Uptake by rat liver of bovine growth hormone free or bound to a monoclonal antibody

Biol Cell (I 994) 82, 45-49 © Elsevier, Paris

45

Original article

Uptake by rat liver of bovine growth hormone free or bound to a monoclonal antibody Christine Tans, Frans Dubois, Zhi-Duan Zhong, Michel Jadot, Robert Wattiaux *, Simone Wattiaux-De Coninck Laboratoire de C h i m i e Physiologique, Facult~s Universitaires N o t r e - D a m e de la Paix, 61, rue de Bruxelles, B-5000, Namur, B e l g i u m

S u m m a r y - In the work reported here, we have compared the elimination from the blood, the uptake by the liver and the intracellular

distribution of bovine growth hormone, free(Gh) or bound to a monoclonal antibody (GhAb). Results show that: a) the elimination from the blood is more rapid for Gh than for GhAb; b) both molecules are quickly taken up by the liver; c) probably after travelling through endosomes, Gh and GhAb get to lysosomes where they are degraded. However, Gh mostly ends in hepatocyte lysosomes while GhAb is recovered to a large extent in sinusoidal cell lysosomes; and d) binding by isolated hepatocytes is markedly less efficient for GhAb than for Gh. rat liver / lysosomes I growth hormone / monodonal antibody / subcellular fractionalion

Introduction

Several works have shown that the binding o f a monoclonal antibody to growth hormone (Gh) enhances its biological activity in vivo [3, 7, 13]. Until now, the mechanism o f this phenomenom remain unknown. The liver is a major site o f action of Gh. After binding to receptors located in hepatocyte plasma membrane, Gh is internalized and to a large extent reaches lysosomes where it is degraded [15]. Nothing has been described concerning the fate o f Gh linked to a monoclonal antibody (GhAb) which is an important question w i t h r e s p e c t to its h o r m o n a l a c t i o n . In the w o r k reported here, we have determined the elimination from the blood, the uptake by the liver and the intracellular distribution o f Gh and G h A b injected in rat. T h e h o r m o n e was labelled with 02sI)tyramine cellobiose to prevent the release o f radioactive degradation products from organelles where the endocytosed protein was digested [2]. Our results show that the binding o f a monoclonal antibody to growth hormone affects the lifetime in blood o f the hormone and its cellular location in the liver.

Materials and methods

Experiments were performed on male Wistar rats weighing 200-250 g. lodination of tyramine-cellobiose and its subsequent binding to growth hormone was carried out by the method of Pittman et al [14] as modified by Hysing and Toileshaug [8]. The method involves the incubation of 10/zl iodogen with t /.tl of 0.01 M tyramine cellobiose in 0.02 M phosphate buffer (pH 7.2) and 10/.tl of Na 125 I at room temperature for 30 min. The reaction was stopped by adding 10/zl of Na2SO 3 followed by 5/11 of 0.1 M KI. The solution was transferred to a second tube contain-

• Correspondence and reprints

ing 30 nmol of trichlorotriazine in 20/.ti acetonitrile. 5/zl of 0.01 M NaOH was added followed by 20/11 of Gh solution (2 mg). After 10 min, the total mixture was fractionated by passage through a Sephadex G-25 column, using 0.1 M phosphate buffer (pH 7.4), containing 1% bovine serum albumin as eluting buffer. For the preparation of GhAb, 0.02 mg of Gh were incubated for 12 h at 4°C with 0.41 mg of monoclonal antibody in 0.75 ml of a medium containing 0.1 ml PBS and 0.1% bovine serum albumin (pH 7.4); the formation of the complex was checked by immunoprecipitation with a goat anti-mouse antiserum and by FPLC with a Superose 12 column. 0.015 mg of hormone (free or bound) was injected intravenously under ether anesthesia. Animals were killed at various times after injection; the liver was perfused with cold 0.15 M NaCI, then removed and homogenised in ice-cold 0.25 M sucrose. The homogenate was fractionated by differential centrifugation as described by de Duve et al [5], giving a nuclear fraction N, a heavy mitochondrial fraction M, a light mitochondrial fraction L, a microsomal fraction P and a soluble fraction S. Density gradient centrifugation was performed according to Beaufay et al [1]. Cathepsin C was measured as described by Jadot et al [9], arylsulfatase following the method of Bowers et al [4], protein by the method of Lowry et al [12]. The proteolytic degradation of the product after its uptake was assessed by measuring the acid-soluble radioactivity in 5% trichloracetic acid. Liver cells were isolated according to Gudmundsen et al [6] using the collagenase perfusion method. The liver perfusion technique consists of a two-step procedure. In a first step, the liver is subjected to a non-recirculating portal vein perfusion with a calcium free buffer; in a second step, the liver is perfused with collagenase in a buffer containing calcium. Separation of the different cells from the liver suspension was performed by differential centrifugation and elutriation. The cells were further purified by a selective attachment method using fibronectin coated dishes. The cell preparations were checked by optical microscopy with peroxidase staining for Kupffer cells. Methionyl Gh(MI L126)was kindly supplied by Monsanto (St Louis, MO, USA). Anti-Gh monoclonal antibody (MAB 6B 1,

46

C T a n s et al

isotype IgG1)produced as described in [13] and goat anti-mouse serum were provided by Dr Portetelle and Dr Renaville (Faculty of Agronomy, Gembloux, Belgium).

60-

Results and discussion '~ 40

Blood clearance and uptake by the liver As shown in figure 1, the elimination of Gh is more rapid than GhAb. It is to be noted that results are similar when labelling is done with 12sI (not shown). In the same figure is illustrated the uptake by the liver of Gh and GhAb. When Gh is injected, liver radioactivity increases for about 20 min, then remains constant until at least 100 rain have elapsed. Radioactivity accumulates more slowly in liver after GhAb injection; at any time, the amount present in this organ is lower than the amount recovered after injecting the free hormone. In both cases, the proportion of acid soluble radioactivity increases with time, reaching about 70% after 100 min.

Intracellular routing of Gh and GhAb The distributions of radioactivity were established after differential centrifugation according to de Duve et al [5], 5 and 80 rain after injection of Gh or GhAb labelled with (usI)tyramine cellobiose. As shown in figure 2, after 5 min radioactivity originating from Gh or GhAb is mainly recovered in microsomal fraction P. Such a distribution corresponds to what is generally observed when an endocytosed compound is present in pinocytic vesicles and in endosomal compartment [11]. Later the distributions become lysosomal, most of the radioactivity is recovered in the mitochondrial fractions and exhibits a peak of relative specific activity in the light mitochondrial fraction L. At that time, a large proportion of the radioactivity is acid-soluble but remains associated with sedimentable structures. To confirm the lysosomal destination of Gh and GhAb, a total mitocho~adrial fraction(M+L) isolated 80 min after injection was analyzed by isopycnic centrifugation in a sucrose gradient. The distribution curves (fig 3) are similar to those of cathepsin C, a reference e n z y m e o f lysosomes. These results suggest that Gh and GhAb follow a similar intracellular pathway, travelling through endosomes before reaching lysosomes where they are degraded.

Cellular location of Gh and GhAb In order to see if intracellular travels of Gh and GhAb take place in the same liver cells (hepatocytes or sinusoisal cells), we used an indirect approach that we have described recently [17]. As we have shown, it is possible to distinguish between lysosomes of hepatocytes and of sinusoidal cells by injecting chloroquine to rat. Indeed, chloroquine decreases the equilibrium density of hepatocyte lysosomes in a sucrose gradient without affecting the density of nonparenchymal cell organdies. Therefore the selective density shift of hepatocyte lysosomes caused by chloroquine can help to distinguish whether a molecule endocytosed by the liver reaches the lysosomes of hepatocytes or of sinusoidal cells [17]. Results obtained after Gh or GhAb injection are illustrated in figure 4. In agreement with our previous observations [17], chloroquine shifts the distribution curve of cathepsin C, an hydrolase which is mostly located in bepatocyte lysosomes, towards lower density regions. In the case of arylsulfatase which is shared between parenchymal

O O

20

!

÷

80

I00

T

0

0

fi0

4b

60

Min.

80 f

20

o

io

I

80

1'00

Fig 1. Radioactivity present in blood and in liver. Radioactivity was measured at increasing times after injection of Gh (0) or GhAb ( O ) labelled with 0~I)tyramine cellobiose and is given as percentages of the injected dose. Means with SD for at least three animals are presented. The total volume of blood was estimated by supposing it represents 6% of the body weight [ 18].

and non-parenchymal cell lysosomes, the distribution becomes bimodal, part of the hydrolase is shifted like cathepsin C and part remains where lysosomes of untreated animal equilibrate. Radioactivity coming from Gh is shifted to a large extent to lower densities after chloroquine injection; on the other hand, a large part of radioactivity originating from GhAb does not exhibit change in distribution and remains like a certain proportion of arylsulfatase where lysosomes unaffected by chioroquine treatment (sinusoidal cell lysosomes) are located. These results illustrate that endocytosed Gh is targeted mainly to hepatocyte lysosomes while GhAb reaches for the major part, sinusoidal cell lysosomes.

Distribution of radioactivity in isolated cells To check the observations reported in the previous section, in one experiment we have established the radioactivity distribution amongst isolated liver cells. Results are given in figure 5. Radioactivity coming from Gh is mainly located in hepatocytes while about 70% of radioactivity originating from Gb_Ab is recovered in sinusoidal cells. On the other

Uptake of growth hormone by liver

Gh

47

Alkaline Phosphodiesterase

GhAb

4

(A) 0

fl

¢9

r~

Gh

12

Cathepsin C

GhAb

8 (B) 4

0

I

N

M

I

L I

0

50

P

S

N

M

I

100 0

L P

S

i

N

M

L P

,

S

I

I

I

I

I

50

100

0

50

100

Protein(%)

2. Distribution of radioactivity after differential centrifugation. The distributions were obtained with livers originating from rats killed 5 min (A) or 80 min fB) after injection. Shaded areas correspond to acid soluble radioactivity. For the sake of comparison, a representative distribution of a plasma membrane enzyme, alkaline phosphodiesterase and of a lysosomal enzyme, cathepsin C are illustrated. N, nuclear fraction; M, heavy mitochondrial fraction; L, light mitoehondrial fraction; P, microsomal fraction and S, soluble fraction. Fig

B

t11

20

.. 0

1.00

I

I

1.10

1.20

ml

I

1.30

1.00

1.10

1.20

1.30

Density ( g/ml ) Fig 3. Density distribution histogram Of radioactivity (I~A) and cathepsin C ([-]) after isopycnic centrifugation of a total mitochondrial fraction (M+L). The particle preparation was isolated 80 min after injection of Gh (A) or GhAb (B). Centrifugation was performed at 39 000 rev/min in the SW 65 Spinet rotor. The time integral of the square angular velocity was 860 rad 2Ins. The sucrose gradient extended from 1.05 g/ml to 1.26 g/ml density. Ordinate: frequency Q/SQ.r where Q represents the activity found in the fraction, SQ, the total activity recovered in the sum of the fractions and r, the increment of density from top to bottom of the fraction.

48

C Tans et al

GhAb

Gh

20-

20 ¸

10.

10

0 1.00

|

!

1.10

1.20

I

1.30

0

! ,

I.i0

1.00

Cathepsin C ~

20

10

10

, 1.10

, 1.20

1.20

!

1.30

Arylsulfatase

20

.0 1.00

j/t

, 1.30

0 1.00

" ........ | 1.10

|

1.20

, 1.30

Density ( g/ml ) Fig 4. Density distribution histogram of cathepsin C, arylsulfatase and radioactivity in a sucrose gradient. Mitochondrial fractions were isolated from a normal rat (f---q) or from a rat having received two chloroquine injections(15 mg IP), one I00 min and a second one 50 rain before being killed (17/~). 130 min before the killing, rats were injected with labelled Gh or GhAb. For the centrifugation conditions and explanation of the graph, see legend to figure 3.

hand, binding by isolated hepatocytes of Gh and GhAb has been determined. As illustrated by figure 6, the association of the monoclonal antibody to Gh considerably decreases the binding of the hormone to these cells.

Conclusions The major information brought about by the results reported in this paper is that the binding of a monoclonal antibody to growth hormone slows down the elimination of the hormone from the blood and causes it to be internalized to a large extent by liver sinusoidal cells and not by hepatocytes as in the case of the free hormone. The persistance of GhAb for a long time in blood probably results from a decrease of uptake by the tissues parti-

cularly the liver, as a result of a lower affinity of the complex to growth hormone receptor as indicated by our binding experiments. With respect to the preferential targeting of GhAb to sinusoidal cells, a plausible explanation is that the complex is preferentially internalized via the Fc receptor present in the plasma membrane of these cells and absent from hepatocytes. Are our observations related to the stimulation of growth hormone activity caused by its binding to a monoclonal antibody? Obviously, the increase of the hormone life time in blood induced by the antibody could lead to a prolongation of the hormone effect. On the other hand, the fact that the antibody decreases the binding and the uptake of Gh by bepatocytes seems unfavorable to hormone action in vivo. Indeed, that action is strongly dependent on the liberation of IGFs by hepatocytes [10, 16]. Information on this paradoxi-

Uptake of growth hormone by liver

References

Gh

GhAb

(b) •

(e)

(c) Fig 5. Distribution of radioactivity in different kinds of liver cells. Separation of liver cells was achieved 15 min after injection of Gh or GhAb; (a) hepatocytes; (b) endothelial cells; (c) Kupffer cells.

20-

0

15"

O

o O

o

10" / .8 o

5"

k-" 0

0

•

49

&

•

i0 Time (min.)

Fig 6. Binding of Gh or GhAb to hepatocytes. Binding experiments were performed as described in [18]. Briefly, freshly isolated hepatocytes were incubated with Gh (O) or with GhAb (O) at 4°C in RPMI 1640 medium. At the indicated times, aliquots containing 10s cells were sampled and laid in a Eppendorf microtube, on the surface of 0.15 ml oil (a mixture of dibutylphthalate and dioctylphthalate of 1.20 g/ml density) and centrifuged at 10 000 g for 30 s. The cell pellet was cut and added to 0.5 ml boiling SDS solution (10 *7o)in a counting vial.

cal situation could be obtained by investigating the effects o f growth hormone on liver sinusoidal cells.

Acknowledgments This work was supported by the Fonds de la Recherche Scientifique, the Fonds de la Recherche Fondamentale Collective (contract n. 2.4555.92) and the Fonds de la Recherche Sciontifique Mtdicale (contract n. 3.4523.91).

I Beaufay H, Jacques P, Baudhuin P, Sellinger OZ, Berthet J de Duve C (1964) Tissue fractionation studies. 18. Resolution of mitochondrial fractions from rat liver into three distinct populations of cytoplasmic particles by means of density equilibration in various gradients. Biochem J 92, 184-205 2 Berg T, Kinberg GM, Ford T, Blomhoff R (1985) Intracellular transport of asialoglycoproteins in rat hepatocytes. Exp Cell Res 161,285-296 3 Bomford R, Aston R (1990) Enhancement of bovine growth hormone activity by antibodies against growth hormone peptides. J Endocrinol 125, 31-38 4 Bowers WE, Finkenstaedt JT, de Duve C (1967) Lysosomes in lymphoid tissue. 1. The measurement of hydrolytic activities in whole homogenates. J Cell Biol 32, 325-327 5 de Duve C, Pressman BC, Gianetto R, Wattiaux R, Appelmans F (1955) Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissue. Biochem J 60, 604---617 6 Gudmundsen O, Berg T, Roos N, Nenseter MS (1993) Hepatic uptake of VLDL in cholesterol fed rabbits. J Lip Res 34, 589-592 7 Holder AT, Aston R, Preece MA, Ivanyi J (1985) Monoclohal antibody-mediated enhancement of growth hormone activity in vivo. Endocrinology 107, 9-12 8 Hysing J, Tolleshaug H (1986) Quantitative aspects of the uptake.and degradation of lysozyme in the rat kidney in vivo. Biochim Biophys Acta 887, 42-50 9 Jadot M, Colmant C, Wattiaux-De Coninck S, Wattiaux R (1984) Intralysosomal hydrolysis of glycyl-L-phenylalanine 2 naphtylamide. Biochem J 219, 965-970 10 Johnson TR, Blossey BK, Denko CW, Ilan J (1989) Expression of insulin-like growth factor I in cultured rat hepatocytes: effects of insulin and growth hormone. Mol Endocrinol 3,580-587 11 Limet JN, Quintart J, Schneider YJ, Courtoy P (1985) Receptor mediated endocytosis of polymeric IgA and galactosylated serum albumin in rat liver. Eur J Biochem 146, 539-548. 12 Lowry OH, Rosebrouh NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193, 265-275 13 Massart S, Maiter D, Portetelle D, Adam E, Renaville R, Ketelslegers JM (1993) Monoclonal antibodies to bovine growth hormone potentiate hormonal activity in vivo by enhancing growth hormone binding to hepatic somatogenic receptors. J Endocrinol 139, 383-393 14 Pittman RC, Careco JE, Glass CK, Green SR, Taylor CA, Attic AD (1983) A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo. Biochem J 212, 791-800 15 Postel-Vinay MC, Kayser C, Desbuquois B (1982) Fate of injected human growth hormone in the female rat liver in vivo. Endocrinology 111,244-251 16 Schwander JC, Hauri C, Zapf J, Froesch ER (1983) Synthesis and secretion of insulin-like growth factor and its binding protein by the perfused rat liver: dependence on growth hormone status. Endocrinology 113, 297-305 17 Wattiaux R, Gentinne F, Jadot M, Dubois F, Wattiaux-De Coninck S (1993) Choroquine allows to distinguish between hepatocytes lysosomes and sinusoidal cell lysosomes. Biochem Biophys Res Commun 190, 808-813 18 Zhong ZD, Wattiaux-De Coninck S, Wattiaux R (1993) Uptake of tyramine by rat hepatocytes. Biochim Biophys Acta 1176, 77-82