Pancreatic beta -Cell Damage Mediated by beta -Cell Production of Interleukin-1

A NOVEL MECHANISM FOR VIRUS-INDUCED DIABETES*

Monique R. Heitmeier, Marc Arnush, Anna L. Scarim, and John A. CorbettDagger

From the Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104

Received for publication, October 6, 2000, and in revised form, November 14, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Viral infection is one environmental factor that may initiate beta -cell damage during the development of autoimmune diabetes. Formed during viral replication, double-stranded RNA (dsRNA) activates the antiviral response in infected cells. In combination, synthetic dsRNA (polyinosinic-polycytidylic acid, poly(I-C)) and interferon (IFN)-gamma stimulate inducible nitric-oxide synthase (iNOS) expression, inhibit insulin secretion, and induce islet degeneration. Interleukin-1 (IL-1) appears to mediate dsRNA + IFN-gamma -induced islet damage in a nitric oxide-dependent manner, as the interleukin-1 receptor antagonist protein prevents dsRNA + IFN-gamma -induced iNOS expression, inhibition of insulin secretion, and islet degeneration. IL-1beta is synthesized as an inactive precursor protein that requires cleavage by the IL-1beta -converting enzyme (ICE) for activation. dsRNA and IFN-gamma stimulate IL-1beta expression and ICE activation in primary beta -cells, respectively. Selective ICE inhibition attenuates dsRNA + IFN-gamma -induced iNOS expression by primary beta -cells. In addition, poly(I-C) + IFN-gamma -induced iNOS expression and nitric oxide production by human islets are prevented by interleukin-1 receptor antagonist protein, indicating that human islets respond to dsRNA and IFN-gamma in a manner similar to rat islets. These studies provide biochemical evidence for a novel mechanism by which viral infection may initiate beta -cell damage during the development of autoimmune diabetes. The viral replicative intermediate dsRNA stimulates beta -cell production of pro-IL-1beta , and following cleavage to its mature form by IFN-gamma -activated ICE, IL-1 then initiates beta -cell damage in a nitric oxide-dependent fashion.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insulin-dependent diabetes mellitus is an autoimmune disease characterized by the selective destruction of insulin-secreting beta -cells found in pancreatic islets of Langerhans (1). Triggering events that precipitate beta -cell damage have remained elusive; however, evidence supports a role for viral infection in the initiation of autoimmune diabetes. Viruses have been isolated from pancreata, and virus-specific IgM antibodies have been identified in newly diagnosed diabetic patients (2, 3). Diabetes can be induced in genetically susceptible strains of mice, rats, and primates by infection with encephalomyocarditis virus, Coxsackie B4 virus, Kilham's rat virus, rubella virus, and retrovirus (3, 4). Encephalomyocarditis-induced diabetes in mice is associated with increased expression of iNOS1 and macrophage-derived cytokines IL-1beta , IL-12, and TNF-alpha . Administration of neutralizing antisera for IL-1beta or TNF or of the iNOS-selective inhibitor aminoguanidine attenuates diabetes development in this mouse model (5). In vitro studies have identified cytokine-stimulated iNOS expression and nitric oxide production by beta -cells as one mechanism by which IL-1 inhibits insulin secretion and induces islet damage (6, 7).

Double-stranded RNA (dsRNA), formed during viral replication, is an active component of a viral infection that triggers antiviral responses in infected cells (8). The antiviral response includes the expression of type 1 interferons (9), nitric oxide production (10), macrophage IL-1 release (11), and a general inhibition of protein translation (12, 13). Similar to a viral infection, the synthetic dsRNA molecule polyinosinic-polycytidylic acid (poly(I-C)) also activates these antiviral responses (14-16). In vivo, administration of poly(I-C) to diabetes-resistant and -prone BioBreeding rats results in the induction and acceleration of diabetes, respectively (17, 18).

Although viral infection has been implicated in the development of autoimmune diabetes, the response of islets and specifically beta -cells to a viral insult has been poorly defined. We have shown that dsRNA, in combination with IFN-gamma , inhibits glucose-stimulated insulin secretion and induces islet degeneration in a nitric oxide-dependent manner (19). Alone, neither poly(I-C) nor IFN-gamma stimulates iNOS expression or inhibits insulin secretion by rat islets (19). In combination with IFN-gamma , dsRNA also activates macrophages, stimulating iNOS expression, nitric oxide formation, and IL-1 release (11). Islets contain 5-10 resident macrophages that express and release IL-1 when activated in response to TNF + LPS. Local accumulation of IL-1 in islets stimulates the expression of iNOS and production of nitric oxide by beta -cells resulting in a potent inhibition of insulin secretion and islet degeneration (20). In this report, we show that the inhibitory and destructive effects of dsRNA + IFN-gamma on insulin secretion and islet viability are mediated by the intra-islet production of IL-1. Furthermore, we show that beta -cells themselves are a source of IL-1 in response to dsRNA and that beta -cell production of IL-1 leads to IL-1- and nitric oxide-dependent inhibition of beta -cell function.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- CMRL-1066 tissue culture medium, L-glutamine, penicillin, streptomycin, and rat recombinant IFN-gamma were from Life Technologies, Inc. Fetal calf serum was obtained from HyClone (Logan, UT). Male Harlan Sprague-Dawley rats (250-300 g) were purchased from Harlan Breeders (Indianapolis, IN). Poly(I-C) and collagenase type XI were from Sigma. [alpha -32P]dCTP and enhanced chemiluminescence (ECL) reagents were purchased from Amersham Pharmacia Biotech. Human recombinant IL-1beta was from Cistron Biotechnology (Pine Brook, NJ). Horseradish peroxidase-conjugated donkey anti-rabbit IgG, FITC-conjugated donkey anti-guinea pig, and CY3-conjugated donkey anti-rabbit secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Rabbit antiserum specific for the C-terminal 27 amino acids of mouse macrophage iNOS was a gift from Dr. Thomas Misko (G. D. Searle, St. Louis, MO). iNOS and cyclophilin cDNAs were gifts from Dr. Charles Rodi (Monsanto Corporate Research, St. Louis, MO) and Dr. Steve Carroll (Department of Pathology, University of Alabama, Birmingham, AL), respectively. Guinea pig anti-human insulin antibody was from Linco Research, Inc. (St. Louis, MO), and goat anti-rat IL-1beta antibody was from R & D Systems (Minneapolis, MN). All other reagents were from commercially available sources.

Islet Isolation and Culture-- Islets were isolated from male Harlan Sprague-Dawley rats by collagenase digestion as described previously (21). Following isolation, islets were cultured overnight in complete CMRL-1066 (CMRL-1066 containing 2 mM L-glutamine, 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin) under an atmosphere of 95% air and 5% CO2 at 37 °C. Prior to each experiment, islets were washed 3 times in complete CMRL-1066, counted, and then cultured for an additional 3 h at 37 °C. Experiments were initiated by the addition of poly(I-C) and IFN-gamma followed by culture for the indicated times. Where indicated, islets were pretreated for 30 min with IRAP prior to the addition of poly(I-C) and IFN-gamma .

The Islet Isolation Core Facility at Washington University School of Medicine and the Diabetes Research Institute at the University of Miami provided human islets. Isolated human islets were cultured for 3 days at 37 °C in complete CMRL-1066 prior to experimentation. Where indicated, human islets were pretreated for 30 min with IRAP prior to incubation with cytokines and poly(I-C).

Purification of beta -Cells by Fluorescence-activated Cell Sorting (FACS)-- Islets isolated from 12 rats were cultured overnight (~1200 islets/3 ml) in complete CMRL-1066 media under an atmosphere of 95% air and 5% CO2 at 37 °C. Islets were then dispersed into individual cells by treatment with trypsin (1.0 mg/ml) in Ca2+- and Mg2+-free Hanks solution at 37 °C for 3 min as stated previously (21). Dispersed islet cells were incubated for 60 min at 37 °C in complete CMRL-1066 prior to cell sorting. Islet cells were purified as described previously (22) using a FACSTAR + flow cytometer (Becton Dickinson, San Jose, CA). The cells were illuminated at 488 nm, and emission was monitored at 515-535 nm. This procedure results in beta - and alpha -cell purity of 90-95 and 80-85%, respectively. For RT-PCR analysis of IL-1alpha and IL-1beta mRNA expression, narrow gated windows were used to enhance beta -cell purity, which was greater than 98% based on post-sort FACS analysis and immunohistochemical analysis of insulin-containing cells (data not shown).

Insulin Secretion-- Islets (220/ml of complete CMRL-1066) were cultured for 40 h with the indicated concentrations of poly(I-C), rat IFN-gamma , and IRAP. The islets were isolated and washed three times in Krebs-Ringer bicarbonate buffer (KRB: 25 mM Hepes, 115 mM NaCl, 24 mM NaHCO3, 5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, and 0.1% bovine serum albumin, pH 7.4) containing 3 mM D-glucose, and insulin secretion was performed as described (23). Medium insulin content was determined by radioimmunoassay (24).

Islet Viability-- Islets (25/500 µl of complete CMRL-1066) were cultured for 96 h in 24-well microtiter plates with the indicated concentrations of poly(I-C), IFN-gamma , and IRAP. Islet degeneration was determined in a double-blind manner by phase-contrast microscopic analysis. Islet degeneration is characterized by the loss of islet integrity, disintegration, and partial dispersion of islets as described previously (23, 25, 26).

Western Blot Analysis-- Rat or human islets (120/400 µl of complete CMRL-1066), cultured for the indicated times with poly(I-C), rat or human IFN-gamma , and IRAP were isolated, lysed, and protein separated by SDS-gel electrophoresis as described (23). Detection of iNOS was by ECL according to the manufacturer's specifications (Amersham Pharmacia Biotech) and as described previously (23).

Northern Blot Analysis-- Rat islets (900/3 ml complete CMRL-1066) were cultured for 18 h at 37 °C with poly(I-C), rat IFN-gamma , IRAP, and cycloheximide (CHX) as indicated. After culture, the islets were washed 3 times with 0.1 M phosphate-buffered saline, pH 7.4, and total RNA was isolated using the RNeasy kit (Qiagen, Inc., Chatsworth, CA). RNA (5-10 µg) was denatured, fractionated, and transferred to Duralon UV nylon membranes (Stratagene, La Jolla, CA) as described (23). Membranes were hybridized to a 32P-labeled probe specific for rat iNOS or cyclophilin (27). The cDNA probe was radiolabeled with [alpha -32P]dCTP by random priming using the Prime-a-Gene nick translation system from Promega (Madison, WI). iNOS cDNA probe corresponds to bases 509-1415 of the rat iNOS coding region. Cyclophilin was used as an internal control for RNA loading. Hybridization and autoradiography were performed as described previously (28).

Nitrite Determination-- Nitrite production was determined by mixing 50 µl of culture medium with 50 µl of Griess reagent (29). The absorbance at 540 nm was measured, and nitrite concentrations were calculated from a sodium nitrite standard curve.

RT-PCR-- RT-PCR analysis of IL-1alpha and IL-1beta mRNA accumulation by rat and human islets (100/condition) and FACS-purified beta - and alpha -cells (100,000 cells/condition) was performed as described previously (20, 30). GAPDH mRNA accumulation was used as a control for PCRs, and total RNA isolated from rat islets treated for 4 h with TNF + LPS was used as a positive control for islet expression of IL-1alpha and IL-1beta . We have previously shown that resident macrophages are the source of IL-1 in response to TNF + LPS (20).

Immunoprecipitation and Immunofluorescence-- For immunoprecipitations, rat islets (500 islets/ml of methionine-deficient minimum Eagle's medium) were treated for 18 h with poly(I-C) + IFN-gamma . [35S]Methionine (500 µCi) was added, and the islets were cultured for 6 additional h. The islets were isolated, and lysed, and IL-1 was immunoprecipitated using hamster anti-IL-1alpha - and hamster anti-IL-1beta -specific antisera as described (20).

Immunohistochemistry was performed as described previously (20). In brief, islets were isolated, dispersed into individual cells by trypsin treatment as stated above, and centrifuged onto slides. The cells were fixed in 4% paraformaldehyde containing 0.1% Triton X-100 for 30 min and then blocked for 1 h with 5% bovine serum albumin (in 0.1 M phosphate-buffered saline). ICE was identified using rabbit anti-mouse ICE (1:40 dilution) antiserum specific for the p10 active form of ICE, IL-1beta was identified using goat anti-rat IL-1beta antiserum (1:20 dilution), and insulin was identified using guinea pig anti-human insulin (1:200 dilution). Secondary antibodies included FITC- or CY3-conjugated donkey anti-rat, donkey anti-guinea pig, and donkey anti-mouse antisera (1:200 dilution). All figures for immunohistochemistry were at a × 40 magnification.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

IL-1 Mediates dsRNA + IFN-gamma -induced iNOS Expression and Nitric Oxide Formation by Rat Islets-- To determine whether the endogenous production of IL-1 is required for dsRNA + IFN-gamma -induced damage, islets were incubated for 40 h with dsRNA and IFN-gamma in the presence or absence of the interleukin-1 receptor antagonist protein (IRAP). IRAP competes with IL-1 for receptor binding and thereby prevents IL-1-induced signaling events (31). Treatment of rat islets with dsRNA + IFN-gamma results in a 2.5-fold increase in nitrite production (poly(I-C) + IFN-gamma treated, 30 pmol/islet; untreated, 12 pmol/islet). In a concentration-dependent manner, IRAP prevents poly(I-C) + IFN-gamma -induced nitrite formation with maximal ~70% inhibition at 1-10 µg/ml (Fig. 1a). We have recently shown that dsRNA + IFN-gamma -induced iNOS mRNA accumulation and protein expression are maximal following 18- and 40-h incubations, respectively, and that concentrations of 50 µg/ml poly(I-C) + 150 units/ml IFN-gamma stimulate maximal iNOS expression and nitrite formation by rat islets (19). As shown in Fig. 1b, IRAP prevents dsRNA + IFN-gamma -induced iNOS mRNA accumulation following an 18-h incubation. IRAP also prevents dsRNA + IFN-gamma -induced iNOS protein expression following a 40-h incubation (Fig. 1c). Alone, neither IFN-gamma nor dsRNA stimulates iNOS expression or nitrite formation by rat islets (Fig. 1b and Ref. 19). Consistent with a requirement for IL-1 production, dsRNA + IFN-gamma -induced iNOS mRNA accumulation requires de novo protein synthesis. CHX, at a concentration (10 µM) that inhibits islet total protein synthesis by greater than 95% (32), prevents dsRNA + IFN-gamma -induced iNOS mRNA accumulation by rat islets (Fig. 1b). Importantly, CHX (at 10 µM) does not alter the levels of iNOS mRNA that accumulate in response to 1 unit/ml IL-1, nor does CHX inhibit glucose-stimulated insulin secretion by rat islets (6, 32). These results show that de novo protein synthesis is required for dsRNA + IFN-gamma -induced iNOS expression by rat islets and suggest that dsRNA + IFN-gamma -induced iNOS expression and nitrite formation is mediated by the intra-islet release of IL-1.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1.   IRAP inhibits dsRNA + IFN-gamma -induced iNOS expression and nitrite formation by isolated rat islets. Islets were pretreated for 30 min with IRAP, and then 50 µg/ml poly(I-C) and 150 units/ml IFN-gamma were added. Following a 40-h incubation at 37 °C, the media were removed, and nitrite production (a) and iNOS protein expression (c) were determined as described under "Experimental Procedures." b, rat islets were pretreated for 30 min with IRAP, followed by the addition of poly(I-C), IFN-gamma , and cycloheximide (CHX), and the islets were cultured for 18 h. iNOS mRNA accumulation was examined by Northern analysis as described under "Experimental Procedures." Results are the average ± S.E. of three independent experiments for nitrite production (a), or representative of three independent experiments for the analysis of iNOS expression (b and c).

IL-1 Mediates dsRNA + IFN-gamma -induced Inhibition of Glucose-stimulated Insulin Secretion and Islet Degeneration-- We have previously shown that dsRNA + IFN-gamma inhibits glucose-stimulated insulin secretion and induces islet degeneration in a nitric oxide-dependent manner (19). To determine whether the inhibitory and destructive effects of dsRNA + IFN-gamma are mediated by the intra-islet release of IL-1, rat islets were incubated for 40 (insulin secretion) or 96 h (islet viability) with 50 µg/ml poly(I-C) + 150 units/ml IFN-gamma in the presence or absence of IRAP. As shown in Fig. 2, dsRNA + IFN-gamma inhibits glucose-stimulated insulin secretion and induces the degeneration of 89% of islets. IRAP prevents the inhibitory effects of dsRNA + IFN-gamma on glucose-stimulated insulin secretion and attenuates islet degeneration by ~75%. These results indicate that dsRNA + IFN-gamma -induced inhibition of insulin secretion, and induction of islet degeneration is mediated by IL-1.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 2.   IRAP prevents dsRNA + IFN-gamma -induced inhibition of glucose-stimulated insulin secretion and islet degeneration. a, isolated rat islets were pretreated for 30 min with or without IRAP, poly(I-C) and IFN-gamma were then added and the islets cultured for 40 h at 37 °C. The islets were isolated, and glucose-stimulated insulin secretion was examined as described under "Experimental Procedures." b, isolated rat islets were pretreated for 30 min with IRAP, poly(I-C) and IFN-gamma were then added, and islet morphological damage was examined following a 96-h culture at 37 °C as described under "Experimental Procedures." Results for a and b are the average ± S.E. of three independent experiments.

IRAP Prevents dsRNA + IFN-gamma -induced iNOS Expression by FACS-purified beta -Cells-- To examine whether beta -cells are a source of IL-1 in response to dsRNA + IFN-gamma , primary beta -cells purified by fluorescence-activated cell sorting (FACS) were incubated with poly(I-C) and IFN-gamma in the presence or absence of IRAP. Following a 40-h incubation, beta -cells express high levels of iNOS in response to dsRNA + IFN-gamma (Fig. 3). IRAP prevents dsRNA + IFN-gamma -induced iNOS expression by FACS-purified beta -cells, indicating that beta -cells may be one islet cellular source of IL-1. Alone, neither dsRNA nor IFN-gamma induces iNOS expression by FACS-purified beta -cells, and dsRNA and IFN-gamma , alone or in combination, fail to induce iNOS expression by FACS-purified alpha -cells (data not shown).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 3.   dsRNA + IFN-gamma -induced iNOS expression by FACS-purified beta -cells is prevented by IRAP. Primary beta -cells purified by FACS were pretreated for 30 min with or without IRAP. Poly(I-C) and IFN-gamma were then added, and the cells were cultured for 40 additional h at 37 °C. The cells were isolated by centrifugation, and iNOS expression was examined by Western blot analysis as described under "Experimental Procedures." Results are representative of three independent experiments.

dsRNA Stimulates IL-1 mRNA Accumulation and Protein Expression in Primary beta -Cells and Rat Islets-- Two isoforms of IL-1 have been identified, IL-1alpha and IL-1beta . To determine the isoform(s) of IL-1 expressed by beta -cells, the effects of dsRNA and IFN-gamma on IL-1alpha and IL-1beta mRNA accumulation were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoprecipitation. Treatment of FACS-purified beta -cells with poly(I-C) or poly(I-C) + IFN-gamma results in IL-1beta mRNA accumulation following an 18-h incubation (Fig. 4a). FACS-purified alpha -cells fail to express either IL-1alpha or IL-1beta (data not shown), and FACS-purified beta -cells fail to express IL-1alpha in response to either poly(I-C), IFN-gamma , or poly(I-C) + IFN-gamma (Fig. 4a). In rat islets, poly(I-C) and poly(I-C) + IFN-gamma stimulate both IL-1alpha and IL-1beta mRNA accumulation that is first apparent following a 12-h incubation and that persists for up to 24 h (Fig. 4b). Resident macrophages appear to be the islet cellular source of IL-1alpha , as dsRNA + IFN-gamma fails to stimulate IL-1alpha mRNA accumulation in islets depleted of resident macrophages (data not shown) and primary beta -cells purified by FACS (Fig. 4a). These results indicate that dsRNA alone stimulates IL-1beta expression by primary beta -cells.



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 4.   dsRNA stimulates IL-1 mRNA accumulation in FACS-purified beta -cells and rat islets. a, FACS-purified beta -cells were cultured for 18 h at 37 °C with poly(I-C) and IFN-gamma , alone or in combination. Total RNA was isolated, and RT-PCR was used to evaluate IL-1alpha and IL-1beta mRNA accumulation as described under "Experimental Procedures." b, IL-1alpha and IL-1beta mRNA accumulation in rat islets treated with 50 µg/ml poly(I-C), 1 unit/ml IL-1, and 150 units/ml IFN-gamma at 37 °C for 12, 18, 24, and 40 h was examined by RT-PCR. GAPDH mRNA accumulation is shown as an internal control for the RT-PCRs. c, isolated rat islets were treated for 18 h with 50 µg/ml dsRNA + 150 units/ml IFN-gamma . [35S]Methionine (500 µCi) was added, and the islets were cultured for an additional 6 h. The islets were isolated; IL-1alpha and IL-1beta were immunoprecipitated; and immunoprecipitates were resolved by SDS-15% polyacrylamide gel electrophoresis and visualized by fluorography as described under "Experimental Procedures." Results for a-c are representative of three independent experiments.

To confirm that IL-1 is expressed at the protein level in response to dsRNA + IFN-gamma , IL-1alpha and IL-1beta were sequentially immunoprecipitated from [35S]methionine-labeled rat islets using hamster monoclonal antisera previously used to immunoprecipitate both the pro-forms and mature forms of IL-1alpha and IL-1beta from activated macrophages (33). Poly(I-C) + IFN-gamma stimulates the expression of immunoprecipitable pro-IL-1beta from isolated rat islets following a 24-h incubation (Fig. 4c). We were unable to immunoprecipitate IL-1alpha from rat islets, a finding that is consistent with IL-1alpha expression by the limited number of macrophages found in rat islets (~10/islet). Also, we were unable to immunoprecipitate the mature form of IL-1beta from islets, consistent with its release following proteolytic processing (Fig. 4c (34)).

Immunohistochemical Identification of Islet Cellular Sources of IL-1beta -- To confirm directly that beta -cells are a source of IL-1beta , islets were treated for 18 h with poly(I-C) or poly(I-C) + IRAP, isolated, and dispersed into individual cells, and IL-1beta expressing cells were identified by immunocytochemistry. As shown in Fig. 5a, treatment of rat islets with poly(I-C) results in IL-1beta expression (red fluorescence), and IL-1beta immunoreactivity localizes with insulin-containing cells (green fluorescence), as indicated by the yellow fluorescence following double exposure. To control for IL-1beta binding to cell membranes, rat islets were treated for 18 h with poly(I-C) + IRAP. Under these conditions, IRAP should antagonize the interactions of IL-1beta with its surface receptors on beta -cells. As shown in Fig. 5b, IL-1beta expression (green fluorescence) localizes to insulin-containing cells (red fluorescence), as indicated by the yellow following double exposure. Similar results were obtained from islets treated for 18 h with poly(I-C) + IFN-gamma and poly(I-C) + IFN-gamma  + IRAP (data not shown). In addition, IL-1beta was not expressed in untreated islets nor was it expressed in islets treated for 18 h with IFN-gamma (data not shown). Whereas beta -cells express high levels of IL-1beta , less than 1% of islet cells express this cytokine in response to poly(I-C).



View larger version (46K):
[in this window]
[in a new window]
 
Fig. 5.   Immunohistochemical identification of beta -cells as an islet cellular source of IL-1beta in response to dsRNA. Rat islets were treated for 18 h with 50 µg/ml poly(I-C) (a), poly(I-C) and 10 µg/ml IRAP (b), or for 5 h with 10 µg/ml LPS + 10 ng/ml TNF (c). The islets were isolated and dispersed into individual cells, and IL-1beta was identified by immunofluorescence using goat anti-rat IL-1beta and CY-3-conjugated donkey anti-goat secondary antisera (red fluorescence, a and c), or FITC-conjugated donkey anti-goat secondary antisera (green fluorescence, b). Insulin was identified using guinea pig anti-human insulin and FITC-conjugated donkey anti-guinea pig secondary antisera (green fluorescence, a and c), or CY-3-conjugated donkey anti-guinea secondary antisera (red fluorescence, b). Results are representative of three independent experiments.

As expected, poly(I-C) also stimulates IL-1beta expression by a second population of islet cells that appear to be resident macrophages. As shown in Fig. 5b, IL-1beta immunoreactivity localizes with a large highly vacuolized cell containing a high degree of macrophage morphology. To confirm that these cells are macrophages, islets were treated for 5 h with TNF + LPS. We have previously shown that macrophages are the sole islet cellular source of IL-1beta under these conditions (20). As shown in Fig. 5c, a 5-h incubation of islets with TNF + LPS results in the expression of IL-1beta (red fluorescence) by resident macrophages. The morphology of the IL-1beta expressing macrophages in Fig. 5, b and c, are nearly identical. Similar numbers of macrophages express IL-1beta in response poly(I-C) as compared with TNF + LPS. These findings provide direct support for beta -cell expression of IL-1beta and that resident islet macrophages also produce this cytokine in response to poly(I-C).

The RINm5F cell IL-1 bioassay, which is specific for active IL-1 (14, 33), was used to quantitate the levels of IL-1 released by islets in response to poly(I-C) and IFN-gamma . In this experiment, islets were treated for 40 h with 50 µg/ml poly(I-C), 150 units/ml IFN-gamma , or both poly(I-C) and IFN-gamma ; the culture supernatant was isolated and IL-1 levels quantitated. Alone, neither IFN-gamma nor poly(I-C) stimulate IL-1 release by islets; however, poly(I-C) + IFN-gamma stimulates the accumulation of 32.6 ± 1.2 pg/ml of IL-1. We have previously shown that this level of IL-1 is sufficient to stimulate iNOS expression by beta -cells in the presence of IFN-gamma (23).

IFN-gamma Induces ICE Activation in beta -Cells-- The interleukin-1beta -converting enzyme (ICE or caspase-1) is required for cleavage of inactive pro-IL-1beta to the active mature cytokine. ICE is present in the cytoplasm of cells as an inactive 45-kDa enzyme that self-cleaves into p10 and p20 subunits to form an active tetramer (34). IFN-gamma has been shown to induce ICE expression by NIT cells (Ref. 35 and data not shown) and activates ICE in beta -cells (Fig. 6a). By using antiserum specific for active ICE (p10 subunit; red fluorescence), we show that a 24-h incubation of rat islets with IFN-gamma stimulates ICE activation. Active ICE (red fluorescence) localizes to insulin-containing cells (green fluorescence) as evidenced by the intense yellow fluorescence upon double exposure (Fig. 6a). IFN-gamma activates ICE in greater than 90% of insulin-containing cells. Active ICE was not detected in untreated or poly(I-C)-treated islet cells, and poly(I-C) did not further enhance or inhibit IFN-gamma -induced ICE expression or activation in beta -cells (data not shown). In addition, we did not detect cross-reactivity or nonspecific binding of the secondary antibodies.



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 6.   ICE activation is required for dsRNA + IFN-gamma -induced iNOS expression by FACS-purified beta -cells. a, rat islets were treated with or without 150 units/ml IFN-gamma for 24 h. The islets were dispersed into individual cells, and active ICE was identified by immunofluorescence using rabbit anti-mouse ICE antiserum specific for the p10 active form of ICE, and CY-3-conjugated donkey anti-rabbit secondary antisera (red fluorescence). Insulin-containing cells were identified using guinea pig anti-human insulin and FITC-conjugated donkey anti-guinea pig secondary antiserum (green fluorescence). b, FACS-purified beta -cells were preincubated for 30 min with the indicated concentrations of the ICE-selective inhibitor YVAD-FMK (Enzyme System Products, Livermore, CA). Poly(I-C) (50 µg/ml) and IFN-gamma (150 units/ml) were then added, and the cells were cultured for 40 h at 37 °C. beta -Cells were isolated, and iNOS expression was evaluated by Western blot analysis as described under "Experimental Procedures." Results for both a and b are representative of three independent experiments.

ICE Activation Is Required for dsRNA + IFN-gamma -induced iNOS Expression by beta -Cells-- Since dsRNA stimulates IL-1beta expression and IFN-gamma activates ICE in beta -cells, we examined whether IL-1beta processing by ICE is required for beta -cell expression of iNOS in response to dsRNA + IFN-gamma . As shown in Fig. 6b, the ICE-selective peptide inhibitor benzyloxycarbonyl-Tyr-Val-ala-Asp(OMe)-fluoromethyl ketone (YVAD-FMK) attenuates poly(I-C) + IFN-gamma -induced iNOS expression by FACS-purified beta -cells in a concentration-dependent manner with a maximal ~80% inhibition observed at 100-200 µM (determined by densitometry). Me2SO, the vehicle control for YVAD-FMK, does not inhibit dsRNA + IFN-gamma -induced iNOS expression (Fig. 6b), and 200 µM YVAD-FMK does not inhibit IL-1-induced iNOS expression by primary beta -cells (data not shown). The latter experiment was used to control for nonspecific inhibitory actions of YVAD-FMK on proteolytic cleavage events that are required for iNOS expression (such as Ikappa B degradation; see Ref. 6). In addition, YVAD-FMK did not alter beta -cell viability as determined by trypan blue exclusion (data not shown). These results indicate that proteolytic processing of IL-1beta by IFN-gamma -activated ICE is required for dsRNA + IFN-gamma -induced iNOS expression by primary beta -cells.

dsRNA + IFN-gamma -induced iNOS Expression and Nitric Oxide Formation by Human Islets Is Dependent on the Intra-islet Production of IL-1-- To determine whether dsRNA + IFN-gamma stimulates iNOS expression and nitric oxide formation, human islets were incubated with poly(I-C), in the presence or absence of IFN-gamma , for 48 h. Alone, neither poly(I-C) (Fig. 7, a and b) nor IFN-gamma (Refs. 30, 36, and 37 and data not shown) stimulate nitric oxide production or iNOS expression by human islets. However, in the presence of IFN-gamma , poly(I-C) stimulates the concentration-dependent production of nitric oxide and expression of iNOS that is maximal at 400 µg/ml (Fig. 7, a and b). The levels of iNOS expressed and nitrite produced in response to 400 µg/ml poly(I-C) + IFN-gamma are similar in magnitude to the levels induced in response to a 48-h incubation with IL-1 + IFN-gamma (minimal combination of cytokines required to stimulate iNOS expression by human beta -cells; see Refs. 30, 36, and 37). Poly(I-C) + IFN-gamma -induced iNOS expression and nitric oxide production appears to require the intra-islet production of IL-1. As shown in Fig. 7, a and b, IRAP prevents dsRNA + IFN-gamma -induced nitric oxide production and iNOS expression by human islets. These findings indicate that poly(I-C) + IFN-gamma stimulates iNOS expression and nitric oxide production by human islets and that nitric oxide production under these conditions is dependent on the local release of IL-1 in human islets.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 7.   IRAP prevents poly(I-C) + IFN-gamma -induced nitrite production and iNOS expression by human islets. Human islets were treated for 48 h with IL-1 (75 units/ml), IFN-gamma (750 units/ml), IRAP (25 µg/ml), and poly(I-C) as indicated. The islets were then isolated, and nitrite production was determined on the culture medium (a), and Western blot analysis was used to examine iNOS expression by the isolated islet (b). Results are the average ± S.E. of three experiments from three independent human islet isolations for nitrite production (a), and representative of three independent experiments from three independent human islet isolations for iNOS expression (b).

dsRNA Stimulates IL-1alpha and IL-1beta mRNA Accumulation in Human Islets-- To determine the isoform(s) of IL-1 expressed in response to dsRNA and IFN-gamma , human islets were incubated for 6, 12, and 24 h with 400 µg/ml poly(I-C) and 750 units/ml IFN-gamma , alone and in combination. The islets were isolated, and IL-1alpha and IL-1beta mRNA accumulation was examined by RT-PCR. As shown in Fig. 8, poly(I-C) and poly(I-C) + IFN-gamma stimulate the mRNA accumulation of both IL-1alpha and IL-1beta in human islets. IL-1alpha and IL-1beta mRNA accumulation is first apparent following a 6-h incubation and persists for up to 24 h. Alone, IFN-gamma fails to stimulate IL-1 expression by human islets (data not shown), a finding that is consistent with the inability of this cytokine to induce iNOS and IL-1 expression by rat islets.



View larger version (46K):
[in this window]
[in a new window]
 
Fig. 8.   Poly(I-C) stimulates IL-1 expression by human islets. Human islets were treated for the indicated times with poly(I-C) (400 µg/ml) or poly(I-C) + IFN-gamma (750 units/ml). Total RNA was isolated, and RT-PCR was used to evaluate IL-1alpha and IL-1beta mRNA accumulation as described under "Experimental Procedures." GAPDH mRNA accumulation is shown as an internal control for the RT-PCRs. Results are representative of three independent experiments from three independent human islet isolations.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A number of mechanisms have been proposed to explain how viral infections may induce autoimmune diabetes. One hypothesis, termed "molecular mimicry" suggests that viruses may contain protein structural motifs that mimic host proteins (38, 39). In this model, an immune response against the invading pathogen will eliminate the pathogen but may also cross-react with one or more host proteins that share similar determinants with the infecting agent. Thus, the disease process would continue even after the invading virus is cleared from the host. A second hypothesis, termed "bystander activation," suggests that viral infection of target tissue may 1) modify surface antigens into immunogenic forms, 2) stimulate the induction of antigen expression, or 3) induce the release of sequestered antigens during target cell lysis (40).

Experimentally, direct evidence supporting molecular mimicry or bystander activation as mechanisms of virally induced insulin-dependent diabetes mellitus has been difficult to obtain, in part, because the host immune response may have already cleared the viral determinant by the time the disease manifests itself. However, viruses such as Coxsackie B4 have been isolated from the pancreata of acutely diabetic individuals (41). Although Coxsackie B4 virus protein P2-C shares significant sequence homology with the islet antigen glutamic acid decarboxylase (GAD65; see Ref. 42), and infection of mice with Coxsackie B4 induces diabetes (43), a recent report by Horwitz et al. (44), supports bystander activation as the mechanism for Coxsackie B4-induced diabetes. Diabetes induced by Coxsackie B4 appears to occur as a result of local inflammation, tissue damage, and the release of islet antigens followed by re-stimulation of autoreactive T-cells.

A common determinant of RNA and DNA genome virus families is the formation of dsRNA during viral replication (45). Virus-derived single-stranded RNAs may also contain extensive dsRNA secondary structure (45). dsRNA is an active component of a viral infection that stimulates antiviral responses in infected cells (8). The synthetic dsRNA molecule poly(I-C) has been shown to induce an antiviral response that is similar to the antiviral response activated following viral infection (8, 16). Since viruses from both RNA and DNA genome families have been associated with the development of autoimmune diabetes (3, 4), the synthetic dsRNA molecule, poly(I-C), was used to examine mechanisms of virally induced islet dysfunction and degeneration. In this study, we show that poly(I-C), in the presence of IFN-gamma , stimulates iNOS expression and nitrite formation, inhibits insulin secretion, and induces rat islet degeneration by an IL-1-dependent mechanism. beta -Cells, selectively destroyed during the development of autoimmune diabetes, are one islet cellular source of IL-1 in response to dsRNA. IFN-gamma appears to participate in dsRNA + IFN-gamma -induced iNOS expression by activating ICE, the enzyme responsible for processing IL-1beta from the inactive pro-form to the active mature cytokine. We show that IFN-gamma stimulates ICE activation (processing from inactive pro-ICE to the active mature enzyme) and that selective inhibition of ICE using YVAD-FMK prevents poly(I-C) + IFN-gamma -induced iNOS expression by FACS-purified beta -cells.

As evidenced by immunohistochemical analysis, poly(I-C) stimulates IL-1beta expression in both insulin-containing cells as well as resident macrophages. It is not surprising that resident macrophages are a source of IL-1 in islets, as we have previously shown that poly(I-C) stimulates IL-1 expression and release from mouse peritoneal macrophages and RAW 264.7 cells (11, 46). Unexpectedly, poly(I-C) also stimulates beta -cells to express and produce this proinflammatory cytokine that has been implicated in beta -cell destruction (6, 7). Less than 1% of islet macrophages and beta -cells appear to express IL-1 in response to poly(I-C). Although this is a small proportion of islet cells, previous studies have shown that activation of resident macrophages (which compose ~0.1% of islet cells) in both rat and human islets results in the local production of IL-1 to levels sufficient to stimulate iNOS expression, nitric oxide production, and inhibit insulin secretion (20, 30, 47). In addition, the amount of IL-1 released by islets treated with poly(I-C) + IFN-gamma (~30 pg/ml) is an amount that we have shown to be sufficient to stimulate beta -cell expression of iNOS in the presence of IFN-gamma (23).

Results presented in this study also indicate that the beta -cells themselves are capable of producing sufficient levels of IL-1 to inhibit beta -cell function and to stimulate iNOS expression. Treatment of FACS-purified beta -cells with poly(I-C) + IFN-gamma results in iNOS expression in an IL-1-dependent manner. In addition, poly(I-C) + IFN-gamma -induced inhibition of glucose-stimulated insulin secretion by FACS-purified beta -cells is dependent on nitric oxide production (19). Although beta -cells are capable of producing sufficient levels of IL-1 to impair beta -cell function (based on the FACS-purified beta -cell studies), both beta -cells and macrophages could contribute to poly(I-C) + IFN-gamma islet damage by releasing IL-1 locally in islets. However, we have previously shown that poly(I-C) + IFN-gamma stimulates iNOS expression by macrophage-depleted rat islets and by FACS-purified beta -cells (19). These findings provide the first evidence that primary endocrine cells are capable of producing the proinflammatory cytokine IL-1 and that the levels of IL-1 produced are sufficient to inhibit beta -cell function (independent of macrophage IL-1 production). In addition, these findings also suggest that IL-1 may participate in the antiviral response activated by dsRNA in beta -cells.

Similar to rat islets, we also show that a combination of dsRNA and IFN-gamma stimulate IL-1alpha , IL-1beta , and iNOS expression and nitric oxide production by human islets. iNOS expression and nitric oxide production in response to dsRNA + IFN-gamma appears to be dependent on the local production of IL-1 in human islets. Antagonism of the IL-1 receptor using IRAP prevents dsRNA + IFN-gamma -induced iNOS expression and nitric oxide production by human islets. Presently, it is not clear if beta -cells, resident macrophages, or both are cellular sources of IL-1 in human islets. However, based on the effects of poly(I-C) + IFN-gamma on IL-1 expression by rat islets, it is likely that both cell types contribute to poly(I-C)-induced IL-1 expression in human islets. Also, since rat and human islets express iNOS in an IL-1-dependent manner, it is likely that the mechanisms by which poly(I-C) + IFN-gamma stimulate iNOS expression by human islets may be similar to those activated in rat islets.

In summary, this study provides direct evidence for endocrine beta -cell expression of IL-1 as a novel mechanism by which viral infection may mediate beta -cell damage in autoimmune diabetes. Our findings suggest that dsRNA, the active component of a viral infection that activates the antiviral response, stimulates IL-1beta expression by beta -cells and IL-1alpha and IL-1beta expression by macrophages. IL-1beta requires proteolytic processing for activation (34), an event that appears to be mediated by IFN-gamma -induced ICE activation in beta -cells. Active ICE cleaves inactive pro-IL-1beta to the active mature cytokine resulting in the release of IL-1beta by beta -cells followed by autocrine or paracrine stimulation of adjacent beta -cells to express iNOS and produce nitric oxide (Fig. 9). The local release of IL-1 by resident macrophages may also contribute to poly(I-C) + IFN-gamma -induced iNOS expression by islets. In support of this model, we have recently shown that Coxsackie B4 infection of isolated rat islets results in iNOS expression.2 Although our findings do not distinguish between bystander activation or molecular mimicry as a mechanism of virally induced autoimmune diabetes, these findings support a novel mechanism by which a viral infection may stimulate beta -cell damage that could result in autoimmunity directed against beta -cells.



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 9.   Schematic model of dsRNA + IFN-gamma -induced beta -cell damage.



    ACKNOWLEDGEMENTS

We thank Jessica Gorman for expert technical assistance and Drs. Paul Lacy, Mark Buller, Michael Moxley, Claudette Klein, and Abdul Waheed for advice and critical review of this manuscript. We also thank Dr. Byeong Chang (Amgen Inc.) for the gift of IRAP; the Islet Isolation Core Facility and the Diabetes Research and Training Center at Washington University School of Medicine for supplying human islets and performing insulin RIAs; and the Diabetes Research Institute at the University of Miami for supplying human islets.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants DK-52194 and AI44458 and a Career Development award from the Juvenile Diabetes Foundation International (to J. A. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, St. Louis University, 1402 South Grand Blvd., St. Louis, MO 63104. Tel.: 314-577-8165; Fax: 314-577-8156; E-mail: corbettj@slu.edu.

Published, JBC Papers in Press, December 6, 2000, DOI 10.1074/jbc.M009159200

2 J. A. Corbett, unpublished observations.


    ABBREVIATIONS

The abbreviations used are: iNOS, inducible nitric-oxide synthase; IL, interleukin; IFN-gamma , interferon-gamma ; dsRNA, double-stranded RNA; TNF, tumor necrosis factor; LPS, lipopolysaccharide; FITC, fluorescein isothiocyanate; RT-PCR, reverse transcriptase-polymerase chain reaction; IRAP, interleukin-1 receptor antagonist protein; ICE, IL-1beta -converting enzyme; FACS, fluorescence-activated cell sorting; CHX, cycloheximide; FMK, fluoromethyl ketone; YVAD-FMK, benzyloxycarbonyl-Tyr-Val-Ala-Asp(OMe)-fluoromethyl ketone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Gepts, W. (1965) Diabetes 14, 619-633[Medline] [Order article via Infotrieve]
2. Bach, J.-F. (1994) Endocr. Rev. 15, 516-541[Abstract]
3. Yoon, J.-W. (1995) Diabetes Metab. Rev. 11, 83-107[Medline] [Order article via Infotrieve]
4. von Herrath, M. G., and Oldstone, M. B. A. (1996) Curr. Opin. Immunol. 8, 878-885[CrossRef][Medline] [Order article via Infotrieve]
5. Hirasawa, K., Jun, H. S., Maeda, K., Kawaguchi, Y., Itagaki, S., Mikami, T., Baek, H. S., Doi, K., and Yoon, J.-W. (1997) J. Virol. 71, 4024-4031[Abstract]
6. Corbett, J. A., and McDaniel, M. L. (1996) in Nitric Oxide: Principles and Action (Lancaster, J. R., Jr., ed) , pp. 177-217, Academic Press, Inc., San Diego, CA
7. Mandrup-Poulsen, T. (1996) Diabetologia 39, 1005-1029[CrossRef][Medline] [Order article via Infotrieve]
8. Jacobs, B. L., and Langland, J. O. (1996) Virology 219, 339-349[CrossRef][Medline] [Order article via Infotrieve]
9. Kerr, I. M., and Stark, G. R. (1992) J. Interferon Res. 12, 237-240[Medline] [Order article via Infotrieve]
10. Karupiah, G., Xie, Q., Buller, M. L., Nathan, C., Duarte, C., and MacMicking, J. D. (1993) Science 261, 1445-1448[Medline] [Order article via Infotrieve]
11. Heitmeier, M. R., Scarim, A. L., and Corbett, J. A. (1998) J. Biol. Chem. 273, 15301-15307[Abstract/Free Full Text]
12. Wu, S., and Kaufman, R. J. (1997) J. Biol. Chem. 272, 1291-1296[Abstract/Free Full Text]
13. de Haro, C., Mendez, R., and Santoyo, J. (1996) FASEB. J. 10, 1378-1387[Abstract/Free Full Text]
14. Kreil, T. R., and Eibl, M. M. (1996) Virology 219, 304-306[CrossRef][Medline] [Order article via Infotrieve]
15. Melkova, Z., and Esteban, M. (1995) J. Immunol. 155, 5711-5718[Abstract]
16. Clemens, M. J., and Elia, A. (1997) J. Interferon Cytokine Res. 17, 503-524[Medline] [Order article via Infotrieve]
17. Ewel, C. H., Sobel, D. O., Zeligs, B. J., and Bellanti, J. A. (1992) Diabetes 41, 1016-1021[Abstract]
18. Sobel, D. O., Newsome, J., Ewel, C. H., Bellanti, J. A., Abbassi, V., Creswell, K., and Blair, O. (1992) Diabetes 41, 515-520[Abstract]
19. Heitmeier, M. R., Scarim, A. L., and Corbett, J. A. (1999) J. Biol. Chem. 274, 12531-12536[Abstract/Free Full Text]
20. Arnush, M. A., Scarim, A. L., Heitmeier, M. R., Kelly, C. B., and Corbett, J. A. (1998) J. Immunol. 160, 2684-2691[Abstract/Free Full Text]
21. McDaniel, M. L., Colca, J. R., Kotagal, N., and Lacy, P. E. (1983) Methods Enzymol. 98, 182-200[Medline] [Order article via Infotrieve]
22. Pipeleers, D. G., Int Veld, P. A., Van De Winkel, M., Maes, E., Schuit, F. C., and Gepts, W. (1985) Endocrinology 117, 806-816[Abstract]
23. Heitmeier, M. R., Scarim, A. L., and Corbett, J. A. (1997) J. Biol. Chem. 272, 13697-13704[Abstract/Free Full Text]
24. Wright, P. H., Makulu, D. R., Vichick, D., and Sussman, K. E. (1971) Diabetes 20, 33-45[Medline] [Order article via Infotrieve]
25. Lacy, P. E., and Finke, E. H. (1991) Am. J. Pathol. 138, 1183-1190[Abstract]
26. Corbett, J. A., and McDaniel, M. L. (1994) Biochem. J. 299, 719-724[Medline] [Order article via Infotrieve]
27. Brown, R., and Mackey, K. (1997) in Current Protocols in Molecular Biology (Ausubel, F. M. , Brent, R. , Kingston, R. E. , Moore, D. D. , Seidman, J. G. , Smith, J. A. , and Struh, L. K., eds), Vol. 2 , pp. 4.9.1-4.9.13, Green Publishing Associates and Wiley-Interscience, New York
28. Burd, P. R., Rogers, H. W., Gordon, J. R., Martin, C. A., Jayaraman, S., Wilson, S. D., Dvorak, A. M., Galli, S. J., and Dorf, M. E. (1989) J. Exp. Med. 170, 245-257[Abstract]
29. Green, L. C., Wagner, D. A., Glogowski, J., Skipper, P. L., Wishnok, J. S., and Tannenbaum, S. R. (1982) Anal. Biochem. 126, 131-138[Medline] [Order article via Infotrieve]
30. Arnush, M., Heitmeier, M. R., Scarim, A. L., Marino, M. A., Manning, P. A., and Corbett, J. A. (1998) J. Clin. Invest. 102, 516-526[Abstract/Free Full Text]
31. Arend, W. P. (1991) J. Clin. Invest. 88, 1445-1451[Medline] [Order article via Infotrieve]
32. Hughes, J. H., Colca, J. R., Easom, R. A., Turk, J., and McDaniel, M. L. (1990) J. Clin. Invest. 86, 856-863[Medline] [Order article via Infotrieve]
33. Hill, J. R., Corbett, J. A., Kwon, G., Marshall, C. A., and McDaniel, M. L. (1996) J. Biol. Chem. 271, 22672-22678[Abstract/Free Full Text]
34. Wilson, K. P., Black, J. A., Thomson, J. A., Kim, E. E., Griffith, J. P., Navia, M. A., Murcko, M. A., Chambers, S. P., Aldape, R. A., Raybuck, S. A., and Livingston, D. J. (1994) Nature 370, 270-274[CrossRef][Medline] [Order article via Infotrieve]
35. Stephens, L. A., Thomas, H. E., Ming, L., Grell, M., Darwiche, R., Volodin, L., and Kay, T. W. (1999) Endocrinology 140, 3219-3227[Abstract/Free Full Text]
36. Corbett, J. A., Sweetland, M. A., Wang, J. L., Lancaster, J. R., Jr., and McDaniel, M. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1731-1735[Abstract]
37. Corbett, J. A., Kwon, G., Marino, M., Rodi, C. P., Sullivan, P. M., and McDaniel, M. L. (1996) Am. J. Physiol. 39, C1581-C1587
38. von Herrath, M. G., Evans, C. F., Horwitz, M. S., and Oldstone, M. B. (1996) Immunol. Rev. 152, 111-143[Medline] [Order article via Infotrieve]
39. Maclaren, N. K., and Atkinson, M. A. (1997) Mol. Med. Today 3, 76-83[CrossRef][Medline] [Order article via Infotrieve]
40. von Herrath, M. G., Homann, D., Gairin, J. E., and Oldstone, M. B. (1997) Biochem. Soc. Trans. 25, 630-635[Medline] [Order article via Infotrieve]
41. Notkins, A. L., and Yoon, J.-W. (1984) in Concepts in Viral Pathogenesis (Notkins, A. L. , and Oldstone, M. B. A., eds) , Springer-Verlag Inc., New York
42. Atkinson, M. A., Bowman, M. A., Campbell, L., Darrow, B. L., Kaufman, D. L., and Maclaren, N. K. (1994) J. Clin. Invest. 94, 2125-2129[Medline] [Order article via Infotrieve]
43. Yoon, J., Onodera, T., and Notkins, A. (1978) J. Exp. Med. 148, 1068-1080[Abstract]
44. Horwitz, M. S., Bradley, L. M., Harbertson, J., Krahl, T., Lee, J., and Sarvetnick, N. (1998) Nat. Med. 4, 781-785[Medline] [Order article via Infotrieve]
45. Lengyel, P. (1987) EMBO J. 19, 3630-3638[Abstract/Free Full Text]
46. Corbett, J. A., and McDaniel, M. L. (1995) J. Exp. Med. 181, 559-568[Abstract]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.