(Received for publication, April 6, 1994; and in revised form, November 21, 1994)
From the
Osteoclastic bone resorption is dependent upon cell-matrix
recognition. This process is mediated by the integrin
whose expression is enhanced, in
avian osteoclast precursors, by bone-seeking steroids. The purpose of
this study was to determine if bone-modulating cytokines impact on
expression by mouse marrow
macrophages (BMMs), known to differentiate into osteoclasts. Of the
cytokines tested. Interleukin-4 (IL-4) is most effective in increasing
mRNA levels by a mechanism involving transactivation
of the
gene. Moreover, IL-4 augmented
mRNA is mirrored by plasma membrane appearance of
. As IL-4 induces
and not
mRNA, the
chain
appears to regulate surface expression of the heterodimer. The
functional significance of IL-4-induced
is underscored by the fact that, while attachment to fibronectin
is unaltered, treatment of BMMs with the cytokine enhances
-mediated binding to vitronectin
5-fold. Expression of this heterodimer by BMMs driven along a
non-osteoclastic lineage suggests
may play a role in the inflammatory response of macrophages.
Integrins are transmembrane heterodimers consisting of
individual and
subunits. Many of these complexes recognize
and anchor cells to extracellular matrices, events associated with
transmission of signals across the plasma
membrane(1, 2) .
In terms of the osteoclast, the
principal if not exclusive resorptive cell of bone, we (3) and
others (4, 5) have shown that the integrin
is pivotal to its matrix recognizing
and degradative activity. This observation prompted us to explore the
effect, on
expression, of agents
known to impact osteoclast function. In this regard, we find the
bone-seeking steroids, 1,25-dihydroxyvitamin D
(6) and retinoic acid (7) , both of which
stimulate osteoclastogenesis, enhance expression by avian osteoclast
precursors of
, an event associated
with accelerated transcription of the
subunit gene.
Cytokines also impact on osteoclast function. For example,
osteoclastogenesis is accelerated by IL-1(8) ,
IL-6(9, 10) , and TNF
(11) and suppressed
by IL-4(12, 13) . Prompted by our observation that
bone-seeking steroids regulate the
integrin subunit
and cell surface appearance of
, we
asked if cytokines effect expression, by murine osteoclast precursors,
of the same heterodimer. We find that a number of cytokines augment
mRNA steady state levels, the most potent being IL-4.
Transactivation of the
gene by IL-4 is followed by
increased plasma membrane appearance of
and specific enhancement of binding, by treated cells, of the
ligand, vitronectin. We believe
these findings are particularly significant as we have recently shown
that overexpression of IL-4 gene in the mouse leads to a form of
osteoporosis similar to that developing in post-menopausal
women(14) .
Figure 1:
Various cytokines modulate integrin mRNA expression. Adherent murine BMMs maintained in
M-CSF (500 units/ml) were treated with various cytokines for 24 h.
Equal amounts of total RNA were probed with rat
cDNA
by Northern analysis. IL-4 increases steady state
message migrating as a major 3.6 kb and minor 4.4 kb species
5-fold. IL-6, TNF
, and GM-CSF also up-regulate
integrin subunit mRNA levels up to 2-fold. IL-1
, IL-3,
IFN
, and hTGF-
, on the other hand, do not alter
message.
Because IL-4 is the most potent of
the agents tested, we further characterized its inductive properties. We find the phenomenon is both dose- (Fig. 2) and time- (Fig. 3) dependent.
mRNA is enhanced after 6 h of treatment at a concentration of
IL-4 as low as 10 units/ml. The effect maximizes (5-fold induction) 24
h after a single addition of 50 units/ml cytokine followed by a decline
to basal levels at 72 h. In contrast to
, IL-4 does
not alter
mRNA expression ( Fig. 2and Fig. 3).
Figure 2:
IL-4 increases , but not
, integrin mRNA expression in a dose-dependent manner.
Murine BMMs were exposed to various concentrations of IL-4 or vehicle
for 24 h, and equal amounts of RNA were analyzed by Northern blotting.
mRNA is augmented by IL-4 in a dose-dependent manner
with maximal induction (5-fold) occurring with 50 units/ml. In
contrast, the cytokine does not impact
mRNA
levels.
Figure 3:
IL-4
increases , but not
, integrin mRNA
levels in a time-dependent manner. BMMs were exposed to a single dose
of IL-4 (50 units/ml) or vehicle at time 0, and after various periods
of culture equal amounts of total RNA analyzed by Northern blotting.
IL-4 increases steady state levels of
integrin
expression within 6 h of treatment. Maximal induction occurs by 24 h,
followed by a decline to basal levels at 72 h. In contrast, the
cytokine does not impact on
mRNA.
The results presented thus far do not distinguish
between accelerated gene transcription and message
stabilization prompting us to perform nuclear run-on experiments. As
shown in Fig. 4, IL-4 treatment of murine marrow macrophages
specifically transactivates the
integrin gene.
Figure 4:
IL-4
transactivates the integrin gene. Equal amounts of
[
-
P]UTP-labeled nascent mRNA chains were
hybridized to slot-blotted vector DNA,
cDNA, and
G3PDH cDNA. IL-4 treatment specifically accelerates
gene transcription.
We
have previously shown that our population of marrow cells consists
exclusively of those dependent on M-CSF(16) . Reflecting
previous reports(20) , we also find IL-4 treatment of these
cells promotes their multinucleation. This observation raised the
possibility that mRNA expression may vary between
mono- and multinucleated cells, respectively. To address this issue, we
performed in situ hybridization on control and IL-4-treated
cells. As seen in Fig. 5, control cultures, containing only
M-CSF, do not form polykaryons and express relatively low levels of
mRNA. While all IL-4-treated macrophages contain more
mRNA than do their untreated counterparts, the
mononuclear population is particularly rich in the message.
Figure 5:
IL-4-treated mononuclear and
multinucleated macrophages express increased levels of integrin mRNA. Digoxigenin-labeled sense and antisense rat
cRNA probes, transcribed from cloned partial rat
cDNA, were used for in situ hybridization
analysis. A, IL-4-treated macrophages form numerous
polykaryons (arrows). While all cells contain more
than do their untreated counterparts, the mononuclear population
is particularly rich in the message (
200). B, control
cultures do not form polykaryons and express relatively low levels of
mRNA in the cytoplasm (
200). C,
IL-4-treated macrophages hybridized with sense cRNA probe, as negative
control, show no significant reaction
(
200).
Figure 6:
IL-4 induces plasma membrane expression of
in a dose-dependent manner. Adherent
population of murine BMMs, treated with various concentrations of IL-4
or vehicle, were surface labeled by
I, lysed, and the
lysates immunoprecipitated with an anti-
antibody. The
immunoprecipitates were analyzed by SDS-polyacrylamide gel
electrophoresis under non-reducing conditions. IL-4 up-regulates plasma
membrane expression of
dose
dependently with maximal induction (3-fold) at 50 units/ml. M
, molecular weight marker.
Figure 7:
IL-4
induces plasma membrane expression of in a time-dependent manner. BMMs were exposed to IL-4 (50
units/ml) at time 0 and
immunoprecipitates prepared
as described in Fig. 6with time. IL-4 augments
surface-expressed
after 12 h of
treatment. Maximal induction occurs by 36 h. M
,
molecular weight marker.
Figure 8:
IL-4 induces integrin
mRNA and
expressed by adherent and
non-adherent macrophages. Plastic adherent BMMs and those cultured in
Teflon-coated plates were exposed to IL-4 (50 units/ml) for 2 days. The
cells were then analyzed for steady state
mRNA levels
and plasma membrane expression of
.
Although less effectively than in adherent population (A),
IL-4 markedly up-regulates
subunit mRNA levels and
surface expression of
in
non-adherent population (NA) maintained in Teflon-coated
plates.
Figure 9:
IL-4 specifically enhances macrophage
attachment to vitronectin. Murine BMMs maintained in Teflon-coated
plates were treated with IL-4 (50 units/ml) or vehicle for 24 h and
added to each well coated with either vitronectin (VN) or
fibronectin (FN). IL-4 enhances vitronectin binding up to
5-fold, while not effecting attachment to fibronectin. Polyclonal
anti-human antiserum completely
blocks IL-4-induced vitronectin binding while non-immune serum has no
effect. All values represent the mean ± S.D. of the ratio of
binding of IL-4-treated cells/binding of the control cells from
triplicate determinations.
The advent of techniques whereby osteoclasts may be isolated or generated and maintained in culture has yielded insights into the mechanisms whereby these cells degrade bone. The resorptive process apparently requires formation, at the cell-bone interface, of an isolated microenvironment into which protons and acidic proteases are secreted(21, 22) . Thus, the means by which osteoclasts recognize and bind to bone are central to their degradative activity.
The integrin is
expressed on mature osteoclasts (23, 24) and we (3) and others (4, 25) have shown that its
occupancy by blocking antibodies dampens the cells' capacity to
bind to and mobilize bone. Recently, these observations have been
extended to the whole animal as Fisher et al.(4) find
that administration of echistatin, an RGD-containing protein recognized
by
, inhibits bone loss in the
oophorectomized rat. Interestingly,
also serves, in avian osteoclasts, to transmit matrix-derived
signals to the cells' interior, an event which may also impact on
the rate of bone degradation(26) .
The importance of
as a mediator of osteoclastic bone
resorption underscores the rationale for exploring the means by which
the heterodimer is regulated. We recently found that the bone-seeking
osteoclastogenic steroids, 1,25-dihydroxyvitamin D
(6) and retinoic acid(7) , enhance surface
expression of
by marrow-derived,
avian osteoclast precursors.
Osteoclast precursors are hematopoietic
and of monocyte/macrophage lineage(27) . These cells acquire
calcitonin receptors and tartrate-resistant acid phosphatase activity
as they mature and diminish expression of the vitamin D receptor(28) , the latter being essential to osteoclast
commitment(29) . With these observations in mind, and the
demonstration that marrow-derived macrophage progenitors differentiate,
in culture, into osteoclasts (28) , attention has turned to
regulated expression, by these mononuclear cells, of
osteoclast-associated markers.
It is now clear that in addition to
steroids cytokines such as IL-1 (8) ,
IL-6(9, 10) , TNF(11) , and IL-4 (12, 13) modulate osteoclastogenesis. These
observations prompted us to ask if, similar to 1,25-dihydroxyvitamin
D
and retinoic acid, resorption-modulating cytokines impact
on
expression by osteoclast
precursors.
Because our steroid-based studies were performed on
cells derived from chicken(6, 7, 30) , a
species for which few cytokines are available, we turned to
M-CSF-dependent murine marrow-residing osteoclast precursors, cells we
have previously shown can be isolated in homogeneity(16) . In
light of our data that mRNA expression parallels, and
may regulate,
plasma membrane
capacity (6, 7) , we performed Northern analysis on
these mouse marrow-derived cells. We used in these experiments a rat
cDNA, highly homologous to its human counterpart,
reasoning that its homology would extend to the mouse. In fact, during
these studies we cloned a mouse
cDNA which is more
than 90% homologous to the rat probe. (
)Northern analysis of
IL-4-treated macrophages is identical whether one uses the murine or
rat
probe (data not shown).
We find that IL-6,
TNF, and GM-CSF, each of which have been implicated in osteoclast
precursor differentiation(9, 10, 31) ,
up-regulate expression of the
integrin subunit. Of
the cytokines tested, however, IL-4 through transactivation of the
gene, most effectively increases steady state levels
of the message, an event confirmed by in situ hybridization.
In contrast, IL-4 has no effect on
mRNA levels.
mRNA induction by IL-4 is paralleled by surface
expression of
, suggesting the event
may be functionally significant. To explore this issue, we developed a
cell-matrix attachment assay using as a substrate, vitronectin, an
RGD-containing protein and established
ligand(1, 2) . Reflecting plasma membrane
capacity, IL-4 enhances
anti-
antibody inhibitable macrophage
binding to vitronectin as much as 5-fold while not altering attachment
to fibronectin, a circumstance mediated through
integrins(1) . This finding is consistent with the fact
that exposure of murine peritoneal macrophages to IL-4 enhances their
adhesive properties to plastic(32) . While this latter study
offers no insight into the underlying mechanisms by which the cytokine
promotes adherence, the experiments were performed in serum-containing
medium, assuring an abundance of the
ligand, vitronectin.
IL-4 is an immunoregulatory cytokine produced by T-lymphocytes and mast cells, exerting a panoply of effects on mononuclear phagocytes(33) . While reports are inconsistent, the body of evidence indicates the cytokine blunts macrophage proliferation(34) . Thus, we were not surprised to discover that IL-4 is anti-osteoclastogenic in vitro(12) . In keeping with this observation, we recently reported that overexpression of the cytokine in transgenic mice induces osteoporosis histologically similar to that often appearing in post-menopausal patients(14) . In this circumstance bone remodeling, a process initiated by osteoclastic activity, is markedly reduced.
While being clearly anti-osteoclastogenic, IL-4 provokes macrophage precursor differentiation along an immunoregulatory pathway into cells primed to participate in an inflammatory response. For example, IL-4 induces major histocompatibility complex antigen (35) and mannose receptor expression (32) by murine macrophages, and as seen in this and another study(20) , prompts their multinucleation. In this regard, IL-4 overexpression in transgenic mice induces a mononuclear inflammatory ocular lesion(36) .
Given the fact that IL-4 is not expressed by murine marrow cells(14) , it seems unlikely that the cytokine plays a significant role in differentiation of resident macrophage precursors. On the other hand, inflammation is characteristically associated with profound cytokine expression, and it is in this milieu that IL-4 may exert its macrophage differentiating effect, prompting these cells along an antigen-presenting phagocytotic pathway and away from the osteoclast phenotype.