The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by De Benedetti, F.
Right arrow Articles by Martini, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by De Benedetti, F.
Right arrow Articles by Martini, A.
Right arrowPubmed/NCBI databases
*Substance via MeSH
Medline Plus Health Information
*Immunization
The Journal of Immunology, 2001, 166: 4334-4340.
Copyright © 2001 by The American Association of Immunologists

In Vivo Neutralization of Human IL-6 (hIL-6) Achieved by Immunization of hIL-6-Transgenic Mice with a hIL-6 Receptor Antagonist1

Fabrizio De Benedetti*, Patrizia Pignatti*, Marina Vivarelli*, Cristina Meazza*, Gennaro Ciliberto{dagger}, Rocco Savino{dagger} and Alberto Martini2,*

* Dipartimento di Scienze Pediatriche, Instituto di Ricerca e Cura a Carattere Scientifico Policlinico San Matteo, Universita’ degli Studi di Pavia, Pavia, Italy; and {dagger} Instituto Ricerche di Biologia Moleculaire, Rome, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neutralization of IL-6 represents an attractive therapeutic option in several diseases, including B cell neoplasia, osteoporosis, and autoimmunity. Therapeutic attempts in humans have shown that administration of injectable doses of a mAb to IL-6 does not provide efficient neutralization of the cytokine in vivo. Therefore, alternative approaches are needed. In this study, we evaluated whether the Ab response to human IL-6 (hIL-6) elicited by vaccination with Sant1 (a hIL-6 variant with seven amino acid substitutions) was able to fully correct in vivo the clinical and biological effects of a chronic endogenous overproduction of hIL-6 in the hIL-6-transgenic NSE/hIL-6 mice. Because of the overexpression of hIL-6, occurring since birth, with circulating levels in the nanogram per milliliter range, NSE/hIL-6 mice have a marked decrease in growth rate, associated with decrease in insulin-like growth factor I levels, and represent an animal model of the growth impairment associated with human chronic inflammatory diseases. Following immunization with Sant1, but not with hIL-6, NSE/hIL-6 mice developed high titers of polyclonal Abs to hIL-6. The Abs, acquired by transplacental transfer, effectively neutralized IL-6 activities in vivo as shown by the complete correction of the growth defect and normalization of insulin-like growth factor levels in the hIL-6-transgenic offspring. Immunization with Sant1 could therefore represent a novel and simple therapeutic approach for the specific neutralization of IL-6 in humans.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleulin 6 is a pleiotropic cytokine with multiple functions on the immune and inflammatory responses. A vast body of evidence, obtained in experimental animals and supported by data in humans, suggests that excessive production of IL-6 is involved in the pathogenesis of various diseases. In B cell malignancies, IL-6 has been shown to promote growth and inhibit apoptosis of multiple myeloma cells and is considered a pivotal factor in the multistep process of the malignant cell transformation (1, 2, 3, 4). Even more compelling evidence of the role of IL-6 has been obtained in Castleman’s disease, both in the localized and multicentric forms, where overproduction of IL-6 appears to play a major role in promoting lymph node hyperplasia and in the clinical manifestations of the disease (5, 6, 7, 8). Based on its role in osteoclast activation and induction of bone resorption (9) and on the observation that IL-6-deficient mice are protected from ovariectomy-induced osteoporosis (10), IL-6 is considered a pivotal mediator of postmenopausal osteoporosis. Evidence in IL-6-deficient mice supports the hypothesis that IL-6 is also involved in the pathogenesis of rheumatoid arthritis and multiple sclerosis (11, 12, 13). Therefore, selective blockage of IL-6 activities in humans represents an attractive therapeutic option in several conditions.

Therapeutic attempts in humans with mAbs to IL-6 have yielded disappointing results. Administration of a single mAb to IL-6 does not lead to efficient neutralization of its biological activities in vivo. This lack of neutralization appears to be secondary to the stabilization of IL-6 in monomeric complexes leading to accumulation of the cytokine in the circulation (14). Indeed, clinical benefit in terminal multiple myeloma patients has been reported only in the patients with low IL-6 production (14). In the majority of patients with diseases that may benefit from anti-IL-6 treatments, IL-6 production is much higher and therefore it is not conceivable that injectable doses of mAbs could lead to an efficient neutralization. Currently available experimental alternative approaches to selective blockage of IL-6 biological activities in vivo are mAbs to its receptor {alpha}-chain or receptor antagonists (15, 16). One of the major limitations to their potential therapeutic use is represented by the need to produce large amounts of these recombinant molecules to allow for administration of high and repeated doses. In addition, these molecules are potentially immunogenic and may elicit Ab responses that may limit the long-term efficacy of these treatments.

We have previously demonstrated that vaccination of human IL-6 (hIL-6)3-transgenic mice with the biologically inactive hIL-6 receptor antagonist Sant1, a hIL-6 variant with seven amino acid substitutions (15), induces a strong polyclonal Ab response cross-reactive with hIL-6 (17). These Abs were shown to neutralize IL-6 bioactivity in vitro on the hepatoma cell line Hep3B (17) and on the myeloma cell line XG1 (our unpublished observation) and the in vivo effect of exogenously administered recombinant hIL-6 on acute phase protein production (17). In the present study, we decided to verify whether the Ab response to hIL-6 elicited by vaccination with Sant1 was sufficient to counteract in vivo the clinical and biological effects of the endogenous chronic overproduction of hIL-6 in the hIL-6-transgenic NSE/hIL-6 mice.

In the NSE/hIL-6-transgenic mice circulating hIL-6 levels in the range of nanogram per milliliter, occurring since birth, cause a marked decrease in growth rate, which is evident during the first 4 wk of postnatal life. The growth defect is associated with a decrease in circulating levels of insulin-like growth factor-I (IGF-I) (18), and IGF-I plays a major role in postnatal growth (10, 20). Growth impairment is a common manifestation of several childhood diseases with chronic inflammation and/or severe recurrent infections, such as juvenile rheumatoid arthritis, Crohn’s disease, cystic fibrosis, and immunodeficiencies, all of which are characterized by elevated IL-6 and decreased IGF-I levels. Therefore, NSE/hIL-6 mice represent a faithful model of an IL-6-mediated common complication of chronic inflammation (18). Possibly due to hIL-6 expression since birth, and similar to other transgenic mice (21, 22), NSE/hIL-6 mice are immunologically tolerant to hIL-6, as shown by the low levels of Abs to hIL-6 elicited by immunization with hIL-6 itself (17). This feature makes the NSE/hIL-6 mice a suitable model for the evaluation of an anti-hIL-6 vaccination protocol.

In the present study, we show that the Ab response to hIL-6 induced by immunization with Sant1 of hIL-6-transgenic mice has sufficient potency to completely neutralize the clinical and biological effects of a chronic endogenous hIL-6 production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and treatments

NSE/hIL-6 mice were generated using a construct carrying the rat neurospecific enolase (NSE) promoter driving the expression of the hIL-6 cDNA, as previously described (18). We used mice of line 26, presenting peripheral expression of the transgene, resulting in measurable circulating levels of hIL-6 in the range of nanogram per milliliter since birth, growth defect, and decreased circulating IGF-I levels (18). Transgenic animals were identified by PCR analysis of DNA extracted from a tail segment, as described elsewhere (23). CB6F1 females, of the same strain of the NSE/hIL-6 mice, were obtained from Charles River Italy (Calco, Italy). Mice were maintained in standard conditions under a 12-h light-dark cycle, provided irradiated food (4RF21; Mucedola, Settimo Milanese, Milan, Italy), and chlorinated water ad libitum. Procedures involving animals and their care were conducted in conformity with national and international laws and policies (24, 25 ; National Institutes of Health Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, 1985). Recombinant hIL-6 and Sant1 were produced as previously described (26). Ags were formulated in Al[OH]3 at 1 mg/ml. Eight-week-old CB6F1 or NSE/hIL-6 females were immunized i.p. with 100 µl of the Ag formulation and boosted 20 and 40 days after the first immunization, as previously described (17). To evaluate serum titers of Ab to hIL-6 and serum hIL-6 levels, immunized females were bled before immunization and 10 days after the third booster. Immunized females were mated 2 days after the third booster. To obtain approximately half of the offspring carrying the hIL-6 transgene, CB6F1 females were mated with NSE/hIL-6-transgenic males, while NSE/hIL-6-transgenic females were mated with nontransgenic CB6F1 males. The offspring was either euthanized at the age of 7 days to obtain serum and plasma for the measurement of Ab titers to hIL-6 and levels of hIL-6 and IGF-I, or followed with measurement of body weight three times a week and bled at 4 wk after birth.

Measurement of Ab titers to hIL-6 by ELISA

Ninety-six-well flat-bottom microtiter plates were coated with 100 µl/well recombinant hIL-6 at 10 µg/ml in PBS (pH 7.4). After blocking for 2 h at room temperature with 200 µl/well PBS, 0.8% BSA, and 0.1% NaN3, 100 µl of serial serum dilutions was incubated for 2 h at room temperature. Plates were washed and then incubated with a peroxidase-labeled rabbit anti-mouse Ig antiserum (Dako, Glostrup, Denmark), diluted 1:100 in PBS, 0.8% BSA, and 0.1% NaN3. After washing, binding was revealed by adding o-phenylenediamine in distilled water for 20 min at room temperature. The color reaction was stopped with 50 µl/well 2 M H2SO4 and the OD was read at 492 nm. Serum titer of Abs to hIL-6 was calculated as the reciprocal of the dilution that yielded an OD five times that of a pool of preimmune sera diluted 1:100.

Measurement of hIL-6 and IGF-I

Serum hIL-6 levels were determined with a commercially available immunoassay according to the instructions provided by the manufacturer (R&D Systems, Minneapolis, MN). IGF-I levels were measured following acid-ethanol extraction of plasma samples (anticoagulated with EDTA, final concentration 5 mg/ml whole blood), using a commercially available radioimmunoassay, as described previously (18).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Study design

The early expression of hIL-6 and the early occurrence of hIL-6 overproduction-mediated manifestations made it difficult to establish an approach that 1) was technically feasible and 2) could allow the evaluation of the effectiveness in correcting the clinical manifestation of the chronic endogenous hIL-6 overexpression. We excluded the possibility of immunizing newborn mice for two reasons. It is very likely that newborn, or very young, mice would not respond satisfactorily to a standard vaccination protocol (R. Savino, unpublished observation and Ref. 27). Moreover, since in NSE/hIL-6 mice the effects of hIL-6 overexpression on the growth rate are evident during the first 4 wk of postnatal life, there would not have been sufficient time to allow induction of the Ab response to hIL-6 and evaluation of its subsequent effects on the growth rate. We decided therefore to take advantage of the transplacental transfer of Igs. However, since NSE/hIL-6-transgenic females are either unfertile or give birth to a low number of offspring (our unpublished observation), possibly due to their small size, we decided to first immunize with Sant-1 CB6F1 adult females, of the same strain of the NSE/hIL-6 transgenics, with two objectives: 1) to validate the feasibility and efficacy of the experimental approach and 2) to obtain NSE/hIL-6-transgenic females of bigger size and, therefore, possibly fertile.

Abs to hIL-6 induced by immunization of CB6F1 females with Sant1 are passively acquired through the placenta by the offspring

Nontransgenic CB6F1 females were immunized i.p. with Sant1 formulated in aluminum hydroxide (Al[OH]3) or with Al[OH]3 alone. Evaluation of serum titers of Abs to hIL-6, 10 days after the third booster, showed that females immunized with Sant-1 developed high titers of Abs to hIL-6 (58,000 ± 19,595), whereas CB6F1 females immunized with Al[OH]3 alone did not (<1000). The immunized CB6F1 females were mated with NSE/hIL-6-transgenic males to obtain approximately half of the offspring carrying the hIL-6 transgene, allowing comparison with the nontransgenic littermates. To verify the occurrence of the transplacental transfer of Ab to hIL-6, we evaluated serum Ab titers to hIL-6 in the offspring. One week-old mice born from Sant1-immunized females had elevated titers of Abs to hIL-6, while mice born from Al[OH]3-immunized mice had undetectable levels. As expected, since the offspring had not been immunized, but received the Abs by transplacental transfer, serum titers of Abs to hIL-6 declined with age, being ~3-fold lower in 4-wk-old animals and decreasing to undetectable levels in 2-mo-old mice (Table IGo). No significant differences in Ab titers were found between the transgenic and the nontransgenic offspring (data not shown).


View this table:
[in this window]
[in a new window]
  		Table I. Serum titers of Abs to hIL-6 and serum levels of hIL-6 in the transgenic offspring of CB6F1 females immunized with Al[OH]3 or with Sant1 at the indicated ages1

 
It has been previously shown that in the NSE/hIL-6 mice the polyclonal Ab response to hIL-6, induced by immunization with Sant1, caused a marked decrease in circulating hIL-6 levels detectable by ELISA (17). When sera of Sant1-immunized animals were subjected to dissociation of the hIL-6-Ab complexes and subsequent gel filtration, serum levels of hIL-6 were found to be in the range of nanogram per milliliter and similar to those measured before the immunization, therefore demonstrating that the decrease in hIL-6 levels measurable by ELISA was due to masking of the circulating cytokine (17). To evaluate whether the same masking of hIL-6 occurred in the offspring after acquirement of the Abs to hIL-6 by transplacental transfer, we measured circulating hIL-6 levels by ELISA in the transgenic offspring of Sant1-immunized animals and compared them to those of the offspring of Al[OH]3-immunized mice. One-week-old transgenic NSE/hIL-6 mice born from Al[OH]3-immunized females had nanograms per milliliter of circulating hIL-6, which increased with age. On the contrary, hIL-6 was not detectable by ELISA in the 1-wk-old transgenic NSE/hIL-6 mice born from Sant1-immunized CB6F1 females (Table IGo). In the transgenic offspring of Sant1-immunized CB6F1 females, the decrease with age in serum titers of Abs to hIL-6 was associated with an increase in the circulating levels of hIL-6 detectable by ELISA (Table IGo). At 2 mo of age, transgenic mice born from Sant1-immunized females had circulating levels of hIL-6 comparable to those of the transgenic offspring of Al[OH]3-immunized females. These results show that the Abs to hIL-6 elicited by Sant1 immunization are passively acquired through the placenta by the offspring and mask circulating hIL-6.

Passively acquired Abs to hIL-6 induce normalization of the growth rate and of IGF-I levels in the offspring of Sant1-immunized CB6F1 females

To evaluate whether passively acquired Abs to hIL-6 could neutralize the effects of hIL-6 overexpression, we evaluated the growth rate and circulating IGF-I levels in the transgenic and nontransgenic mice born from CB6F1 females immunized with either Sant1 or Al[OH]3 alone. As previously mentioned, since the immunized CB6F1 females were mated with NSE/hIL-6-transgenic males, approximately half of the offspring was expected to carry the hIL-6 transgene and, therefore, could be compared with their nontransgenic littermates. As expected (13), the transgenic offspring of females immunized with Al[OH]3 alone showed a marked decrease in growth rate, evident in the first 4 wk of postnatal life, compared with their nontransgenic littermates, resulting in mice with significantly reduced weight (Fig. 1Go, A and B). On the contrary, the growth rate of the transgenic offspring of Sant1-immunized females was comparable to that of their nontransgenic littermates (Fig. 1GoC). This normalization of the growth rate resulted in transgenic NSE/hIL-6 mice the same size as their nontransgenic littermates (Fig. 1GoD). Passively acquired Abs to hIL-6 do not cause any significant effect on growth rate of nontransgenic mice, as shown by the comparable growth of the nontransgenic offspring of Sant1-immunized and Al[OH]3-immunized females.



View larger version (48K):
[in this window]
[in a new window]
  		FIGURE 1. Effect of Abs to hIL-6 on the growth of the nontransgenic ({square}) and the transgenic () offspring of CB6F1 females immunized with Al[OH]3 (A and B) or with Sant1 (C and D). Growth rates (mean gram increase in body weight per day) in the indicated age periods are shown in A and C. Body weights are shown in B and D. Results are shown as means ± SD deviations. Transgenic offspring of Al[OH]3-immunized CB6F1 females, n = 14; nontransgenic offspring of Al[OH]3-immunized CB6F1 females, n = 20; transgenic offspring of Sant1-immunized CB6F1 females, n = 26; and nontransgenic offspring of Sant1-immunized CB6F1 females, n = 15. *, p < 0.001 vs corresponding nontransgenic. All other comparisons with corresponding nontransgenic offspring were not statistically significant (p > 0.05)

 
As previously mentioned, the growth defect of the transgenic NSE/hIL-6 is associated with a decrease in circulating levels of IGF-I; this decrease plays an important role in the growth impairment (19). Measurement of plasma IGF-I showed that the transgenic offspring of CB6F1 females immunized with Al[OH]3 alone had IGF-I levels significantly lower than those of their wild-type littermates. In the offspring of Sant1-immunized females, circulating levels of IGF-I were similar between transgenic and nontransgenic littermates at 1 wk of age; at 4 wk of age, circulating IGF-I levels were lower than those of their nontransgenic littermates, reflecting the decrease in Ab titers and the subsequent presence of detectable serum hIL-6 (Fig. 2Go). These results show that immunization of wild-type CB6F1 females with Sant1 induces an Ab response to hIL-6, which, following transplacental transfer to the offspring, is able to normalize growth and IGF-I levels of the NSE/hIL-6 mice.



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 2. Circulating IGF-I levels in 1-wk-old nontransgenic ({square}) and transgenic () offspring of CB6F1 females immunized with Al[OH]3 or with Sant1. Results are shown as means ± SD. Nontransgenic offspring of Al[OH]3-immunized CB6F1 females, n = 10 at 1 wk, n = 9 at 4 wk; transgenic offspring of Al[OH]3-immunized CB6F1 females, n = 7 at 1 wk, n = 9 at 4 wk; nontransgenic offspring of Sant1-immunized CB6F1 females, n = 7 at 1 wk, n = 13 at 4 wk; and transgenic offspring of Sant1-immunized CB6F1 females, n = 7 at 1 wk, n = 10 at 4 wk. *, p < 0.001 vs nontransgenic offspring of Al[OH]3-immunized CB6F1 females; **, not significant (p > 0.05) vs 1-wk-old nontransgenic offspring of Sant1-immunized CB6F1 females; and ***, p < 0.05 vs 4-wk-old nontransgenic offspring of Sant1-immunized CB6F1 females.

 
Passively acquired Abs to hIL-6 induce normalization of the growth rate and of IGF-I levels in the offspring of Sant1-immunized NSE/hIL-6 females

The above-described experiments were performed by immunizing CB6F1 females, which are not transgenic for hIL-6, and are, therefore, not immunologically tolerant to hIL-6. Since Sant1 is a human IL-6 variant, it was reasonable to expect a potent immune response toward an heterologous protein. We wanted to investigate whether the same effectiveness of the immunization with Sant1 on the growth and IGF-I levels could be observed following immunization of the hIL-6-transgenic mice, which might develop an Ab response quantitatively and/or qualitatively different from that of nontransgenic mice. Therefore, we immunized with Sant1 or with recombinant hIL-6, as a control, the NSE/hIL-6-transgenic females of normal size born from Sant1-immunized CB6F1 females. Immunization with Sant1 of NSE/hIL-6-transgenic females induced an Ab response cross-reactive with hIL-6, with Ab titers to hIL-6 that were 5–10 times higher than those elicited by hIL-6 (Table IIGo). In Sant1-immunized females, hIL-6 levels measurable by ELISA dropped to below 0.1 ng/ml, while the immunization with hIL-6 resulted only in a 2- to 3-fold decrease in circulating levels of hIL-6 (Table IIGo). These results reproduced what was previously reported (17). It should be noted that the Ab titers obtained in the NSE/hIL-6-transgenic mice were higher than those obtained in the nontransgenic CB6F1 females. It is tempting to speculate that this might be secondary to the continuous antigenic stimulation provided by the endogenous expression of hIL-6 in the hIL-6-transgenic mice.


View this table:
[in this window]
[in a new window]
  		Table II. Serum titers of Abs to hIL-6 and serum hIL-6 levels of NSE/hIL-6-transgenic females before and after immunization with recombinant hIL-6 or with Sant11

 
After the third booster, the NSE/hIL-6 females immunized with either Sant1 or hIL-6 were mated with nontransgenic CB6F1 males to obtain part of the offspring (approximately half) carrying the transgene that could be compared with their nontransgenic littermates. Also, in this case the offspring of the immunized NSE/hIL-6 females acquired, by transplacental transfer, Abs to hIL-6, whose serum titer declined progressively with age. However, as expected because of the high titers in the Sant-1-immunized NSE/hIL-6 females, serum titers of Abs to hIL-6 were significantly higher in the offspring of Sant1-immunized NSE/hIL-6 females compared with that of the offspring of hIL-6-immunized NSE/hIL-6 females (Table IIIGo). No differences in serum titers of Abs to hIL-6 were observed between the transgenic and nontransgenic offspring (data not shown). In the offspring of hIL-6-immunized NSE/hIL-6 females, the low titers of Abs to hIL-6 were not sufficient to cause efficient masking of circulating hIL-6, as shown by the presence of nanogram per milliliter levels detectable by ELISA. On the contrary, in the offspring of the Sant1-immunized NSE/hIL-6 females, efficient masking of circulating hIL-6 was observed (Table IIIGo).


View this table:
[in this window]
[in a new window]
  		Table III. Serum titers of Abs to hIL-6 and serum levels of hIL-6 in the transgenic offspring of NSE/hIL-6-transgenic females immunized with hIL-6 or with Sant1 at the indicated ages1

 
As expected, because of the low titer of Abs to hIL-6, the growth rate in the first 4 wk of postnatal life of the transgenic offspring of hIL-6-immunized NSE/hIL-6 females was significantly lower than that of their wild-type littermates, resulting in transgenic mice of significantly reduced weight (Fig. 3Go, A and B). On the contrary, the transgenic offspring of Sant1-immunized NSE/hIL-6 females showed a growth rate that was comparable to that of their wild-type littermates (Fig. 3Go, C and D). Measurement of circulating IGF-I levels in the offspring of Sant1-immunized NSE/hIL-6 females showed comparable levels between the transgenic and nontransgenic offspring (Fig. 4Go).



View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 3. Growth rates and body weight of the nontransgenic ({square}) and transgenic () offspring of NSE/hIL-6-transgenic females immunized with hIL-6 (A and B) or with Sant1 (C and D). Growth rates (mean gram increase in body weight per day) in the indicated age periods are shown in A and C. Body weights are shown in B and D. Results are shown as means ± SD. Transgenic offspring of hIL-6-immunized NSE/hIL-6 females, n = 12; nontransgenic offspring of hIL-6-immunized NSE/hIL-6 females, n = 27; transgenic offspring of Sant1-immunized NSE/hIL-6 females, n = 18; and nontransgenic offspring of Sant1-immunized NSE/hIL-6 females, n = 17. *, p < 0.001 vs corresponding nontransgenic offspring. All other comparisons with corresponding nontransgenic offspring were not statistically significant (p > 0.05).

 


View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 4. Circulating IGF-I levels in the nontransgenic ({square}) and transgenic () offspring of NSE/hIL-6 females immunized with Sant1. Results are shown as means ± SD. Transgenic offspring of Sant1-immunized NSE/hIL-6 females, n = 8 at 1 wk, n = 7 at 4 wk; nontransgenic offspring of Sant1-immunized NSE/hIL-6 females, n = 8 at 1 wk, n = 7 at 4 wk.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously shown that immunization with the hIL-6 receptor antagonist Sant1 induced Abs to hIL-6 which were able to efficiently bind hIL-6 and to neutralize hIL-6 bioactivities in vitro (17). In this study, we demonstrated that the Ab response to hIL-6 induced by immunization of hIL-6-transgenic mice is able to neutralize in vivo the clinical and biological effects of the chronic endogenous overproduction of hIL-6.

As previously mentioned, NSE/hIL-6 mice represent a model of the growth impairment associated with childhood chronic inflammatory diseases. The complete correction of the growth defect, as well as the normalization of the IGF-I levels, obtained in this study demonstrate that the growth impairment of the NSE/hIL-6 mice is entirely due to high levels of circulating hIL-6, implementing previous data obtained with the neutralization of hIL-6 with a mAb to the murine IL-6 receptor (18). As previously mentioned, NSE/hIL-6 females are either infertile or give birth to a low number of offspring. The NSE/hIL-6 females of normal size born from Sant1-immunized CB6F1 were fertile and gave birth to a number of offspring comparable to that of wild-type CB6F1 (data not shown). In addition, no differences in the number of offspring were observed between NSE/hIL-6 females immunized with Sant1 or with hIL-6 (data not shown). This finding suggests that the low fertility of NSE/hIL-6-transgenic females may be secondary to their small size. An alternative explanation may be represented by a possible effect of high levels of circulating hIL-6 on the generation of ovules. Additional experiments are needed to clarify this issue.

As shown by the low levels of Abs to hIL-6 elicited by immunization with hIL-6, and similarly to other transgenic mice (21, 22), NSE/hIL-6 mice show, at least partial, immunological tolerance to hIL-6. The NSE/hIL-6-transgenic females of normal size used in the second set of experiments were born from Sant1-immunized CB6F1 females and were therefore exposed to neutralizing Abs to hIL-6 before birth and early in life. It should be noted that in these animals low levels of Abs to hIL-6 were induced by immunization with hIL-6 itself; moreover, the decline in the levels of measurable circulating hIL-6, induced by immunization with hIL-6, was similar to that previously reported in NSE/hIL-6-transgenic mice that were not exposed to neutralizing Abs to hIL-6 (17), therefore suggesting that IL-6 tolerance was unaffected by the exposure to IL-6 Abs in prenatal life and early in life.

Immunization with Sant1 appears to break this tolerance. Since other hIL-6 variants without the three substitutions in the COOH terminus (Q175I/S176R/Q183A) are poorly immunogenic in the NSE/hIL-6 mice (data not shown), it is reasonable to hypothesize that these mutations cause the formation of a new immunodominant epitope. In this respect, it is interesting to note that Dalum et al. (28) have recently demonstrated that insertion of a T cell immunodominant epitope in the murine TNF-{alpha} generates a molecule which is highly immunogenic in mice and that immunization with this molecule induces, in a manner similar to what we found in our study, a polyclonal response to murine TNF-{alpha} that neutralizes TNF-{alpha} in vivo.

In contrast with the administration of mAb to TNF-{alpha}, administration of a single mAb to IL-6 both in animals and humans leads to stabilization of the circulating cytokine, preventing efficient in vivo neutralization of IL-6 bioactivities (14). The polyclonal response generated by immunization with Sant1 appears to overcome this problem; indeed, we have previously demonstrated that when sera of Sant1-immunized animals were subjected to dissociation of the hIL-6-Abs complexes and subsequent gel filtration, serum levels of hIL-6 were found to be similar to those measured before immunization (17). These findings appear to reproduce the observation of a pharmacokinetic study in mice that showed that, although the injection of a single mAb to IL-6 led to stabilization of the circulating IL-6, simultaneous treatment with a mixture of three different Abs induced a marked acceleration in IL-6 clearance (29).

Immunization with Sant1 could be proposed as a therapeutic approach to IL-6-mediated diseases. It is worthy of note that in our experiments we used Al(OH)3, an adjuvant commonly used for vaccination in humans. Major advantages of this immunization approach are simplicity, cheapness, and the use of a molecule devoid of any residual IL-6 agonistic activity. Since, following Sant1 immunization of the NSE/hIL-6 mice, high titers of Abs to hIL-6 persist for at least several weeks (data not shown), one could envisage periodic boosters as a simple means to obtain a stable neutralization of IL-6 activities rather than repeated parenteral injections of mAbs to the IL-6 receptor or receptor antagonists. This will be a major advantage for the patients and will also overcome the need to produce, at high monetary costs, large amounts of materials with recombinant technology.

Although Sant1-immunized NSE/hIL-6 animals appear to be healthy and have not so far developed any pathological signs (data not shown), immunization with Sant1 produces an Ab response directed toward a self-protein (i.e., IL-6), and this in itself could theoretically cause morbidity secondary to autoimmunity to IL-6. It should however be pointed out that ~15% of healthy humans have detectable levels of autoantibodies to hIL-6 that are able to neutralize IL-6 activity (30) without being significantly associated with pathological manifestations. Therefore immunization with Sant1 could reproduce, albeit at a higher level, what is considered a physiological mechanism of regulation of IL-6 activity (31). A potential limitation of this treatment concerns the possible untoward side effects of long-term neutralization of IL-6. IL-6-deficient mice develop normally and can be bred in standard animal facilities. Challenge with pathogens of IL-6-deficient mice has shown increased sensitivity to some, but not all, of the infectious agents examined (32, 33, 34, 35, 36, 37, 38). In a recent trial in multicentric Castleman’s disease with a humanized Ab to the IL-6 receptor, Nishimoto et al. (8) did not report significant side effects, in particular no infectious episodes, even in patients treated for as long as 40 wk.

While further assessing its long-term safety, immunization with Sant1 could be initially proposed as a possible effective treatment for otherwise fatal IL-6-mediated diseases, such as multiple myeloma and multicentric Castleman’s disease. Despite aggressive chemotherapy, median survival of patients with multiple myeloma is ~5 years. In two-thirds of the patients, Castleman’s disease is refractory to chemotherapy and to corticosteroid treatment and therefore the prognosis for these patients is poor (39). Moreover, proof of the therapeutic efficacy of IL-6 neutralization in multicentric Castleman’s disease has already been demonstrated (8).


   Acknowledgments
 
We thank Nicola Corea for technical assistance.


   Footnotes
 
1 This work was supported by Instituto di Ricerca e Cura a Carattere Scientifico Policlinico San Matteo, Pavia (Grants 390RCR96/02 and 020RFM96/02) and by the Ministero Italiano della Università e della Ricerca Scientifica e Tecnologica. Back

2 Address correspondence and reprint requests to Dr. Alberto Martini, Dipartimento di Scienze Pediatriche, Universita’ degli Studi di Pavia, Instituto di Ricerca e Cura a Carattere Scientifico Policlinico San Matteo, Piazzale le Golgi 2, 27100 Pavia, Italy. Back

3 Abbreviations used in this paper: hIL-6, human IL-6; IGF-I, insulin-like growth factor I; NSE, neuro-specific enolase; Al[OH]3, aluminum hydroxide. Back

Received for publication October 19, 2000. Accepted for publication January 22, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hilbert, D. M., M. Kopf, B. A. Mock, G. Köhler, S. Rudikoff. 1995. Interleukin 6 is essential for in vivo development of B lineage neoplasm. J. Exp. Med. 182:243.[Abstract/Free Full Text]
  2. Lattanzio, G., C. Libert, M. Aquilina, M. Cappelletti, G. Ciliberto, P. Musiani, V. Poli. 1997. Defective development of pristane-oil-induced plasmacytomas in interleukin-6-deficient BALB/c mice. Am. J. Pathol. 151:689.[Abstract]
  3. Hallek, M., P. Leif Bergsagel, K. C. Anderson. 1998. Multiple myeloma: increasing evidence for a multistep transformation process. Blood 91:3.[Free Full Text]
  4. Sporeno, E., R. Savino, L. Ciapponi, G. Paonessa, A. Cabibbo, A. Lahm, K. Pulkki, R. X. Sun, C. Toniatti, B. Klein, G. Ciliberto. 1996. Human interleukin-6 receptor super-antagonist with high potency and wide spectrum on multiple myeloma cells. Blood 87:4510.[Abstract/Free Full Text]
  5. Yoshizaki, K., T. Matsuda, N. Nishimoto, T. Kuritani, L. Taheo, K. Aozasa, T. Nakahata, H. Kaway, H. Tagoh, T. Kimori. 1989. Pathogenic significance of interelukin-6 (IL-6/BSF-2) in Castleman’s disease. Blood 74:1360.[Abstract/Free Full Text]
  6. Brandt, S. J., M. D. Bodine, C. E. Dunbar, A. W. Nienhuis. 1990. Dysregulated interleukin 6 expression produces a syndrome resembling Castleman’s disease in mice. J. Clin. Invest. 86:592.
  7. Beck, J. T., S. M. Hsu, J. Wijdenes, R. Bataille, B. Klein, D. Vesole, K. Hayden, S. Jagannath, B. Barlogie. 1994. Alleviation of systemic manifestation of Castleman’s disease by monoclonal anti-interleukin-6 antibody. N. Engl. J. Med. 330:602.[Free Full Text]
  8. Nishimoto, N., M. Sasai, Y. Shima, M. Nakagawa, T. Matsumoto, T. Shirai, T. Kishimoto, K. Yoshizaki. 2000. Improvement in Castleman’s disease by humanized anti-interleukin-6 receptor antibody therapy. Blood 95:56.[Abstract/Free Full Text]
  9. Tamura, T., N. Udagawa, N. Takahashi, C. Miyaura, S. Tanaka, Y. Yamada, Y. Koishihara, Y. Ohsugi, K. Kumaki, T. Taga. 1993. Soluble interleukin 6 receptor triggers osteoclast formation by interleukin 6. Proc. Natl. Acad. Sci. USA 90:11924.[Abstract/Free Full Text]
  10. Poli, V., R. Balena, E. Fattori, A. Markatos, M. Yamamoto, H. Tanaka, G. Ciliberto, G. A. Rodan, F. Costantini. 1994. Interleukin-6 deficient mice are protected from bone loss caused by estrogen depletion. EMBO J. 13:1189.[Medline]
  11. Alonzi, T., E. Fattori, D. Lazzaro, P. Costa, L. Probert, G. Kollias, F. De Benedetti, V. Poli, G. Ciliberto. 1998. Interleukin-6 is required for the development of collagen-induced arthritis. J. Exp. Med. 187:461.[Abstract/Free Full Text]
  12. Eugster, H. P., K. Frei, M. Kopf, H. Lassmann, A. Fontana. 1998. IL-6-deficient mice resist myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis. Eur. J. Immunol. 28:2178.[Medline]
  13. Mendel, I., A. Katz, N. Kozak, A. Ben-Nun, M. Revel. 1998. Interleukin-6 functions in autoimmune encephalomyelitis: a study in gene-targeted mice. Eur. J. Immunol. 28:1727.[Medline]
  14. Lu, Y. Z., H. Brailly, J. Wijdenes, R. Bataille, J. F. Rossi, B. Klein. 1995. Measurement of whole body interleukin-6 (IL-6) production: prediction of the efficacy of anti-IL-6 treatments. Blood 86:3123.[Abstract/Free Full Text]
  15. Savino, R., L. Ciapponi, A. Lahm, A. Demartis, A. Cabibbo, C. Toniatti, P. Delmastro, S. Altamura, G. Ciliberto. 1994. Rational design of receptor super-antagonist of human interleukin-6. EMBO J. 13:5863.[Medline]
  16. Sato, K., M. Tsuchiya, J. Saldanha, Y. Koishihara, Y. Ohsugi, T. Kishimoto, M. M. Bendig. 1993. Reshaping a human antibody to inhibit the interleukin 6 dependent tumor cell growth. Cancer Res. 53:851.[Abstract/Free Full Text]
  17. Ciapponi, L., D. Maione, A. Scoumanne, P. Costa, M. B. Hansen, M. Svenson, K. Bendtzen, T. Alonzi, G. Paonessa, R. Cortese, et al 1997. Induction of interleukin-6 (IL-6) autoantibodies through vaccination with an engineered IL-6 receptor antagonist. Nat. Biotechnol. 15:997.[Medline]
  18. De Benedetti, F., T. Alonzi, A. Moretta, D. Lazzaro, P. Costa, V. Poli, A. Martini, G. Ciliberto, E. Fattori. 1997. Interleukin 6 causes growth impairment in transgenic mice through a decrease in insulin-like growth factor-I. J. Clin. Invest. 99:643.[Medline]
  19. Baker, J., J. P. Liu, E. J. Robertson, A. Efstratiatis. 1993. Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75:73.[Medline]
  20. Woods, K. A., C. Camacho-Hubner, M. O. Savage, A. J. L. Clarck. 1996. Intrauterine growth retardation and postnatal growth failure associated with deletion of the insulin-like growth factor I gene. N. Engl. J. Med. 335:1363.[Free Full Text]
  21. Poplonski, L., L. Vukusic, J. Pawling, S. Caploff, J. Roder, N. Hozumi, J. Wither. 1996. Tolerance is overcome in beef insulin-transgenic mice activation of low-affinity autoreactive T cells. Eur. J. Immunol. 26:601.[Medline]
  22. Borchers, A., J. Braspenning, J. Meijer, W. Osen, L. Gissmann, I. Jochmus. 1999. E7-specific cytotoxic tolerance in HPV-transgenic mice. Arch. Virol. 144:1539.[Medline]
  23. Fattori, E., D. Lazzaro, P. Musiani, A. Modesti, T. Alonzi, G. Ciliberto. 1995. IL-6 expression in neurons of transgenic mice causes reactive astrocytosis and increase in ramified microglial cells but no neuronal damage. Eur. J. Neurosci. 7:2441.[Medline]
  24. European Economic Community Council Directive 86/609, OJ L 358, 1, Dec. 12, 1987.
  25. Italian Legislative Decree 116/92, Gazzetta Ufficiale della Repubblica Italiana No. 40, Feb. 18, 1992.
  26. Arcone, R., P. Pucci, F. Zappacosta, V. Fontaine, A. Malorni, G. Marino, G. Ciliberto. 1991. Single step purification and structural characterization of human interleukin-6 produced in Escherichia coli from a T7 RNA polymerase chain reaction vector. Eur. J. Biochem. 198:541.[Medline]
  27. Adkins, B.. 1999. T-cell function in newborn mice and humans. Immunol. Today 20:330.[Medline]
  28. Dalum, B., D. M. Butler, M. R. Jensen, P. Hinderson, L. Steinaa, A. M. Waterston, S. N. Grell, M. Feldman, H. I. Elsner, S. Mouritsen. 1999. Therapeutic antibodies elicited by immunization against TNF-{alpha}. Nat. Biotechnol. 17:666.[Medline]
  29. Montero-Julian, F. A., B. Klein, E. Gautherot, H. Brailly. 1995. Pharmacokinetic study of anti-interleukin-6 (IL-6) therapy with monoclonal antibodies: enhancement of IL-6 clearance by cocktails of anti-IL-6 antibodies. Blood 85:917.[Abstract/Free Full Text]
  30. Hansen, M. B., M. Svenson, K. Abell, K. Yasukawa, M. Diamant, K. Bendtzen. 1995. Influence of interleukin-6 (IL-6) autoantibodies on IL-6 binding to cellular receptors. Eur. J. Immunol. 25:348.[Medline]
  31. Hansen, M. B., M. Svenson, M. Diamant, K. Abell, K. Bendtzen. 1995. Interleukin-6 autoantibodies: possible biological and clinical significance. Leukemia 9:1113.[Medline]
  32. Anguita, J., M. Rincòn, S. Samanta, S. W. Barthold, R. A. Flavell, E. Fikrig. 1998. Borrelia burgdorferi-infected, interleukin-6-deficient mice have decreased Th2 responses and increased Lyme arthritis. J. Infect. Dis. 178:1512.[Medline]
  33. Bromander, A. K., L. Ekman, M. Kopf, J. G. Nedrud, N. Y. Lycke. 1996. IL-6-deficient mice exhibit normal mucosal IgA responses to local immunizations and Helicobacter felis infection. J. Immunol. 156:4290.[Abstract]
  34. Kopf, M., G. h. Baumann, M. Freer, M. Freudenberg, T. Lamers, R. Kishimoto, H. Bluethmann Zinkernagel, G. Köhler. 1994. Impaired immune and acute-phase responses in interleukin-6-deficient mice. Nature 368:339.[Medline]
  35. Ladel, C. H., C. Blum, A. Dreher, K. Reifenberg, M. Kopf, S. H. E. Kaufmann. 1997. Lethal tuberculosis in interleukin-6-deficient mutant mice. Infect. Immun. 65:4843.[Abstract]
  36. Moskowitz, N. H., R. B. Brown, L. R. Steven. 1997. Efficient immunity against Leishmania major in the absence of interleukin-6. Infect. Immun. 65:2448.[Abstract]
  37. Romani, L., A. Mencacci, F. Cenci, R. Spaccapelo, C. Toniatti, P. Puccetti, F. Bistoni, V. Poli. 1996. Impaired neutrophil response and CD4+ T helper cell 1 development in interleukin 6-deficient mice infected with Candida albicans. J. Exp. Med. 183:1345.[Abstract/Free Full Text]
  38. van der Poll, T., V. C. Keogh, X. Guirao, W. A. Buurman, M. Kopf, S. F. Lowry. 1997. Interleukin-6 gene-deficient mice show impaired defense against pneumococcal pneumonia. J. Infect. Dis. 176:439.[Medline]
  39. Herrada, J., F. Cabanillas, L. Rice, J. Manning, W. Pugh. 1998. The clinical behaviour of localized multicentric Castleman disease. Ann. Intern. Med. 128:657.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Rheumatology (Oxford)Home page
V. E. MacRae, C. Farquharson, and S. F. Ahmed
The pathophysiology of the growth plate in juvenile idiopathic arthritis
Rheumatology, January 1, 2006; 45(1): 11 - 19.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
A. R. Cappola, Q.-L. Xue, L. Ferrucci, J. M. Guralnik, S. Volpato, and L. P. Fried
Insulin-Like Growth Factor I and Interleukin-6 Contribute Synergistically to Disability and Mortality in Older Women
J. Clin. Endocrinol. Metab., May 1, 2003; 88(5): 2019 - 2025.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by De Benedetti, F.
Right arrow Articles by Martini, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by De Benedetti, F.
Right arrow Articles by Martini, A.
Right arrowPubmed/NCBI databases
*Substance via MeSH
Medline Plus Health Information
*Immunization

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS