1 Laboratory for Experimental Medicine and Endocrinology (LEGENDO), University Hospital Gasthuisberg, Catholic University of Leuven, Leuven, Belgium
2 Clinic of Endocrinology, University Hospital "St. Spiridon," University of Medicine and Pharmacy "Gr. T. Popa," Iasi, Romania
3 Laboratory for Immunology, University Hospital Gasthuisberg, Catholic University of Leuven, Leuven, Belgium
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ABSTRACT |
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Type 1 diabetes is caused by immune destruction of insulin-producing ß-cells in the pancreatic islets of Langerhans (1). In the NOD mouse, a widely used animal model for the study of type 1 diabetes, lymphocytic infiltration into the pancreatic islets (insulitis) is the primary lesion that eventually progresses to overt diabetes. Evidence is accumulating that type 1 diabetes is mediated by T-helper 1 (Th1) cells and that protection or prevention of the disease can be achieved by shifting the immune response toward Th2 activation (2,3).
Several diabetogenic autoantigens have been described, such as GAD, insulin, and heat shock protein, in both animal models and human disease (4,5). Furthermore, epitope spreading and molecular mimicry is enlarging their spectrum during disease evolution (5). The biochemical properties of antigens together with the genetic background of the immune system significantly influence the type (Th1 or Th2) of immune activation (6). Many studies have investigated the effects of administration of diabetes relevant autoantigens or their immunodominant peptides in the NOD mouse model. Most studies convincingly demonstrate a prevention or delayed onset of disease, associated with a shift toward a more Th2-biased response (5,7). Recently, also in type 1 diabetic patients, peptide treatment with specific autoantigens has been shown to prevent or delay the disease and was concluded to be safe (8).
This vaccination-triggered shift from autoreactive Th1 toward Th2 cells has not only been observed in NOD mice (9) but also in experimental autoimmune encephalomyelitis (EAE), another Th1-mediated autoimmune disease (10). Nevertheless, the administration of self-antigen in the EAE model has lead to unforeseen consequences of Th2 activation. Indeed, vaccination with the myelin proteolipid peptide PLPp(139151) in SJL/J mice under certain conditions, essentially depending on the time point of peptide rechallenge, unexpectedly resulted in lethal anaphylaxis (11). Also in the human situation, a recent phase II trial in which multiple sclerosis (MS) patients received an altered peptide ligand from a myelin basic protein epitope, repeated injections with the peptide induced immediate hypersensitivity reactions in 9% of the subjects (12). These reports suggest that an autovaccine-triggered immune response may produce extremely undesirable allergic reactions. To what extent anaphylaxis depends on the genetic background, the antigen type, or the timing of vaccination is not yet completely known.
The aim of the present study was to test whether vaccination in NOD mice, animals having an immune system genetically prone to destroy pancreatic islets through Th1-mediated mechanisms, is perfectly safe or whether life-threatening reactions can be induced by manipulating the immune response. Indeed, the NOD mouse strain also displays immune system particularities beyond Th1-dependent autoimmune islet destruction because they seem to be highly susceptible to anaphylactic shock under certain specified conditions (13,14). NOD mice might therefore behave differently from other mouse strains in certain situations of immune challenge (13). To investigate whether vaccination could induce lethal shock in NOD mice, we tested a wide panel of antigens. We compared the effects of several diabetes-relevant autoantigens and diabetes-irrelevant peptides or proteins in NOD mice to the effects obtained in two other mouse strains (BALB/c and C57BL/6 mice), in which a Th1-activated immune system is absent. Based on our previous findings, we also treated mice from various strains with an immunomodulatory agent known to favor an autoantigen-specific Th2 shift in NOD mice, namely 1,25(OH)2D3, and examined its effect on the safety profile of vaccination.
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RESEARCH DESIGN AND METHODS |
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Proteins and peptides.
Hen egg white lysozyme (HEL) (94% purity), ovalbumin (OVA) (Grade V, 98% purity), keyhole limpet hemocyanine (KLH), bovine insulin B (ins-B) chain, and PLP peptide (amino acids 135151) were purchased from Sigma (Bornem, Belgium). GAD65 (p524542) (SRLSKVAPVIKARMMEYGTT), heat shock protein (hsp)-65 peptide (PALDSLTPANED), and tetanus toxin (TT) peptide (p830843) (QYIKANSKIGITE) contained a COOH-terminal amidation (CONH2) group and were >75% pure (Eurogentec, Luik, Belgium).
Vaccination and intervention regimens.
The 8-week-old NOD, BALB/c, and C57BL/6 mice were immunized by injection of 100 µg antigen (HEL, GAD65, ins-B, hsp65, PLP, OVA, KLH, and TT) emulsified at a 1:1 concentration in complete Freunds adjuvant (CFA) or incomplete Freunds adjuvant (Difco Laboratories, Detroit, MI) in the hind footpads. NOD-scid mice were injected with 100 µg HEL antigen suspended in CFA. All mice were reinjected with the same antigen 3 weeks later in a similar manner.
NOD, BALB/c, and C57BL/6 mice were treated with 1,25(OH)2D3 from 3 weeks of age until the end of the experiment. The drug was suspended in arachis oil and administered at a dose of 5 µg/kg i.p. every 2 days. Control NOD, BALB/c, and C57BL/6 mice received the treatment vehicle.
Dexamethasone (50 µg dissolved in 0.9% NaCl; Aacidexam, NV Organon, Oss, the Netherlands) was injected intraperitoneally in NOD mice 48, 24, and 2 h before the booster injection. WEB2086 (platelet-activating factor receptor [PAF-R] antagonist; Boehringer Mannheim, Mannheim, Germany; a gift from Prof. Vermylen, University of Leuven, Leuven, Belgium), 500 µg dissolved in PBS by sonication, was injected intraperitoneally in NOD mice 10 min before the HEL booster injection.
Follow-up of mice.
Clinical evolution and survival rate after sensitization with various peptides were monitored in different mouse strains. Shock was characterized by pilo-erection; prostration; erythema of the tail, ears, and footpads; and dyspnea with shallow breathing. Serum for antibody measurement and spleens for quantification of mRNA levels were collected before immunization and again before booster administration.
Measurement of antibody production.
HEL-specific IgG1, IgG2a, and IgE antibodies were measured in mouse sera by enzyme-linked immunosorbent assay (ELISA). Briefly, for IgG1 and IgG2a ELISA, 96-well microtiter plates were coated overnight with HEL (10 µg/ml). Samples for IgG1 and IgG2a measurement were diluted at 1:3,000 and 1:800, respectively. Bound IgG1 and IgG2a were detected using biotinylated anti-mouse IgG1 or anti-mouse IgG2a, respectively. The plates were further processed with horseradish peroxidaseconjugated streptavidin and tetramethylbenzidine as substrate. To measure HEL-specific IgE antibody, plates were coated with anti-mouse IgE antibody. After incubation with diluted mouse sera (1:20), bound IgE was incubated with HEL. The plates were then processed with peroxidase-conjugated anti-HEL antibody, and tetramethylbenzidine was used as a substrate. Plates were read at 450 nm on a MultiSkan ELISA reader (ThermoLab Systems, Brussels, Belgium).
Total IgE was measured by sandwich ELISA (BD Biosciences, Erembodegem, Belgium) according to the manufacturers instructions. Serum samples were analyzed in duplicate at a 1:10 dilution.
Real-time RT-PCR for cytokines, PAF-acetylhydrolase, and PAF-R.
Total RNA from spleen was extracted using the SV total RNA isolation system (Promega, Madison, WI). A 1-µg target RNA was reverse-transcribed as described previously (16).
Real-time quantitative RT-PCR was performed for ß-actin, interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-, IL-12,
-interferon (IFN-
), IL-4, IL-13, PAF-acetylhydrolase (AH), and PAF-R as previously described (16). The primers and fluorogenic probe for PAF-AH and PAF-R were designed based on the published sequences and are listed in Table 1.
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RESULTS |
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Th2 shift parallels shock.
Because the clinical picture was compatible with a state of anaphylaxis, rescue treatment with corticosteroids was attempted. As shown in Fig. 2, pretreatment of NOD mice with 50 µg dexamethasone, injected intraperitoneally 48, 24, and 2 h before the HEL booster, rescued all NOD micecontrol as well as 1,25(OH)2D3-treated (n = 8 for each group). This result suggests involvement of the immune system in the shock phenomenon. To further investigate the mechanism involved, antibody production was measured. Therefore, HEL-specific serum IgG1 and IgG2a and total serum IgE levels of the NOD and C57BL/6 mice, treated with 1
,25(OH)2D3 or vehicle, were measured 3 weeks after the first HEL immunization (just before HEL rechallenge).
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Involvement of platelet activation in shock.
Because the clinical picture of lethal shock was accompanied by hypercoagulation, compatible with anaphylaxis leading to death by disseminated intravasal coagulation, we questioned the involvement of platelet activation in induced shock. We investigated if factors involved in this pathway showed an abnormal expression profile in the NOD mouse or were influenced by 1,25(OH)2D3. Therefore, mRNA levels for PAF-R and PAF-AH, the enzyme most responsible for degradation of PAF, were quantified by real-time RT-PCR in the spleens of NOD and C57BL/6 mice 3 weeks after the first footpad injection with HEL.
Interestingly, a clear inter-strain difference in the expression of PAF-R mRNA was observed, with 2.5-fold higher levels in NOD mice than in C57BL/6 mice (P < 0.05, Fig. 5A). Additionally, 1,25(OH)2D3 administration further increased the expression PAF-R mRNA in NOD as well as C57BL/6 mice by 1.5- and 2.5-fold, respectively (P < 0.05, Fig. 5A). These results, showing a regulation of the PAF-R in the analyzed system, are highly indicative for an important role of the PAF pathway in induction and sensitivity for anaphylaxis.
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To conclusively demonstrate the involvement of the platelet activation pathway in the shock phenomenon, we attempted to rescue the HEL-immunized NOD mice from lethal shock by administration of a PAF-R antagonist. Indeed, mortality was completely abrogated by administration of a single dose of 500 µg of the PAF-R antagonist WEB2086 10 min before the HEL booster in all HEL-immunized NOD mice, irrespective of 1,25(OH)2D3 treatment (Fig. 6). Only mild nonlethal shock symptoms were observed in 75% of the rescued mice; in each group, shock was completely prevented in two of eight mice, whereas the other six developed milder shock symptoms, from which they all recovered (Fig. 6).
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DISCUSSION |
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NOD mice are animals that have a special immune system, with particularities of both T-cells and antigen-presenting cells (2). Although their most known immune feature is the Th1-driven destruction of pancreatic islets (4), literature data about their Th2 activity are conflicting (25,26). The initial aim of our study was to check the safety of antigenic vaccination in this particular strain with respect to anaphylaxis. The set of antigens tested included diabetes-relevant autoantigens (GAD65, mouse insulin, and hsp65) and diabetes-irrelevant self (PLP) or nonself (HEL, OVA, KLH, and TT) antigens. The behavior of NOD mice to vaccination was compared with that of two other mouse strains: C57BL/6 and BALB/c mice. To force T-cell differentiation toward a Th2 setting, we submitted groups of mice from the three strains to therapy with 1,25(OH)2D3. The active form of vitamin D is known as a powerful modulator of the immune system (27). The 1
,25(OH)2D3-induced shift toward Th2 differentiation can even prevent the onset of Th1-mediated autoimmune diseases, such as diabetes in NOD mice (28) or EAE in SJL/J mice (29).
The only antigen that provoked anaphylactic reactions in our experiments was HEL. However, mice from various strains behaved in different ways. C57BL/6 mice were completely resistant to HEL-induced anaphylaxis, irrespective of 1,25(OH)2D3 administration. This outcome was not unexpected because C57BL/6 mice are known for having a predominantly Th1-driven immune response, though not accompanied by autoimmune phenomena (30). Although T-cell lymphocyte differentiation is mostly Th2-directed in BALB/c mice (30), HEL vaccination by itself did not evolve to shock. When the immune system was further stressed toward Th2 responses by 1
,25(OH)2D3 administration, all BALB/c mice nevertheless developed a mild reversible form of shock. Unexpectedly, NOD mice, known for their Th1-mediated immune reactivity, also developed shock consecutive to HEL sensitization. Moreover, shock appeared in NOD mice even without any modulation of the immune system with 1
,25(OH)2D3 and was rather aggressive (even lethal for certain animals). 1
,25(OH)2D3 administration further increased mortality in HEL-sensitized NOD mice.
We were surprised by the strain-selective anaphylactic shock induced by HEL in NOD mice. This mouse strain is indeed known to react through Th1-mediated mechanisms (24,25) and was moreover described as having a defect in antigen presentation (31,32). An exacerbation of anaphylaxis caused by the administration of 1,25(OH)2D3 was also not immediately clear because previous studies suggested that a 1
,25(OH)2D3-dependent differentiation shift toward Th2 takes place exclusively in the presence of diabetes-relevant autoantigens (33). We decided therefore to search for the mechanisms underlying the strain-specific behavior of the NOD immune system.
Complete rescue by therapy with dexamethasone demonstrated undoubtedly that HEL-induced shock in NOD mice depended on immune effectors. Furthermore, the complete absence of symptoms in NOD-scid mice lacking T- and B-cells (34) proved the indispensable role of T-cells in HEL-induced shock. Cytokine modifications caused by 1,25(OH)2D3 in NOD mice were partially suggestive for a Th1 to Th2 shift, with a decrease in Th1-secreted IFN-
and a concomitant increase in Th2-specific IL-13. However, no differences in IL-12 and IL-4 levels were found between the 1
,25(OH)2D3-treated and nontreated HEL-sensitized NOD mice. Thus, these changes did not completely overlap the 1
,25(OH)2D3-dependent modifications found when the immune system was activated by autoantigens (33), suggesting antigen-specific immune modulation by 1
,25(OH)2D3. Comparison of cytokine mRNA spectra between HEL-immunized shock-sensitive NOD mice and shock-resistant C57BL/6 mice showed differences in IL-13 mRNA levels, which were further accentuated by 1
,25(OH)2D3 administration. Therefore, our data supported that the cytokine has a critical role in the particular behavior of NOD mice toward HEL sensitization. IL-13 is a Th2-specific cytokine closely related to IL-4 (35) and synthesized in both humans and mice (36). IL-13 has been alleged to be importantly involved in Th2-initiated allergic reactions, including anaphylaxis (37). The significant difference in serum IgE levels between the two mouse strains provided evidence of IgE-mediated anaphylactic shock in NOD mice. IgE is an immunoglobulin involved in most allergic reactions (13,38). The parallelism between IL-13 mRNA levels and serum IgE levels assessed in our mice stresses a causal relationship between the two immune parameters. Direct stimulation of IgE synthesis by IL-13 has been actually described (38). Certain NOD mice also displayed higher levels of Th2-dependent IgG1. IgG1 may by itself induce anaphylactic shock in mice (11,13).
PAF is an important mediator of immune, hemostatic, and allergic reactions (39). This bioactive phospholipid can be regarded as a cytokine because of its multiple actions on a wide range of cells (including neutrophils, monocytes, and platelets) and tissues that possess specific membrane receptors (PAF-R) (40). The acute inflammatory effects of PAF are largely due to the stimulation of neutrophilic granulocytes as the first line of defense (40). Exaggerated synthesis of PAF or defective mechanisms of PAF inactivation can however lead to pathologic inflammation, shock, and thrombosis (39). There are data in the literature suggesting significant interference of 1,25(OH)2D3 with PAF actions (41) or metabolism (42). Because 1,25(OH)2D3 had such a powerful impact on shock incidence and severity, we suspected an interference of PAF in HEL-induced shock. PAF-R mRNA levels were significantly higher in NOD mice, pleading in favor of an increased sensitivity to PAF inflammatory actions in this strain. In concordance with other studies (43), 1,25(OH)2D3 therapy caused a further increase in the PAF-R expression, a phenomenon that might partly explain the increase in shock severity. Surprisingly, PAF-AH mRNA levels in HEL-sensitized NOD mice were significantly higher than those in C57BL/6 mice. PAF-AH is the main enzyme involved in PAF degradation. The increase in PAF-AH mRNA levels after HEL sensitization could be the expression of a natural feedback reaction against exaggerated PAF synthesis (44). Contrary to another study (42), 1,25(OH)2D3 therapy did not significantly inhibit PAF-AH mRNA expression in HEL-sensitized NOD mice. However, the minor interstrain difference of PAF-AH levels was no longer significant in the 1,25(OH)2D3-treated groups. The direct proof of PAF involvement in the etiopathogenesis of strain-specific HEL-induced shock was obtained when rescue therapy with a PAF-R antagonist (WEB2086) showed to be efficient in preventing shock or diminishing its severity. The activation of the Th2 arm of the immune attack is probably linked to the activation of the PAF system. IgE, for example, can undeniably cause direct stimulation of PAF synthesis by various cells, such as mastocytes (41). Moreover, a recent article (45) suggested that murine antigen-induced anaphylaxis is primarily mediated by the PAF pathway and not by the classic IgE pathway.
Finally the intriguing question remains why HEL induces anaphylaxis in the NOD mouse model, as opposed to the other antigens tested. An extensive GenBank search did not reveal any peptide homology between HEL and any of the known diabetes-relevant autoantigens, not excluding, however, a possible similarity with an as yet undefined autoantigen.
In conclusion, our data clearly demonstrate the appearance of a severe Th2-biased immune reaction after challenge with a widely used control antigen in a mouse strain known mainly for its genetic propensity toward Th1-mediated autoimmune destruction of ß-cells. Strain specificity of this reaction points out that immune systems with a disturbed genetic background may reply unpredictably to an immune challenge, even with "innocent" antigens. Autoimmunity should therefore not be simply viewed as an isolated particularity of the immune response but rather as a general disturbance of the immune system. It is currently accepted that Th1-oriented autoimmunity is involved in the pathogenesis of human type 1 diabetes (15). Shifting the activation of the immune system toward the Th2 differentiation pathway has been proven efficient in preventing diabetes in animal models (2,3,5) and might be regarded as an interesting alternative to type 1 diabetes prevention in humans (5,9). The immune system of type 1 diabetic patients may, however, manifest changes beyond the local islet-destructive effect (46). Our data complete the conflicting experience already existing with vaccination strategies in EAE and human MS (11,21,24). Extreme caution is therefore needed when adopting immune-modulating strategies, taking into consideration that a shift in T-helper differentiation might always represent a transfer of risk from a Th1-mediated type IV delayed immune attack to a Th2-mediated anaphylactic reaction, especially in situations with an already-activated Th2 system, such as a coincidental infection or allergy.
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ACKNOWLEDGMENTS |
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The expert technical assistance of Jos Laureys is gratefully acknowledged.
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FOOTNOTES |
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Received for publication 13 May 2002 and accepted in revised form 5 November 2002.
L.O. and B.D. contributed equally to this work.
AH, acetylhydrolase; CFA, complete Freunds adjuvant; EAE, encephalomyelitis; ELISA, enzyme-linked immunosorbent assay; HEL, hen egg white lysozyme; hsp, heat shock protein; IFN-,
-interferon; IL, interleukin; ins-B, insulin B; KLH, keyhole limpet hemocyanine; MS, multiple sclerosis; OVA, ovalbumin; PAF-R, platelet-activating factor receptor; Th1, T-helper 1; TNF, tumor necrosis factor; TT, tetanus toxin.
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REFERENCES |
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