0013-7227/91/1284-2103$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 4 Printed in U.S.A.
Passive Immunization against Insulin-Like Growth Factor-I Does not Inhibit Growth HormoneStimulated Growth of Dwarf Rats G. S. G. SPENCER*, S. C. HODGKINSON, AND J. J. BASS Ruakura Agricultural Centre, Private Bag, Hamilton, New Zealand
ABSTRACT. Passive immunization against insulin-like growth factor-I (IGF-I) was undertaken in GH-deficient rats in an attempt to elucidate the relative importance of the endocrine vs. autocrine/paracrine actions of IGF-I in stimulating growth. Antiserum against IGF-I was raised in sheep and purified by affinity chromatography. The ability of the purified antibodies to neutralize the actions of IGF-I in vitro and bind IGF-I in vivo were extensively tested using L6 myoblast and cartilage bioassays. Four groups of male rats with isolated GH deficiency were used in the study. At 49 days of age the rats received 100 pi normal saline given sc each day for 10 days, 2 mg/kg recombinant bovine GH (bGH) given in 100 ft\, sc, each day, 2 mg/kg bGH, sc, and 300 nl immunoglobulin G purified from normal sheep serum given daily ip, or 2 mg/kg bGH plus 300 (A anti-IGF-I immunoglobulin G daily, ip (a dose that was able to completely
H
YPOPHYSECTOMIZED (hypox) rats fail to grow, but growth can be restored by the administration of GH (1). Similarly, synthesis of cartilage matrix glycosaminoglycans, such as chondroitin sulfate, is reduced in hypox animals, but is restored to normal by GH treatment (2). Although plasma from GH-treated hypox animals stimulates sulfate uptake into cartilage in vitro, direct addition of GH to hypox serum has usually been ineffective (3). These observations suggest that there is a GH-dependent serum factor that mediates the effects of GH on cartilage sulfation. From this grew the belief that all of the anabolic actions of GH were mediated by this sulfation factor (and a similar thymidine factor which stimulated thymidine uptake into cells in like fashion). These factors were later called somatomedins (4) and, ultimately, insulin-like growth factors (IGFs). Supporting the concept that IGFs are the mediators of the action of GH is a weight of circumstantial evidence that low levels of IGF are often associated with low growth rates, and (less consistently) higher levels of IGFs may be associated with overgrowth. Purified preparations of IGFs have also been shown to stimulate growth Received September 24,1990. * To whom requests for reprints should be addressed.
inhibit IGF-I actions on sulfate uptake into cartilage). Treatment with GH significantly increased growth rates (P < 0.001) in the rats, but there was no difference between any of the three GH-treated groups; passive immunization against IGF-I did not diminish the GH-stimulated growth in these rats. Excess antibody could be detected in the plasma of all anti-IGF-I-treated rats at the conclusion of the experiment, and the antibody was capable of sequestering both free and binding protein-bound IGF-I. The absence of even a slight retardation of GH-stimulated growth in the anti-IGF-I-treated rats suggests that circulating IGF-I may not be important in mediating the growth-promoting actions of GH, although the immunoneutralization probably does not affect GH stimulation of tissue IGF-I production. (Endocrinology 128: 2103-2109, 1991)
in hypopituitary or hypox animals (5, 6), but the responses have never been as great as those obtained with GH treatment. On the face of it, these data might suggest that plasma IGF-I does not play a large part in stimulating growth, although the lack of effect is likely to be partly due to factors such as impurity of the preparations used, lack of cofactors, accelerated clearance in the absence of binding proteins, or inappropriate administration schedules. It has been reported that GH can stimulate IGF-I production from a number of different tissues (7), and it may be via this locally produced IGF that GH has its actions. If this is so, what is the role.of the circulating IGF? Does it reflect a sink for clearance of excess IGF-I produced for autocrine/paracrine actions or is circulating IGF-I required for growth? In an attempt to answer this question we have raised large quantities of high affinity antibodies specific to IGF-I and used them to immunoneutralize plasma IGFI. These studies extend a contemporary study of passive immunization against IGF-I that has been recently reported (8), but in that study it was not shown that the antibody used could immunoneutralize IGF action, and the choice of an animal model in which growth is not GH dependent (guinea pig) was unfortunate. The use of 2103
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PASSIVE IMMUNIZATION AGAINST IGF-I
an animal with an isolated GH deficiency and demonstration of inhibition of IGF action by the antibodies should provide a more reliable indication of the role of IGF-I in growth.
Materials and Methods GH-deficient rats The rats used in this study were bred at Ruakura from the strain of rats with an isolated GH deficiency originally identified and reported by Charlton et al. (9). These rats have been shown to respond to exogenous GH treatment with marked increases in growth rate (10). Forty-eight male rats were used in the growth trial which started at 49 days of age; this has previously been identified as an age at which the rats were particularly responsive to exogenous GH treatment (10), but was free from possible interference associated with puberty. The rats were housed in the same room in groups of eight, with treatments spread at random throughout the groups. Ad libitum access to food and water were available to all rats at all times; temperature and humidity were controlled and a 12-h light, 12h dark regimen was in place. Generation and purification of the IGF-I antiserum Antisera were raised in sheep to an egg albumin conjugate of recombinant human IGF-I (courtesy of Dr. B. D. Burleigh, IMC/Pitman Moore, Terre Haute, IN). The conjugates were prepared by coupling IGF-I to carrier protein at a molar ratio of 10:1 using l-ethyl-3-(3-dimethylamino-propyl)carbodiimide HC1 and stored at -20 C until use. Animals were immunized by im injection at four sites with conjugate (equivalent to 200 ng IGF-I/injection) emulsified in Freund's Complete Adjuvant (for the primary immunization) and in Freund's Incomplete Adjuvant on subsequent occasions; booster injections were given at monthly intervals. Antibody production was monitored by measuring IGF-I tracer binding (as outlined below) in blood samples collected 2 weeks after each booster injection. Purification of the IGF-I antibody Specific anti-IGF-I antibody was purified by an automated affinity chromatographic method modified from that of Hodgkinson et al. (11). Briefly, specific anti-IGF-I immunoglobulin (Ig) was purified from an Ig fraction of whole serum (12) by cycling batches of 300-400 ml through an IGF-I Sepharose column ( 1 x 5 cm; 4 mg IGF-I/g CNBr-Sepharose). After affinity adsorption the column was washed to remove extraneous protein, and specific anti-IGF-I immunoglobulin was eluted using acidic buffers containing acetonitrile (20%, vol/ vol). The purification was monitored by antiserum dilution/ IGF-I tracer binding. One hundred and eighty milligrams of specific anti-IGF-I were recovered from a 2-liter pool of antiserum. Antibody was resalted into 0.15 M NaCl using an Amicon ultrafiltration system (HIP10 hollow fiber cartridge), concentrated to 1 mg/ml, and stored at -20 C. Immunoneutralizing capacity of the Ig After purification, the ability of the anti-IGF-I antibody preparation to neutralize the effects of IGF-I was measured in
Endo»1991 Vol 128* No 4
vivo and in vitro. The functional binding capacity of the purified anti-IGF-I antibody in vitro was calculated from its titer as 7.5 Mg IGF-I/mg purified antibody (7.5 /ig IGF-I/ml solution). The ability of different doses of the anti-IGF-I antibody to neutralize sulfate uptake into rat costal cartilage in vitro was measured. Normal rat plasma (NRP) was used as the standard, and the same plasma with different amounts of anti-IGF-I antibody added before serial dilution was used to assess the ability of the antibody preparation to inhibit or neutralize sulfate uptake in a normal rat cartilage bioassay (13). Similar evaluation was made by RRA with confluent cultured L6 myoblasts (14-16). Assays were performed in quadruplicate, with sequential addition of radioiodinated IGF-I (50,000 cpm/ 200 n\) and 200 /A antibody at various dilutions from 10"3-10~5. After incubation for 2 h at 22 C, the cells were washed three times, and bound label was solubilized using sodium hydroxideTriton X-100. Using the information obtained from the in vitro evaluation of immunoneutralizing capacity of the anti-IGF-I antibody preparation, an assessment of the quantities of antibody needed for in vivo immunoneutralization was made. Different quantities of the anti-IGF-I antibody were administered ip to rats, and the animals were bled 24 h after injection. The ability of the plasma from these rats to stimulate sulfate uptake in the in vitro bioassay was investigated. Having established that the antibody was capable of completely neutralizing the sulfate uptake when given at less than 3 mg/kg BW, this dose was used in the passive immunization growth trial. Passive immunization growth trial At 49 days of age the rats were weighed and allocated to groups. One group acted as a control group (normal dwarves; n = 8), another group (n = 8) received daily injections of 2 mg/ kg, sc, recombinant bGH (Cyanamid), a third group (n = 16) received a similar daily injection of bGH and also a daily injection of 300 /xl IgG, ip, prepared from normal sheep serum (NSS) at a concentration of 956 Mg/ml, while the last group (n = 16) received bGH and a 300 /tl ip injection of the anti-IGF-I IgG (at 956 /ig/ml) each day. The animals were weighed and injected between 0830-0930 h each day. Treatment continued for 10 days, and 24 h after the last injection the animals were anesthetized with CO2, bled by cardiac puncture, and killed. Plasma was separated and frozen for later evaluation of residual excess antibody-binding capacity and plasma IGF-I levels. Body weights and the weights of liver, spleen, kidney, and thymus were measured immediately after slaughter. IGF-I tracer binding to terminal plasma Plasma dilutions (100 n\) were incubated for 24 h at 4 C with 200 /il radioiodinated IGF-I (20,000 cpm; 120 tiC\/ng) prepared as previously described (17). Diluent for all assay reagents and dilutions was 64 raM phosphate buffer (pH 7.4) containing 0.25% (wt/vol) BSA. Antibody-bound and free tracer were separated by centrifugation after precipitation of the bound fraction using polyethylene glycol 6000 (20%, wt/vol) with the addition of carrier IgG (0.6 mg/tube). Data are expressed as the percentage of tracer specifically bound by the antibody.
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PASSIVE IMMUNIZATION AGAINST IGF-I Plasma IGF-I extraction and RIA Before measurement in a RIA, samples were extracted by reversed phase HPLC (C18 Microbondapak, Waters Associates, Millford, MA; Alltech, Deerfield, IL) and lyophilized. Removal of binding proteins and antibody from IGF-I fractions was confirmed before assay by IGF-I tracer binding at neutral pH and size-exclusion chromatography (11, 18). IGF-I was estimated by a limited reagent RIA (Hodgkinson, S. C, J. J. Bass, and P. D. Gluckman, submitted) modified from the method of Gluckman et al. (20) as validated for rat plasma (18). Briefly, recombinant human IGF-I (courtesy of Dr. A. Skottner, KabiVitrum, Stockholm, Sweden) was used as standard and, after iodination (21), as radioligand. Antiserum to IGF-I was raised in-house and used at an initial dilution of 1:10,000. Cross-reactivity with IGF-2 was less than 1.0%, and that with insulin was less than 0.1%. Assays were performed by incubation of standard or sample with tracer and antiserum for 24 h at 4 C. Separation of bound and free fractions was achieved using a magnetizable solid phase (Advanced Magnetics, Inc., Cambridge, MA) second antibody. Estimates of interand intraassay variation were 10.2% and 7.8%, respectively. Perturbation of IGF-I-binding protein interaction by the antibody It was considered possible that the IGF-I-binding proteins may protect the IGF-I from the antibody, thus enabling delivery of the IGF-I to peripheral sites just as in nonimmunized animals. To examine this possibility the antibody binding to carrier protein-bound IGF was studied. NRP (200 /xl) was incubated overnight at 4 C with IGF-I tracer (100,000 cpm) and subjected to size-exclusion chromatography at neutral pH, as previously described. Further samples of the preincubated plasma were then incubated for a further 4 h at 4 C together with 200 n\ terminal plasma from rats given either normal sheep IgG or anti-IGF-I antibody, followed by size-exclusion chromatography.
Results Immunoneutralizing ability of the antiserum and IgG preparations Initial assays using addition of unpurified antiserum to the costal cartilage bioassay in vitro showed that the antiserum was capable of inhibiting sulfate uptake into cartilage (Fig. 1); the higher dose of antibody producing an 85% inhibition (potency ratio, 0.156). Subsequently, it was shown that the affinity-purified IgG fraction of these antisera was also capable of inhibiting sulfation in a dose-dependent manner when added to an in vitro costal cartilage bioassay (Fig. 2), and finally, it was demonstrated that administration of the IgG fraction to GH-treated dwarf rats in vivo was able to reverse the effect of the GH treatment, as reflected by the ability of the plasma to stimulate cartilage sulfation in vitro (Fig. 3). Purified antibody effectively blocked IGF-I binding to
9
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% Plasma FIG. 1. Dose-response curves of the 35SO4-incorporating ability (bioassayable somatomedin/IGF-I) of NRP ( ), the same NRP to which 7.6 n\ anti-IGF-I serum were added before dilution for use in the assay (- -), and NRP containing 76 n\ anti-IGF-I serum (- - -).
4
-
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FIG. 2. Dose-response curves of the 35 SO 4 -incorporating ability (bioassayable somatomedin/IGF-I) of NRP ( ) and the same N R P to which 1.5 mg/ml affinity-purified IgG from anti-IGF-I serum were added before dilution for use in the assay ( - - - ) .
receptors. Thus, the presence of IGF-I antibody resulted in a dose dependent inhibition of IGF-I tracer binding to L6 myoblasts (ED50 = 5 x 10"3) (Fig. 4). Effects of passive immunization on growth GH treatment of the dwarf rats resulted in a significant (P < 0.001) increase in growth velocity compared with
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PASSIVE IMMUNIZATION AGAINST IGF-I
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in the size of liver, kidney, spleen, and thymus were 99%, 97%, 92%, and 96% of controls, respectively.
-i
Effects of passive immunization on blood hormone and IgG levels 8 a o
5
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7
-
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% Plasma FlG. 3. Dose-response curves of the 35SO4-incorporating ability (bioassayable somatomedin/IGF-I) of serum from a rat receiving NSS IgG ( ) and a rat receiving the affinity-purified fraction of anti-IGF-I serum (- - -).
100
o
Large amounts of ovine IgG were found in the plasma from all rats injected with antibodies, but in none of the control rats or those receiving GH alone. These data show that the IgG from the ip injection reached the blood and remained in the circulation for at least 24 h. Labeled IGF-I was significantly sequestered by the plasma from the IGF-I antibody-treated rats, but not by that from the control rats receiving normal sheep serum IgG, indicating that there was sufficient antiserum present to effectively bind additional IGF-I (Fig. 6). Binding of tracer to 1:100 dilutions of treatment plasmas (collected 24 h after antibody injection) was 65 ± 3.8% (mean ± SEM; n = 15). Binding of tracer to other (non-IGF-I IgG treatment) plasma (12 ± 2.4%) was not significantly different from estimates of nonspecific tracer precipitation (Fig. 6). GH treatment resulted in a 100% increase in total plasma IGF-I levels over the levels in the control dwarves. Levels of IGF-I in the plasma of the control dwarves ranged from 50-60 ng/ml, while levels in rats treated with 200 Mg/kg bGH all fell within the range of 100-130 ng/ml. There was no difference in total IGF-I in the terminal plasma from any of the three GH-treated groups of rats.
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Perturbation of IGF-I-binding protein interaction by antibody 60
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Log Antibody dilution 125
FlG. 4. The inhibition of [ I]IGF-I binding to L6 myoblasts in culture (expressed as a percentage of maximum binding, Bo) by varying dilutions of anti-IGF-I serum added to the incubation medium.
untreated dwarves. Concurrent administration of either NSS IgG or anti-IGF-I antibody failed to have any effect on GH-stimulated growth (Fig. 5); all three GH-treated groups grew at the same rate. Although liver weights in the GH-treated animals were 10% heavier than in the controls, this was not statistically significant, and the increase was in proportion to total body size. There were also no significant differences between the groups with regard to the weight of any of the organs measured (Table 1). When corrected for body weight, the mean changes
An aliquot of terminal plasma from the rats was incubated with IGF-I tracer, and the equilibrated mixture was subjected to size-exclusion chromatography. Normal dwarf rat plasma (Fig. 7, top panel) and plasma from GH- plus NSS-treated rats (Fig. 7, middle panel) showed similar profiles. By contrast, secondary incubation with plasma from anti-IGF-I-treated rats resulted in a marked high mol wt shift of eluted radioactivity (Fig. 7, bottom panel). Further tracer (25%) was identified in an insoluble fraction that was removed before chromatography. These data provide strong evidence that the anti-IGF-I antibody sequestered the IGF-I when it was prebound to its binding proteins. Discussion The results of the present study showed that immunoneutralization of circulating IGF-I did not inhibit GHstimulated growth in GH-deficient rats. In all studies in which passive immunization is used as a means of eliciting a physiological response, the neutralizing capability of the antiserum is crucial. In our experiment, the neutralizing ability of the preparation was extensively tested to ensure it was adequate. The
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PASSIVE IMMUNIZATION AGAINST IGF-I
2107
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FIG. 5. a, Growth curves for control dwarf rats (- - -) and for dwarf rats receiving 2 mg/kg day bGH, bGH plus NSS IgG, or bGH plus anti-IGFI serum daily from day 0 ( ). b, Growth velocities (mean ± SEM) in control dwarves and those receiving bGH, bGH plus NSS IgG, or bGH plus anti-IGF-I serum. ***, P < 0.001 compared with control animals. TABLE 1. Average daily weight gain (ADWG) and liver, kidney, spleen, and thymus weights in dwarf rats receiving saline, bGH (2 mg/kg day), 2 mg/kd-day bGH plus 287 ng normal sheep IgG, or 2 mg/kg day bGH plus 287 ng anti-IGF-I IgG ADWG (g) Saline GH GH + IgG GH + anti-IGF-I
2.75 ± 4.12 ± 4.13 ± 4.01 ±
0.11 0.15 0.18 0.18
Liver wt (g) 5.60 ± 0.21 6.12 ± 0.17 6.20 ± 0.23 6.00 ± 0.23
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6
7
8 9 10 11 12 13 14 15
Animal number 125
FIG. 6. Amount of [ I]IGF-I (expressed as a percentage of the total counts added) bound by a 1:1000 dilution of terminal plasma from rats passively immunized against IGF-I (•); control dwarves, GH-treated, and GH- plus NSS IgG-treated dwarf rat plasmas bound only 12 ± 2% of the label (M).
ability of both the antiserum and the purified IgG fraction of the anti-IGF-I antiserum to inhibit binding of IGF-I to receptors on myoblasts in culture and to inhibit the specific biological action of IGF-I in stimulating sulfate incorporation into cartilage in vitro was demonstrated. The affinity of the anti-IGF-I antibody preparation in vitro (Ka, 2 x 1010 mol/liter) was similar to the expected Ka of both IGF-I receptors and IGF-binding proteins (22-24). Although the antibodies were clearly capable of neutralizing the action of IGF-I in vitro, it was important to demonstrate that the dose given was sufficient to completely immunoneutralize circulating IGF-I in vivo. The dose of anti-IGF-I was estimated by calculation and confirmed by preliminary experimentation. The com-
Kidney wt (g) 1.06 ± 0.05 1.09 ± 0.01 1.07 ± 0.02 1.11 ± 0.03
Spleen wt 0.40 ± 0.42 ± 0.42 ± 0.43 ±
0.05 0.01 0.02 0.02
Thymus wt (g) 0.41 ± 0.04 0.37 ± 0.03 0.43 ± 0.06 0.38 ± 0.03
plete inhibition of plasma sulfation factor activity by this dose when given to rats in vivo confirms the adequacy of the neutralizing ability of this dose of antibody. Furthermore, the presence of excess immunoneutralizing capacity was supported by the finding of free anti-IGF-Ibinding capacity in terminal plasmas. The anti-IGF-I antibody was not only able to sequester free IGF-I, but was also able to bind IGF-I when prebound to plasma binding proteins. This was illustrated by the finding that trace labeled IGF-binding protein complexes demonstrated a high mol wt shift on incubation with terminal plasmas. Thus, not only did the antibodies have sufficient capacity to bind circulating IGFI, but it appears that their epitope specificity is such that they interact with precomplexed IGF-I. It would seem unlikely that such a large mol wt complex would readily cross from the plasma to the extracellular space where cell receptors might have an opportunity to compete with the antibody complex for the IGF-I. Transfer of small amounts of antibody (probably as free Ig) out of the bloodstream has previously been reported in rats (25). Quantification of the amount of antibody in the tissues and whether this was sufficient to neutralize tissue IGFI was not possible in this study. However, the affinities of the antibodies and receptors appear comparable and the presence of small amounts of antibody in tissues may also have been sufficient to perturb the activity of locally produced IGF-I. Certainly in vitro, antibody could inhibit binding of IGF-I to receptors at high dilution. A recent report has shown that passive immunization against IGF-I has no effect on growth in guinea pigs (8). The GH-deficient dwarf rat used in the present study is
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PASSIVE IMMUNIZATION AGAINST IGF-I
2108 20000 i •«-*
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Fraction number FIG. 7. FPLC profiles of sera from control dwarf rats (top panel), GHtreated dwarf rats (middle panel), and GH-treated dwarf rats (bottom panel) passively immunized with anti-IGF-I serum, after incubation with [126I]IGF-I tracer. Clearly, administration of anti-IGF-I serum resulted in displacement of all of the IGF-I label into a high (>150 kDa) mol wt fraction.
more suitable for this type of experiment than the guinea pig, since elevations in plasma IGF-I and growth rates can clearly be achieved by GH administration (9,10, 26, 27); growth of the guinea pig is not GH dependent (2830), and it is also not certain that IGF-I is GH dependent in the guinea pig. It has been suggested that the growth of the adult guinea pig may resemble the growth of fetuses, in that the GH-IGF-I axis may not be of great importance (31). The GH-deficient dwarf rat has an additional advan-
Endo • 1991 Voll28«No4
tage over animals with normal GH secretion for use in these studies, in that positive feedback, by removing IGFI, cannot occur through increased GH secretion (although effects on other pituitary hormones may be possible). In contrast to our results, Kerr et al. (8) reported increased levels of IGF-I in their passively immunized animals. They suggested that this was a result of either increased production of IGF-I through diminished negative feedback or decreased metabolic clearance of the antibody-bound hormone. The question arises as to whether IGF-I plays a role in mediating the growth-promoting activity of GH by endocrine or autocrine/paracrine actions. Plasma levels of IGF-I have been correlated with growth and used as a marker for GH deficiency. Modest reductions in plasma IGF-I levels (50%) lead to severe dwarfism, yet the levels of IGF-I, as measured by bioassay, are much lower than those in these passively immunized animals. This suggests that the major effect of GH is by local release of IGF-I for autocrine/paracrine actions. If this is so, it raises the question of the role of circulating IGF-I; if this is not so, then it questions the role of IGF-I in growth at any level. No data are available from this study on the levels of IGF-I in the tissues of these animals, but such measurements could help determine the relative contributions of tissue and plasma levels of IGF-I in growth. A less likely explanation for the lack of effect of immunoneutralization of circulating IGF-I on growth could be that (particularly in a GH-deficient animal) there may have been an increase in the number of IGFI receptors in the target tissues, thus allowing more of any transiently unneutralized circulating IGF-I to have its biological effect. This proposal stems from extrapolation of the report on receptor down-regulation by IGFI (32). Finally, the most logical explanation of these results is that, at least in the GH-treated dwarf rat, IGF-I circulating in plasma plays only a small part in stimulating growth. This, however, does not preclude the possibility that blood-borne IGF-I may fulfill specific endocrine functions in the support of growth processes. Examples of this may be whole body protein and glucose metabolism (33), erthyropoiesis (34, 35), and renal function (36). Alternatively, it is possible that in the GHtreated dwarf rat, circulating IGF-I represents a clearing system for local IGF-I that is produced in excess of autocrine/paracrine requirements.
Acknowledgments We are grateful to P. Dobbie for help with the animal management, and to J. Napier for purification of the antibodies. Dr B. D. Burleigh kindly provided the IGF-I for antibody production and affinity purification, and the recombinant bGH was a gift from Cyanamid.
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PASSIVE IMMUNIZATION AGAINST IGF-I
References 1. Evans HM, Simpson ME, Marx W, Kibrick E 1943 Bioassay of pituitary growth hormone. Width of proximal epiphyseal cartilage in tibia in hypox rats. Endocrinology 32:13-16 2. Ellis S, Huble J, Simpson ME 1953 Influence of hypophysectomy and growth hormone on cartilage sulphate metabolism. Proc Soc Exp Biol Med 84:603-605 3. Salmon WD, Daughaday WH 1957 Hormonally controlled serum factor which stimulates sulphate incorporation by cartilage in vitro. J Lab Clin Med 49:825-836 4. Daughaday WH, Hall K, Raben MS, Salmon WD, Van den Brande JL, Van Wyk JJ 1972 A proposed designation for a sulphation factor. Nature 235:107 5. Van Buul-Offers S, Van den Brande JL 1980 Effect of growth hormone and peptide fractions containing somatomedin activity on growth and cartilage metabolism of Snell dwarf mice. Acta Endocrinol (Copenh) 92:242-257 6. Schoenle E, Zapf J, Humbel RE, Froesch ER 1982 Insulin-like growth factor-1 stimulates growth in hypophysectomized rats. Nature 296:252-253 7. D'Ercole AJ, Applewhite GT, Underwood LE 1980 Evidence that somatomedin is synthesised by multiple tissues in the fetus. Dev Biol 75:315-328 8. Kerr DE, Laarveld B, Manns JG 1990 Effects of passive immunization of growing guinea-pigs with an insulin-like growth factor-I monoclonal antibody. J Endocrinol 124:403-415 9. Charlton, HM, Clark RG, Robinson ICAF, Porter Goff AE, Cox BS, Bugnon C, Bloch BA 1988 Growth hormone deficient dwarfism in the rat: a new mutation. J Endoorinol 119:51-58 10. Dobbie P, Bass J, Hodgkinson S, Spencer S, Clark R, Growth studies on a growth hormone-deficient dwarf rat and the effect of long term GH treatment. Program of the Combined Australian/ New Zealand Endocrine Societies, Melbourne, Australia, 1989 (Abstract 201) 11. Hodgkinson SC, Moore LG, Napier JR, Davis SR, Bass JJ, Gluckman PD 1988 Characterisation of insulin-like growth factor binding proteins in ovine tissue fluids. J Endocrinol 120:429-438 12. Kekwick RA 1940 The serum proteins in multiple myelomatosis Biochem J 34:1248-1257 13. Yde H 1968 A simplified technique for the determination of growth hormone dependent sulphation factor using an intact animal. Acta Endocrinol (Copenh) 57:557-564 14. Ballard FJ, Read LC, Francis GL, Bagley CJ, Wallace JC 1986 Binding properties and biological potencies of insulin-like growth factors in L6 myoblasts. Biochem J 233:223-230 15. Ballard FJ, Francis GL, Ross N, Bagley CJ, May B, Wallace JC 1987 Natural and synthetic forms of insulin-like growth factor-I (IGF-I) and the potent derivative Des tripeptide IGF-I: biological properties and receptor binding. Biochem Biophys Res Commun 149:398-402 16. Hodgkinson SC, Napier JR, Davis SR, Patel B, Gluckman PD 1989 Binding protein, radioreceptor and biological activities of methionyl insulin-like growth factor-1 variants. Mol Cell Endocrinol 66:37-44 17. Hodgkinson SC, Lowry PJ 1982 Selective elution of immunoadsorbed anti (human) prolactin immunoglobulin with enhanced immunochemical properties. Biochem J 205:535-541 18. Brier BH, Gallaher BW, Gluckman PD, The radioimmunoassay
19. 20.
21.
22. 23.
24.
25. 26. 27.
28. 29. 30.
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for insulin-like growth factor-1: a re-assessment. J Endocrinol, in press Deleted in proof Gluckman PD, Johnson-Barrett JJ, Butler JH, Edgar B, Gunn TR 1983 Studies of insulin-like growth factor-I II by specific radioligand assays in umbilical cord blood. Clin Endocrinol (Oxf) 19:405413 Salacinski PR, McLean C, Sykes JEC, Clement-Jones VV, Lowry PJ 1981 Iodination of proteins, glycoproteins and peptides using a solid-phase oxidising agent l,3,4,6,-tetrachloro-3..,6..diphenyl glyouril (Iodogen). Anal Biochem 117:136-146 Martin JL, Baxter RC 1986 Insulin-like growth factor-binding protein from human plasma: purification and characterization. J Biol Chem 261:8754-8760 Tramontano D, Moses AC, Picone R, Ingbar SH 1986 Characterization and regulation of the receptor for insulin-like growth factor1 in the FRTL-5 rat thyroid follicular cell line. Endocrinology 120:785-790 Adashi EY, Resnick CE, Hernadez ER, Svoboda ME, van Wyk JJ 1988 Characterization and regulation of a specific cell membrane preparation for somatomedin-C/insulin-like growth factor-I in cultured rat granulosa cells. Endocrinology 122:194-201 Worrel NR, Cumber AJ, Parnell GD, Ross WCJ, Forrester JA 1986 Fate of an antibody-ricin A chain conjugate administered to normal rats. Biochem Pharmacol 35:417-423 Robinson G, Hodgkinson SC, Spencer GSG, Bass JJ, IGF-1 response to GH in the GH-deficient dwarf rat. Program of the New Zealand Society for Endocrinology, 1990, p 1 (Abstract) Skottner A, Clark RG, Fryklund L, Robinson ICAF 1989 Growth responses in a mutant dwarf rat to human growth hormone and recombinant human insulin-like growth factor-I. Endocrinology 124:2519-2562 Mitchell ML, Guillemin R, Seyle H 1954 The effect of somatotropic hormone in the growth of normal and hypophysectomized guineapigs. Endocrinology 54:111-114 Knobil E, Greep RO 1959 The physiology of growth hormone with particular reference to its action in the rhesus monkey and the 'species specificity' problem. Recent Prog Horm Res 15:1-69 Clayton BE, Worden JA 1960 Growth in young hypophysectomized guinea-pig. J Endocrinol 20:30-47
31. Knobil E, Hotchkiss J 1964 Growth hormone. Annu Rev Physiol 26:47-74 32. Rosenfeld RG, Hintz RL 1980 Characterization of a specific receptor for somatomedin-C (SM-C) on cultured human lymphocytes: evidence that SM-C modulates homologous receptor concentrations. Endocrinology 107:1841-1848 33. Jacob R, Barrett E, Plewe G, Fagin KD, Sherwin RS 1989 Acute effects of insulin-like growth factor-1 on glucose and amino-acid metabolism in the awake fasted rat. J Clin Invest 83:1717-1723 34. Kurtz A, Matter R, Eckdardt KU, Zapf J 1990 Erthyropoiesis, serum erthyropoietin and serum IGF-1 in rats during accelerated growth. Acta Endocrinol (Copenh) 122:323-328 35. Phillips AF, Persson B, Hall K 1988 The effects of biosynthetic insulin-like growth factor-1 supplementation on somatic growth, maturation and erthyropoiesis in the neonatal rat. Pediatr Res 23:298-305 36. Guler H-P Eckardt K-U, Zapf J, Bauer C, Froesch ER 1989 Insulinlike growth factor-I increases glomerular filtration rate and renal plasma flow in man. Acta Endocrinol (Copenh) 121:101-106
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Vol. 128, No. 4 Printed in U.S.A.
Passive Immunization against Insulin-Like Growth Factor-I Does not Inhibit Growth HormoneStimulated Growth of Dwarf Rats G. S. G. SPENCER*, S. C. HODGKINSON, AND J. J. BASS Ruakura Agricultural Centre, Private Bag, Hamilton, New Zealand
ABSTRACT. Passive immunization against insulin-like growth factor-I (IGF-I) was undertaken in GH-deficient rats in an attempt to elucidate the relative importance of the endocrine vs. autocrine/paracrine actions of IGF-I in stimulating growth. Antiserum against IGF-I was raised in sheep and purified by affinity chromatography. The ability of the purified antibodies to neutralize the actions of IGF-I in vitro and bind IGF-I in vivo were extensively tested using L6 myoblast and cartilage bioassays. Four groups of male rats with isolated GH deficiency were used in the study. At 49 days of age the rats received 100 pi normal saline given sc each day for 10 days, 2 mg/kg recombinant bovine GH (bGH) given in 100 ft\, sc, each day, 2 mg/kg bGH, sc, and 300 nl immunoglobulin G purified from normal sheep serum given daily ip, or 2 mg/kg bGH plus 300 (A anti-IGF-I immunoglobulin G daily, ip (a dose that was able to completely
H
YPOPHYSECTOMIZED (hypox) rats fail to grow, but growth can be restored by the administration of GH (1). Similarly, synthesis of cartilage matrix glycosaminoglycans, such as chondroitin sulfate, is reduced in hypox animals, but is restored to normal by GH treatment (2). Although plasma from GH-treated hypox animals stimulates sulfate uptake into cartilage in vitro, direct addition of GH to hypox serum has usually been ineffective (3). These observations suggest that there is a GH-dependent serum factor that mediates the effects of GH on cartilage sulfation. From this grew the belief that all of the anabolic actions of GH were mediated by this sulfation factor (and a similar thymidine factor which stimulated thymidine uptake into cells in like fashion). These factors were later called somatomedins (4) and, ultimately, insulin-like growth factors (IGFs). Supporting the concept that IGFs are the mediators of the action of GH is a weight of circumstantial evidence that low levels of IGF are often associated with low growth rates, and (less consistently) higher levels of IGFs may be associated with overgrowth. Purified preparations of IGFs have also been shown to stimulate growth Received September 24,1990. * To whom requests for reprints should be addressed.
inhibit IGF-I actions on sulfate uptake into cartilage). Treatment with GH significantly increased growth rates (P < 0.001) in the rats, but there was no difference between any of the three GH-treated groups; passive immunization against IGF-I did not diminish the GH-stimulated growth in these rats. Excess antibody could be detected in the plasma of all anti-IGF-I-treated rats at the conclusion of the experiment, and the antibody was capable of sequestering both free and binding protein-bound IGF-I. The absence of even a slight retardation of GH-stimulated growth in the anti-IGF-I-treated rats suggests that circulating IGF-I may not be important in mediating the growth-promoting actions of GH, although the immunoneutralization probably does not affect GH stimulation of tissue IGF-I production. (Endocrinology 128: 2103-2109, 1991)
in hypopituitary or hypox animals (5, 6), but the responses have never been as great as those obtained with GH treatment. On the face of it, these data might suggest that plasma IGF-I does not play a large part in stimulating growth, although the lack of effect is likely to be partly due to factors such as impurity of the preparations used, lack of cofactors, accelerated clearance in the absence of binding proteins, or inappropriate administration schedules. It has been reported that GH can stimulate IGF-I production from a number of different tissues (7), and it may be via this locally produced IGF that GH has its actions. If this is so, what is the role.of the circulating IGF? Does it reflect a sink for clearance of excess IGF-I produced for autocrine/paracrine actions or is circulating IGF-I required for growth? In an attempt to answer this question we have raised large quantities of high affinity antibodies specific to IGF-I and used them to immunoneutralize plasma IGFI. These studies extend a contemporary study of passive immunization against IGF-I that has been recently reported (8), but in that study it was not shown that the antibody used could immunoneutralize IGF action, and the choice of an animal model in which growth is not GH dependent (guinea pig) was unfortunate. The use of 2103
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2104
PASSIVE IMMUNIZATION AGAINST IGF-I
an animal with an isolated GH deficiency and demonstration of inhibition of IGF action by the antibodies should provide a more reliable indication of the role of IGF-I in growth.
Materials and Methods GH-deficient rats The rats used in this study were bred at Ruakura from the strain of rats with an isolated GH deficiency originally identified and reported by Charlton et al. (9). These rats have been shown to respond to exogenous GH treatment with marked increases in growth rate (10). Forty-eight male rats were used in the growth trial which started at 49 days of age; this has previously been identified as an age at which the rats were particularly responsive to exogenous GH treatment (10), but was free from possible interference associated with puberty. The rats were housed in the same room in groups of eight, with treatments spread at random throughout the groups. Ad libitum access to food and water were available to all rats at all times; temperature and humidity were controlled and a 12-h light, 12h dark regimen was in place. Generation and purification of the IGF-I antiserum Antisera were raised in sheep to an egg albumin conjugate of recombinant human IGF-I (courtesy of Dr. B. D. Burleigh, IMC/Pitman Moore, Terre Haute, IN). The conjugates were prepared by coupling IGF-I to carrier protein at a molar ratio of 10:1 using l-ethyl-3-(3-dimethylamino-propyl)carbodiimide HC1 and stored at -20 C until use. Animals were immunized by im injection at four sites with conjugate (equivalent to 200 ng IGF-I/injection) emulsified in Freund's Complete Adjuvant (for the primary immunization) and in Freund's Incomplete Adjuvant on subsequent occasions; booster injections were given at monthly intervals. Antibody production was monitored by measuring IGF-I tracer binding (as outlined below) in blood samples collected 2 weeks after each booster injection. Purification of the IGF-I antibody Specific anti-IGF-I antibody was purified by an automated affinity chromatographic method modified from that of Hodgkinson et al. (11). Briefly, specific anti-IGF-I immunoglobulin (Ig) was purified from an Ig fraction of whole serum (12) by cycling batches of 300-400 ml through an IGF-I Sepharose column ( 1 x 5 cm; 4 mg IGF-I/g CNBr-Sepharose). After affinity adsorption the column was washed to remove extraneous protein, and specific anti-IGF-I immunoglobulin was eluted using acidic buffers containing acetonitrile (20%, vol/ vol). The purification was monitored by antiserum dilution/ IGF-I tracer binding. One hundred and eighty milligrams of specific anti-IGF-I were recovered from a 2-liter pool of antiserum. Antibody was resalted into 0.15 M NaCl using an Amicon ultrafiltration system (HIP10 hollow fiber cartridge), concentrated to 1 mg/ml, and stored at -20 C. Immunoneutralizing capacity of the Ig After purification, the ability of the anti-IGF-I antibody preparation to neutralize the effects of IGF-I was measured in
Endo»1991 Vol 128* No 4
vivo and in vitro. The functional binding capacity of the purified anti-IGF-I antibody in vitro was calculated from its titer as 7.5 Mg IGF-I/mg purified antibody (7.5 /ig IGF-I/ml solution). The ability of different doses of the anti-IGF-I antibody to neutralize sulfate uptake into rat costal cartilage in vitro was measured. Normal rat plasma (NRP) was used as the standard, and the same plasma with different amounts of anti-IGF-I antibody added before serial dilution was used to assess the ability of the antibody preparation to inhibit or neutralize sulfate uptake in a normal rat cartilage bioassay (13). Similar evaluation was made by RRA with confluent cultured L6 myoblasts (14-16). Assays were performed in quadruplicate, with sequential addition of radioiodinated IGF-I (50,000 cpm/ 200 n\) and 200 /A antibody at various dilutions from 10"3-10~5. After incubation for 2 h at 22 C, the cells were washed three times, and bound label was solubilized using sodium hydroxideTriton X-100. Using the information obtained from the in vitro evaluation of immunoneutralizing capacity of the anti-IGF-I antibody preparation, an assessment of the quantities of antibody needed for in vivo immunoneutralization was made. Different quantities of the anti-IGF-I antibody were administered ip to rats, and the animals were bled 24 h after injection. The ability of the plasma from these rats to stimulate sulfate uptake in the in vitro bioassay was investigated. Having established that the antibody was capable of completely neutralizing the sulfate uptake when given at less than 3 mg/kg BW, this dose was used in the passive immunization growth trial. Passive immunization growth trial At 49 days of age the rats were weighed and allocated to groups. One group acted as a control group (normal dwarves; n = 8), another group (n = 8) received daily injections of 2 mg/ kg, sc, recombinant bGH (Cyanamid), a third group (n = 16) received a similar daily injection of bGH and also a daily injection of 300 /xl IgG, ip, prepared from normal sheep serum (NSS) at a concentration of 956 Mg/ml, while the last group (n = 16) received bGH and a 300 /tl ip injection of the anti-IGF-I IgG (at 956 /ig/ml) each day. The animals were weighed and injected between 0830-0930 h each day. Treatment continued for 10 days, and 24 h after the last injection the animals were anesthetized with CO2, bled by cardiac puncture, and killed. Plasma was separated and frozen for later evaluation of residual excess antibody-binding capacity and plasma IGF-I levels. Body weights and the weights of liver, spleen, kidney, and thymus were measured immediately after slaughter. IGF-I tracer binding to terminal plasma Plasma dilutions (100 n\) were incubated for 24 h at 4 C with 200 /il radioiodinated IGF-I (20,000 cpm; 120 tiC\/ng) prepared as previously described (17). Diluent for all assay reagents and dilutions was 64 raM phosphate buffer (pH 7.4) containing 0.25% (wt/vol) BSA. Antibody-bound and free tracer were separated by centrifugation after precipitation of the bound fraction using polyethylene glycol 6000 (20%, wt/vol) with the addition of carrier IgG (0.6 mg/tube). Data are expressed as the percentage of tracer specifically bound by the antibody.
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PASSIVE IMMUNIZATION AGAINST IGF-I Plasma IGF-I extraction and RIA Before measurement in a RIA, samples were extracted by reversed phase HPLC (C18 Microbondapak, Waters Associates, Millford, MA; Alltech, Deerfield, IL) and lyophilized. Removal of binding proteins and antibody from IGF-I fractions was confirmed before assay by IGF-I tracer binding at neutral pH and size-exclusion chromatography (11, 18). IGF-I was estimated by a limited reagent RIA (Hodgkinson, S. C, J. J. Bass, and P. D. Gluckman, submitted) modified from the method of Gluckman et al. (20) as validated for rat plasma (18). Briefly, recombinant human IGF-I (courtesy of Dr. A. Skottner, KabiVitrum, Stockholm, Sweden) was used as standard and, after iodination (21), as radioligand. Antiserum to IGF-I was raised in-house and used at an initial dilution of 1:10,000. Cross-reactivity with IGF-2 was less than 1.0%, and that with insulin was less than 0.1%. Assays were performed by incubation of standard or sample with tracer and antiserum for 24 h at 4 C. Separation of bound and free fractions was achieved using a magnetizable solid phase (Advanced Magnetics, Inc., Cambridge, MA) second antibody. Estimates of interand intraassay variation were 10.2% and 7.8%, respectively. Perturbation of IGF-I-binding protein interaction by the antibody It was considered possible that the IGF-I-binding proteins may protect the IGF-I from the antibody, thus enabling delivery of the IGF-I to peripheral sites just as in nonimmunized animals. To examine this possibility the antibody binding to carrier protein-bound IGF was studied. NRP (200 /xl) was incubated overnight at 4 C with IGF-I tracer (100,000 cpm) and subjected to size-exclusion chromatography at neutral pH, as previously described. Further samples of the preincubated plasma were then incubated for a further 4 h at 4 C together with 200 n\ terminal plasma from rats given either normal sheep IgG or anti-IGF-I antibody, followed by size-exclusion chromatography.
Results Immunoneutralizing ability of the antiserum and IgG preparations Initial assays using addition of unpurified antiserum to the costal cartilage bioassay in vitro showed that the antiserum was capable of inhibiting sulfate uptake into cartilage (Fig. 1); the higher dose of antibody producing an 85% inhibition (potency ratio, 0.156). Subsequently, it was shown that the affinity-purified IgG fraction of these antisera was also capable of inhibiting sulfation in a dose-dependent manner when added to an in vitro costal cartilage bioassay (Fig. 2), and finally, it was demonstrated that administration of the IgG fraction to GH-treated dwarf rats in vivo was able to reverse the effect of the GH treatment, as reflected by the ability of the plasma to stimulate cartilage sulfation in vitro (Fig. 3). Purified antibody effectively blocked IGF-I binding to
9
-i
3
-
2105
o
10
30
% Plasma FIG. 1. Dose-response curves of the 35SO4-incorporating ability (bioassayable somatomedin/IGF-I) of NRP ( ), the same NRP to which 7.6 n\ anti-IGF-I serum were added before dilution for use in the assay (- -), and NRP containing 76 n\ anti-IGF-I serum (- - -).
4
-
a
E o U
0
3
10 30 % Plasma
FIG. 2. Dose-response curves of the 35 SO 4 -incorporating ability (bioassayable somatomedin/IGF-I) of NRP ( ) and the same N R P to which 1.5 mg/ml affinity-purified IgG from anti-IGF-I serum were added before dilution for use in the assay ( - - - ) .
receptors. Thus, the presence of IGF-I antibody resulted in a dose dependent inhibition of IGF-I tracer binding to L6 myoblasts (ED50 = 5 x 10"3) (Fig. 4). Effects of passive immunization on growth GH treatment of the dwarf rats resulted in a significant (P < 0.001) increase in growth velocity compared with
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PASSIVE IMMUNIZATION AGAINST IGF-I
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in the size of liver, kidney, spleen, and thymus were 99%, 97%, 92%, and 96% of controls, respectively.
-i
Effects of passive immunization on blood hormone and IgG levels 8 a o
5
t
o U
7
-
I
10
% Plasma FlG. 3. Dose-response curves of the 35SO4-incorporating ability (bioassayable somatomedin/IGF-I) of serum from a rat receiving NSS IgG ( ) and a rat receiving the affinity-purified fraction of anti-IGF-I serum (- - -).
100
o
Large amounts of ovine IgG were found in the plasma from all rats injected with antibodies, but in none of the control rats or those receiving GH alone. These data show that the IgG from the ip injection reached the blood and remained in the circulation for at least 24 h. Labeled IGF-I was significantly sequestered by the plasma from the IGF-I antibody-treated rats, but not by that from the control rats receiving normal sheep serum IgG, indicating that there was sufficient antiserum present to effectively bind additional IGF-I (Fig. 6). Binding of tracer to 1:100 dilutions of treatment plasmas (collected 24 h after antibody injection) was 65 ± 3.8% (mean ± SEM; n = 15). Binding of tracer to other (non-IGF-I IgG treatment) plasma (12 ± 2.4%) was not significantly different from estimates of nonspecific tracer precipitation (Fig. 6). GH treatment resulted in a 100% increase in total plasma IGF-I levels over the levels in the control dwarves. Levels of IGF-I in the plasma of the control dwarves ranged from 50-60 ng/ml, while levels in rats treated with 200 Mg/kg bGH all fell within the range of 100-130 ng/ml. There was no difference in total IGF-I in the terminal plasma from any of the three GH-treated groups of rats.
80
Perturbation of IGF-I-binding protein interaction by antibody 60
40
20
10
10
10
Log Antibody dilution 125
FlG. 4. The inhibition of [ I]IGF-I binding to L6 myoblasts in culture (expressed as a percentage of maximum binding, Bo) by varying dilutions of anti-IGF-I serum added to the incubation medium.
untreated dwarves. Concurrent administration of either NSS IgG or anti-IGF-I antibody failed to have any effect on GH-stimulated growth (Fig. 5); all three GH-treated groups grew at the same rate. Although liver weights in the GH-treated animals were 10% heavier than in the controls, this was not statistically significant, and the increase was in proportion to total body size. There were also no significant differences between the groups with regard to the weight of any of the organs measured (Table 1). When corrected for body weight, the mean changes
An aliquot of terminal plasma from the rats was incubated with IGF-I tracer, and the equilibrated mixture was subjected to size-exclusion chromatography. Normal dwarf rat plasma (Fig. 7, top panel) and plasma from GH- plus NSS-treated rats (Fig. 7, middle panel) showed similar profiles. By contrast, secondary incubation with plasma from anti-IGF-I-treated rats resulted in a marked high mol wt shift of eluted radioactivity (Fig. 7, bottom panel). Further tracer (25%) was identified in an insoluble fraction that was removed before chromatography. These data provide strong evidence that the anti-IGF-I antibody sequestered the IGF-I when it was prebound to its binding proteins. Discussion The results of the present study showed that immunoneutralization of circulating IGF-I did not inhibit GHstimulated growth in GH-deficient rats. In all studies in which passive immunization is used as a means of eliciting a physiological response, the neutralizing capability of the antiserum is crucial. In our experiment, the neutralizing ability of the preparation was extensively tested to ensure it was adequate. The
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PASSIVE IMMUNIZATION AGAINST IGF-I
2107
160 -i
140
4 -
120 •
3 •
100
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80 -
1 -
*.? :
60
0 -8
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0
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Day of Treatment
Gil + NSS
Gil +
an(i-IGFl
FIG. 5. a, Growth curves for control dwarf rats (- - -) and for dwarf rats receiving 2 mg/kg day bGH, bGH plus NSS IgG, or bGH plus anti-IGFI serum daily from day 0 ( ). b, Growth velocities (mean ± SEM) in control dwarves and those receiving bGH, bGH plus NSS IgG, or bGH plus anti-IGF-I serum. ***, P < 0.001 compared with control animals. TABLE 1. Average daily weight gain (ADWG) and liver, kidney, spleen, and thymus weights in dwarf rats receiving saline, bGH (2 mg/kg day), 2 mg/kd-day bGH plus 287 ng normal sheep IgG, or 2 mg/kg day bGH plus 287 ng anti-IGF-I IgG ADWG (g) Saline GH GH + IgG GH + anti-IGF-I
2.75 ± 4.12 ± 4.13 ± 4.01 ±
0.11 0.15 0.18 0.18
Liver wt (g) 5.60 ± 0.21 6.12 ± 0.17 6.20 ± 0.23 6.00 ± 0.23
100 •O
c o
f
80 60 40 20 0 1 2
3
4
5
6
7
8 9 10 11 12 13 14 15
Animal number 125
FIG. 6. Amount of [ I]IGF-I (expressed as a percentage of the total counts added) bound by a 1:1000 dilution of terminal plasma from rats passively immunized against IGF-I (•); control dwarves, GH-treated, and GH- plus NSS IgG-treated dwarf rat plasmas bound only 12 ± 2% of the label (M).
ability of both the antiserum and the purified IgG fraction of the anti-IGF-I antiserum to inhibit binding of IGF-I to receptors on myoblasts in culture and to inhibit the specific biological action of IGF-I in stimulating sulfate incorporation into cartilage in vitro was demonstrated. The affinity of the anti-IGF-I antibody preparation in vitro (Ka, 2 x 1010 mol/liter) was similar to the expected Ka of both IGF-I receptors and IGF-binding proteins (22-24). Although the antibodies were clearly capable of neutralizing the action of IGF-I in vitro, it was important to demonstrate that the dose given was sufficient to completely immunoneutralize circulating IGF-I in vivo. The dose of anti-IGF-I was estimated by calculation and confirmed by preliminary experimentation. The com-
Kidney wt (g) 1.06 ± 0.05 1.09 ± 0.01 1.07 ± 0.02 1.11 ± 0.03
Spleen wt 0.40 ± 0.42 ± 0.42 ± 0.43 ±
0.05 0.01 0.02 0.02
Thymus wt (g) 0.41 ± 0.04 0.37 ± 0.03 0.43 ± 0.06 0.38 ± 0.03
plete inhibition of plasma sulfation factor activity by this dose when given to rats in vivo confirms the adequacy of the neutralizing ability of this dose of antibody. Furthermore, the presence of excess immunoneutralizing capacity was supported by the finding of free anti-IGF-Ibinding capacity in terminal plasmas. The anti-IGF-I antibody was not only able to sequester free IGF-I, but was also able to bind IGF-I when prebound to plasma binding proteins. This was illustrated by the finding that trace labeled IGF-binding protein complexes demonstrated a high mol wt shift on incubation with terminal plasmas. Thus, not only did the antibodies have sufficient capacity to bind circulating IGFI, but it appears that their epitope specificity is such that they interact with precomplexed IGF-I. It would seem unlikely that such a large mol wt complex would readily cross from the plasma to the extracellular space where cell receptors might have an opportunity to compete with the antibody complex for the IGF-I. Transfer of small amounts of antibody (probably as free Ig) out of the bloodstream has previously been reported in rats (25). Quantification of the amount of antibody in the tissues and whether this was sufficient to neutralize tissue IGFI was not possible in this study. However, the affinities of the antibodies and receptors appear comparable and the presence of small amounts of antibody in tissues may also have been sufficient to perturb the activity of locally produced IGF-I. Certainly in vitro, antibody could inhibit binding of IGF-I to receptors at high dilution. A recent report has shown that passive immunization against IGF-I has no effect on growth in guinea pigs (8). The GH-deficient dwarf rat used in the present study is
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PASSIVE IMMUNIZATION AGAINST IGF-I
2108 20000 i •«-*
S C
u
10000
a. in
C
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•
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1
20000 i
a 10000 -
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a 5000 -
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20
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40
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60
Fraction number FIG. 7. FPLC profiles of sera from control dwarf rats (top panel), GHtreated dwarf rats (middle panel), and GH-treated dwarf rats (bottom panel) passively immunized with anti-IGF-I serum, after incubation with [126I]IGF-I tracer. Clearly, administration of anti-IGF-I serum resulted in displacement of all of the IGF-I label into a high (>150 kDa) mol wt fraction.
more suitable for this type of experiment than the guinea pig, since elevations in plasma IGF-I and growth rates can clearly be achieved by GH administration (9,10, 26, 27); growth of the guinea pig is not GH dependent (2830), and it is also not certain that IGF-I is GH dependent in the guinea pig. It has been suggested that the growth of the adult guinea pig may resemble the growth of fetuses, in that the GH-IGF-I axis may not be of great importance (31). The GH-deficient dwarf rat has an additional advan-
Endo • 1991 Voll28«No4
tage over animals with normal GH secretion for use in these studies, in that positive feedback, by removing IGFI, cannot occur through increased GH secretion (although effects on other pituitary hormones may be possible). In contrast to our results, Kerr et al. (8) reported increased levels of IGF-I in their passively immunized animals. They suggested that this was a result of either increased production of IGF-I through diminished negative feedback or decreased metabolic clearance of the antibody-bound hormone. The question arises as to whether IGF-I plays a role in mediating the growth-promoting activity of GH by endocrine or autocrine/paracrine actions. Plasma levels of IGF-I have been correlated with growth and used as a marker for GH deficiency. Modest reductions in plasma IGF-I levels (50%) lead to severe dwarfism, yet the levels of IGF-I, as measured by bioassay, are much lower than those in these passively immunized animals. This suggests that the major effect of GH is by local release of IGF-I for autocrine/paracrine actions. If this is so, it raises the question of the role of circulating IGF-I; if this is not so, then it questions the role of IGF-I in growth at any level. No data are available from this study on the levels of IGF-I in the tissues of these animals, but such measurements could help determine the relative contributions of tissue and plasma levels of IGF-I in growth. A less likely explanation for the lack of effect of immunoneutralization of circulating IGF-I on growth could be that (particularly in a GH-deficient animal) there may have been an increase in the number of IGFI receptors in the target tissues, thus allowing more of any transiently unneutralized circulating IGF-I to have its biological effect. This proposal stems from extrapolation of the report on receptor down-regulation by IGFI (32). Finally, the most logical explanation of these results is that, at least in the GH-treated dwarf rat, IGF-I circulating in plasma plays only a small part in stimulating growth. This, however, does not preclude the possibility that blood-borne IGF-I may fulfill specific endocrine functions in the support of growth processes. Examples of this may be whole body protein and glucose metabolism (33), erthyropoiesis (34, 35), and renal function (36). Alternatively, it is possible that in the GHtreated dwarf rat, circulating IGF-I represents a clearing system for local IGF-I that is produced in excess of autocrine/paracrine requirements.
Acknowledgments We are grateful to P. Dobbie for help with the animal management, and to J. Napier for purification of the antibodies. Dr B. D. Burleigh kindly provided the IGF-I for antibody production and affinity purification, and the recombinant bGH was a gift from Cyanamid.
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PASSIVE IMMUNIZATION AGAINST IGF-I
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