Oral insulin for diabetes prevention in NOD mice: potentiation by enhancing Th2 cytokine expression in the gut through bacterial adjuvant

Oral administration of insulin suppresses the development of diabetes in nonobese diabetic (NOD) mice and deviates the cytokine balance in the islets of Langerhans from a Th1 to a Th2 type cytokine pattern. However, the effect of oral insulin is limited and disease suppression is limited to a narrow dose range. Therefore we tried to improve the outcome of suboptimal insulin dosing by bacterial adjuvant. Mice treated with a suboptimal dose of oral insulin showed no change in diabetes incidence although a shift from Th1 towards Th2 cytokine expression occurred in inflamed islets. Significant suppression of diabetes development was only seen in NOD mice receiving both, insulin and the bacterial preparation OM-89 as adjuvant. OM-89 is a protein extract of Escherichia coli, with nonspecific immunostimulatory properties. Potentiation of the effect of oral insulin by the adjuvant was associated with upregulation of interleukin (IL)-4 Th2 cells in infiltrated islets and sustained local IL-2 gene expression. RT PCR analyses of cytokine expression in the gut showed a clear deviation to Th2 type reactivity and downregulation of inducible nitric oxide (NO) synthase (iNOS) expression by the bacterial adjuvant but not by oral insulin alone. Since macrophages are the primary target cells of adjuvant action we tested its effect on mouse macrophages in vitro. Treatment with OM-89 induced transient release of tumour necrosis factor alpha and nitrite but rendered macrophages refractory to restimulation by the potent macrophage activator lipopolysaccharide. In conclusion, the protective effect of oral insulin can be potentiated by pretreatment with the bacterial adjuvant OM-89. This effect correlates with enhanced Th2 cytokine and decreased iNOS gene expression in the gut, probably due to the downregulation of proinflammatory mediators by exposure to the adjuvant.     

Tissue microarrays for high-throughput molecular profiling of tumor specimens

The impact of oral treatment with insulin on disease development was studied in diabetes prone BB rats. Because of the positive outcome of a prior study in non obese diabetic (NOD) mice, BB rats received insulin in combination with a bacterial adjuvant. Porcine insulin was given orally twice weekly from 35-100 days of age, the E. coli preparation OM-89 was fed on alternate days. Other groups received vehicle, the bacterial adjuvant, or insulin alone. Both insulin containing oral dosing regimens induced a transient non significant delay in diabetes onset. Insulin alone, however did not decrease the final diabetes incidence. Oral dosing with insulin plus adjuvant caused exacerbation of disease development as judged from the decreased survival rate in comparison with the insulin treated group (p < 0.05). Intra-islet infiltration also increased (p < 0.005) compared with the insulin or vehicle treated groups. The effect correlated with enhanced interferon gamma (IFNgamma) and decreased interleukin 10 (IL-10) gene expression in the gut suggesting a shift towards proinflammatory T helper 1 (Th1) reactivity (p < 0.01). Although treatment with adjuvant alone also increased the degree of insulitis, an enhanced incidence of diabetes and a shift in cytokine expression was only seen in the group receiving insulin plus adjuvant. Taken together, the data suggest that treatment with a bacterial adjuvant and oral insulin may alter the gut immunoregulatory state such that disease promoting rather than protective immune responses are induced.     

An update on cytokines in the pathogenesis of insulin-dependent diabetes mellitus

Correlation studies between cytokines expressed in islets and autoimmune diabetes development in NOD mice and BB rats have demonstrated that beta-cell destructive insulitis is associated with increased expression of proinflammatory cytokines (IL-1, TNF alpha, and IFN alpha) and type 1 cytokines (IFN gamma, TNF beta, IL-2 and IL-12), whereas non-destructive (benign) insulitis is associated with increased expression of type 2 cytokines (IL-4 and IL-10) and the type 3 cytokine (TGF beta). Cytokines (IL-1, TNF alpha, TNF beta and IFN gamma) may be directly cytotoxic to beta-cells by inducing nitric oxide and oxygen free radicals in the beta-cells. In addition, cytokines may sensitize beta-cells to T-cell-mediated cytotoxicity in vivo by upregulating MHC class I expression on the beta-cells (an action of IFN gamma), and inducing Fas (CD95) expression on beta-cells (actions of IL-1, and possibly TNF alpha and IFN gamma). Transgenic expression of cytokines in beta-cells of non-diabetes-prone mice and NOD mice has suggested pathogenic roles for IFN alpha, IFN gamma, IL-2 and IL-10 in insulin-dependent diabetes mellitus (IDDM) development, and protective roles for IL-4, IL-6 and TNF alpha. Systemic administrations of a wide variety of cytokines can prevent IDDM development in NOD mice and/or BB rats; however, a given cytokine may retard or accelerate IDDM development, depending on the dose and frequency of administration, and the age and the diabetes-prone animal model studied (NOD mouse or BB rat). Islet-reactive CD4+ T-cell lines and clones that adoptively transfer IDDM into young NOD mice have a Th1 phenotype (IFN gamma-producing), but other islet-specific Th1 clones that produce TGF beta can adoptively transfer protection against IDDM in NOD mice. NOD mice with targeted deletions of IL-12 and IFN gamma genes still develop IDDM, albeit delayed and slightly less often. In contrast, post-natal deletions of IL-12 and IFN gamma, also IL-1, TNF alpha, IL-2, and IL-6--by systemic administrations of neutralizing antibodies, soluble receptors and receptor antagonists, and receptor-targeted cytotoxic drugs--significantly decrease IDDM incidence in NOD mice and/or BB rats. These cytokine deletion studies have provided the best evidence for pathologic roles for proinflammatory cytokines (IL-1, TNF alpha, and IL-6) and type 1 cytokines (IFN gamma, IL-2 and IL-12) in IDDM development.     

In vivo study on the immunomodulating effects of OM-85 BV on survival, inflammatory cell recruitment and bacterial clearance in Klebsiella pneumonia

Oral administration of the bacterial extract OM-85 BV has been shown to prime alveolar macrophages (AM) in such a way that they secrete significantly more nitric oxide, tumor necrosis factor-alpha and interleukin-1beta upon in vitro stimulation with lipopolysaccharide (LPS). As increased cytokine secretion by AM may account for the therapeutic effect of OM-85 BV in respiratory tract infections, we studied the effect of orally administered OM-85 BV on the outcome of Klebsiella pneumoniae-induced pneumonia. Mice received a daily oral dose of OM-85 BV (350 mg/kg body weight) for 5 days and were intratracheally infected with 333, 1000 or 3333 CFU K. pneumoniae on day 8. It was shown that OM-85 BV pretreatment of mice has no effect on bacterial clearance, neutrophil recruitment and survival in this acute respiratory tract infection. Also, OM-85 BV treatment had no protective effect in a recurrent infection with K. pneumoniae. It is concluded that AM activation by oral treatment with OM-85 BV is not sufficient to play a protective role in respiratory tract infection with K. pneumoniae.     

Functional consequences of a decreased potassium affinity in a potassium channel pore. Ion interactions and C-type inactivation

We have shown previously that the inactivation of macrophages in nonobese diabetic (NOD) mice results in the prevention of diabetes; however, the mechanisms involved remain unknown. In this study, we found that T cells in a macrophage-depleted environment lost their ability to differentiate into beta cell-cytotoxic T cells, resulting in the prevention of autoimmune diabetes, but these T cells regained their beta cell-cytotoxic potential when returned to a macrophage-containing environment. To learn why T cells in a macrophage-depleted environment lose their ability to kill beta cells, we examined the islet antigen-specific immune response and T cell activation in macrophage-depleted NOD mice. There was a shift in the immune balance, a decrease in the T helper cell type 1 (Th1) immune response, and an increase in the Th2 immune response, due to the reduced expression of the macrophage-derived cytokine IL-12. As well, there was a deficit in T cell activation, evidenced by significant decreases in the expression of Fas ligand and perforin. The administration of IL-12 substantially reversed the prevention of diabetes in NOD mice conferred by macrophage depletion. We conclude that macrophages play an essential role in the development and activation of beta cell-cytotoxic T cells that cause beta cell destruction, resulting in autoimmune diabetes in NOD mice.     

Oral dosing of rats with OM-89 results in the appearance of specific OM-89 antibodies of the IgG2a isotype: possible significance in the treatment of rheumatoid arthritis

OM-89 is a glycoprotein-rich extract of Escherichia coli shown to be effective in the treatment of rheumatoid arthritis (RA). It has been reported that oral dosing of animals results in the appearance of specific OM-89 antibodies. In the current study we have investigated some of the immunoglobulin isotypes that may be involved. OM-89 antibodies of IgG1, IgG2a and IgM isotypes were measured by ELISA in serum from rats dosed three times a week for 3 weeks at 4 or 40 mg kg(-1). The results showed a small but significant rise in IgM and a greater rise in IgG2a. The possibility that antigens within OM-89 (e.g. hsp65) may have homology with antigens involved in RA raises the possibility that OM-89 antibodies, particularly of the IgG2 class, may block pathogenic antigens from being recognized by T cells.     

The immunomodulator OM-89 induces ACTH and glucocorticoid secretion in rats through an IL-1 dependent pathway

The immunomodulator OM-89 (bacterial extract from E. coli), known to act on the immune system by modulating both humoral and cellular responses, significantly increases ACTH and glucocorticoids secretion in normal Wistar rats. A comparative administration of IL-1 displays a similar pattern of release. Moreover, OM-89-induced responses are blocked by IL-1ra, the soluble receptor antagonist of IL-1. The effect of OM-89 can thus be explained, at least in part, via IL-1 which directly enhances the secretion of both stress hormones. Finally, a comparative experiment between control and stressed rats (by immobilization) shows that the responses to OM-89 measured in this study (ACTH and corticosterone secretion) are stress-modulated.     

Deviation of pancreas-infiltrating cells to Th2 by interleukin-12 antagonist administration inhibits autoimmune diabetes

Nonobese diabetic (NOD) mice develop spontaneous insulin-dependent diabetes mellitus (IDDM), and the pancreas-infiltrating T cells invariably show a Th1 phenotype. We demonstrated here that the interleukin (IL)-12 antagonist (p40)2 can deviate the default Th1 development of naive T cell receptor (TCR)-transgenic CD4+ cells to the Th2 pathway in vitro. Although (p40)2 does not modify the cytokine profile of polarized Th1 cells, it prevents further recruitment of CD4- cells into the Th1 subset. To study the involvement of Th1 and Th2 cells in the initiation and progression of IDDM, we targeted endogenous IL-12 by administration of (p40)2 in NOD mice. (p40)2 administration to NOD mice inhibits interferon-gamma but not IL-10 production in response to lipopolysaccharide (LPS) or to the putative autoantigen IA-2. Serum immunoglobulin isotypes determined after (p40)2 treatment indicate an increase in Th2 and a decrease in Th1 helper activity. Administration of (p40)2 from 3 weeks of age onwards, before the onset of insulitis, results in the deviation of pancreas-infiltrating CD4+ but not CD8+ cells to the Th2 phenotype as well as in the reduction of spontaneous and cyclophosphamide-accelerated IDDM. After treating NOD mice with (p40)2 from 9 weeks of age, when insulitis is well established, few Th2 and a reduced percentage of Th1 cells are found in the pancreas. This is associated with a slightly decreased incidence of spontaneous IDDM, but no protection from cyclophosphamide-accelerated IDDM. In conclusion, deviation of pancreas-infiltrating CD4+ cells to Th2 is associated with protection from IDDM. However, targeting IL-12 after the onset of insulitis, when the pancreas contains polarized Th1 cells, is not sufficient to induce an effective immune deviation able to significantly modify the course of disease.     

Beta-cell destruction may be a late consequence of the autoimmune process in nonobese diabetic mice

The NOD mouse is an animal model of IDDM that shows many of the characteristics of human IDDM. It has been proposed that beta-cell destruction in IDDM progresses over time in a linear manner. Recently, we and others have demonstrated that T helper type 1 (Th1) cells have pathogenic roles in the NOD model and proposed that cytokine balances change as the disease progresses. However, it has not been demonstrated how or when the cytokine balances change or how the beta-cell destruction progresses. We have recently demonstrated that the cytokine profiles of CD45RB(low) CD4+ cells correlate either with their pathogenic or with their protective roles in the NOD mouse. To further analyze this apparent correlation between the shift in cytokine level and IDDM, we examined the anti-CD3-induced cytokine profiles of this subset from NOD mice of various ages compared with that from age-matched I-Ak transgenic NOD and BALB/c mice as controls. A significantly higher ratio of anti-CD3-induced interferon-gamma/interleukin-4 was found in diabetic NOD mice (P < 0.0001) but not in age-matched nondiabetic NOD mice. This cytokine ratio did not change significantly until the onset of diabetes in NOD mice. Based upon these results, we propose that IDDM in the NOD mouse progresses as a predominant inflammatory beta-cell dysfunction without actual beta-cell destruction until late in the disease process. This supports the possibility that late-stage immunotherapy may preserve islet beta-cell mass.     

Human 4-1BB regulates CD28 co-stimulation to promote Th1 cell responses

Our present study provides evidence that the 4-1BB signal is critical to CD28 co-stimulation in maintaining T cell activation when CD28 has been down-regulated because of repeated stimulation. The 4-1BB signal synergized with CD28 co-stimulation by lowering the threshold of anti-CD28 required to sustain proliferation and IL-2 production. The 4-1BB signal also modulated CD28-mediated cytokine profiles by markedly enhancing Th1 but suppressing Th2-type cytokine production. The 4-1BB signal generated Th1-type cells, as identified by intracellular IFN-gamma production. IFN-gamma induction was detected preferentially in 4-1BB-expressing cells, but not in those expressing CD30. 4-1BB and CD30 were induced in both CD4+ and CD8+ cells, but the location of the two molecules was mutually exclusive in each T cell subset. Our study suggests that the 4-1BB signal regulates CD28 co-stimulation in the targeted subset cells to favor Th1 development and maintain long-term cell growth.     

Exogenous bovine surfactant suppresses tumor necrosis factor-alpha release by murine macrophages stimulated by genital mycoplasmas

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that appears to play a significant role in the development of neonatal chronic lung disease (CLD). Inflammation and CLD are also associated with respiratory tract colonization with genital mycoplasmas. The possible protective roles of surfactant in mitigating the inflammatory response to these microbes were investigated. Murine RAW 264.7 macrophages were preincubated with an exogenous surfactant and exposed overnight to sterile media, lipopolysaccharide (LPS), Mycoplasma hominis, or Ureaplasma urealyticum. Macrophages released TNF-alpha in response to challenge with LPS, U. urealyticum, and M. hominis in a concentration-dependent fashion. Surfactant suppressed LPS and M. hominis induced TNF-alpha production in a dose-dependent manner but suppressed U. urealyticum-mediated TNF-alpha production only at the higher dose tested. Similar effects were seen in hyperoxia (95% O2). Thus, exogenous bovine surfactant significantly inhibits the production of TNF-alpha by murine macrophages stimulated with genital mycoplasmas and bacterial LPS.     

TGF-beta1 alters APC preference, polarizing islet antigen responses toward a Th2 phenotype

TGF-beta1, expressed in the pancreatic islets, protects the nonobese diabetic (NOD) mouse from insulin-dependent diabetes mellitus (IDDM). The islet antigen-specific T cell response of ins-TGF-beta1 mice relied on different antigen-presenting cells (APC) from those used by NOD T cells. T cells from NOD mice utilized B cells to present islet antigen, whereas T cells from ins-TGF-beta1 mice utilized macrophages. In addition, the islet antigen-specific T cell repertoire of ins-TGF-beta1 mice was distinct and deviated toward an IL-4-producing Th2 phenotype. When ins-TGF-beta1 mice were treated with anti-iL-4 antibody, islet antigen-specific IFNGamma-producing Th1 cells were unleashed, and the incidence of diabetes increased to the level of NOD mice. This suggests active suppression of a diabetogenic T cell response. This study describes a novel mechanism in which expression of TGF-beta1 in the context of self-antigen shifts APC preference, deviating T cell responses to a Th2 phenotype, preventing IDDM.     

Lipopolysaccharide-binding protein is present in effluents of patients with Gram-negative and Gram-positive CAPD peritonitis

BACKGROUND: Bacterial peritonitis is a frequent complication during treatment of end-stage renal failure by continuous ambulatory peritoneal dialysis. Local host defence mechanisms including the secretion of proinflammatory cytokines by peritoneal macrophages are of particular importance in the pathogenesis of infectious complications. LPS-binding protein (LBP) and soluble CD14 (sCD14) are serum factors known to regulate the endotoxin-induced cellular immune response. However, it is still unknown whether LBP and sCD14 are also present in the peritoneal effluent of CAPD patients. METHODS: Using specific immunoassays, we examined the concentration of LBP, sCD14 and the proinflammatory cytokines TNF-alpha, IL-1beta and IL-6 in the dialysis effluents of 31 patients with CAPD-associated peritonitis. Twenty patients without peritonitis served as controls. Intraperitoneal LPS concentrations were determined using the limulus amebocyte lysate assay. RESULTS: Bacterial lipopolysaccharide could be detected in 42% of the infected dialysis effluents. In comparison to controls (0.2 +/- 0.05 microg/ml), LBP was significantly elevated in both gram-negative/LPS-positive (1.03 +/- 0.3 microg/ml) and gram-positive infections (0.5 +/- 0.14 microg/ml) (P<0.05). No significant differences were detected concerning the intraperitoneal sCD14 levels in the three patient groups. Levels of TNF-alpha, IL-1beta and IL-6 were significantly increased in the effluents of patients with bacterial peritonitis compared to noninfected controls. Moreover the respective cytokine concentrations were significantly higher in the gram-negative/LPS-positive compared to the gram-positive bacterial infections (P<0.01). CONCLUSION: Our data demonstrate that LBP is significantly elevated in the dialysis effluents of patients with CAPD-associated peritonitis caused by both gram-negative and gram-positive bacteria and might be used as a marker of intraperitoneal infection. Moreover, our findings support the concept that LBP enhances the effects of LPS on cytokine production by peritoneal macrophages. The function of LBP in gram-positive infection remains to be further elucidated.     

Effect of oral and intravenous insulin and glutamic acid decarboxylase in NOD mice

Islet cell antigens have been administered orally and intravenously (I.V.) to NOD mice to assess their abilities to protect from or delay the onset of diabetes, and thereby provide insights that may have therapeutic implications in human trials. Whereas we and others have observed a delay in the onset of diabetes in NOD mice that have been fed with insulin from early life, we report here for the first time that feedings with porcine GAD65 alone (p = 0.226) or in combination with insulin (p = 0.011), have anti-diabetic effects in a prolonged study period (>400 days). While antigen-specific inhibitions of in vitro lymphocytic proliferation responses were seen (p < 0.05), antibody levels were unaffected by oral antigen treatments. IFN-gamma mRNA levels were downregulated in the islet infiltrates following oral antigen treatments while IL-2 and TNF-beta were expressed in all instances. We also observed that I.V. human recombinant GAD65, and porcine GAD given at weaning, delayed diabetes onset (p = 0.004) while similar treatments with a variety of inactive insulin preparations were generally ineffective. These findings thus indicate varying effects of oral and I.V. autoantigen administrations on the development of diabetes in NOD mice, and describe the immunological processes induced by oral autoantigen treatments.     

Prevention of autoimmunity in nonobese diabetic (NOD) mice by neonatal transfer of allogeneic thymic macrophages

Nonobese diabetic (NOD) mice spontaneously develop insulin dependent diabetes mellitus. The disease results from an autoimmune process which involves mononuclear cells surrounding and eventually infiltrating the pancreatic islets of Langerhans. Macrophages are thought to be the first cells to infiltrate the islets and are actively involved in the disease process because diabetes is prevented if host macrophages are depleted or inactivated. Several lines of evidence also suggest that NOD macrophages are phenotypically and functionally abnormal. In this study, allogeneic (CBA) macrophages derived from the thymus were inoculated into newborn NOD mice and these were followed for more than 250 days. Spontaneous diabetes was significantly reduced in female NOD mice (6% diabetic versus 45% of controls). Insulitis was also significantly reduced in both male and female mice compared to their control counterparts, and in most cases there were virtually no inflammatory cells in the pancreas. Allogeneic skin grafting and mixed leukocyte cultures indicated that the recipients were not tolerant of donor antigens, and donor-derived cells were not detected in the lymphoid tissues by either flow cytometry or immunohistochemistry. The results show that macrophages from diabetes-resistant donors will prevent insulitis and diabetes in most recipients, however, the mechanism for the protection is unclear, but does not appear to be due to long-term tolerance induction.     

Endogenous immune response to glutamic acid decarboxylase (GAD67) in NOD mice is modulated by adjuvant immunotherapy

We have shown that immunization of non-obese diabetic (NOD) mice with adjuvants (CFA or BCG) prevents the onset of diabetes by induction of regulatory cells. Since autoimmune responses to glutamic acid decarboxylase (GAD) are up-regulated in insulin-dependent diabetes mellitus (IDDM), in this study GAD67-specific antibody, T cell proliferation and lymphokine production patterns were analysed in the adjuvant-treated mice to characterize the regulatory mechanisms underlying the protection. We used both spontaneous diabetes and syngeneic islet transplantation models in NOD mice. Protection against spontaneous diabetes and prevention of syngeneic islet graft rejection by CFA or BCG treatment was found to be accompanied by the production of long lasting and high titre anti-GAD67 antibody of IgG1 isotype in the sera. Upon in vitro stimulation with GAD67, draining lymph node and spleen cells from CFA-immunized NOD mice or syngeneic islet-grafted and BCG-protected NOD mice produced much more IL-4, whereas there was no significant change in IFN-gamma production. The strong early T cell proliferative response to GAD67 in CFA or BCG-immunized NOD mice was followed by a low or unresponsiveness state. Taken together, these results suggest a shift in Th1/Th2 balance in the GAD67-specific endogenous immune response to a change in Th2 levels after adjuvant treatment. We postulate that the protective effect of CFA or BCG is due to the diversion of GAD-specific endogenous cellular immune response to a non-pathogenic humoral response.     

The biphasic mRNA expression pattern of bovine interleukin-8 in Pasteurella haemolytica lipopolysaccharide-stimulated alveolar macrophages is primarily due to tumor necrosis factor alpha

Pasteurella haemolytica serotype 1 is the bacterial agent responsible for the pathophysiological events associated with bovine pneumonic pasteurellosis. Our previous studies support a role for the lipopolysaccharide (LPS) from P. haemolytica in the induction of proinflammatory cytokines. One of the pathological hallmarks of bovine pneumonic pasteurellosis is an influx of neutrophils into the alveolar spaces. This pronounced influx suggests the local production of a chemotactic factor(s) such as interleukin-8 (IL-8). In the context of the lung, the alveolar macrophage appears to be the major producer of IL-8, a proinflammatory cytokine with potent neutrophil chemotactic activity. By using Northern blot analysis, we have examined the kinetics of IL-8 mRNA expression in P. haemolytica LPS-stimulated bovine alveolar macrophages and found that 1 ng of LPS per ml induces maximal expression of IL-8 mRNA. The results also indicate a biphasic time course expression pattern in which IL-8 mRNA levels peak between 1 and 2 h in the first phase and between 16 and 24 h in the second phase (P < 0.01). In addition, monospecific polyclonal antibodies were used to demonstrate the role of tumor necrosis factor alpha (TNF-alpha) in the second phase of IL-8 mRNA expression. Our findings support a role for P. haemolytica LPS and TNF-alpha in the induction of IL-8 from bovine alveolar macrophages.