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Dipartimento di Endocrinologia e Metabolismo, Sezione Metabolismo, Università di Pisa (L.M., M.M., R.L., S.D.G., R.N., P.M.), 56100 Pisa; Dipartimento di Scienze Cliniche, Università La Sapienza, Roma (F.D., C.S., M.R.), Roma; Cattedra di Endocrinologia e Patologia Costituzionale, Università di Catania (S.P., F.P.), Catania; and Dipartimento di Oncologia, Università di Pisa (F.M., U.B.), Pisa, Italy
Address all correspondence and requests for reprints to: Piero Marchetti, M.D., Dipartimento di Endocrinologia e Metabolismo, Ortopedia e Traumatologia, Medicina del Lavoro (Sezione Metabolismo), via Paradisa 2, Ospedale Cisanello, 56100 Pisa, Italy. E-mail: marchant{at}immr.med.unipi.it
Abstract
Studies in rodents have suggested that Th2 and Th3 cytokines can be
effective in reducing proinflammatory and Th1 cytokine-induced islet
damage. Whether this is the case with human islets and might be due to
a direct action of Th2 and Th3 cytokines is not known. In the present
study, we evaluated whether Th2 (500 U/ml IL-4 plus 100 U/ml IL-10) or
Th3 (5 ng/ml TGF-1ß) cytokines may prevent the derangements induced
on isolated human islets by prolonged (12 or 72 h) exposure to
combined proinflammatory (50 U/ml IL-1ß, 1000 U/ml TNF 
) and Th1
(1000 U/ml interferon 
) cytokines. Compared with control islets,
cells preincubated for 12 or 72 h with proinflammatory and Th1
cytokines showed a significant decrease of glucose-stimulated insulin
secretion and a significant increase of nitrites production. The
addition of IL-4 plus IL-10 or TGF-1ß in the medium prevented these
cytostatic effects in the 12-h incubation experiments, but not after
the 72-h exposure period. IL-1ß, interferon , and TNF caused
no major change in either islet cell survival or Bcl-2 and Bax mRNA
expression after a 12-h incubation; however, a marked increase in the
amount of dead cells, with a major decrease of Bcl-2 mRNA expression,
was observed after 72 h. The presence of Th2, but not of Th3,
cytokines significantly reduced ß-cell death, without any major
effect on Bcl-2 and Bax mRNA expression. These results suggest that Th2
and (at lower extent) Th3 cytokines may have a partial, direct
protective effect on isolated human islets exposed to the cytostatic
and cytotoxic action of proinflammatory and Th1 cytokines.
CYTOKINES ARE CHEMICAL messenger molecules
produced by a variety of activated cells that act as regulators and
mediators of immune responses (1). Recently, cytokines
have been classified into several different groups (2):
proinflammatory cytokines, such as IL-1ß, TNF , and interferon
(IFN) ; Type 1 cytokines (Th1), such as IL-12, IFN , IL-2, and
TNF ß; Type 2 cytokines (Th2), such as IL-4, IL-6, IL-10; and Type 3
cytokines (Th3), such as TGF ß.
Autoimmune diabetes (Type 1 or insulin dependent) results from a
selective destruction of pancreatic insulin-producing ß cells,
which occurs in genetically predisposed subjects (3, 4).
It is believed that certain cytokines (in particular some
proinflammatory and Th1 cytokines) could play an important role in
immune-induced damage of islet ß cells (5). IL-1
(produced by activated islet infiltrating macrophages), TNF
(produced by macrophages and activated T lymphocytes), and IFN
(produced by T lymphocytes) can bind to specific receptors of ß cells
and elicit signals (which have been only partially characterized) able
to activate several different pathways (5). In particular,
through the activation of phospholipases and proteases, and an
increased synthesis of nitric oxide (NO) synthase, production of
oxygen-free radicals (6) and NO takes place
(7). These radicals can inactivate mithochondrial and
cytosolic enzymes, leading to decreased ATP levels and impaired insulin
secretion (8). If the production of free radicals is
prolonged, they can damage some cellular constituents, including
membrane phospholipids and DNA, eventually causing ß-cell death
(9).
The evidence that in rodent models of autoimmune diabetes the
expression of Th2 and Th3 cytokines in the local inflammatory reaction
in and around the islets (insulitis) is associated with reduced or
absent ß-cell damage has recently led to the concept that these
cytokines may counterbalance the deleterious effects of proinflammatory
and Th1 cytokines. Indeed, transgenic expression of IL-4 in ß cells
of NOD mice completely protected the animals from insulitis and
diabetes (10), and transgenic expression of IL-10 or
TGF-1ß induced either a peri-islet infiltration without progression
to diabetes (11) or chronic pancreatitis without effect on
ß cells number or function (12). In addition, systemic
administration of IL-4 may prevent the development of diabetes in NOD
mice (13), and the combined administration of IL-4 and
IL-10 has been reported to avoid recurrence of autoimmune diabetes in
NOD mice transplanted with syngeneic islets (14). However,
to our knowledge, no study has directly evaluated the effect of Th2
and/or Th3 cytokines on isolated islets exposed to combined cytostatic
and cytotoxic cytokines. In addition, whether and at which extent the
laboratory animal findings previously discussed can be applied to the
human setting is far from being elucidated. In human pancreatic samples
from patients with Type 1 diabetes, IL-1, -4, and -10 expression has
been reported not to correlate with either destructive or benign
insulitis, whereas IFN is associated with destructive islet
infiltration (2). In vitro, IL-1 and, more
dramatically, a combination of IL-1ß, IFN , and TNF can
markedly impair isolated human islet function and survival (15, 16), possibly (16, 17), but not definitely
(15, 18), through mechanisms involving NO production. At
the present time, it is not known if these in vitro effects
of proinflammatory and Th1 cytokines on human islets can be affected by
Th2 or Th3 cytokines.
In the present study, we investigated whether Th2 (IL-4 plus IL-10) or
Th3 (TGF-1ß) cytokines may counteract the derangements induced on
isolated human islets by a 12- or 72-h exposure to a combination of
IL-1ß, IFN , and TNF . We assessed islet secretory function in
response to glucose, nitrites concentration, islet cell survival and
Bcl-2 and Bax mRNA expression. The results suggest that Th2 and (at a
lower extent) Th3 cytokines may have a partial, direct protective
effect on human islets exposed to a combination of proinflammatory and
Th1 cytokines.
Materials and Methods
The islets were prepared and characterized as described previously (19, 20). To complete this study, the islets from 18 different pancreases were used, with the approval of our local Ethics Committee.
Within 7–10 d from the isolation, aliquots of approximately 300 hand
picked islets were cultured free-floating for 12 or 72 h in Petri
dishes containing 6 ml supplemented M 199 culture medium under the
following conditions: 1) control M 199 medium; 2) M 199 medium
containing combined 50 U/ml IL-1ß, 1000 U/ml TNF , and 1000 U/ml
IFN ; 3) same as 2), with the addition of 500 U/ml IL-4 and 100 U/ml
IL-10; and 4) same as 2), with the addition of 5 ng/ml TGF-1ß. IL-4
plus IL-10 and TGF-1ß were also tested in the absence of other
cytokines. All the cytokines were of human recombinant origin, were
used at concentrations previously used in this kind of experiments
(15, 21, 22, 23), and had been purchased from
Roche Diagnostics (Mannheim, Germany).
At the end of the culture periods, the islets were evaluated for their glucose-stimulated insulin secretion, as described previously (19, 20, 24). Following a 45-min preincubation period in 3.3 mmol/liter glucose, batches of approximately 50 islets of comparable size were kept at 37 C for 45 min in Krebs-Ringer bicarbonate solution and 0.5% albumin (pH 7.4), containing 3.3 mmol/liter glucose. At the end of this challenge, the medium was completely removed and replaced with Krebs-Ringer bicarbonate solution containing 16.7 mmol/liter glucose. After an additional 45-min incubation, the medium was removed and assayed. One to three replicates from eight pancreases were used and averaged when needed, so that the number of islet preparations obtained from independent donors in this part of the study is 8. Insulin concentrations in the samples were measured by immunoradiometric assay (Pantec Forniture Biomediche, Turin, Italy).
Nitrite, the stable end product of NO, was determined (25) in five preparations by incubating 150 islets in 0.6-ml culture medium for 12 or 72 h under the varying conditions. Triplicate samples of 90 µl were then removed from the medium and added to 10 µl 0.5% N-(1-naphtyl)ethylendiamine dihydrochloride and 5% sulfanilamide in a 25% H3PO4 solution, prepared less than 12 h before use. The reaction was carried out at 60 C for 2 min, and the absorbance at 560 nm was measured in a Packard spectrophotometer (Packard, Groningen, The Netherlands) against a standard curve. Five islet preparations were used in this part of the study. The variability of the assay was less than 5%.
Islet cell death/survival was assessed by the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) technique (In Situ Cell Death Detection system; Roche Diagnostics) and the Cell Death Detection ELISAPLUS assay (Roche Diagnostics), both handled according to the procedure indicated by the manufacturer. Electron microscopy evaluation (26, 27) was performed in control and cytokine-exposed islets to estimate the cellular composition of the human islet preparations and to assess the effects of the varying cytokine combinations (see Results for details).
The expression of mRNA encoding for Bcl-2 and Bax, two molecules involved in determining the fate of cells (survival or death), was evaluated by RT-PCR in four separate experiments. Total RNA was extracted from purified human pancreatic islets with Trizol (Life Technologies, Inc., Grand Island, NY) according to the manufacturer’s instructions and quantitated by optical density (O.D.). First-strand cDNA synthesis was performed in a total volume of 20 µl, using 2 µg of each RNA sample primed with random examers with 200 U Superscript II (Life Technologies, Inc.); cDNA aliquots corresponding to 200 ng RNA were subsequently amplified in a 100-µl reaction volume containing 20 pmol upstream and downstream specific primers, 2.5 U Taq DNA polymerase (Life Technologies, Inc.), 200 µM of each deoxynucleoside triphosphate, and 1.5 mM MgCl2. Human Bcl-2 primer pair (5'-ACAACATCGCCCTGTGGATGAC-3' and 5'-ATAGCTGATTCGACGTTTTGCC-3') and Bax primer pair (5'-GGCCCACCAGCTCTGAGCAGA-3' and 5'-GCCACGTGGGCGTCCCAAAGT-3') generated a 408-bp and a 477-bp product, respectively. Expression of ß-actin as RNA control was analyzed using the following primers, generating a 354-bp product (5'-ACCAACTGGGAGGAGATGGAG-3' and 5'-CGTGAGGATCTTCATGAGGTAAGTC-3'). Multiple exons spanning primers have been used to avoid detection of genomic DNA. cDNA was amplified in serial cycles from 20–41 cycles for Bcl-2, Bax, and ß-actin, and linearity was demonstrated within this range. According to this result, 35 cycles were used for Bcl-2 and Bax and 25 cycles for ß-actin. All PCR products have been electrophoresed on 1.2% Separide agarose gel and bands visualized by ethidium bromide staining. To confirm the identity of the bands, amplified products have been sequenced with the Ampli-Taq cycle method using an automated sequencer (ABI373A; Perkin-Elmer Corp., Norwalk, CT).
Results are given as mean ± SE. Statistical comparison of data were performed by the t test or the ANOVA test (plus the Bonferroni correction when needed).
Results
Figure 1
shows islet insulin
secretion after a 12-h incubation. Glucose-stimulated hormone release
was 1229 ± 146 pmol/liter from control islets and decreased
significantly (511 ± 116 pmol/liter, P < 0.05)
from the islets cultured for 12 h in the presence of IL-1ß, IFN
, and TNF . The release of insulin from the islets precultured
with these latter cytokines plus IL-4 and IL-10 (934 ± 140
pmol/liter) or TGF-1ß (1043 ± 192 pmol/liter) was not
significantly different from that of control islets.
|
In the 12-h experiments, nitrite concentration (µmol/liter) was significantly (P < 0.05) higher after preculture with the proinflammatory and Th1 cytokines (2.1 ± 0.6) compared with control islets (0.7 ± 0.3); the addition to the former of IL-4 and IL-10 (1.2 ± 0.8), but not that of TGF-1ß (1.9 ± 0.3 P < 0.05 vs. control islets), prevented the increase of nitrite levels.
Results obtained after a 72-h incubation are given in Table 1. Again, glucose-stimulated insulin
release was suppressed after exposure to the "toxic" cytokines;
however, in contrast with data obtained after a 12-h incubation, the
presence of Th2 or Th3 cytokines did not protect the islets from the
cytostatic effect of IL-1ß, TNF , and IFN . Accordingly, the
addition of Th2 or Th3 cytokines did not cause any change to the
significant increase of nitrites concentration induced by IL-1ß, TNF
, and IFN (Table 1).
|
). The increased
death of islet cells was significantly prevented by the presence of Th2
cytokines, but not by the addition of Th3 cytokines (Table 2).
|
shows a typical appearance of control
islets (A) and cytotoxic cytokines exposed islets (B). In this latter
case, morphological changes typical of apoptotic phenomena (such as
nuclear condensation and chromatin clumping) were observed in the ß
cells. Quantitative assessment of ß cells was performed in six
control preparations, five preparations exposed to the toxic cytokines,
and three preparations exposed to the toxic cytokines plus either Th2
or Th3 cytokines. A significant difference in the amount of apoptotic
ß cells was observed between the experimental groups by ANOVA
(P < 0.01). Apoptotic ß-cell percentage (calculated
as number of apoptotic ß cells over apoptotic plus nonapoptotic
ß cells x 100) was 11 ± 3.2% in control islets and
increased to 38 ± 3.2% in cytotoxic cytokine-exposed islets
(P < 0.05 by the Bonferroni correction). The presence
of IL-4 plus IL-10 cytokines (amount of apoptotic ß cells: 16.3
± 6%, NS vs. control islets), but not the presence of
TGF-1ß (amount of apoptotic ß cells: 30 ± 1.4%,
P < 0.05 vs. control islets), prevented
this deleterious effect.
|
). However, after a 72-h exposure,
IL-1ß, TNF , and IFN caused a marked decrease of Bcl-2
expression, which did not seem to be further affected by the presence
of Th2 or Th3 cytokines (Fig. 3B).
|
The results of the present in vitro study show that
IL-4 plus IL-10 may counteract the cytostatic and cytotoxic effect
induced by prolonged exposure of human islets to combined IL-1ß, TNF
, and IFN . At our experimental conditions, TGF-1ß (Th3
cytokine) prevented the cytostatic action of the proinflammatory and
Th1 cytokines after a 12-h exposure, but not their cytotoxic
effect.
The action of Th2 cytokines on insulin secretion seems to be mediated,
at least in part, by a reduced production of NO, as documented by
decreased levels of nitrites in the incubation media. Indeed, it has
been previously shown (and confirmed in the present study) that NO
production increases after culturing human islets with IL-1ß, TNF
, and IFN and that increased levels of this free radical are
associated with inhibition of glucose-stimulated insulin release
(16). A reduction of IL-1-induced NO production due to the
action of IL-4 (21) and IL-10 (22) has been
previously observed with rat islets, even though the mechanisms remain
unknown. The effect of TGF-1ß on glucose-stimulated insulin release
was accompanied by no significant change of nitrites concentration,
suggesting that this cytokine possibly acts on pathways different from
NO production. In this regard, we must keep in mind that this latter is
just one (even though one of the most important and the most largely
studied) of the several mechanisms through which cytostatic cytokines
exert their action on ß cells (28). TGF-1ß might also
have a direct stimulatory effect on insulin release (23, 29), thus counterbalancing the inhibitory action of combined
IL-1, TNF , and IFN . This latter finding, however, has not been
confirmed in the present study by using human islets.
The protective effect of Th2 and Th3 on proinflammatory and Th1-induced
insulin secretion damage was lost after a 72-h incubation. However, at
our experimental conditions IL-4 plus IL-10 were able to reduce the
cytotoxic effect of combined IL-1ß, TNF , and IFN , an effect
that electron microscopy showed to involve islet ß cells. This was
not due to reduced NO production, since nitrite levels were not
affected by the presence of Th2 cytokines. These results provide
evidence that IL-4 and IL-10 may protect human islets by the cytotoxic
damage induced by proinflammatory and Th1 cytokines and support the
view that islet cell death induced by IL-1ß, TNF , and IFN is
due only in part, if any, to NO production (30).
The cytotoxic effect of proinflammatory and Th1 cytokines was associated with a marked decrease of Bcl-2 expression, and the protective effect of Th2 cytokines was not accompanied by any further change of Bcl-2 mRNA. Although in some experimental models IL-4 and IL-10 have been reported to be able to maintain expression of Bcl-2 following apoptotic insults (31, 32), these cytokines might also act at a level(s) different from Bcl-2. In this regard, IL-10 was shown to promote cell survival without any modification of Bcl-2 molecules (33).
Pancreatic islet ß-cell destruction in Type 1 diabetes is due, at
least in part, to the damage induced by some proinflammatory and Th1
cytokines and, in particular, IL-1ß, TNF , and IFN
(5). Investigations performed in rodent models have
demonstrated that Th2 and Th3 cytokines may reduce ß-cell damage,
through indirect mechanisms (10, 11, 12). In the present work,
we show that IL-4 plus IL-10 and, at a lower extent, TGF-1ß may have
a partial, direct protective role on human islets exposed to IL-1ß,
TNF , and IFN . Although this information needs to be confirmed
in additional experimental settings, and the precise mechanisms
investigated more in depth, evidence has now been provided to support
the concept that Th2 cytokines can reduce the cytostatic and cytotoxic
damage induced by proinflammatory and Th1 cytokines on human pancreatic
islet cells.
Acknowledgments
Footnotes
This work was supported by grants from the Italian National Research Council, Ministero Università e Ricerca Scientifica e Tecnologica (COFIN 2000), Regione Toscana, by Telethon Grant E660, and by the Juvenile Diabetes Foundation International (1-1999-679).
Abbreviations: IFN, Interferon; NO, nitric oxide; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling.
Received August 15, 2000.
Accepted April 26, 2001.
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