JOURNAL OF CELLULAR PHYSIOLOGY 148:430-439(1991)

Clearance and Fate of Leukemia-Inhibitory Factor (LIF) After Injection Into Mice DOUGLAS J. HILTON,* NlCOS A. NICOLA, PAUL M. WARINC, AND DONALD METCALF The Walter and Eliza Ha// lnstitute of Medical Research, Royal Meibourne Hospital, Melbourne 3050, Victoria, Australia Leukemia-inhibitory factor (LIF) elicits effects on a broad range of cell types, including cells of the monocytic and megakaryocytic series, embryonal stem cells, hepatocytes, adipocytes, and osteoblasts. Native and recombinant LIF, injected intravenously into adult mice, had an initial half-life of 6-8 min and a more prolonged second clearance phase. Clearance of '''I-LIF from the circulation was paralleled by a rapid accumulation in the kidneys, liver, lungs, and spleen and a more gradual accumulation in the thyroid gland. Labeling of the renal glomerular tufts, parenchymal hepatocytes, splenic red pulp, alveolar pneumocytes, and thyroid follicular cells as well as of megakaryocytes and osteoblasts in the bone cavities, placental trophoblasts, and cells of the choroid plexus was demonstrable autoradiographically. The appearance of a large amount of nonprecipitable I z 5 1 in the urine suggested that the kidneys were the major route of LIF clearance from the body.

Leukaemia-inhibitory factor (LIF) is a glycoprotein that was originally characterized and purified (Tomida et al., 1984a,b; Hilton et al., 1988a,b; Metcalf et al., 19881, sequenced a t the amino acid level (Simpson et al., 19881, and genetically cloned (Gearing e t al., 1987; Gough et al., 1988a) on the basis of its ability to induce the differentiation, and suppress the clonogenicity, of the murine myeloid leukaemic cell line M1. Subsequently, it has become apparent from studies in vitro that LIF is pleiotropic in its actions. LIF is able to inhibit the differentiation and thereby maintain the pluripotentiality of embryonal stem (ES) cells (Williams et al., 1988; Smith et al., 19881, to stimulate acute-phase protein synthesis by hepatocytes (Baumann and Wong, 19891, to enhance osteoblast anabolism and indirectly promote bone resorption by osteoclasts (Abe et al., 1986; Reid et al., 1989; Allan et al., 1990), to induce a switch in the type of neurotransmitter synthesized by sympathetic nerves (Yamamori et al., 1989), and to inhibit the activity of lipoprotein lipase in the adipocytic cell line 3T3-Ll (Mori et al., 1989). Chronic elevation of LIF levels in mice results in a complex pathology that, in part, reflects the known actions of LIF in vitro (Metcalf and Gearing, 1989; Metcalf et al., 19901. Such mice undergo a profound reduction in weight due to a n almost total loss of subcutaneous fat and calcium metabolism is dysregulated, with a n increased serum calcium concentration, calcium deposition in skeletal muscle, heart, and liver and excessive production of new bone, with severely reduced medullary haemopoiesis. Classical signs of a n acute-phase response are also observed, with decreased serum albumin levels and a n increased erythrocyte sedimentation rate. In addition, organs such as the thymus, adrenal glands, pancreas, and gonads develop C 1991 WILEY-LISS. INC

structural changes, and the mice become moribund within 4-5 weeks. To understand better the role of LIF in vivo and to substantiate whether the observed pathological effects of elevated LIF levels are direct or indirect, radioiodinated LIF (1251-LIF) was used to determine those organs that are accessible to systemically administered LIF. In addition, the clearance rate and fate of glycosylated and nonglycosylated 1251-LIFand the distribution of '251-LIF injected into normal male mice, gravid and nongravid female mice, and mice with disease resulting from increased LIF levels were compared.

MATERIALS AND METHODS Purification of LIF Native murine LIF was purified from medium conditioned by lipopolysaccharide-stimulated Krebs I1 ascites tumor cells using sequential chromatography on DEAE-Sepharose CL-6B, lentil lectin-Sepharose 4B, CM-Sepharose CL-GB, and a phenyl-silica-based reverse-phase high-performance liquid chromatography (HPLC) matrix, as described in detail previously (Hilton et al., 1988a). The LIF used was pure, as judged by the presence of a single silver-staining species of a n apparent molecular weight (Mr) of 50,000-60,000. Recombinant murine LIF was produced in Escherichia coli as a fusion product with Schistosoma japonicum glutathione S transferase, to which it was linked via a thrombin cleavage sequence. Recombinant LIF was purified by chromatography on glutathione-agarose (from which it was eluted using thrombin) and subsequently by reverse-phase HPLC. The LIF was Received December 26, 1990.

*To whom reprint requestsicorrespondence should be addressed.

CLEARANCE AND FATE OF INJECTED LIF

43 1

pure a s judged by the presence of a single silver suboccipital puncture of the exposed posterior atlanstaining band with a n apparent Mr of 20,000 after tooccipital membrane. Approximately 2 pl of CSF was electrophoresis on a polyacrylamide gel in the presence thus obtained from the cerebellomedullary cistern. of sodium dodecyl sulfate (SDS) (Gearing et al., 198913). Visibly blood-stained samples of CSF (1115) were excluded from the study. Urine, when available, was collected directly from the bladder using a 30 gauge Radioiodination of proteins needle. In each case, the amount of lZ5Ipresent in a fluid was Proteins were radioiodinated as described previously (Hilton et al., 1988c, 1990). Briefly, purified LIF (1-2 quantitiated in a gamma counter (Packard Crystal pg) in 100 pl of 40% (vol/vol) CH,CN/O.l% (viv) Tween Multi-Detector; Packard, Downers Grove, IL) for a t 20 or ovalbumin (ICN ImmunoBiologicals, Lisle, IL) in least 1 min or until the standard deviation was 1%.In phosphate-buffered saline (PBS), O.l%(v/v) Tween 20, some cases the proportion of lZ5Iin a macromolecular were radioiodinated by the addition of 2.7 pl (1mCi; 1 form was determined by the addition of three volumes Ci = 37 GBq) of carrier-free Na lZ5I (New England of ice-cold 20% (wiv) trichloroacetic acid to samples, Nuclear) and, while vortex mixing, 5 pl of 0.2 mM which after 10 min were centrifuged. The resultant iodine monochloride in 2 M NaCl, as described by precipitate was recounted in a gamma counter. After mice had been sacrificed by exsanguination, Contreras et al. (1983). After 1 rnin at room temperature, 10 p1 of 1 M KI was added and lZ5I-LIF was organs were removed and placed into preweighed 10 ml separated from unincorporated lZ5Iby sequential gel tubes containing 3.0% (viv) formalin in 20 mM sodium phosphate, 0.15 M NaCl buffered a t pH 7.4. The weight filtration and cation-exchange chromatography. of tissue was determined and the content of '251was Characterization of radioiodinated LIF ('251-LIF) measured using a gamma counter. After radioiodination, incorporation of lZ5I was asElectrophoresis sessed by precipitation with cold 20% (wiv) trichloroTwo microliters of blood, CSF, bile, yolk fluid, or acetic acid and was greater than 98%. The capacity of '"I-LIF to induce M1 differentiation was found to be urine were added to 18 ~1 of 50 mM Tris HC1,4% (wiv) indistinguishable from that of unlabeled LIF (Hilton SDS, 12% (viv) glycerol, 50 mM 2-mercaptoethanol, and et al., 1 9 8 8 ~ )The . proportion of '"1 present that was 0.01% (w/v) Serva blue G a t pH 6.8 and loaded onto gels capable of binding specifically to M1 cells was deter- 0.7-mm-thick TrisiTricine polyacrylamide mined to be between 85% and 100%. The specific (Schagger and Jagow, 1987) composed of a 2 cm 4% T, radioactivity of the preparation was assessed by self- 3%C stacking gel; a 2 cm 10%T, 3% C "spacer gel"; and displacement analysis (Calvo et al., 1983) and was in a 10 cm 16.5% T, 6% C separating gel containing 6 M general from 8 x lo5 to 1.3 x lo6 cpmipmole as urea. Electrophoresis was carried out a t a t 50 V constant voltage overnight, and gels were fixed and described previously (Hilton e t al., 1 9 8 8 ~ ) . stained in 40% (viv) methanol, 10% (viv) acetic acid, Injection of mice 0.05% (wiv) Coomassie brilliant blue in water for 1 h r Eight-week-old male and female (gravid and non- a t room temperature, destained, dried, and exposed to gravid) C57BLi6J mice or DBAiBJ female mice, en- a phosphorimager screen (Molecular Dynamics) for 72 grafted 3 weeks previously with 1 x lo6 FDC-P1 cells h r . that had been engineered to produce LIF constitutively Autoradiography (referred to subsequently a s FDiLIF mice; Metcalf and Organs or whole neonatal mice that had been fixed Gearing, 1989), were injected into the tail vein with '"I-LIF or 1251-ovalbumin[8 x lo5-4 x lo6 cpm in 200 for 48 h r in formalin were dehydrated and embedded in p1 of Dulbecco's modified Eagle's medium (DME) sup- paraffin wax and sections 1-1.5 pm in thickness were plemented with 10% (viv) fetal calf serum (FCSII or cut and placed on gelatin-coated slides. Slides were unlabeled E. coli-derived recombinant murine LIF in dipped in Kodak NTB2 photographic emulsion a t 42°C 0.2 ml of 0.15 M NaC1, containing 5% (viv) FCS. and exposed for 2-12 weeks at 4"C, after which slides Neonatal mice were injected into the umbilical vein were developed for 3 min in 8% (w/v) Kodak D19 with 2 x 105-1 x lo6 cpm 1251-LIFin 50 ~1 of DME developer in water, washed for 1 min on 0.3% (viv) acetic acid in water, and fixed in Agfa G333c X-ray containing 10% (viv) FCS. fixer for 3 min. Sections were stained with haematoxCollection and processing of fluids and tissues ylin and eosin and mounted in Depex. Autoradiographs One minute after injection, mice were anesthetized were photographed under oil a t x400. with Penthrane (Abbott Laboratories, Chicago, IL) and Preparation of tissue homogenates and binding were bled from the retroorbital plexus using a ca illary of T-LIF 2 tube. The volume of blood and its content of l 2 I were Tissues were removed from 8-week-old male C57BLi6 determined. At various times after injection, mice were again anesthetized and exsanguinated from severed mice that had been sacrificed by exsanguination and axillary vessels. Serum was obtained by allowing blood placed directly into twice the mass of ice-cold 10 mM to stand for 1h r at room temperature and removing the Tris HCl, pH 7.0, containing 1 mM phenyl methyl resultant clot by centrifugation. Fluids were collected sulfonyl fluoride (PMSF),50 pgiml leupeptin, 50 Fg/ml using finely drawn capillary tubes. Yolk fluid was aprotinin, and 1 mM EGTA. Homogenization was obtained from exposed embryos; bile from the gall carried out in a Dounce homogenizer, using 20 strokes bladder and cerebrospinal fluid (CSF) was obtained by of a loose fitting pestle followed by 20 strokes of a

432

HILTON ET AL.

tight-fitting pestle. Forty microliters of tissue homogenate was incubated in Falcon 2054 tubes (Becton Dickinson, Lincoln Park, NJ) for 16 h r on ice with 40 pl of Iz51-LIF 1 x lo5 cpm, with a specific radioactivity of 1.1 x lo6 cpmipmole; Hilton et al., 1991) and 20 pl of RPMI 1640 medium buffered a t pH 7.4 with 20 mM Hepes and containing 10% (viv) FCS (RHF), with or without 10 p iml of unlabeled LIF. Membrane-associated and free '251-LIF were separated by centrifugation through 180 p1 of chilled FCS in flexible tapered plastic tubes. The tip of the tube, containing the membranes, was cut off, and the pellet and supernatant were counted in a gamma counter. Specific binding was defined as the difference between that observed in the absence and presence of a n excess of unlabeled LIF.

Bioassay for LIF Bioassays for LIF were performed in 1 ml cultures (DME, 20% (viv) FCS and 0.3% (wiv) agar] containing 300 M1 leukaemic cells. Serum samples were assayed in serial twofold dilution by addition of 0.1 ml of sample to the culture dish. After 7 days of incubation at 37°C in a fully humidified atmosphere of 10% CO, in air, cultures were scored for the frequency of differentiated colonies. Assays were standardized by inclusion of a sample of purified recombinant murine LIF of known activity.

RESULTS C57BLi6 male mice were injected intravenously with 2 x lo6 cpm of radioiodinated native Krebs 11-derived LIF (Mr = 50,000-60,000), recombinant E . coli-derived LIF (Mr = 20,000), or ovalbumin, and the decrease in the concentration of trichloroacetic acid (TCA)-preciptable lz5I was monitored (Fig. 1).A biphasic clearance pattern was observed for both forms of LIF, in which the initial phase was rapid (t ,h 6-10 min) and resulted in a marked reduction in the circulating 1251levels. The second phase was slower for &xosylated Iz5I-LIF (t, 6-8 hr) than nonglycosylated I-LIF (t1,?3-4 hr). The clearance pattern for ovalbumin was also biphasic (data not shown); however, the initial phase was slower than observed for either LIF species (t 1,,20-30 m i d , while the subsequent phase was more rapid (t gl-2 hr). In parallel studies, mice were injected with unlabeled recombinant E . coli-derived LIF, and clearance was monitored by biological assay of the capacity of serum to induce differentiation in MI colonies. A quite different clearance rate was observed (Fig. 2). As with radiolabeled LIF, there was a n initial rapid phase of clearance with a half-life of 3-5 min, but, in stark contrast, the following second phase was also rapid (t 8-20 rnin), suggesting that much of the TCA-precipitable radioiodinated material detected in the second phase, although macromolecular, was not biologically active. The fate of glycosylated and nonglycosylated Iz5I-LIF and 1251-ovalbuminfollowing clearance from the serum of male mice was examined 60 min after intravenous injection. It was ap arent that, in mice injected with either form of LIF, E5I levels in the liver, lung, spleen, thymus, and pancreas were higher than for the corresponding organs of ovalbumin-injected mice (Table 1). Although there was no differential accumulation of lZ5I

in the kidneys, salivary gland, or thyroid of LIFinjected mice compared with ovalbumin-injected mice, these organs contributed markedly to the clearance of 1251-LIF.The organ distribution pattern was similar in normal male, gravid and nongravid female, and female FD/LIF mice (Fig. 3). In each type of animal, high levels of Iz5Iwere present in kidney, liver, spleen, thymus, pancreas, salivary gland, adrenals, and thyroid. The kinetics of the accumulation of lZ5I-LIFin these organs after intravenous injection was examined in greater detail. Twenty C57BLi6 male mice were injected with 4 x lo6 cpm of Iz5I-LIF. At various times thereafter mice were sacrificed, organs were removed, and the content of 1251was determined by counting in a gamma counter. The decrease in lz5I levels in the serum was paralleled by a rapid increase in the content of 1251within the liver, spleen, lung, and kidneys. Maximal levels of lz5I were present in these organs between 5 and 10 min after injection, then declined rapidly to less than 10% of the peak value within 1 hr (Fig. 4A-D). A similar kinetic pattern was observed for the accumulation of 1251in the thymus and adrenals (data not shown). Accumulation of Iz5Iin the pancreas and salivary gland was significantly slower than in the liver, kidneys, lung, or spleen; however, elevated levels were maintained for more than 3 h r (Fig. 4E,F). Levels of lz5I in the gastrointestinal tract were also high in comparison to tissues such as skeletal muscle. This was most pronounced in the stomach; however, no clear kinetic pattern of accumulation could be discerned (data not shown). Licking of the site of injection, the tail, and resultant ingestion of blood, containing lZ5I,may explain this finding and might also account for the variability noted between mice. Within the thyroid, Iz5I accumulated more slowly than in other tissues (Fig. 4G), presumably reflecting the incorporation of 1251 into thyroglobulin. Very low levels of Iz5I were detected in the brain, skeletal muscle, and fat, with little or no variation over time (Fig. 4G,H). As described, injection of 1251-LIF into mice was followed by a rapid decline in the concentration of Iz5I present in the serum (Fig. 5A). The clearance of lZ5ILIF was also examined electrophoretically, and it was apparent that the initial phase of clearance was marked by a reduction in the concentration of intact '251-LIFthat paralleled the kinetics associated with the clearance of biologically active LIF from the circulation (Fig. 2). Over the time period examined (1min to 24 hr), no lz5I was detected in CSF using either a gamma counter (Fig. 5B) or the phosphorimager after electrophoresis (data not shown). In contrast, TCA-nonprecipitable, low-molecular-weight species containing 1251 were readily detected in the bile and to a greater extent in the urine of mice. In both bile and urine maximal levels were present 2-7 h r after injection and had declined after 18-24 h r (Fig. 5C,D). The distribution pattern of 1251-labeled cells in of tissues of mice that had been injected with 4 x lo6 cpm of lZ5I-LIFwas identified by autoradiography of tissue sections. In the liver, labeling of the parenchymal hepatocytes was heavy (Fig. 6A), and, in young mice (e.g., 2 days postpartum), labeling of hepatic megakaryocytes but not erythroid cells was also seen (data not shown). In the lung, alveolar pneumocytes were labeled

433

CLEARANCE AND FATE OF INJECTED LIF

I

I

I

1

60

120

180

240

1 300

Time (minutes)

Fig. 1. Clearance of glycosylated '"I-nmLIF and nonglycosylated '"I-emLIF from the circulation of mice. C57BL male mice were injected IV with 2 x lo6 cpm of '"I-nmLIF produced by Krebs I1 cells (0) or 12sI-emLIF produced as a recombinant protein in E. coli ( 0 ) .At various times thereafter blood was taken under anesthetia from the retroorbital plexus, and the concentration of TCA-precipitable "'1 was determined. The data shown are combined from four independent experiments using glycosylated '"I-LIF and three using nonglycosylated 12'II-LIF.

TABLE 1. Distribution of Iz5I 2 hr after IV injection of glycosylated and nonglycosylated '"I-LIF and "'1-ovalbumin'

io,oo

-E L

a VI

t 3

LL J

5

ioa

la51(cpm/mg tissue) '2511-nmLIF '"I-rmLIF

Blood Kidney Liver Lung Spleen Pancreas Salivary gland Brain Skeletal muscle Thyroid2

48.3 f 0.4 71.5 f 3.5 174.2 i 46.9 53.2 i 11.8 65.9 5 24.6 96.8 f 11.7 74.2 k 34.2 8.5 f 1.3 13.7 2~ 1.4 4,903 5 22

56.1 i 16.4 105.2 f 19.6 156.3 28.5 59.5 k 21.8 108.0 31.7 60.1 f 23.4 79.3 i 14.2 8.6 i 1.1 7.1 !I 0.1 16.010 i 9.410

+ +

'2511-ovalhumin 40.8 i 12.3 37.4 i 5.4 14.3 f 0.0 21.9 i 7.6 19.7 i 4.1 24.5 i 3.5 105.4 i 22.2 2.1 f 0.2 6.4 f 2.3 6.357 f 1.993

'C57BL/6 male mice wereinjected IV with 1.2X106cpmofglycosylated"'I-nmLIF, nonglycosylated "%mLIF or '"I-ovalbumin. After 90 min, mice were exsanguinated and organs were removed, weighed, and counted in a gamma counter. Data are mean and range of the Concentrationof lia1 per milligram of tissue in two mice. 'The concentration u f I2',I in the thyroid is expressed per organ. The results shown are typical of three independent experiments.

k v)

1c

Tissue

I

I

I

I

I

I

2 4 6 810 20 Minutes after injection

I

30

Fig. 2. Half-life of LIF after IV injection of unlabeled LIF into mice. Adult C57BL mice were injected intravenously with 100,000 units of recombinant E. coli-derived murine LIF. After the specified time mice were sacrificed by exsanguination and the concentration of circulating LIF was monitored by biological assay of serial twofold dilutions of sera. Each point represents assay data from a different mouse.

(Fig. 6C). In the spleen, labeling of the red pulp was light; however, the marginal zone surrounding the lymphoid follicles was more intensely labeled (Fig. 6D). Little or no labeling of cells of the lymphoid follicles was noted. Within the thymus, endothelial cells of cortical and outer medullary vessels were labeled, but

lymphoid tissue was not (Fig. 6E). Labeling of the mesenchymal cells of the villi of the small intestine (Fig. 6F) was present. Although the pancreas and salivary gland accumulated 1251,the former to a reater extent after injection of lZ5I-LIFthan with l2 I-ovalbumin (Fig. 31, little or no autoradiographic labeling above background of cells was observed within these tissues (data not shown). Within bone (femur, tibia, ribs, vertebrae, and cranium) labeling of osteoblasts was prominent. Labeling was most pronounced in regions of active bone formation, for example, in the trabeculae and zones of hypertrophy of vertebrae (Fig. 7A) of neonatal mice and in the trabeculae of the femurs of mice with excess circulating levels of LIF (data not shown) and exhibiting a pathological increase in bone deposition. En-

F

434

HILTON ET AL.

"T

gl 30

-

0

0

3

0.5

0

Liver

Lung

Spleen

Thymus

Heart

Sm. bowel Colon

Kidney

Thyroid

Pancreas Salivary glMd

1.0

LcJ

E

P

u8

O Thigh

Stomach Skin

Femur

Cronium

N

2.5.

d C57BL.'*51-LIF Q non-gravid C578L,u51-LIF

2.0

0

0

gravid C57BL,'251-LIF FDC-PVLIF engrafted, "'I-LIF E4 d C57BL.'zSI-ovalbumin

1.5

0.5

&&Sternum

Adipose Mes.LN Brain

Adrenal Ovary

Testis

Uterus

Fig. 3. Fate of nonglycosylated IZ5I-emLIFinjected into male mice, gravid and nongravid female mice, and FDC-PlILIF engrafted mice. Mice were injected IV with 2-8 X lo6 cpm of '2sII-emLIF or '"'I-ovalbumin and after 90 min were sacrificed by exsanguination. The concentration of IZ5Iin various tissues was then determined as a ratio of that present in the blood and the mean and range of duplicate determinations are shown.

hanced numbers of megakaryocytes were also evident in these mice, and these too were labeled with 1251-LIF (Fig. 7B). Cells of organs involved in the clearance of serum proteins or the metabolism of iodine were also labeled, notably the renal glomerular tufts (Fig. 6B) and thyroid follicular cells (data not shown). Cells within the choroid plexus (Fig. 7C) and placental trophoblasts (Fig. 7D), tissues that are known to exclude or transport serum proteins, were also labeled. In the latter case, it is apparent that labeling was primarily restricted to the surface of trophoblasts lining the maternal circulation. To attempt to correlate the accumulation of lZ5Iupon in'ection of lz5I-LIFin vivo, with the capacity to bind l2 I-LIF specifically in vitro, homogenates of a number of tissues were incubated with approximately 1.0 nM lz5I-LIF in the presence or absence of 600 nM of unlabeled LIF. It was apparent that specific LIF recep-

d

tors were present in liver, spleen, placenta, and lung homogenates suspensions but that little or no specific binding was observed to kidney, salivary gland, pancreas, brain, or skeletal muscle homogenates (Table 2).

DISCUSSION The present data indicate that, like multi-CSF (IL-3) (Metcalf and Nicola, 19881, granulocyte-macrophage colony-stimulating factor (Metcalf, 1984), macrophage colony-stimulating factor (Shadduck e t al., 1979), erythropoietin (Fukuda et, al., 19891, and IL-2 (Cheever et al., 1985), LIF is cleared rapidly from the circulation upon intravenous injection into mice. Clearance was biphasic, with a rapid initial phase that exhibited a half-life of 5-10 min. The length of the second phase appeared longer when TCA-precipitable 1251, rather than biologically active LIF, was measured. This may reflect the presence of macromolecular but biologically

435

CLEARANCE AND FATE OF INJECTED LIF

fi

1200 800 LOO 0

LJ

:$Lmi

'""it

~

A

900

600

t .\. Cerebrospinol fluid

I'

I

Bile

C

6 12 18 24

O O

Time after injection 1 hours)

Time ofter infection (hours)

Fig. 5. Accumulation of la51in the cerebrospinal fluid, bile, and urine after IV injection of '251-emLIF.Male mice were injected IV with 4 x lo6 cpm of '"I-emLIF and at various times thereafter were exsanguinated under anesthesia, The concentration of '"1 in the serum (A), cerebrospinal fluid (B), bile (C), and urine (D) was determined. Data from two independent experiments are shown.

I

~200,000

O r

I

Thyrald -

'

Time after injection (hours)

Fig. 4. Kinetics of accumulation of 12'1 in the tissues of mice injected with '251-LIF. Male mice were injected IV with 4 x 10" cpm of 125 I-emLIF and at various times thereafter were exsanguinated under anesthesia. The concentration of Iz5I in the liver (A), lung (B), spleen (C), kidney (D), pancreas (E), salivary gland (F), fat ( G ) ,brain (H), and thyroid (I) was then determined. Data from two independent experiments are shown.

inactive products of LIF metabolism in the serum of mice. A similar phenomenon was noted in a study of IL-3 clearance (Metcalf and Nicola, 1988). It has been suggested that glycosylation may retard the clearance and therefore extend the half-life of polypeptides in vivo (Ashwell and Harford, 1982); however, little difference was noted between the clearance rate or fate of glycosylated LIF produced by Krebs I1 cells (M, 50,000-60,000) and nonglycosylated E .

coli-derived recombinant LIF (M, 20,000), although the second phase of clearance of the former was slower (t l/i 6-8 h r vs. t .3-4 hr). In the case of erythropoietin, sialic acid has been claimed to play a special role in clearance behavior, with asialoerythropoietin being cleared rapidly in the liver via the galactose binding protein of hepatocytes (Fukuda et al., 1989). The glycosylated species of LIF used in the studies described here was the most basic form produced by Krebs I1 cells and hence may not have contained sialic acid. Whether the more acidic, sialyated forms of LIF produced by Krebs I1 cells (Gough et al., 1988b) are cleared in an analagous fashion remains to be determined. The fate of lZ5I-LIFwas also examined in the present study. The majority of the lZ51-LIFaccumulated in the kidneys, liver, spleen, and lungs within 10 min of injection. Subsequently, concentrations within the liver spleen and lungs declined to 10% of their peak value after 2 hr. Removal of lz5Ifrom the kidney was slower with 10% of the peak level remaining after 7 hr. The notion that the kidney was responsible for the ultimate clearance of LIF was supported by the accumulation, 2-7 h r after injection, of large quantities of lZ5I in the urine of mice in a form that was not TCA-precipitable. The labeling pattern within the kidney was striking and selectively involved the glomerular tufts. A different picture was observed in mice injected with 1251-IL-3,in which the Bowman's capsule and proximal tubules were heavily labeled (Metcalf and Nicloa, 1988) and mice injected with 1251-ovalbu-

436

HILTON ET AL.

Fig. 6. Autoradiographs of tissues from mice injected IV with 1251-emLIF.Mice were injected with 4 x loG cpm of 1251-emLIFand after 90 min were exsanguinated under anesthesia. The liver (A),kidneys (B), Iungs (C), spleen (D), thymus (E), and small intestine (F) were then removed, fixed, sectioned, and exposed to autoradiographic emulsion for 60 days. After development, autoradiographs were stained with haematoxylin and eosin, mounted in DePex, and photographed at x400.

CLEARANCE AND FATE OF INJECTED LIF

437

Fig. 7. Autoradiographs of tissues from mice injected IV with 1251-emLIF.Male mice (A-C) or gravid female mice 12 days postcoital (D) were injected with 4 X 10' cpm of "'I-emLIF and after 90 min were exsanguinated under anesthesia. The femur (A, B), brain (C), and placenta (D) were then removed and processed as for Figure 6.

min, in which labeling of the tubules was prominent. Autoradiography revealed labeling of the red pulp and the marginal zone within the spleen, parenchymal hepatocytes, and alveolar pneumocytes. The labeling of parenchymal hepatocytes by lZ5I-LIFadministered at a peripheral location confirms the potential of LIF as a regulator of the acute-phase response to tissue injury (Baumann and Wong, 1989) and is consistent with the decrease in serum albumin levels and increase in erythrocyte sedimentation rate observed in mice with elevated circulating LIF levels (Metcalf et al., 1990). The basis of apparently selective routes of renal clearance for different hormones is not understood;

however, in the case of LIF, it is unlikely to be via specific, high-affinity receptors of the kind found on LIF-responsive cells such as M1 cells and ES cells (Hilton et al., 1988c, 1990; Williams et al., 19881, since no binding could be detected to a kidney homogenate. Specific and saturable binding to liver, lung, and spleen homogenates was observed, suggesting that such receptors might be responsible for the accumulation of "'I-LIF in these organs. In the case of the liver, this observation is consistent with the previous in vitro demonstration that high-affinity receptors for LIF are present o n isolated adult and foetal parenchymal hepatocytes (Hilton et al., 1991).

438

HILTON ET AL

TABLE 2. Binding of '"I-rmLIF to tissue homogenates in vitro' Tissue Kidney Liver Lung Spleen Pancreas Salivary gland Brain Skeletal muscle Placenta

Bound "'1-rmLIF (cum) - Competitor t Competitor 5,245 i 607 9,866 47 5,994 f 190 4,933 i 319 5,734 i 242 4,441 i 496 6,479 k 95 3,164 f 411 7,904 k 829

*

5,448 +_ 117 3,300 I 143 3,263 i 23 2,327 f 320 5,303 i 487 4,031 f 212 6,562 f 411 3,318 f 195 3,146 f 34

'Tissues were removed from C57BL/6 mice and homogenised as described in Materialsand Methods.Fortymicroliter aliquotswereincuhatedon ice for 12 hrin a volume of 100 fil, with 100,000 cpm of '""IrmLIF, in the presence or absence of 600 nM unlabeled rmLIF as a competitor, Subsequently the reaction mixture was centrifuged through FCS, and the amount of membraneassociated and free lZsI. rmLIF were determined using a gamma counter. The mean and range of replicate analyses are shown and are typical of three separate experiments.

The concentration of lZ5Iin the pancreas and salivary glands of lZ5I-LIF-injected mice was considerably higher than for mice injected with 1251-ovalbumin. Maximal concentrations of 1251in these organs were reached more slowly and declined more slowly than observed for the kidney, liver, lungs, or spleen. As with the kidney, lz5I-LIFdid not bind specifically to homogenates of these tissues. Whether peptides produced from the metabolism of lZ5I-LIFin vivo have a n affinity for cells within these tissues is not known. As might be expected, from the role of the thyroid in iodine metabolism, mice injected with 1251-LIF incorporated lZ5I slowly into this organ and no diminuition in content was apparent even after 24 hr. Labeling of cells of the choroid plexus and the trophoblasts of the placenta were also observed. These cell types have been implicated in the regulation of the passage of proteins from blood to CSF and to the foetal circulation, respectively. While the transport of immunoglobulins and hormones across the placenta and neuromodulators across the blood-CSF barrier is well established, the absence of intact lZ5I-LIF in either fluid compartment indicates that passage of LIF is prevented rather than facilitated by these cell types. Interestingly, receptors for a number of hormones, including epidermal growth factor, platelet-derived growth factor, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and erythropoietin, are expressed by the placenta (Maros and Mochizuki, 1987; Taylor and Williams, 1988; Arceci et al., 1989; Gearing et al., 1989a; Sawyer et al., 1989; Umuzaki et al., 1989). Whether such receptors represent a general means of regulating the passage of hormones between the mother and the fetus is yet to be established. The rapid clearance of LIF from the circulation and the exclusion of LIF from certain fluid compartments may represent mechanisms by which the potentially broad actions of LIF are limited in vivo. Local and transient production of low concentrations of LIF that acts via high-affinity receptors might also serve to reduce any pathological manifestation of prolonged and elevated circulating LIF levels.

ACKNOWLEDGMENTS The authors thank Sandra Misfud, Dale Cary, and Audrey Szalai for their excellent technical assistance and Steven Mihajlovic, Viki Likiardopolous, and Tamara Bucci for the preparation of histological sections and are indebted to Meredith Layton and Jeffery Rosenfeld for their technical advice. This work was supported by the Anti-Cancer Council of Victoria, Australia; AMRAD Corporation; The National Health and Medical Research Council; The J D & L Harris Trust; and The National Institute of Health, Bethesda, Maryland (grant CA-22556).

LITERATURE CITED Abe, E., Tanaka, H., Ishimi, Y., Miyaura, C., Hayashi, T., Nagasawa, H., Tomida, M., Yamaguchi, Y., Hozumi, M., and Suda, T. (1986) Differentiation-inducing factor purified from conditioned medium of mitogen-treated spleen cell cultures stimulates bone resorption. Proc. Natl. Acad. Sci. USA, 83,5958-5962. Allan, E.H., Hilton, D.J., Brown, M.A., Evely, R.S., Yumita, S., Metcalf, D., Gough, N.M., Ng, K.W., Nicola, N.A., and Martin, T.J. (1990) Osteoclasts display receptors for and responses to leukemia inhibitory factor. J. Cell. Physiol. 145t110-119. Arceci, R.J., Shanahan, F., Stanley, E.R., and Pollard, J.W. (1989) Temporal expression and location of colony-stimulating factor 1 (CSF-1) and its receptor in the female reproductive tract are consistent with CSF-1 regulated placental development Proc. Natl. Acad. Sci. USA, 86:881&8822. Ashwell, G., and Harford, J. (1982) Carbohydrate-specific receptors of the liver Annu. Rev. Biochem., 51531-534. Baumann, H., and Wong, G.G. (1989) Hepatocyte-stimulating factor 111 shares structural and functional identity with leukemia inhibitory factor. J. Immunol., 143t1163-1167. Calvo, J.C., Radicella, J.P., and Charreau, E.H. (1983) Measurement of specific radioactivity in labelled hormones by self-displacement analysis. Biochem. J., 212959-264. Chewer, M.A., Thompson, J.A., Kern, D.E., and Greenberg, P.D., (1985) Interleukin 2 (IL 2) administered in vivo: influence of IL 2 route and timing on T cell growth J. Immunol., 134t3895-3900. Contreras, M.A., Bale, N.F., and Spar, I.L. (1983)Iodine monochloride (ICl) iodination techniques. Methods Enzymol., 92.277-292. Fukuda, M., Sasaki, H., Lopez, L., and Fukuda, M. (1989) Survival of recombinant erythropoietin in the circulation: The role of carbohydrates. Blood, 73r8P89. Gearing, D.P., Gough, N.M., King, J.A., Hilton, D.J., Nicola, N.A., Simpson, R.J., Nice, E.C., Kelso, A., and Metcalf, D. (1987) Molecular cloning and expression of a cDNA encoding a murine myeloid leuemia inhibitory factor (LIF).EMBO J., 6t3995-4002. Gearing, D.P., King, J.A., Gough, N.M., and Nicola, N.A. (1989a) Expression cloning of a receptor for human granulocytemacrophage colony-stimulating factor EMBO J., 8,3667-3676. Gearing, D.P., Nicola, N.A., Metcalf, D., Foote, S., Willson, T.A., Gough, N.M., and Williams, R.L. (1989133 Production of leukemia inhibitory factor in Escherichia coli by a novel procedure and its use in maintaining embryonic stem cells in culture. Biotechnology, 7:1157-1161. Gough, N.M., Gearing, D.P., King, J.A., Willson, T.A., Hilton, D.J., Nicola, N.A., and Metcalf, D. (1988a)Molecular cloning and expression of the human homologue of the murine gene encoding murine myeloid leukemia inhibitory factor. Proc. Natl. Acad. Sci. USA, 85r2623-2627. Goueh. N.M.. Hilton. D.J.. Gearing. D.P.. Willson. T.A., Kine. J.A.. NFcola, N.A., and Metcalf, D. ( 1 9 k b ) Biochemical characteryzation of murine leukaemia inhibitory factor produced by Krebs ascites and by yeast cells. Blood Cells, 14:431442. Hilton, D.J., Nicola, N.A., Gough, N.M., and Metcalf, D. (1988a) Resolution and purification of three distinct factors produced by Krebs ascites cells which have differentiation-inducing activity on murine myeloid cell lines. J. Biol. Chem., 263:9238-9243. Hilton, D.J., Nicola, N.A., and Metcalf, D. (1988133 Purification of a murine leukemia inhibitory factor from Krebs ascites cells. Anal. Biochem., 173t359-367. Hilton, D.J., Nicola, N.A., and Metcalf, D. (1988~) Specific binding of murine leukemia inhibitory factor to normal and leukemic monocvtic cells. Proc. Natl. Acad. Sci. USA. 8,5:5971-5975.

CLEARANCE AND FATE OF INJECTED LIF Hilton, D.J., Nicola, N.A., and Metcalf, D. (1991) Distribution and comparison of receptors for leukemia inhibitory factor on murine hemopoietic and hepatic cells. J. Cell. Physiol. 146207-215. Maros, T., and Mochizuki, M. (19A7)Immunohistochemicallocalization of the epidermal growth factor receptor and myc oncogene product in human placenta: Implication for trophoblast proliferation and differentiation Am. J. Obstet. Gynecol., 156:721-727. Metcalf, D. (1984) The Hemopoietic Colony Stimulating Factors. Elsevier, Amsterdam. Metcalf, D., and Gearing, D.P.(1989) Fatal syndrome in mice engrafted with cells producing high levels of the leukemia inhibitory factor. Proc. Natl. Acad. Sci. USA, 86.5948-5952. Metcalf, D., Hilton, D.J., and Nicola, N.A. (1988) Clonal analysis of the actions of the murine leukemia inhibitory factor on leukemic and normal hemopoietic cells. Leukaemia, 2:216221. Metcalf, D., and Nicola, N.A. (1988) Tissue localization and fate in mice of injected multi-potential colony stimulating factor. Proc. Natl. Acad. Sci. USA, 85.3160-3164. Metcalf, D., Nicola, N.A., and Gearing, D.P., (1990) Effects of injected leukemia inhibitory factor on hematopoietic and other tissues. Blood, 76t50-56. Mori, M.. Yamaeuchi. K.. and Abe, K. (1989) Purification of a lipoprotein lipakinhibitory protein produced by a melanoma cell line associated with cancer cachexia. Biochem. Biophys. Res. Commun., 16Ot1085-1092. Reid, I.R., Lowe, C., Cornish, J., Skinner, J.J.M., Hilton, D.J., Willson, T.A.. Gearing. D.P.. and Martin. T.S. (1990) Leukemia inhibitory factor: A novel bone-active cytokine. Endocrinology (in press). Sawyer, S.T., Krantz, S.B., and Sawada, K. (1989) Receptors for erytropoietin in mouse and human erythroid cells and placenta. Blood, 74.103-109. Schagger, H., and von Jagow, G. (19871 Tricine-sodium dodecylsulphate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem., 166t368-379. Shadduck, R.K., Waheed,A., Porcellini, A., Rizzoli, V., and Pigoli, G. "/

439

(1979) Physiological distribution of colony-stimulating factor in vivo. Blood, 54.894-905. Simpson, R.J., Hilton, D.J., Nice, E.C., Rubira, M.R., Metcalf, D., Gearing, D.P., Gough, N.M., and Nicola, N.A. (1988) Structural characterization of a murine myeloid leukemia inhibitory factor. Eur. J . Biochem., 175t541-547. Smith, A.G., Heath, J.K.,Donaldson, D.D., Wong, G.G.,Moreau, J.-F., Stahl, M., and Rogers, D. (1988) Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature, 336:688-690. Taylor, R.N., and Williams, L.T. (1988) Developmental expression of platelet-derived growth factor and its receptor in the human placenta. Mol. Endocrinol., 2t627-632. Tomida, M., Yamamoto-Yamaguchi, Y., and Hozumi, M. (1984a) Purification of a factor inducing the differentiation of mouse myeloid leukemic M1 cells from conditioned medium of mouse fibroblast L929 cells. J . Biol. Chern., 259t10978-10982. Tomida, M., Yamamoto-Yamaguchi, Y., and Hozumi, M. (1984b) Characterization of factor inducing differentiation of mouse myeloid leukemic cells purified from conditioned by mouse Ehrlichs ascites turnour cells. FEBS Lett., 178.291-296. Umuzaki, H., Okabe, T., Sasaki, N., Hagiwara, K., Takaku, F., Tobita, M., Yasukawa, K., Ito, S., and Umezawa, Y. (1989) Identification and characterization of receptors for granulocyte colony-stimulating factor on human placental and trophoblastic cells Proc. Natl. Acad. Sci. U S A , 86:9323-9326. Williams, R.L., Hilton, D.J., Pease, S., Willson, T.A., Stewart, C.L., Gearing, D.P., Wagner, E.F., Metcalf, D., Nicola, N.A., and Gough, N.M. (1988) Myeloid leukemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature, 3 3 6 t 6 8 6 687. Yamamori, T., Fukada, K., Aebersold, R., Korsching, S., Fann, M.-J., and Patterson, P.H. (1989)The cholinergic neuronal differentiation factor from heart cells is identical to leukemia inhibitory factor. Science, 246:1412-1416.