©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interleukin-4 Induces Expression of the Integrin via Transactivation of the Gene (*)

(Received for publication, April 6, 1994; and in revised form, November 21, 1994)

Sohei Kitazawa F. Patrick Ross Kevin McHugh Steven L. Teitelbaum (§)

From the Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Osteoclastic bone resorption is dependent upon cell-matrix recognition. This process is mediated by the integrin alpha(v)beta(3) 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 alpha(v)beta(3) expression by mouse marrow macrophages (BMMs), known to differentiate into osteoclasts. Of the cytokines tested. Interleukin-4 (IL-4) is most effective in increasing beta(3) mRNA levels by a mechanism involving transactivation of the beta(3) gene. Moreover, IL-4 augmented beta(3) mRNA is mirrored by plasma membrane appearance of alpha(v)beta(3). As IL-4 induces beta(3) and not alpha(v) mRNA, the beta(3) chain appears to regulate surface expression of the heterodimer. The functional significance of IL-4-induced alpha(v)beta(3) is underscored by the fact that, while attachment to fibronectin is unaltered, treatment of BMMs with the cytokine enhances alpha(v)beta(3)-mediated binding to vitronectin 5-fold. Expression of this heterodimer by BMMs driven along a non-osteoclastic lineage suggests alpha(v)beta(3) may play a role in the inflammatory response of macrophages.


INTRODUCTION

Integrins are transmembrane heterodimers consisting of individual alpha and beta 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 alpha(v)beta(3) is pivotal to its matrix recognizing and degradative activity. This observation prompted us to explore the effect, on alpha(v)beta(3) expression, of agents known to impact osteoclast function. In this regard, we find the bone-seeking steroids, 1,25-dihydroxyvitamin D(3)(6) and retinoic acid (7) , both of which stimulate osteoclastogenesis, enhance expression by avian osteoclast precursors of alpha(v)beta(3), an event associated with accelerated transcription of the beta(3) subunit gene.

Cytokines also impact on osteoclast function. For example, osteoclastogenesis is accelerated by IL^1-1(8) , IL-6(9, 10) , and TNFalpha (11) and suppressed by IL-4(12, 13) . Prompted by our observation that bone-seeking steroids regulate the beta(3) integrin subunit and cell surface appearance of alpha(v)beta(3), we asked if cytokines effect expression, by murine osteoclast precursors, of the same heterodimer. We find that a number of cytokines augment beta(3) mRNA steady state levels, the most potent being IL-4. Transactivation of the beta(3) gene by IL-4 is followed by increased plasma membrane appearance of alpha(v)beta(3) and specific enhancement of binding, by treated cells, of the alpha(v)beta(3) 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) .


MATERIALS AND METHODS

Cytokines

Recombinant mouse IL-1beta, IL-3, IL-4, IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF), TNFalpha, interferon (IFN) and recombinant human transforming growth factor beta(2) (hTGF-beta(2)) were purchased from Genzyme (Cambridge, MA). Mouse macrophage colony stimulating factor (M-CSF) was purified to Stage 1, by a batch calcium phosphate method (15) , from serum-free medium conditioned by the mouse fibroblastic cell line L929.

Mouse Bone Marrow Macrophages

Bone marrow cells were obtained from 6-week-old male A/J mice (Jackson Laboratories, Benton Harbor, MI) by flushing femora and tibiae with alpha-modified Eagle's medium (alpha-MEM). The cells were cultured in alpha-MEM supplemented with 10% fetal bovine serum in the presence of 500 units/ml M-CSF for 24 h. The non-adherent population was harvested and incubated in Pronase solution (0.02% w/v Pronase, B grade, Calbiochem, 1.5 mM EDTA in phosphate-buffered saline (PBS)) for 15 min at 37 °C. The suspension was layered on horse serum and incubated for 10 min at 4 °C. The bone marrow macrophage (BMM)-enriched population was recovered from the upper layer by centrifugation (1500 revolutions/min for 5 min) and plated at 1.5 times 106 cells/ml in alpha-MEM with 10% fetal bovine serum and 500 units/ml M-CSF for 2 days which yields, as we have described(16) , a pure population of M-CSF-dependent macrophage precursors. The adherent population of BMMs was then treated with cytokines at concentrations known to be biologically effective (IL-1beta 10 units/ml, IL-3 200 units/ml, IL-4 50 units/ml, IL-6 5000 units/ml, GM-CSF 500 units/ml, TNFalpha 105 units/ml, IFN 100 units/ml, and hTGF-beta(2) 500 units/ml) or vehicle for up to 3 days in the presence of 500 units/ml M-CSF. After Pronase treatment, the non-adherent population to be used in the cell attachment assay was maintained in Teflon-coated plates (Nalge, Rochester, NY) in conditions described above.

Northern Blot Analysis

A partial, rat integrin beta(3) subunit cDNA (exons III-XIII) 93% homologous to its human counterpart (17) and less than 50% homologous to any other known beta subunit sequence, kindly provided by Dr. M. Poncz (Children's Hospital of Philadelphia, PA) was [P]dCTP-labeled by the random priming method (Boehringer Mannheim). A partial, chicken integrin alpha(v) subunit cDNA(18) , a kind gift of Dr. B. Bossy (University of California, San Francisco, CA), was used to reprobe the same membrane after striping the beta(3) cDNA. Total cellular BMM RNA was isolated by single-step acid guanidinium thiocyanate-phenol-chloroform (AGPC) extraction. Equal amounts of RNA (10 mg/lane) were electrophoresed in 1.0% agarose gels and transferred to nylon membranes with a vacuum blotter. The membranes were prehybridized with hybridization buffer (5 times SSPE, 5 times Denhardt's solution, 50% formamide, 0.1% SDS, 1 times Background Quencher, Tel-Test, Inc., Friendswood, TX) for 2 h at 42 °C, and hybridized in hybridization buffer with P-labeled probe for 16 h at 42 °C. The membranes were then washed with 2 times SSC, 0.1% SDS for 30 min at 42 °C and 0.2 times SSC, 0.1% SDS for 30 min at 60 °C and exposed to Kodak X-OMAT film for 2 days at -80 °C.

Nuclear Run-on Assay

BMMs were washed twice with ice-cold PBS and collected by scraping in 1 times SSC followed by centrifugation at 1500 revolutions/min for 5 min. Nuclei were extracted in lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl(2), 0.5% Nonidet P-40) for 10 min on ice, and centrifuged at 3000 revolutions/min for 5 min. Pellets were washed twice with lysis buffer and resuspended with 0.5 ml of suspension buffer (50 mM Tris-HCl, pH 8.3, 40% glycerol, 5 mM MgCl(2), 0.1 mM EDTA). Suspended nuclei were placed in 100 ml of reaction mixture (10 mM Tris-HCl, pH 8.0, 5 mM MgCl(2), 300 mM KCl, 0.5 mM each of ATP, CTP, GTP, 100 mCi of [alpha-P]UTP 3000 Ci/mM), and transcribed in vitro for 30 min at 30 °C. After DNase I treatment (final concentration 20 mg/ml) for 30 min at 30 °C, transcription was halted with 200 ml of stop solution (20 mM Tris-HCl, pH 7.4, 2% SDS, 10 mM EDTA, 200 mg/ml of proteinase K) and the nuclei incubated for 30 min at 42 °C. Following extraction in phenol-chloroform, 50 mg of carrier RNA and 5 ml of ice-cold 5% trichloroacetic acid were added to the supernatant, and the solution was incubated for 30 min on ice and filtered through a nitrocellulose membrane (2.5 cm in diameter). The blotted membrane was washed three times with 3% trichloroacetic acid and incubated with 0.9 ml of incubation solution (20 mM HEPES pH 7.5, 5 mM MgCl(2), 1 mM CaCl(2), 25 mg/ml of DNase I) for 30 min at 37 °C. Labeled RNA was eluted by incubation in 30 ml of 0.5 M EDTA and 100 ml of 10% SDS for 10 min at 60 °C and purified by proteinase K (25 mg/ml) treatment for 30 min at 37 °C and ethanol precipitation and finally dissolved in 50 ml of 10 mM Tris-HCl, pH 7.5 and 2 mM EDTA. 10 mg each of rat beta(3) cDNA, mouse glyceraldehyde-3-phosphate dehydrogenase cDNA, and vector DNA were denatured in 50 ml of 0.2 N NaOH for 30 min at 27 °C, neutralized with 500 ml of 6 times SSC, slot-blotted on a nylon membrane saturated with H(2)O and 6 times SSC, and probed with transcribed RNA in hybridization solution (50% formamide, 5 times SSC, 50 mM sodium phosphate, pH 6.5, 1 times Denhardt's solution, 1 times Background Quencher) mixed 50% (v/v) with dextran sulfate for 48 h at 42 °C. The membranes were washed with 2 times SSC, 0.1% SDS for 15 min at 27 °C and with 0.2 times SSC, 0.1% SDS for 30 min at 65 °C and exposed to Kodak X-OMAT film for 2 days at -80 °C.

In Situ Hybridization

The beta(3) cDNA fragment was subcloned into pGEM-3Z vector (Promega, Madison, WI). Digoxigenin-labeled sense and antisense cRNA were transcribed in vitro using Riboprobe Gemini II transcription system (Promega) and digoxigenin-UTP (Boehringer Mannheim) and used as hybridization probes. Treated BMMs cultured in chamber slides (Nunc, Naperville, IL) were washed three times with 100 mM phosphate buffer (PB,pH 7.4), and fixed with 4% paraformaldehyde in 0.1 M PB for 30 min at 4 °C. Slides were washed in 100 mM PB for 10 min and treated with proteinase K (5 mg/ml) for 10 min at 37 °C, followed by refixation with 4% paraformaldehyde in 100 mM PB for 10 min at 27 °C, and acetylation by 100 mM triethanolamine, pH 8.0, with 0.25% glacial acetic acid. After washing twice with 100 mM PB for 1 min each, specimens were dehydrated through graded ethanol and air-dried. Each specimen was hybridized with 100 ml of hybridization solution (50% formamide, 10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 600 mM NaCl, 1 times Denhardt's solution, 0.25% SDS, 10% dextran sulfate, and labeled probe) and incubated at 50 °C for 16-20 h in a moist chamber saturated with 50% formamide 4 times SSC. After hybridization, the specimens were washed with 50% formamide 2 times SSC for 30 min at 50 °C, twice with 2 times SSC for 15 min each at 50 °C, and twice with 0.2 times SSC for 15 min at 50 °C. After washing briefly with 100 mM Tris-HCl, pH 7.5, 150 mM NaCl (TS) with 1.5% non-fat dry milk for 30 min at 27 °C, the specimens were incubated with 1:1000-fold dilution of alkaline phosphatase-conjugated anti-digoxigenin antibody (Boehringer Mannheim) for 45 min at 27 °C, washed twice with TS for 15 min at 27 °C, and briefly with 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl2 (TSM). Development was carried out using Nitro-blue tetrazolium (175 ng/ml) and 5-bromo-4-chloro-3-indolyl phosphate (340 ng/ml) in TSM for 3 h at 27 °C in the dark. After the reaction was stopped with 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, the specimens were mounted with GEL/MOUNT mount (Biomedia, Foster City, CA).

Immunoprecipitation

Treated BMMs were rinsed free of culture medium with PBS and surface labeled by I-lactoperoxidase (19) for 1 h at 27 °C. The cells were lysed in a minimal volume of lysis buffer (10 mM Tris, pH 7.2, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 0.025% NaN(3), 5 mM iodoacetamide, 1 mM CaCl(2), 1 mM MgCl(2), 4 mM phenylmethylsulfonyl fluoride and aprotinin 0.25 units/ml). Each lysate, containing equal trichloroacetic acid-precipitable counts, was precleared with protein G-Sepharose, (Pharmacia Biotech, Inc.) and immunoprecipitated with a polyclonal rabbit anti-beta(3) integrin subunit antibody raised against synthetic peptides corresponding to the cytoplasmic portion of beta(3) integrin-specific amino acid sequence (kindly donated by Dr. L. F. Reichardt, Howard Hughes Medical Institute, University of California, San Francisco, CA). Preliminary studies were performed to determine antibody excess (10 mg/sample) which was used to quantitatively immunoprecipitate cell surface alpha(v)beta(3). The precipitate, recovered with excess protein G-Sepharose, was boiled in electrophoresis sample buffer and subjected to 6% SDS-polyacrylamide gel electrophoresis under non-reducing conditions. The gels were dried and exposed at -80 °C to Kodak X-OMAT film for 2 days and band intensity determined by photospectrometry.

Cell Attachment Assay

96-well flat bottom plates (ICN Titertek, Costa Mesa, CA) were coated with 100 ml of either mouse fibronectin (10 mg/ml, Telios, San Diego, CA) or vitronectin (10 mg/ml, Telios) for 16 h at 4 °C. After washing twice with PBS and blocking with 100 ml of 10 mg/ml of heat denatured bovine serum albumin fraction V (Sigma) for 30 min at 27 °C, 100 ml of suspended BMMs, containing 4 times 106 cell/ml, previously maintained in Teflon-coated plates, were preincubated with either non-immunized rabbit serum or polyclonal antihuman alpha(v)beta(3) antiserum (^2)(catalog no. 12119-012, Life Technologies, Inc.) (dilution 1:200) for 30 min and added to either vitronectin- or fibronectin-coated wells. Attached cells were washed three times with PBS, fixed with 2% formaldehyde for 20 min, rinsed twice with 10 mM borate buffer, pH 8.4, and stained with 1% methylene blue for 10 min. After washing three times with tap water, 100 ml of 0.1 M HCl were added to each well which was incubated for 1 h at 27 °C. Absorbance at 650 nm was determined, which, as established in our laboratory, serves as a function of attached cell number(3) .


RESULTS

Regulation of Expression of beta(3) Subunit mRNA by Various Cytokines

As shown in Fig. 1, beta(3) integrin mRNA, extracted from M-CSF-dependent murine macrophages maintained in that cytokine (500 units/ml) for 24 h (control), migrates as a major 3.6 kb and minor 4.4 kb species. IL-4 increases steady state beta(3) message expression 5-fold. IL-6, GM-CSF, and TNFalpha also up-regulate beta(3) integrin subunit mRNA levels but are less effective, enhancing expression no more than 2-fold. IL-1beta, IL-3, IFN, and hTGF-beta(2) on the other hand do not alter beta(3) message.


Figure 1: Various cytokines modulate beta(3) 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 beta(3) cDNA by Northern analysis. IL-4 increases steady state beta(3) message migrating as a major 3.6 kb and minor 4.4 kb species 5-fold. IL-6, TNFalpha, and GM-CSF also up-regulate beta(3) integrin subunit mRNA levels up to 2-fold. IL-1beta, IL-3, IFN, and hTGF-beta(2), on the other hand, do not alter beta(3) message.



Because IL-4 is the most potent of the agents tested, we further characterized its beta(3) inductive properties. We find the phenomenon is both dose- (Fig. 2) and time- (Fig. 3) dependent. beta(3) 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 beta(3), IL-4 does not alter alpha(v) mRNA expression ( Fig. 2and Fig. 3).


Figure 2: IL-4 increases beta(3), but not alpha(v), 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. beta(3) 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 alpha(v) mRNA levels.




Figure 3: IL-4 increases beta(3), but not alpha(v), 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 beta(3) 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 alpha(v) mRNA.



The results presented thus far do not distinguish between accelerated beta(3) 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 beta(3) integrin gene.


Figure 4: IL-4 transactivates the beta(3) integrin gene. Equal amounts of [alpha-P]UTP-labeled nascent mRNA chains were hybridized to slot-blotted vector DNA, beta(3) cDNA, and G3PDH cDNA. IL-4 treatment specifically accelerates beta(3) 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 beta(3) 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 beta(3) mRNA. While all IL-4-treated macrophages contain more beta(3) 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 beta(3) integrin mRNA. Digoxigenin-labeled sense and antisense rat beta(3) cRNA probes, transcribed from cloned partial rat beta(3) cDNA, were used for in situ hybridization analysis. A, IL-4-treated macrophages form numerous polykaryons (arrows). While all cells contain more beta(3) than do their untreated counterparts, the mononuclear population is particularly rich in the message (times200). B, control cultures do not form polykaryons and express relatively low levels of beta(3) mRNA in the cytoplasm (times200). C, IL-4-treated macrophages hybridized with sense cRNA probe, as negative control, show no significant reaction (times200).



IL-4-induced Increase in beta(3) Subunit mRNA Levels Is Paralleled by Surface Expression of alpha(v)beta(3) Integrin

Having documented an agonistic effect of the IL-4 on beta(3) subunit mRNA expression, we asked if this event is paralleled by surface expression of alpha(v)beta(3) integrin. As seen in Fig. 6, IL-4 up-regulates plasma membrane expression of alpha(v)beta(3) dose dependently with maximal induction (3-fold) seen with 50 units/ml. Similarly, IL-4 augments surface expressed alpha(v)beta(3) after 12 h of treatment with peak induction occurring by 36 h (Fig. 7). Because of apparent molecular mass and, more importantly, the fact that except in platelets, beta(3) associates only with alpha(v), we reason that the beta(3)-associated band migrating at 145 kDa is, in fact, alpha(v). The greater intensity of the alpha(v) as compared to the beta(3) band ( Fig. 6and 7) reflects the fact that the former is more abundant in tyrosine residues(1) .


Figure 6: IL-4 induces plasma membrane expression of alpha(v)beta(3) 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-beta(3) antibody. The immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. IL-4 up-regulates plasma membrane expression of alpha(v)beta(3) dose dependently with maximal induction (3-fold) at 50 units/ml. M(r), molecular weight marker.




Figure 7: IL-4 induces plasma membrane expression of alpha(v)beta(3) in a time-dependent manner. BMMs were exposed to IL-4 (50 units/ml) at time 0 and beta(3) immunoprecipitates prepared as described in Fig. 6with time. IL-4 augments surface-expressed alpha(v)beta(3) after 12 h of treatment. Maximal induction occurs by 36 h. M(r), molecular weight marker.



IL-4-induced Expression of alpha(v)beta(3) Is Associated with Enhanced Attachment to Vitronectin

We next asked if enhanced expression of alpha(v)beta(3) integrin under the influence of IL-4 is functionally significant. To this end, we compared the capacity of IL-4-treated and -untreated cells cultured in Teflon-coated plates to attach to immobilized vitronectin. Albeit less effectively than in adherent macrophages, IL-4 profoundly up-regulates beta(3) subunit mRNA levels and surface expression of alpha(v)beta(3) in cells maintained in Teflon-coated plates (i.e. non-adherent) (Fig. 8). Mirroring the increase in alpha(v)beta(3), IL-4 enhances vitronectin binding by previously non-adherent cells up to 5-fold, while not effecting attachment to fibronectin (Fig. 9). Importantly, anti-alpha(v)beta(3) antiserum specifically blocks IL-4-induced vitronectin binding.


Figure 8: IL-4 induces beta(3) integrin mRNA and alpha(v)beta(3) 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 beta(3) mRNA levels and plasma membrane expression of alpha(v)beta(3). Although less effectively than in adherent population (A), IL-4 markedly up-regulates beta(3) subunit mRNA levels and surface expression of alpha(v)beta(3) 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 alpha(v)beta(3) 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.




DISCUSSION

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 alpha(v)beta(3) 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 alpha(v)beta(3), inhibits bone loss in the oophorectomized rat. Interestingly, alpha(v)beta(3) 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 alpha(v)beta(3) 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(3)(6) and retinoic acid(7) , enhance surface expression of alpha(v)beta(3) 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(3) 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) , TNFalpha(11) , and IL-4 (12, 13) modulate osteoclastogenesis. These observations prompted us to ask if, similar to 1,25-dihydroxyvitamin D(3) and retinoic acid, resorption-modulating cytokines impact on alpha(v)beta(3) 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 beta(3) mRNA expression parallels, and may regulate, alpha(v)beta(3) plasma membrane capacity (6, 7) , we performed Northern analysis on these mouse marrow-derived cells. We used in these experiments a rat beta(3) 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 beta(3) cDNA which is more than 90% homologous to the rat probe. (^3)Northern analysis of IL-4-treated macrophages is identical whether one uses the murine or rat beta(3) probe (data not shown).

We find that IL-6, TNFalpha, and GM-CSF, each of which have been implicated in osteoclast precursor differentiation(9, 10, 31) , up-regulate expression of the beta(3) integrin subunit. Of the cytokines tested, however, IL-4 through transactivation of the beta(3) 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 alpha(v) mRNA levels. beta(3) mRNA induction by IL-4 is paralleled by surface expression of alpha(v)beta(3), 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 alpha(v)beta(3) ligand(1, 2) . Reflecting plasma membrane alpha(v)beta(3) capacity, IL-4 enhances anti-alpha(v)beta(3) antibody inhibitable macrophage binding to vitronectin as much as 5-fold while not altering attachment to fibronectin, a circumstance mediated through beta(1) 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 alpha(v)beta(3) 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.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants DE05413 (to S. L. T.), AR42404 (to F. P. R.), and a grant from the Shriner's Hospital for Crippled Children, St. Louis Unit (to S. L. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pathology, Washington University School of Medicine, 216 S. Kingshighway, St. Louis, MO 63110.

(^1)
The abbreviations used are: IL, interleukin; BMM, bone marrow macrophage; M-CSF, macrophage colony stimulating factor; GM-CSF, granulocyte-macrophage colony stimulating factor; TNFalpha, tumor necrosis factor alpha; IFN, interferon ; hTGF-beta(2), human transforming growth factor beta(2); alpha-MEM, alpha-modified Eagle's medium; PBS, phosphate-buffered saline; AGPC, acid guanidinium thiocyanate-phenol-chloroform; PB, phosphate buffer; kb, kilobase(s).

(^2)
The antibody, raised against vitronectin receptor isolated from human placenta recognizes alpha(v)beta(3) and alpha(v)beta(5). We find, however, that IL-4 does not increase beta(5) mRNA expression by mouse BMMs (M. Inoue, unpublished observations). Hence, inhibition of IL-4-induced vitronectin binding reflects, in this circumstance, blocking of up-regulated alpha(v)beta(3).

(^3)
S. Kitazawa, F. P. Ross, K. McHugh, and S. L. Teitelbaum, unpublished data.


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