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Antibody
Institutes of Microbiology (F.N.) and Internal Medicine, Infectious Diseases, and Immunopathology (P.M.), University of Milan, Milan; and the Institutes of Internal Medicine, Endocrinology, and Metabolism (F.N., M.L.), Microbiology (P.Z., R.D.M.), and Anatomo-Pathology (G.M., S.G.), University of Catania, Catania, Italy; and Human Genome Sciences (G.G.), Rockville, Maryland 20850
Address all correspondence and requests for reprints to: Ferdinando Nicoletti, M.D., Via Luigi Sturzo n.3, 95021 Cannizzaro, Catania, Italy.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(IFN) in the development
of insulin-dependent diabetes mellitus (IDDM) in diabetes-prone BB rats
was evaluated. Several groups of these animals were treated under
different experimental conditions with a purified polyclonal antibody
(Ab), antirat IFN. The results show that when administered at doses
of 100 or 200 µg/week from the 30/33th until the 105th day of age,
the anti-IFN Ab reversibly reduced the incidence of IDDM compared to
that in control rats treated with either irrelevant rabbit IgG or PBS.
Moreover, when given up to the 105th day of age, these doses of
anti-IFN Abs exerted comparable preventive effects regardless of
whether application started as early as within 24 h after birth or
at the end of the prediabetic period (e.g. 70/75 days).
In contrast, under none of the above experimental conditions did larger
doses of anti-IFN Ab (500 µg or 1 mg/week) exert antidiabetogenic
effects in the BB rats. Apparently, this was due to the exuberant
production of neutralizing Abs elicited by the large amount of the
xenogeneic Ab injected. At histoimmunological analyses, the BB rats
treated with 200 µg/week anti-IFN Abs from 30–80 days of age
exhibited a milder insulitic process along with diminished spleen
frequency of activated lymphoid cells (MHC class II and interleukin-2
receptor positive). Taken together, these results provide further
in vivo evidence for the central pathogenic role of
IFN in BB rat IDDM and anticipate the usefulness of specific IFN
inhibitors in the prevention of the disease in the clinical setting.
Defining novel and less immunogenic forms of specific IFN inhibitors
than xenogeneic Abs is important for improving the efficiency of
anti-IFN-oriented approaches. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and interferon- (IFN) (4, 5, 6).
Regardless of the precise pathogenic mechanism (see Ref. 6 for a
review), substantial evidence supports the key role of IFN in IDDM.
The serum levels of IFN are increased at IDDM onset in both humans
(7) and BB rats (8), and IFN has been detected in
ß-cell-infiltrating lymphocytes in recently diagnosed IDDM patients
(9) and in the insulitic lesions of BB rats (10, 11) and NOD mice
(12, 13, 14). Moreover, IDDM may be transferred to naive NOD mice by
IFN-secreting CD4+ T cells (15), and diabetes and
insulitis develop in transgenic mice that express the IFN gene at
the level of the insulin promoter (16, 17). Finally, in vivo
treatment with either anti-IFN monoclonal antibodies (mAbs) or the
soluble IFN receptor (sIFNR) prevents diabetes in the NOD mouse
(18, 19, 20, 21) and the mouse made diabetic with multiple low doses of
streptozotocin (22, 23).
In a previous study conducted in the BB rat we also observed that
prophylactic treatment with the antirat IFN monoclonal antibody
(mAb) (DB-1) reduced the incidence of IDDM in these animals (24). As
the DB-1 mAb was only available from commercial sources, we could not
afford to extend this preliminary observation. Therefore, several
questions remained unsettled as to the preventive action of anti-IFN
mAb in the BB rat, the dose dependency, the reversibility of the
action, and the effect on the development of insulitis. Moreover, as we
and others have shown that, depending on the tempo of administration in
relation to the autoimmune process, anti-IFN treatment may exert
biphasic effects in other rodent models of autoimmune diseases
(25, 26, 27), it would have also been important that BB rats had been
treated with anti-IFN Ab at different times during their life.
To address these questions, a rabbit polyclonal Ab that neutralizes rat
IFN was produced, purified, and administered in vivo to
BB rats under different experimental conditions. Prolonged prophylactic
treatment with low (100–200 µg/week) doses of anti-IFN Ab
abrogated histoimmunological signs of autoimmune diabetogenesis, thus
preventing in a reversible fashion the development of the disease. At
doses of 500 µg/week or higher, the anti-IFN Ab induced the
production of neutralizing Abs capable of abolishing the preventive
action.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reagents and chemicals
Recombinant rat IFN was produced as previously described
(28). Complete and incomplete Freund’s adjuvants, irrelevant mouse
IgG, and Concanavalin A (Con A) were provided from Sigma Chimica
(Milan, Italy). Irrelevant rabbit IgG were obtained from Rockland
(Gilbertsville, PA). Mouse fluorescein isothiocyanate (FITC)-conjugated
mAb W3/13, reacting with rat with T lymphocytes, polymorphonuclear
cells, hemopoietic cells, and plasma cells, but not with B lymphocytes;
W3/25, reacting with helper T lymphocytes and macrophages; OX-8,
reacting with cytotoxic suppressor T lymphocytes and natural killer
cells; and OX-6, reacting with the Ia-A nonpolymorphic determinant of
the major histocompatibility complex class II locus, were obtained from
Sera-Lab (Crawley Down, UK). Mouse FITC-conjugated OX-39 mAb antirat
IL-2R was obtained from Biosource (Camarillo, CA). The enzyme-linked
immunosorbent assay (ELISA) kit for detection of rat IFN was
purchased from Biosource. [3H]Thymidine was obtained from
Amersham International (Aylesbury, UK). Ficoll-Hypaque, RPMI 1640, PBS,
heat-inactivated FBS, and L-glutamine were purchased from
Life Technologies (Paisley, Scotland). Cyclosporin A (CSA) was provided
by Sandoz (Basel, Switzerland).
Polyclonal antirat IFN Ab
An emulsion consisting of a mixture of complete Freund’s
adjuvant and rat IFN was injected sc into 4-month-old female New
Zealand White rabbits (0.5 mg/rabbits). Two, 4, 8, 12, 21, and 24 weeks
later, the rabbits were boosted sc with an emulsion composed of rat
IFN in incomplete Freund’s adjuvant. The rabbits were exsanguinated
2 weeks after the last immunization. The anti-IFN Ab was then
purified by affinity chromatography on a protein A-Sepharose gel after
salting with 50% saturated
(NH4)2SO4 and depletion of lipids
by trichlorotrifluoroethane. Their capacity to neutral the antiviral
activity of rat IFN was assessed using L929 cell lines.
Experimental design
Several groups of euglycemic male and female BB rats were
randomly created that received anti-IFN Ab according to the doses
and administration schedule reported in Tables 1
, 2, and 3. For the
neonatal experiments, pups from each litter were randomly assigned to
control or experimental groups, marked, and kept with the mother.
Control BB rats were treated with irrelevant rabbit IgG or PBS.
Treatments were given twice a week through ip injection in a final
volume between 100–500 µl. Moreover, to evaluate whether anti-IFN
Ab exerted synergistic antidiabetogenic effects with CSA, an anti-T
cell drug known to prevent IDDM development in BB rats (3), other
experiments were performed in which subtherapeutic doses of CSA were
coadministered with anti-IFN Ab (see Table 4).
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Immunofluorescence analysis
SLC were incubated with appropriate concentrations of
FITC-conjugated mouse mAb W3/13, W3/25, OX-8, OX-6, and OX-39.
Appropriate mAbs were used as the control in each analysis. The
fluorescence of cells was evaluated by flow cytometry (Ortho
Diagnostics, Raritan, NJ) by an observer unaware of the treatment of
the animals. At least 10,000 events were counted for each sample.
Ex vivo lymphoproliferative response
SLC, plated in triplicate in flat-bottomed microtiter plates at
a concentration of 106/ml, were cultured in complete medium
(RPMI 1640, 5% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and
2 mM L-glutamine) for 72 h at 37 C with
5% CO2in the presence or absence of 4 µg/ml Con A. SLC
were pulsed with [3H]thymidine for the last 24 h and
counted by liquid scintillation. Results are expressed as counts per
min and are the mean (±SD) of triplicate
determinations.
IFN secretion
SLC were cultured in complete medium at a concentration of
2 x 106/ml for 48 h in the presence or absence
of 4 µg/ml Con A at a final volume of 2 ml/vial. At the end of the
culture period, the cells were centrifuged, and the supernatants were
collected and assayed for the presence of IFN. The sensitivity of
the assay ranged from 13–1400 pg/ml.
Serum IFN levels
Fasting venous blood samples from healthy and diabetic BB rats
were obtained between 1100–1200 h from selected experimental and
control groups. Blood samples were allowed to clot at room temperature,
and serum was immediately separated by centrifugation at 1000 x
g and stored at -20 C until assayed for the measurement of
circulating levels of IFN. These were measured using the ELISA
previously described.
Measurement of antirabbit IgG
The blood level of rat Abs to rabbit IgG was studied by an ELISA
similar to that described by Finck et al. (30) In brief,
microtiter plates were coated for 1 h at room temperature with
chromatographically purified rabbit IgG in 0.1 M Tris-HCl
(0.5 µg/well). After washing with tap water, blockage with 2% BSA
for 30 min, and further washing, PBS-diluted (1:10, 1:100) serum
samples from individual rats treated with rabbit antirat IFN Ab,
irrelevant rabbit IgG, or PBS were added and kept at room temperature
for 1.5 h. After washing, horseradish peroxidase-conjugated goat
antirat Ig was used as second antibody at a 1:50 dilution in PBS-1%
BSA (75 µl/well) and kept at room temperature for 1 h. After
washing, the colorimetrical reaction was developed by adding
o-phenylenediamine in PBS for 10 min. Readings have been
performed at 490 nm (Titertek, Flow Labs, Rockville, MD) after stopping
the reaction with 50 µl 2 N H2SO4.
Histological examination of pancreatic islets
Histological examination of the pancreatic islets was performed
in a blind fashion by two pathologists unaware of the status and/or the
treatment of the animals, as described previously (29). The degree of
mononuclear cell infiltration was graded as follows: 0, no infiltrate;
1, periductular infiltrate; 2, periislet infiltrate; 3, intraislet
infiltrate; and 4, intraislet infiltrate associated with ß-cell
destruction. At least 12 islets were counted for each rat. The mean
score for each pancreas was calculated by dividing the total score by
the number of islets examined.
Statistical analysis
Results are shown as the mean ± SD.
Statistical analysis was performed by the appropriate use of Fisher’s
exact test and ANOVA with Bonferroni’s adjustment. P
< 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ab temporarily reduces IDDM development in BB rats Ab at
doses of 100 and 200 µg/week reduced in a dose-dependent fashion the
number of rats becoming diabetic by this age and delayed the age of
diabetes onset in those animals that developed IDDM (Tables 1–4
). When
anti-IFN Ab was administered continuously up to 105 days of age, its
preventive action was almost comparable regardless of whether treatment
was started as early as within 24 h after birth, after weaning
(30/33 days), or at the end of the prediabetic period (Tables 1–3).
Moreover, continuous application seems mandatory for the
antidiabetogenic effects to occur, as BB rats that received the
anti-IFN Ab for only 30 days after birth developed diabetes with an
incidence similar to that in control animals (Table 1). The anti-IFN
Ab also lacked its preventive effects when administered at the larger
doses of 0.5 and 1 mg/week (Tables 1–4). In a similar manner,
combining anti-IFN Ab prophylaxis with subtherapeutic doses of CSA
failed to potentiate the preventive effects of the former (Table 4).
Prolonged treatment with anti-IFN Ab was well tolerated, as judged
by both the general appearance of the animals and their body weight,
which was very similar to that of the control animals (data not shown).
However, 5 of 28 rats that received the highest dose (1 mg) of
anti-IFN Ab from 30/33 days of age died after 40–65 days of
treatment. Although the reasons for this high rate of mortality have
not been ascertained, the presence of hematuria in 3 of these 5 animals
suggests that these rats suffered from nephropathy, perhaps
consequential to serum-like disease.
Anti-IFN Ab does not afford long lasting protection from IDDM
development, nor does it act through generation of suppressor cells
To gain insight into the cellular mechanism of action of
anti-IFN Ab, other experiments were performed that aimed at
evaluating whether this treatment afforded long lasting protection from
IDDM development or if it favored the development of antidiabetogenic
suppressor cells. In one experiment, two groups of BB rats were treated
with either 200 µg/week anti-IFN Ab or PBS from 30/33 until 105
days of age, a regimen that we previously found to diminish the
incidence of IDDM in BB rats (Table 2). At the end of the treatment,
the remaining euglycemic rats from the two groups were examined for
later development of IDDM for an additional 75 days. As shown in the
Fig. 1, the preventive action of this treatment, clearly
evident at 105 days of age, was lost within 2 months after treatment
withdrawal, indicating the short term reversibility of the
antidiabetogenic action of anti-IFN Ab.
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Ab (200
µg/week) or PBS from 30–105 days. At the end of the experiment,
euglycemic BB rats from the two groups were killed, and their spleens
were collected for transfer studies. SLC from individual rats, obtained
as described above, were injected ip into 23-day-old syngeneic
recipients at a concentration of 200,000 x 106, and
these animals were examined once a week for diabetes development until
105 days of age. Further control was constituted from age-matched,
untransferred BB rats. There were differences in neither the incidence
nor the kinetics of IDDM appearance in the three groups of rats (seven
rats per group; data not shown).
Anti-IFN Ab prophylaxis reduces histological signs of insulitis
and diminishes the abnormal percentage of MHC class II- and
IL-2R-positive SLC in BB rats
Along with hyperglycemia, BB rats develop a selective mononuclear
cell infiltration of the ß-cells that precedes by 10–15 days the
onset of overt IDDM, and they show multiple abnormalities of SLC
subsets (31). To examine the impact of anti-IFN application on the
development of these histoimmunological pathways, BB rats were treated
with anti-IFN Ab (200 µg/week), irrelevant rabbit IgG (200
µg/week), or PBS from 30–80 days of age, that is for the period of
life that we had previously found to be associated with moderate to
severe insulitis in the majority of the BB rats of our colony. At this
point, eight euglycemic BB rats from each group were killed, and their
pancreata specimens and spleens were collected for histoimmunological
analyses. BB rats becoming diabetic before this age were killed and not
included in the study. It was found that relative to control BB rats,
anti-IFN Ab treatment significantly reduced the severity of
insulitis (Fig. 2) and decreased the high percentage of
MHC class II- and IL-2R-positive SLC (Fig. 3). The lack
of double staining experiments did not permit us to precisely define
the cell type among B and T lymphocytes and macrophages on which
anti-IFN Ab is acting. However, inasmuch as an exuberant expression
of MHC class II antigens and IL-2R has been reported to occur on
splenic T cells from BB rats (31), it seems possible that this cell
population has been the primary target of anti-IFN Ab.
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Ab treatment affects neither the ex vivo
lymphoproliferative response nor the IFN secretory capacity of BB
rat SLC Ab ensued from a generalized immunosuppressive effect on
lymphoid cells. For this reason, SLC obtained from PBS- and
anti-IFN-treated rats were used to study the effects of
anti-IFNAb on the ex vivo lymphoproliferative response or the IFN
secretory capacity of these cells. Table 5 shows that
relative to SLC from control BB rats, SLC from anti-IFN-treated BB
rats maintained a normal lymphoproliferative response and IFN
secretory capacity.
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Ab induces a dose-dependent
formation of antirabbit IgG Ab
generated antixenogeneic responses in BB rats, the formation of
antirabbit IgG Ab was studied in BB rats treated with different doses
of anti-IFN Ab, rabbit IgG, or PBS. As expected, a dose-dependent
increase was found in the production of antirabbit IgG in the rats
treated with anti-IFN Ab. The formation of antirabbit IgG was
slightly, but not significantly, reduced by the adjunct of
subtherapeutic doses of CSA (data not shown and Table 6).
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Ab suppress the appearance of circulating
levels of IFN in acutely diabetic BB rats occurred in anti-IFN-treated BB rats.
Low serum levels of IFN were detectable in the majority of acutely
diabetic BB rats, but in none of the healthy BB rats from the control
group (Fig. 4). The results obtained in the rats treated
with anti-IFN Ab were of interest; whereas serum IFN was
undetectable in both healthy and diabetic rats treated with the
effective dose of 200 µg/week, the diabetic rats treated with the
high and ineffective dose of 500 µg/week had serum IFN levels
comparable to those in the diabetic control rats (Fig. 4). The low
limit of sensitivity of our ELISA (13 pg/ml) allowed us to detect
circulating levels of IFN in most of acutely diabetic BB rats. This
finding, confirming and extending another report in which the higher
limit of sensitivity of that ELISA (75 pg/ml) only allowed us to detect
circulating levels of IFN in 20.6% of recently diabetic BB rats
(8), strengthens another immunopathogenic similarity with the human
disease counterpart, in which disease onset is also associated with
increased serum levels of IFN (7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ab on the development of
diabetes in BB rats provides evidence for the key pathogenic role of
IFN in this model of human IDDM. Our present findings complement and
extend a preliminary observation by our group that the incidence of the
disease was reduced by treatment with the antirat IFN DB-1 mAb (24).
The greater efficiency with which diabetes was prevented in that study
vs. the present one (14% vs. 38%) can probably
be accounted for by the different Abs used and the much larger number
of rats presently considered. The present study helps better define the
mode of action of anti-IFN Ab in the BB rat model.
IFN stimulates the functional activities of both cytotoxic T cells
and macrophages (33), which can mediate ß-cell destruction through
MHC class II-restricted attack and nonspecific immunoinflammation,
respectively (34). Conceivably, when IFN was antagonized with
neutralizing Abs, its stimulatory activities on these cells were
reduced, and cellular diabetogenic pathways were consequently
down-regulated. Nonetheless, although this view accords with the
observation that the rats treated with anti-IFN Ab were virtually
free from insulitis (a macrophage/T cell-dependent event in this
model), the exact immunomodulatory pathways elicited by selective
blockade of endogenous IFN remain to be defined. For example, rather
than reflecting a possible action of this treatment on T cells or
macrophages, the reduced percentages of MHC class II- and
IL-2R-positive SLC of the rats that received anti-IFN Ab could
simply be secondary to the diminished release of antigenic material
from the less injured ß-cells. In a similar manner, the potential
impact of anti-IFN Ab on T cells should not be minimized on the
basis of the normal lymphoproliferative response and IFN secretory
capacity maintained in anti-IFN-treated T cells from BB rats under
ex vivo conditions. Because SLC were washed and then
cultured for several days with mitogen, but without anti-IFN Ab, the
possibility cannot be ruled out that anti-IFN application might have
suppressed T cell function in vivo; therefore, caution would
be exercised to extrapolate more from these data than the prompt
reversibility of a hypothetical impairment of T cell function.
That IDDM occurred in many of the BB rats treated with anti-IFN Abs
despite the effective removal of IFN from serum suggests that IFN
is important, although not necessary, for diabetes development in BB
rats. This observation concurs with recent reports by Hultgren et
al. (35), who found that absence of the IFN gene, achieved by
IFN knockout, delays, but does not prevent, diabetes in NOD mice.
Perhaps diabetogenic pathways may be sufficiently activated in the
absence of IFN by other proinflammatory cytokines implicated in the
pathogenesis of BB rat IDDM, such as IFN (36), IL-1 (37), IL-2 (10),
or IL-12 (10). Moreover, because splenic cells from BB rats that were
given anti-IFN Ab did not protect syngeneic recipients from IDDM
upon transfer, and the antidiabetogenic action of anti-IFN Ab
rapidly ceased after treatment withdrawal, it seems unlikely that the
Ab acted through either induction of suppressor cells or elimination of
autoreactive cells. On the contrary and fitting in with the lack of
effect of short courses of treatment, continuous binding and
neutralization of IFN by the anti-IFN Ab are envisionable that
temporarily prevent the proinflammatory action of the cytokine on the
target cells. Moreover, that the preventive effects of prolonged
anti-IFN-administration were comparable regardless of whether
treatment was started early (e.g. within 24 h from
birth or at 30/33 days of age) during prediabetes or just before
disease development (e.g. 70/75 days) shows that IFN also
plays a key role in the late diabetogenic events, perhaps after islet
antigens have been engaged from autoreactive cells. This observation
may be important for the clinical setting; if IFN is also
pathogenically important in the efferent phase of human IDDM, specific
IFN inhibitors can then be expected to halt/delay the autoreactive
process even when first given during actively ongoing diabetogenesis,
as is the case for newly diagnosed IDDM patients and prediabetic
subjects. Finally, that neonatal pulse treatment with anti-IFN Ab
did not prevent later appearance of IDDM in the adult rats suggests
that IFN does not exert its diabetogenic potential in this model by
disrupting intrathymic education processes, whose malfunctioning in BB
rats (38) has been implicated in the maintenance of autoreactive
cells.
The protective effect of anti-IFN prophylaxis in the BB rat was only
observed when the Ab was continuously administered at 100 and 200
µg/week until 105 days of age. It disappeared when given under the
same experimental conditions at the two larger doses of 0.5 or 1 mg.
The reasons for this inverse dose-dependent effect of anti-IFN Ab
are not completely understood. However, the cytokine network is finely
regulated in vivo, and profound alterations of this
homeostatic system can provoke paradoxical responses. For example,
inverse dose-dependent effects have been observed when a recombinant
dimeric tumor necrosis factor receptor (Fc) was administered to healthy
volunteers who received endotoxin iv, with high doses being less
immunosuppressive than low doses on secondary cytokine levels,
leukocyte margination, and neutrophil migration (39). In addition,
because the two high doses of anti-IFN Abs induced the formation of
antirabbit IgG to a much greater extent than the low effective doses in
the BB rat, it might also be that these antixenogeneic Abs impeded the
bioactivity of the rabbit anti-IFN Ab. The above agrees with the
observation that unlike the diabetic BB rats treated with low doses of
anti-IFN Ab, which did not have circulating IFN in their sera,
those diabetic rats treated with higher doses (0.5 mg/week) showed the
same elevated serum levels of IFN as the diabetic control rats.
The detrimental effect of antixenotypic and antiidiotypic Abs formed
when these heterologous Abs are used in the clinical setting is well
known (40). Because CSA reduces the formation of antimouse IgG in
transplanted patients treated with OKT3 mouse mAb (40), we combined
subtherapeutic doses of CSA with anti-IFN Ab. Presently, this
allowed only a slight and not significantly reduced formation of
antirabbit IgG without potentiating the antidiabetogenic effect of the
anti-IFN Ab. Whether this was due to the low dose of CSA used (2
mg/kg) may be difficult to prove, as we found CSA to exert appreciable
antidiabetogenic effects when administered at 3–3.5 mg/kg in
monotherapeutic regimen. Although other pharmacological approaches may
be used to reduce the immunogenicity of anti-IFN Ab, such as short
courses of tolerizing anti-CD4 mAb (30) or anti-B lymphocyte drugs such
as deoxyspergualin (41), another alternative may rely on using specific
IFN inhibitors less immunogenic than mAbs. One such example is the
sIFNR that was recently produced and characterized in its murine (as
well as human) form (42, 43, 44, 45), which 1) neutralizes IFN in
vitro and in vivo with a greater efficiency than that
of mAb (46), 2) does not induce Ab formation in BALB/c mice (45), 3)
prevents both multiple low doses of streptozotocin-induced (23) and
spontaneous diabetes (21) in mice (23), and 3) is therapeutically
effective in the (New Zealand BlackxNew Zealand White)F1 mouse model
of system lupus erythematosus (46).
Along with the preventive effect of anti-IFN mAb in the NOD mouse
model (19, 20, 21, 22), present results in the BB rat further underscore the
potential utility of specific IFN inhibitors in the prevention and
possibly the treatment of human IDDM. Defining novel and less
immunogenic forms of specific IFN inhibitors than heterologous Abs
can help improve the efficiency of anti-IFN-oriented approaches.
Received July 22, 1996.
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