Interleukin-6 (IL-6) is overproduced in the joints of patients with rheumatoid arthritis (RA)
and, based on its multiple stimulatory effects on cells of the immune system and on vascular endothelia, osteoclasts, and synovial fibroblasts, is believed to participate in the development and
clinical manifestations of this disease. In this study we have analysed the effect of ablating cytokine production in two mouse models of arthritis: collagen-induced arthritis (CIA) in DBA/1J
mice and the inflammatory polyarthritis of tumor necrosis factor
(TNF-
) transgenic mice.
IL-6 was ablated by intercrossing an IL-6 null mutation into both arthritis-susceptible genetic
backgrounds and disease development was monitored by measuring clinical, histological, and
biochemical parameters. Two opposite responses were observed; while arthritis in TNF-
transgenic mice was not affected by inactivation of the IL-6 gene, DBA/1J, IL-6
/
mice were
completely protected from CIA, accompanied by a reduced antibody response to type II collagen and the absence of inflammatory cells and tissue damage in knee joints. These results are
discussed in the light of the present knowledge of cytokine networks in chronic inflammatory
disorders and suggest that IL-6 receptor antagonists might be beneficial for the treatment of RA.
 |
Introduction |
Rheumatoid arthritis (RA)1 is a common human autoimmune disease characterized by chronic inflammation of the sinovial joints and by subsequent progressive
destruction of articular tissue. Although the etiology and
pathogenesis of RA are not yet fully understood, it has become increasingly clear that a series of locally produced cytokines play a central role in disease progression. Indeed,
cytokines are responsible both for the mobilization and continuous activation of the inflammatory cell infiltrate and for inducing production of the enzymes that destroy bone
and cartilage (for review see reference 1).
The current view of the cytokine network in rheumatoid joints supports the notion that TNF-
activates a cytokine cascade characterized by the simultaneous production
of proinflammatory cytokines such as IL-1, IL-6, several
chemokines, GM-CSF, and of antiinflammatory factors
such as IL-10, IL-1RA, and soluble TNF receptor (for review see reference 2). Disease progression/reactivation or,
on the contrary, its silencing, are likely to be due to a dynamic and unstable equilibrium in the production of pro-
and antiinflammatory cytokines.
From among these cytokines, IL-6 has been proposed to
contribute to the development of arthritis. IL-6 is present at
very high levels in serum and synovial fluids of RA and of
juvenile RA patients (3). Soluble forms of the specific IL-6
receptor subunit
(sIL-6R
) are elevated (7, 8) and these
are known to potentiate IL-6 activity by forming IL-6-sIL-6R
complexes that bind and homodimerize the signaling-competent transmembrane receptor glycoprotein (gp)130 (9).
Increased IL-6 bioactivity during RA is believed to be
responsible for local and systemic effects. IL-6 acts as a
stimulator of both B and T cell functions because it promotes proliferation of plasmablastic precursors in the bone
marrow and their final stage of maturation into immunoglobulin-producing plasma cells and participates in the activation and proliferation of T cells (for review see reference
10). Moreover, IL-6, in conjunction with sIL-6R
, has
been recently shown to: (a) activate endothelial cell production of a subset of chemokines and adhesion molecules, thus contributing indirectly to recruitment of leukocytes at
inflammatory sites (11); and (b) induce synovial fibroblast
proliferation (12) and osteoclast formation and activation
(13), therefore suggesting a role in synovial fibrosis and in
bone resorption in inflamed joints. IL-6 is also known to be
an endogenous pirogen (14) and the major cytokine-inducing activation of liver acute phase response genes (15).
However, despite all this indirect and circumstantial evidence, a comprehensive investigation of the role of IL-6 in
the development of arthritis in animal models of RA has
not yet been reported.
The generation of knockout mice for different cytokine
genes has led to the conclusion that in most circumstances,
ablation of a single cytokine is compatible with normal life
(16). Knockout mice can, therefore, be used to evaluate the
role played by a specific cytokine in disease development.
Using appropriate disease models, IL-6-deficient mice (IL-6
/
) have been shown to be resistant to bone loss after
ovariectomy (17), and when bred into the Balb/c genetic
background, to be protected from the development of
pristane oil-induced plasmacytomas (18). We have hence
adopted the same genetic approach to analyze the effects of
ablating IL-6 activity in two available mouse arthritis models: collagen-induced arthritis (CIA) in DBA/1J mice and
the inflammatory polyarthritis of TNF-
transgenic mice
(19). Although the disease's primary cause differs in the two
models, they both share numerous RA histological features. Here we provide evidence that IL-6 plays a crucial
role in the development of autoimmune CIA, but not in
the TNF-
-dependent inflammatory arthritis.
 |
Materials and Methods |
Mice.
IL-6-deficient mice (17) were backcrossed into the
DBA/1J genetic background (H-2q, Jackson Laboratory, Bar
Harbor, ME) for five generations, obtaining DBA/1J, IL-6 mice
that were named DIL-6 mice. In all experiments, only DBA/1J,
IL-6
/
(DIL-6
/
) and DBA/1J, IL-6+/+ (DIL-6+/+) littermate
mice were used.
TNF, IL-6 mice were obtained by intercrossing TNF-
transgenic mice (19) with IL-6
/
mice. Mice carrying the TNF-
transgene and heterozygous for the IL-6 null mutation were intercrossed to obtain TNF, IL-6
/
(TIL-6
/
) and TNF, IL-6+/+
(TIL-6+/+) mice.
DIL-6 and TIL-6 mice were bred at IRBM (Rome, Italy) in a
specific pathogen-free animal facility, whereas the parental DBA/1J strain was obtained from Jackson Laboratory. 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.
Induction of CIA, Antibody Treatment, and Clinical Assessment of
Arthritis.
Male 8-wk-old DBA/1J and DIL-6 mice were immunized intradermally at the base of the tail with 100 µg of bovine
type II collagen (CII; M.M. Griffiths, University of Utah, Salt
Lake City, UT) in 0.05 M acetic acid, emulsified with an equal
volume of complete Freund's adjuvant, containing 100 µg of
H37RA Mycobacterium tuberculosis (Difco, Detroit, MI). On day
21, mice were boosted by intradermal injection with 100 µg of
bovine CII in 0.05 M acetic acid emulsified with an equal volume of incomplete Freund's adjuvant (Difco).
Starting from time of the CII booster injection, DBA/1J mice
were treated subcutaneously once a week for 6 wk with 0.5 or 1 mg/mouse of the following antibodies: (a) the rat mAb 15A7
(20), which neutralizes the murine IL-6 receptor alpha chain, (b)
an isotype-matched IgG2b rat mAb against dinitrophenyl hapten
(LO-DNP-57; provided by H. Bazin, University of Louvain,
Brussels, Belgium), and (c) total rat IgGs (Sigma Chemical Co.,
St. Louis, MO).
CIA development was inspected three times per week and inflammation of the four paws was graded from 0 to 4: grade 0,
paws with no swelling and focal redness; grade 1, paws with
swelling of finger joints; grade 2, paws with mild swelling of ankle or wrist joints; grade 3, paws with severe inflammation of the
entire paw; and grade 4, paws with deformity or ankylosis. Each
paw was graded and the four scores were totaled so that the possible maximum score per mouse was 16.
The arthritis index of TIL-6 mice was determined by observation of the hindpaw deformation twice a week and was scored as follows: 1, phalanx deformity; 2-6, different grades of limbs deformity; 7, ankylosis detected on flexion and severely impaired movement.
Serum Measurements.
Serum samples of DIL-6 mice were obtained before and 10 wk after the first CII immunization. Analysis
of total IgG anti-CII antibodies was performed by ELISA. Microtiter wells were coated with bovine CII dissolved in potassium
phosphate buffer, pH 7.6, (0.5 µg/well) overnight at 4°C. Wells
were blocked with Superblock Blocking Solution in PBS (Pierce,
Rockford, IL) 9 h at 4°C. Sera were diluted in 1% BSA in PNT
(1× PBS containing 0.05% Tween 20, and 0.2 M NaCl) and incubated overnight at 4°C. Antibodies anti-bovine CII were revealed by an alkaline-phosphatase-conjugated goat anti-mouse IgG (Promega, Madison, WI). The concentration of antibodies
was calculated by comparison with a standard positive control
mAb (M.M. Griffiths, University of Utah, Salt Lake City, UT).
ELISA to detect antigen-specific IgG isotypes was performed
with a modified protocol of the assay described above. In brief, after incubation wells were washed and different rabbit anti- mouse IgG isotype antibodies (Dako A/S, Glostrup, Denmark)
were used. Wells were washed and incubated with an alkaline-phosphatase-conjugated goat anti-rabbit IgG (Promega).
IL-6 serum levels were determined by the hybridoma growth
factor assay as previously described (21). Human TNF-
serum
levels were measured by ELISA kits (Medgenix EASIA) according to the manufacturer's specifications.
Histology.
DIL-6 mice were killed 7 wk after the first CII immunization and hind limbs were fixed with 4% paraformaldehyde
in 1× PBS overnight at 4°C. The joints were decalcified for 12 h
(De Castro's buffer: 0.3 M chloral hydrate, 0.72 M HNO3, and
30% ethanol) and then embedded in paraffin blocks. Joint sections
were collected and processed with Villanueva staining.
Statistical Analysis.
Results were analyzed using ANOVA,
chi-square analysis, and the Spearman correlation coefficient, as
appropriate (Statview program, Abacus Concept 1992). A P value
of <0.05 was considered significant.
 |
Results |
Serum IL-6 Levels Increase in Parallel with the Development
of CIA in DBA/1J Mice.
CIA is an animal model for rheumatoid arthritis in which the disease is elicited by immunization of genetically susceptible DBA/1J mice with type II
collagen, and it bears many of the histological features and
both cellular and humoral immune responses characteristically found in RA (22). To establish possible correlations
between IL-6 levels and the severity of arthritis, serum IL-6
levels were evaluated in parallel with disease severity expressed as arthritis index of the affected joints. Mice with
macroscopic joint involvement (arthritis index of >1) had
serum IL-6 levels (52.2 ± 45.8 U/ml) significantly higher
than those of mice without macroscopic involvement (12.5 ± 6.3 U/ml; P = 0.0033) and those of nonimmunized animals (6.3 ± 0.7 U/ml; P = 0.00l). In addition, in mice
with macroscopic joint involvement (arthritis index of >1) a
significant correlation (regression correlation coefficient of
Spearman [Rs] = 0.694; P = 0.008) between serum IL-6 levels and the arthritis index was found (Fig. 1), suggesting a direct correlation between IL-6 production and disease severity.

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Fig. 1.
Serum levels of IL-6 in DBA/1J mice with CIA correlated
with the arthritis index. Type II collagen immunized mice were bled 6 wk
after CII immunization. IL-6 activity was measured by hybridoma growth
assay and the arthritis index evaluated as described in Materials and Methods. Results were analyzed using the Spearman correlation coefficient.
Rs = 0.694; P = 0.008.
|
|
Treatment of CIA with an mAb Neutralizing IL-6 Activity.
To investigate the pathogenic role of IL-6 in CIA, we
first attempted neutralization of IL-6 in vivo using the mAb
15A7, directed against the murine IL-6 receptor alpha
chain (IL-6R
; reference 20, 23). Both 15A7 and control antibodies were administered subcutaneously at weekly
intervals starting from the time of the boosting CII injection. In a first experiment the isotype-matched rat mAb
LO-DNP-57 (20, 23) was used as a control. Surprisingly, both the 15A7 mAb and the LO-DNP-57 control mAb
were able to significantly decrease disease severity, as shown
in Fig. 2 A, when used at the dose of 1 mg/mouse but not
at the dose of 0.5 mg/mouse. As this result may be due to
some unexpected interference with CIA development of
the particular antibody used as control, we repeated the
treatment using total rat IgGs as control (Fig. 2 B). Again, the 15A7 mAb significantly decreased arthritis development (P <0.02 in weeks 9, 10, and 11 after the first CII
immunization), although the total IgG treatment also considerably decreased CIA symptoms, albeit with a statistically
significant decrease (P <0.04) only at week 9. These results
do not allow any conclusions to be drawn about the role of
IL-6 in CIA.

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Fig. 2.
Effects of antibody treatment on arthritis index of CII-immunized DBA/1J mice. (A) Mice (n = 9 for each group) were treated once per week with 1 mg/mouse (white symbols) or 0.5 mg/mouse (black
symbols) of either anti-IL-6R antibody 15A7 (circles), anti-dinitrophenyl
hapten antibody LO-DNP (triangles), or left untreated (squares). *P <0.05
either 15A7 or LO-DNP versus none. (B) Mice were treated with 1 mg/
wk/mouse of either anti-IL6-R antibody 15A7 (circles; n = 9), rat total
IgG antibodies (diamonds; n = 8), or left untreated (squares; n = 8). *P < 0.02 15A7 versus none; #P <0.04 total IgG versus none. Results were reported as mean ± SD. The weeks after the first immunization with CII
and the duration of antibody treatment are indicated on the abscissa.
|
|
IL-6 Production Is Absolutely Necessary for CIA.
An alternative strategy to test the role of IL-6 in CIA is ablation of the
IL-6 gene in mice genetically susceptible to CIA. We have
therefore introduced an IL-6 null mutation (17) into the
CIA-susceptible DBA/1J genetic background. After five
consecutive backcrosses both DIL-6
/
and DIL-6+/+
mice were generated and used for CIA studies. As the
MHC H-2 locus is known to play a major role in the genetic susceptibility to CIA (22), we first confirmed that
both DIL-6
/
and DIL-6+/+ mice carried the same H-2
allele (H-2q) as the parental DBA/1J strain (data not shown).
In two independent experiments, a total number of 16 DIL-6
/
and 17 DIL-6+/+ mice were immunized with
CII and joint swelling was monitored in the weeks after the
injection of the antigen. The results, reported in Fig. 3,
show that while DIL-6+/+ mice displayed the expected
rate of arthritis development (12/17 animals; 70.6%), DIL-6
/
mice were completely protected (0/16; P <0.0001 by
chi-square analysis), suggesting that IL-6 activity is essential
for CIA to develop. This conclusion was further supported
by histological analysis of the joints. Fig. 4 shows representative sections of six knee joints analyzed for each genotype
in which evident inflammatory alterations with proliferating pannus, bone erosion, and mononuclear cell infiltrate
were evident only in DIL-6+/+, whereas in DIL-6
/
no
pathological alteration was detectable; the joint and the articular discs were normal and no signs of pannus formation
and/or inflammatory infiltrate were present.

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Fig. 3.
IL-6 is absolutely required for the development of CIA.
DIL-6+/+ (squares; n = 17) and DIL-6 / (diamonds; n = 16) mice were
observed for arthritis lesions and the percentage of mice that developed
CIA is reported. The figure represents two independent experiments.
Weeks after the first immunization with CII are indicated.
|
|

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Fig. 4.
DIL-6 / mice do not show any histological lesion of the
joints. Representative examples of six knee joints for each genotype are
shown: DIL-6 / (A) and DIL-6+/+ (B). In A no signs of inflammatory
lesions are detectable. In B severe signs of inflammatory process are depicted; the pannus completely infiltrated the articular cartilage (arrowheads); severe signs of cartilage and bone destruction and mononuclear
cells infiltration are shown. Mice were killed 7 wk after first immunization with CII. Paraffin sections were stained with Villanueva staining. T,
Tibia; F, femur. Bars: 50 µm.
|
|
CIA development has been shown to be dependent on
both cellular and humoral immune responses to CII (22).
Since IL-6 is a major factor in the growth and differentiation of B cells into antibody-producing cells (10), we measured the level of the total anti-CII IgG. Although DIL-6
/
mice did develop appreciable levels of anticollagen IgGs,
they were significantly lower than those found in both
DIL-6+/+ mice (Fig. 5 A) and in the parental DBA/1J (not
shown). Previous studies have also suggested that antibodies of the IgG2a isotype play a major pathogenic role in
CIA development (26, 27). We therefore evaluated the anticollagen IgG isotype distribution in both DIL-6+/+ and
DIL-6
/
(Fig. 5 B). Interestingly, although in DIL-6+/+
mice IgG1, IgG2a, and IgG2b were equally present, in
DIL-6
/
mice a predominant IgG2a production was observed. Therefore, although the response to CII is quantitatively reduced in the absence of IL-6, it appears qualitatively compatible with disease development.

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Fig. 5.
DIL-6 / mice
have abnormal humoral response
to type II collagen. DIL-6+/+
(black bars; n = 9) and DIL-6 /
(white bars; n = 8) mice were
bled 10 wk after the first immunization with CII, and levels of
anti-CII antibodies were measured by ELISA. (A) Total IgG
anti-CII antibodies were reported as mean ± SD (*P <0.02). (B) Isotype percentages
of total IgG antibodies anti-CII were reported as mean ± SD.
IgG isotypes are indicated.
|
|
Inflammatory Polyarthritis in TNF-
Transgenic Mice Is IL-6
Independent.
TNF-
transgenic mice overexpress a 3
-untranslated region-modified form of the human TNF-
messenger RNA that is responsible for the development of
chronic inflammatory polyarthritis (19). Since TNF-
is
known to be an inducing factor of IL-6 synthesis (28), we
measured IL-6 levels in vivo to monitor possible correlation with disease development. As shown in Table 1, IL-6
serum levels were constantly elevated in TNF-
transgenic
mice independently from their age, and did not increase
with the development of the articular disease, in contrast
with what we found in the CIA model.
To assess if higher IL-6 production was critical for the
development of arthritis, we have generated transgenic
TIL-6
/
and TIL-6+/+ mice by genetic intercrosses. A total number of 16 TIL-6
/
and 11 TIL-6 +/+ mice were
analyzed for timing and severity of arthritis. The results
(Fig. 6) clearly show that both groups of animals displayed identical disease progression and severity. In agreement
with these data, hind paw histological analysis showed
comparable signs of arthritic lesions in mice belonging to
the two groups (data not shown). Since IL-6 is known to
downregulate TNF-
production in both in vitro and in
vivo models of inflammation (29, 30), the development of
arthritis in TIL-6
/
mice could be at least in part due to
TNF-
upregulation in the absence of IL-6. However, at 8 wk of age, TIL-6
/
mice showed lower serum TNF-
levels when compared to those of the TIL-6+/+ mice,
whereas 3 wk later, this difference had been eliminated (Table 2), indicating that TNF-
production is differently
regulated during the LPS-induced acute inflammation and
the chronic inflammation in TNF-
transgenic mice. Taken
together, these data indicate that IL-6 does not play a
pathogenic role in the onset and development of this TNF-
-dependent arthritis.

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Fig. 6.
The lack of IL-6 activity does not influence the onset and the
development of arthritis in TNF- transgenic mice. TIL-6 / (squares; n = 16) and TIL-6+/+ (diamonds; n = 11) mice were scored for arthritic lesions as described in Materials and Methods. Arthritis index is expressed as
mean values ± SD. The differences were not statistically significant.
|
|
 |
Discussion |
The goal of the present study was to further understand
the role played by IL-6 in the pathogenesis of different arthritic diseases by interfering with this cytokine's activity.
In a first attempt we made use of mAb 15A7 directed
against the IL-6-specific receptor subunit gp80. This particular antibody was chosen for its demonstrated efficacy in
other IL-6-dependent murine models (20, 23) and because in contrast with anti-IL-6 mAbs it is not expected to
stabilize the cytokine in vivo thus prolonging its half life
(21, 31). Treatment with 15A7 effectively decreased CIA
incidence and morbidity only at the dose of 1 mg/wk/
mouse, a dosage which has also been demonstrated as effective in several other models (20, 23). The requirement
for these amounts of 15A7 mAb may be explained by the
peculiar properties of the IL-6 receptorial system, with the
soluble form of the IL-6R
acting as a cytokine agonist
and its levels increasing considerably during inflammatory
diseases in both animals and humans (2, 8, 32). At this dosage the isotype-matched mAb used as a control showed a
similar therapeutic effect, and even total rat IgGs exerted a
protective activity, although less pronounced. The most
likely interpretation of the effect of unrelated antibodies on
CIA is that this relatively high dose of exogenous Ig may
cause nonspecific deviation of the immune system, similar
to what is observed in patients with autoimmune diseases
treated with high dose intravenous Ig (33). Since these results did not provide unequivocal evidence of the role of
IL-6 in CIA, we genetically ablated IL-6 activity, and found
that this resulted in total protection from CIA and in the
absence of inflammatory cell infiltrates in the joints.
Although knockout mice have previously been used to
evaluate the role of a variety of immune response-related
molecules, including cytokines, in CIA (34), this is the
first time that total protection is reported. This result is
therefore of particular relevance and should promote future
investigation as to the role of this cytokine in autoimmune
arthritis. It may be argued that the protection from CIA afforded by IL-6 gene inactivation could be at least partly
due to a higher presence of non-DBA genes physically
linked to the IL-6 locus in the DIL-6
/
mice. Although
this possibility cannot be ruled out, it is extremely unlikely
to account for total protection in 100% of the mice. In addition, none of the non-MHC genes that have been previously identified as contributing to genetic susceptibility to
CIA map close to the IL-6 locus (22, 40, 41).
The multiple biological activities of IL-6 make it difficult
to unravel the mechanism by which absence of this cytokine offers protection from CIA. We have found that although anticollagen IgG production had decreased in DIL-6
/
mice, it was predominantly the IgG2a subclass that
exerts a pathogenic role in CIA (26, 27). In addition, as the
production of IgG2a isotypes is driven by a polarized T
helper 1 response, this finding indirectly suggests that a
pathogenic polarized Th1 response to collagen still occurs
in the absence of IL-6. It is therefore likely that the weaker
anti-CII response in DIL-6
/
mice is but one of the factors contributing to protection from the disease, also in the
light of results obtained using IL-12 p40
/
mice (35),
which showed impaired anti-CII humoral response but still
developed arthritis, albeit at a reduced level. Since IL-6
functions as a late-acting killer helper factor in the differentiation of CTLs (42), and CD8+ T cells play an important
role in CIA initiation (39, 43), IL-6 ablation may, for example,
provoke a decrease in MHC class I-restricted anti-CII
cytotoxic activity. In addition, the defective chemokine
production and leukocyte recruitment at inflammatory sites
caused by IL-6 deficiency (11) may represent an important factor, as the continuous influx of activated cells to inflamed joints is known to be responsible for disease perpetuation
through increased cytokine production, tissue destruction,
and enhancement of the immune response. IL-6 inactivation may therefore exert its effects on CIA development by
acting at several levels. Further studies are needed to clarify
this issue.
Ablation of IL-6 production did not, in contrast, affect
the development of arthritis in TNF-
transgenic mice.
This difference to CIA might be due to a differentiated involvement of the immune system in the two models, as
suggested by the finding that TNF-
transgenic mice bearing a RAG null mutation develop arthritis in the absence of
functional lymphocytes (44). Moreover, it has been recently shown that most of the TNF-
activity in this model is mediated by its capacity to induce IL-1
and to
synergize with it (45), and the constitutive expression of
3
-untranslated region-modified TNF-
may not easily come
under the control of regulatory factors, hence leading to an
unbalanced cytokine cascade (44). Taken together, our results demonstrate that a given cytokine can have varying
relevance in different arthritis models, further corroborating previous observations showing different effects of the
neutralization of TNF-
in CIA or streptococcal cell wall- induced arthritis (46), and of the IL-1 receptor antagonist in
CIA and antigen-induced arthritis in rabbits or mice (47, 48). The same concept may also be true for human arthritides: although anti-TNF-
mAbs markedly ameliorate joint
involvement in the majority of patients with RA (49), administration of the same antibody did not affect arthritis in
one patient with severe systemic juvenile RA (50).
Our data support the idea that IL-6 blockade could be
beneficial for the treatment of human autoimmune arthritis. Administration of an mAb to IL-6 in an open pilot trial
of five patients with RA led to clinical and biological improvements (51). An alternative approach is the recently
generated human IL-6 receptor superantagonist Sant7, an
IL-6 variant that binds the human IL-6R
receptor chain at high affinity but no longer binds to the gp130 receptor
subunit. This molecule has been shown to inhibit the effects of IL-6 with great efficacy on a variety of human cell
lines, including myeloma cells (for review see reference 52).
In contrast to antibodies directed against IL-6, Sant7 is not
expected to prolong IL-6 half-life. Its potential for use in
therapy of chronic arthritis awaits further testing in suitable
animal models, an approach that so far has not been implemented in the mouse and rat systems due to lack of binding
to rodent IL-6R
(Ciliberto, G., unpublished observation).
Received for publication Received for publication 15 September 1997 and in revised form 26 November 1997..
We wish to thank Mr. M. Aquilina and Mrs. S. Germoni (Istituto Ricerche di Biologia Molecolare; IRBM)
for animal care, H.
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