Cyclophosphamide treatment of female non-obese diabetic mice causes enhanced expression of inducible nitric oxide synthase and interferon-gamma, but not of interleukin-4

Abstract

In pancreatic lesions of non-obese diabetic (NOD) mice the expression of inducible nitric oxide synthase (iNOS) and of the cytokines interferon-gamma and interleukin-4 were studied. Strong iNOS expression as determined at the level of transcription, translation and of enzyme activity was associated with destructive insulitis as seen 8-10 days after cyclophosphamide treatment of 70- to 80-day-old female NOD mice. Immunohistochemistry showed iNOS associated with infiltrating macrophages but not in endocrine cells. The enhancement of iNOS after cyclophosphamide correlated with an increase of T-helper type 1 (Th1) associated interferon-gamma expression while T-helper type 2 (Th2) associated interleukin-4 was the dominant cytokine prior to cyclophosphamide and after diabetes onset. We conclude that insulitis in young NOD mice is carried by Th2 cells while cyclophosphamide enhanced insulitis is determined by Th1 cells. Macrophages show two different functional states in insulitis; strong iNOS expression in macrophages is associated with destructive insulitis.

Immunization of non-obese diabetic (NOD) mice with glutamic acid decarboxylase-derived peptide 524–543 reduces cyclophosphamide-accelerated diabetes

“…In our study, an increase in IFN-production after CY treatment alone was found. Such up-regulation of IFN-and a shift in the balance towards Th1 predominance after CY treatment are consistent with previous reports [27,28]. In comparison, subsets of Th2 cells were markedly stimulated by peptide 524-543 immunization, which was characterized by a strong peptide-specific production of IL-4 and IL-10.…”

“…4). This indicates, as previously reported [27,28], that a potentially pathogenic Th1-type T cell population was stimulated by CY treatment. GAD peptide-challenged splenocytes isolated from NOD mice immunized with peptide 524-543 exhibited peptide 524-543-specific production of IFN-and IL-2 (Fig.…”

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

“…Sections for histology were stained with haematoxylin and eosin and were evaluated for mononuclear infiltration using the following semiquantitative rating system: score 0, no periductular infiltrates and less than 5 immune cells peri-and intrainsular; score 1, periductular infiltrates only; score 2: periductular and peri-insular infiltrates only; score 3, intraislet infiltration of less than 20 % of the islet area; score 4, intraislet infiltration of more than 20 % of the islet area. Buffers for cytokine-staining [3] were supplemented with 0.05 % saponine (Sigma, Deisenhofen, Germany). Monoclonal antibodies against murine IL-4 and Il-2 were obtained from Pharmingen (clone BVD6-24G2 and clone S4B6; San Diego, Calif, USA).…”

“…Total RNA was isolated from whole small bowel by Trizol Reagent (Gibco BRL, Berlin, Germany). Determination and quantification of specific mRNA was performed by reverse transcriptase polymerase chain reaction (RT-PCR) as described elsewhere [3,24]. Control experiments to check for linear relationship between amount of PCR product and amplification cycle number were performed.…”

“…Signals were quantified by measuring the 32 P stimulated luminescence (PSL) by a phosphoimager. Relative PSL was calculated by normalization of the measured PSL to the strength of the b -actin signal [3].…”