be performed. The remaining hormone assays require 50 
.mu.l of serum per test. Serum calcium, phosphorus, creatinine, total 
protein concentrations, and alkaline phosphatase enzyme activity are 
measured using a Kodak EktaChem 700XR Analyzer. The analyzer requires 10 
.mu.l of serum or plasma per test. Blood (0.20 ml) can be drawn from the 
retro-orbital plexus of transgenic and littermate control mice every two 
weeks until sufficient serum is available for these studies. Serum is 
isolated from blood using Microtainer serum separators (Beeton Dickinson), 
a system which maximizes serum recovery from small blood volumes. Blood 
draws of this amount and frequency are well-tolerated and do not result in 
anemia. Serum for PTH assays is obtained from animals which are to be 
sacrificed for histomorphometric studies. Approximately 400-500 .mu.l of 
serum can be obtained from a single adult mouse by cardiac puncture 
immediately after euthanasia. Biologically active murine PTH is measured 
using an RIA developed for the rat which crossreacts with murine PTH. This 
assay requires 200 .mu.l of serum per test. 
K. Bone histomorphometric analysis. Mice receive intraperitoneal injection 
of 30 mg/kg of tetracycline hydrochloride in sterile normal saline days 5 
and 2 before sacrifice. All mice are injected at approximately 10 AM to 
avoid possible interference with skeletal circadian rhythmicity (115). 
Mice are euthanized and fixed in cold neutral formalin. The third caudal 
vertebrae is taken, and nondecalcified sections histomorphometrically 
quantitated according to the method of Marie et al. (119). 
L. Microradiography. Formalin-fixed mice or disarticulated mouse skeletal 
tissues are washed, patted dry, and placed on a thin sheet of polyethylene 
film that is in contact with either Kodak high-resolution film or type 1-A 
glass plates. Radiographs are exposed in a Hewlett-Packard Faxatron 805 
unit at 90 kV for 90 min at a focal film distance of 60 cm (120,121). 
Microradiographs are photographed with a Wild-Heebruge M-400 
Photomakroskop camera system using Tech-Pan film. The film is developed in 
HC-110 developer, solution B for 5 min at 68.degree. F. 
M. Bone marrow chimeras and adoptive transfer. Mice are irradiated with 
1000 Rads from a cesium source and on the same day are injected 
intravenously with 1.times.10.sup.7 bone marrow cells. Chimeras are 
analyzed 6-12 weeks later for reconstitution by immunofluorescent staining 
of peripheral blood cells for T and B lymphocyte markers. These studies 
are performed using transgenic (lck-IL-4) donors and wild-type recipients 
congenic for the C57BL/6 background. If disease is transferred by bone 
marrow transplantation, some wild type recipients are thymectomized prior 
to transplantation to determine if the osteoporotic phenotype requires the 
presence of donor-derived T-lineage cells. In addition, unirradiated 
wild-type mice are injected with 5.times.10.sup.6 purified peripheral T 
cells from lck-IL-4 mice, to determine if this is sufficient to cause 
osteoporosis. The converse experiment; transplantation of normal (C57BL/6) 
bone marrow into lck-IL-4 mice; is also performed, to determine if this 
procedure cures them of the osteoporotic phenotype. 
N. In vivo antibody treatments. Mice are given weekly 10 mg injections of 
purified monoclonal antibodies (mAbs against IL-4, IL-4 receptor, NK1.1, 
or Qa-2) or asialo-GM.sub.1 antisera (78,85,87-89). The asialo-GM.sub.1 
antisera is available from commercial sources (Wake Chemicals, Richmond, 
Va.). For mice younger than three weeks of age the intraperitoneal route 
is used. For mice older than three weeks of age, the intravenous route is 
used, since this is technically feasible and insures that systemic levels 
of antibody are achieved. Most of the perturbations of thymic development 
in lck-IL-4 mice are reversed by four weekly injections of anti-IL-4 mAb 
given either IV or IP (35). 
O. Cloning and characterization of the transgenic integration site. In the 
event that the osteoporotic phenotype of the #1315 line of lck-IL-4 mice 
is unique to this line, and does not appear to be IL-4-mediated, the 
transgenic integration site in these animals is cloned. A genomic library 
for the #1315 line is prepared by standard methods: High-molecular weight 
genomic DNA is isolated from splenocytes by digestion in proteinase K (100 
.mu.g/ml) in digestion buffer (122) for 12-18 hr at 50.degree. C. The 
digest is sequentially extracted with phenol, phenol-chloroform, and 
chloroform. The aqueous phase is dialyzed against 100 volumes and two 
changes of TE buffer for 24 hrs at 4.degree. C. Restriction digestion of 
the DNA is necessary for it to be ligated into phage vectors. To determine 
the most appropriate restriction enzyme for this purpose, Southern blots, 
in which the genomic DNA has been digested with various enzymes which cut 
the lck-IL-4 transgenic construct internally, are hybridized with an hGH 
segment probe. Genomic DNA is digested with a restriction enzyme which 
yields hGH-genome fragments between 10-20 kb in size and for which there 
is a site in a .lambda. cloning vector. Ideally, the enzyme yields 
fragments in this size range for both sides of the integration site. A 
variety of .lambda. vectors with various cloning sites are commercially 
available for this purpose (e.g., Stratagene). The restricted DNA is 
size-fractionated on sucrose gradients to enrich for the desired fragment 
(123). This size-fractionated DNA is ligated to a compatibly-digested 
.lambda. vector to produce a subgenomic DNA library. Small-scale test 
ligations are initially performed using a set amount of vector and varying 
amounts of insert. The conditions which result in the greatest number of 
phage clones when packaged and plated on host bacteria are then used in a 
large scale reaction. A library of about 500,000 clones should be more 
than sufficient to include the integration site. Phage plaques bound to 
nitrocellulose filters are denatured in 0.2M NaOH/1.5M NaCl, neutralized 
with 0.4M Tris-Cl/2.times. SSC (pH 7.6) and then 2.times. SSC. The filters 
will be baked for 90 min in vacuo at 80.degree. C., and then hybridized 
with an hGH segment probe. Hybridization conditions are as described above 
for tailblots. Positive plaques are confirmed and purified by repetitive 
screening. 
DNA inserts in positive plaques are subcloned into plasmids or, if 
applicable, directly excised as phagemids by co-infection with helper 
phage, e.g., in .lambda. Zap vectors (Stratagene). Subcloned inserts are 
characterized by restriction mapping and by dideoxynucleotide chain 
termination sequencing. Genomic fragment segments in these inserts are 
used to probe Southern blots of genomic DNA from transgenic and 
nontransgenic #1315 littermates to determine if genomic DNA flanking the 
integration site has been deleted and/or rearranged. The isolation of 
these deleted sequences requires additional screening of genomic libraries 
from normal mice using genomic DNA probes contiguous to the transgene 
integration site. Normal murine genomic phage libraries suitable for this 
purpose are available from commercial sources. Once sequences which are 
contiguous to the transgene or have been deleted by its integration are 
isolated in phage clones, they are analyzed for the presence of coding 
sequences using the exon amplification strategy of Buckler et al. (103). 
This is a PCR-based procedure which identifies exon sequences within 
genomic DNA which have functional 5' and 3' splice acceptor sites. Genomic 
DNA sequences to be analyzed are subcloned into the cloning site of the 
pSPL1 vector (103). The vector is electroporated into COS-7 cells. 
Cytoplasmic RNA is isolated from the cells 48-72 hrs later. The RT 
procedure is performed as described above in section F, except that a 3' 
primer contained in the vector is substituted for random hexamers. The RT 
reaction is subjected to PCR amplification using appropriate 5' and 3' 
vector exon primers as described (103). PCR products are analyzed by 
agarose gel electrophoresis and ethidium bromide staining. Products which 
contain potential exon inserts are cloned and sequenced. If the obtained 
sequences are novel or incompletely characterized for their expression in 
bone tissue, they are .sup.32 P-labeled by the random primer method and 
used to probe RNA blots containing samples from various tissues, including 
bone marrow and whole bone tissue, as well as osteoblast and osteosarcoma 
cell lines. In the event that this PCR procedure does not identify 
functional genes in flanking DNA, an alternative approach is to screen for 
transcripts using probes derived from nonrepetitive regions of genomic DNA 
which flank or are deleted at the integration site. These probes are also 
used to test for the presence of homologous sequences in human genomic DNA 
using the Southern Blot technique. This approach contributed importantly 
to the successful cloning of the cystic fibrosis gene by positional 
methods (124). The integration site is also mapped using in situ 
chromosomal analysis (see below); (125). Once the chromosomal region of 
transgene integration is determined, linkage analysis is performed by 
crossing lck-IL-4 mice with available informative strains of mice whose 
mutations map to this region, to permit more detailed mapping of the 
integration site. 
Those probes which detect novel RNA transcripts are then used to screen 
appropriate cDNA libraries to obtain the complete cDNA coding sequence. If 
novel cDNAs are found which have been disrupted or deleted by transgene 
integration, polyclonal antiserum against these gene products is generated 
to help further characterize their expression in tissues. The amino acid 
sequence of the gene product is deduced from the cDNA sequence. Peptides 
of the predicted protein which are hydrophilic and 10-15 residues in 
length are made using an automated solid-phase peptide synthesizer. The 
peptides are conjugated with and used to immunize rabbits as previously 
described (126). Subsequent experiments to determine the role of these 
gene products in bone remodeling depend, in large part, on clues to their 
function provided by previously cloned homologous gene products. 
P. Chromosomal in situ hybridization. Metaphase chromosome spreads are 
prepared from PHA-stimulated C57BL/6 murine lymph node cells using 
standard cytogenetic methods (127). Chromosomal in situ hybridization is 
performed according to the method of Morton et al. (128) using 
high-specific activity tritiated DNA probes. After washing, slides are 
stained with 0.005% quinacrine mustard dihydrochloride. Metaphases are 
evaluated for hybridization by microscopy using a combination of incident 
ultraviolet and transmitted visible light (128). 
Q. Vertebrate animals. 
(1) Mice may be used exclusively in these experiments. The following is a 
listing of the strains, ages, sex, and numbers of animals which can be 
used for the described work: 
a) C57BL/6 mice bearing the lck-IL-4 and, potentially, the ost-IL-4 
transgene, are bred. 
b) C57BL/6.times.DBA/2 F.sub.1 ; both sexes; 5-20 weeks old. These mice are 
bred together. F.sub.2 mouse zygotes obtained from these mice are 
microinjected with transgene constructs and then transferred to the 
oviducts of pseudopregnant female Swiss-Webster mice. 
c) Swiss-Webster; females; 5-20 weeks old. These mice are used as surrogate 
mothers for embryos after their microinjection. They are induced into a 
pseudopregnant state by mating with vasectomized SJL male mice. 
d) SJL; male; 5-20 weeks old. These mice are vasectomized and then mated 
with Swiss-Webster mice to induce a pseudopregnant state suitable for 
embryo transfer. 
e) C57BL/6; both sexes; 5-20 weeks old. These mice are bred with transgenic 
founders and progeny to maintain transgenic lines. 
f) BALB/c nude (nu/nu); females; 5-20 weeks of age. These mice are used to 
produce ascites containing high titers of anti-IL-4, anti-IL-4 receptor, 
anti-Qa-2, anti-NK1.1 monoclonal antibodies. These antibodies are then 
purified from ascites and used for in vivo treatments as discussed above. 
(2) The lck-IL-4 murine model of osteoporosis is unique, since to our 
knowledge, no other animals develop severe generalized osteoporosis on a 
genetic basis. Mice are also ideal animals for performing studies in which 
various components of the immune system, e.g., T-lineage cells and/or NK 
cells, are depleted. The monoclonal antibody reagents and procedures 
necessary for these in vivo experiments have been extensively developed 
for use in rodents. 
Citations 
1. Whyte MP, et al. Amer J Med 72:193-202, 1982. 
2. Culliton BJ. Science 235:833-834, 1987. 
3. Stevenson JC. Obstet & Gynecol 75(S):36S-41S, 1990. 
4. Schot LPC, et al. J Steroid Biochem Molec Biol 37:167-182, 1990. 
5. Consensus Development Conference. Amer J Med 90:107-110, 1991. 
6. Jackson JA, et al. Medicine 69:137-152, 1990. 
7. Parfitt AM. Calcif Tissue Int 36:S37-S45, 1984. 
8. Mundy GM. Recent Prog Hormone Res 45:507-531, 1989. 
9. Parfitt AM, et al. In Osteoporosis, C. Christiansen et al. (eds). 
Proceedings from the Copenhagen Intl Symp on Osteoporosis, Jun. 3-8, 1984, 
Aalborg Stiftsbogtrykkeri, pp. 111-120. 
10. Mundy GR. Bone 8(Suppl 1):S9-S16, 1987. 
11. Yoshida H, et al. Nature 345:442-443, 1990. 
12. Soriano P, et al. Cell 64:693-702, 1991. 
13. Nowak R. J NIH Res 3:54-58, 1991. 
14. Thomson BM, et al. J Exp Med 164:104-112, 1986. 
15. Thomson BM, et al. J Immunol 138:775-779, 1987. 
16. McSheehy PMJ, et al. J Clin Invest 80:425-429, 1987. 
17. Mundy GR, et al. Ann NY Acad Sci 593:91-97, 1990. 
18. Canalis E, et al. Annu Rev Med 42:17-24, 1991. 
19. Stein GS, et al. FASEB J 4:3111-3123, 1990. 
20. Robey PG. Endocrinol Metabol Clin N Amer 18:859-902, 1989. 
21. Van PT, et al. Cell Tissue Res 225:283-292, 1982. 
22. Malone JD, et al. J Cell Biol 92:227-230, 1982. 
23. Saffar JL, et al. Bone 11:369-372, 1990. 
24. Fallon MD, et al. Calcif Tissue Int 35:29-31, 1983. 
25. Silberstein R, et al. Bone 12:227-236, 1991. 
26. Galli SJ. Lab Invest 62:5-33, 1990. 
27. Witte ON. Cell 63:5-6, 1990. 
28. Paul WE, et al. Ann Rev Immunol 5:429-459, 1987. 
29. Jansen JH, et al. Blut 60:269-274, 1990. 
30. Postlethwaite AE, et al. J Clin Invest 87:2147-2152, 1991. 
31. Howells G, et al. Eur J Immunol 21:97-101, 1991. 
32. Satoh T, et al. Proc Natl Acad Sci USA 88:3314-3318, 1991. 
33. Finkelman FD, et al. Proc Natl Acad Sci USA 83:9675, 1986. 
34. Finkelman FD, et al. J Immunol 141:2335, 1988. 
35. Lewis DB, et al. J Exp Med 173:89-100, 1991. 
36. Garvin AM, et al. Mol Cell Biol 8:3058-3064, 1988. 
37. Perlmutter RM, et al. Biochem Biophys Acta 948:245-262, 1988. 
38. Perlmutter RM, et al. J Cell Biochem 38:117-126, 1988. 
39. Marth JD, et al. Cell 43:393-404, 1985. 
40. Carding SR, et al. Proc Natl Acad Sci USA 86:3342, 1989. 
41. Palacios R, et al. EMBO J 16:91, 1987. 
42. Barcena A, et al. J Exp Med 172:439, 1990. 
43. Garvin AM, et al. Internatl Immunol 2:173, 1990. 
44. Watanabe K, et al. Biochem Biophys Res Commun 172: 1035-1041, 1990. 
45. Shioi A, et al. J Cell Biochem, in press, 1991. 
46. Hart PH, et al. Proc Natl Acad Sci USA 86:3803-3807, 1989. 
47. Standiford TJ, et al. J Immunol 145:1435-1439, 1990. 
48. Lehn M, et al. J Immunol 143:3020-3024, 1989. 
49. Standiford TJ, et al. Biochem Biophys Res Commun 171:531-536, 1990. 
50. Rennick D, et al. Proc Natl Acad Sci USA 84:6889-6893, 1987. 
51. Lacey DL, et al. J Bone Mineral Res 6(supplement 1):S255, 1991. 
52. Imai Y, et al. J Bone Mineral Res 5:393-399, 1990. 
53. Rosen CJ, et al. J Bone Mineral Res 5:851-855, 1990. 
54. Ernst DN, et al. J Immunol 145:1295-1302, 1990. 
55. Lewis DB, et al. Proc Natl Acad Sci USA 85:9743-9747, 1988. 
56. Lewis DB, et al. J Clin Invest 87: 194-202, 1991. 
57. Pacifici R, et al. Proc Natl Acad Sci USA 84:4616-4620, 1987. 
58. Killar LM, et al. Eur J Immunol 19:2205-2210, 1989. 
59. Brown MA, et al. Cell 50:809-818, 1987. 
60. Fallon MD, et al. Human Pathol 12:813-820, 1981. 
61. Harvey JA, et al. Bone 10:237-241, 1989. 
62. Leung DY, et al. Hematol/Oncol Clin N Amer 2:81-100, 1988. 
63. Vercelli D, et al. J Clin Invest 85:1666-1671, 1990. 
64. Kalu DN, et al. Endocrinology 124:7-16, 1989. 
65. Matzsch T, et al. Thrombosis and Haemostasis 56:293-294, 1986. 
66. Glajchen N, et al. Calcif Tissue Int 43:277-280, 1988. 
67. Tsuboyama T, et al. Bone 10:269-277, 1989. 
68. Banchereau J, et al. Bull Cancer (Paris) 78:299, 1991. 
68a. Lian J. et al. Proc Natl Acad Sci USA 86:1143, 1989. 
69. Noma Y, et al. Nature (Lond.) 319:640, 1986. 
70. Chaffin KE, et al. EMBO J 9:3821-3829, 1990. 
71. Maniatis T, et al. Cold Spring Harbor Laboratory Press, Cold Spring 
Harbor, N.Y., 1982. 
72. Seeburg PH. DNA (NY) 1:239, 1982. 
73. Brinster RL, et al. Proc Natl Acad Sci USA 85:836-840, 1988. 
74. Brinster RL, et al. Proc Natl Acad Sci USA 82:4438, 1985. 
74a. Garn SM, et al. Radiology 100:509, 1971. 
74b. Riggs BL, et al. New Eng. J. Med. 314:1676, 1986. 
75. Teitelbaum SL. Human Pathol 15:306-323, 1984. 
75a. Marie P J, et al. Metabol. 34:777, 1985. 
76. Spiegel AM. J. Bone Miner. Res. 6:515, 1991. 
76a. Moss DW. Clin. Biochem. 20:225, 1987. 
76b. Robey PG. Endocrin. Metabol. Clin. N. Amer. 18:859, 1989; Price PA, et 
al. J. Clin. Invest. 66:878, 1980. 
77. Hu-Li J, et al. J Immunol 142:800, 1989. 
78. Ohara J, et al. Nature (Lond) 315:333, 1985. 
79. Kawasaki ES. IN PCR Protocols: A Guide to Methods and Applications, 
Academic Press, 1990, pp. 21-27. 
80. Mohler KM, et al. J Immunol 145:1734-1739, 1990. 
81. Otsuka T, et al. Nucleic Acids Res 15:333-344, 1987. 
82. Brenner CA, et al. BioTechniques 7:1096-1103, 1989. 
83. Tepper RI, et al. Cell 62:457-467, 1990. 
83a. Burstein HJ, et al. J Immunol 147:2950-2956, 1991. 
83b. Muller, et al. Eur J Immunol 21:921-925, 1991. 
84. Biondi A, et al. Eur J Immunol 21:843-846, 1991. 
85. Beckmann MP, et al. J Immunol 144:4212-4217, 1990. 
86. Fowlkes BJ, et al. Adv Immunol 44:207-262, 1989. 
87. Sharabi Y, et al. J Exp Med 171:211-219, 1990. 
88. Gately MK, et al. J Immunol 141:189-200, 1988. 
89. Koo GC, et al. Hybridoma 3:301, 1984. 
90. Kerner SA, et al. Proc Natl Acad Sci USA 86:4455, 1989. 
91. Schule R, et al. Cell 61:497-504, 1990. 
92. Woychik RP, et al. Nature 318:36, 1985. 
93. Ratty AK, et al. Molecular Brain Res 8:355-358, 1990. 
94. Bier DR, et al. Genomics 4:498-504, 1989. 
95. Xiang X, et al. Science 247:967-969, 1991. 
96. McNeish JD, et al. Science 241:837-839, 1988. 
97. Covarrubias L, et al. Mol Cell Biol 7:2243-2247, 1987. 
98. Woychik RP, et al. Nature 346:850, 1990. 
99. Maas RL, et al. Nature 346:853, 1990. 
100. Bishop JO, et al. Mol Biol Med 6:283-298, 1989. 
101. Covarrubias L, et al. Proc Natl Acad Sci USA 83:6020-6024, 1986. 
102. Forrester WC, et al. Genes Develop 4:1637-1649, 1990. 
103. Buckler AJ, et al. Proc Natl Acad Sci USA 88:4005-4009, 1991. 
104. Fallen D, et al. Calcif Tissue Int 33:281-282, 1981. 
105. Liu C, et al. Histochem 86:559-565, 1987. 
106. Chappard D, et al. Basic Appl Histochem 27:75-85, 1983. 
107. Ohta T, et al. Virchows Arch A Pathol Anat 415:459-466, 1989. 
108. Lian JB, et al. J Clin Orthop Rel Res 226:267-291, 1988. 
109. Depaolo LV, et al. In The Clinical Chemistry of Laboratory Animals, WF 
Loeb et al. (eds), Pergamon Press, p 279-308, 1989. 
110. Karanth S, et al. Proc Natl Acad Sci 88:2961-2965, 1991. 
111. Idzerda RL, et al. Molec Cell Biol 9:5154-5162, 1989. 
112. Palmiter RD, et al. Annu Rev Genetics 20:61, 1986. 
113. Cosman D, et al. Nature (Lond) 312:768-771, 1984. 
114. Boyum A. Tissue Antigens 4:269, 1974. 
115. Mishell BB, et al. Selected Methods in Cellular Immunology, WH Freeman 
and Co Pub, NY, 1980, pp. 3-27. 
116. Julius MH, et al. Eur J Immunol 3:645-649, 1973. 
117. Hathcock K. In Current Protocols in Immunology, J Coligan, et al. 
(eds), J Wiley & Sons, 1991, p. 3.3.1. 
118. Glisin V, et al. Biochemistry 13:2633, 1974. 
119. Marie PJ, et al. Metabolism 34:777-783, 1985. 
120. Effman EL. In Models and Techniques in Medical Imaging Research, E 
Milne et al. (eds), Praeger, 1983, p. 164-181. 
121. Effman EL. Invest Radiol 17:529-538, 1982. 
122. Strauss WM. In Current Protocols in Molecular Biology, FM Ausubel et 
al. (eds), J Wiley & Sons, 1991, p 2.2.1. 
123. Weis JH, et al. Ibid, p 5.3.1. 
124. Rommens JM, et al. Science 245:1059-1065, 1989. 
125. Marth JD, et al. Proc Natl Acad Sci 83:7400-7404, 1986. 
126. Marth JD, et al. EMBO J 6:2727-2734, 1987. 
127. Moorhead, et al. Exp Cell Res 20:613-616, 1960. 
128. Morton CC, et al. Am J Hum Genet 36:576-585, 1984. 
While the preferred embodiment of the invention has been illustrated and 
described, it will be appreciated that various changes can be made therein 
without departing from the spirit and scope of the invention.