MK2/3 Are Pivotal for IL-33-Induced and Mast Cell-Dependent Leukocyte Recruitment and the Resulting Skin Inflammation.

The IL-1R family member IL-33R mediates Fcε-receptor-I (FcεRI)-independent activation of mast cells leading to NF-κB activation and consequently the production of cytokines. IL-33 also induces the activation of MAPKs, such as p38. We aimed to define the relevance of the p38-targets, the MAPK-activated protein kinases 2 and 3 (MK2 and MK3) in IL-33-induced signaling and the resulting mast cell effector functions in vitro and in vivo. We demonstrate that the IL-33-induced IL-6 and IL-13 production strongly depends on the MK2/3-mediated activation of ERK1/2 and PI3K signaling. Furthermore, in the presence of the stem cell factors, IL-33 did induce an MK2/3-, ERK1/2- and PI3K-dependent production of TNF-α. In vivo, the loss of MK2/3 in mast cells decreased the IL-33-induced leukocyte recruitment and the resulting skin inflammation. Therefore, the MK2/3-dependent signaling in mast cells is essential to mediate IL-33-induced inflammatory responses. Thus, MK2/3 are potential therapeutic targets for suppression of IL-33-induced inflammation skin diseases such as psoriasis.

Amelioration of experimental arthritis by stroke-induced immunosuppression is independent of Treg cell function.

OBJECTIVES: Clinical evidence suggests that neurological lesions can protect from arthritis. Acute cerebral ischaemia induces severe immunosuppression, resulting in enhanced susceptibility to infections. We aimed to determine if stroke-induced immunosuppression can ameliorate arthritis and to delineate the immunological mechanisms involved. METHODS: Unilateral cerebral ischaemia was induced in mice by occlusion of one middle cerebral artery (MCAO) at different time points after induction of G6PI-induced arthritis in mice. Clinical and histological signs of arthritis were assessed. Regulatory T cells were specifically depleted by injection of diphtheria toxin into transgenic DEREG mice. Immunological correlates of MCAO were determined by flow cytometry and serological methods. RESULTS: MCAO reduced the clinical and histological signs of arthritis significantly. To be effective, stroke had to be induced during the induction phase or the early clinical stage of arthritis. MCAO induced a global loss of leucocytes. Despite the reduced absolute number of lymphocytes, the functional differentiation of T helper cells into Th1/17 cells and the production of autoantibodies were unimpaired. Depletion experiments showed that regulatory T cells were dispensable for the protective effect of MCAO. CONCLUSIONS: MCAO ameliorates arthritis. The correlate of protection from arthritis is not the reduction of a particular pathogenic leucocyte subset or the preferential expansion or emergence of a protective cell population but the global reduction of leucocytes during arthritis.

Quantification of arthritic bone degradation by analysis of 3D micro-computed tomography data.

The use of animal models of arthritis is a key component in the evaluation of therapeutic strategies against the human disease rheumatoid arthritis (RA). Here we present quantitative measurements of bone degradation characterised by the cortical bone profile using glucose-6-phosphate isomerase (G6PI) induced arthritis. We applied micro-computed tomography (µCT) during three arthritis experiments and one control experiment to image the metatarsals of the hind paws and to investigate the effect of experimental arthritis on their cortical bone profile. For measurements of the cortical profile we automatically identified slices that are orthogonal to individual metatarsals, thereby making the measurements independent of animal placement in the scanner. We measured the average cortical thickness index (CTI) of the metatarsals, as well as the thickness changes along the metatarsal. In this study we introduced the cortical thickness gradient (CTG) as a new measure and we investigated how arthritis affects this measure. We found that in general both CTI and CTG are able to quantify arthritic progression, whilst CTG was found to be the more sensitive measure.

Automated Quantification of Early Bone Alterations and Pathological Bone Turnover in Experimental Arthritis by in vivo PET/CT Imaging.

The assessment of bone damage is required to evaluate disease severity and treatment efficacy both in arthritis patients and in experimental arthritis models. Today there is still a lack of in vivo methods that enable the quantification of arthritic processes at an early stage of the disease. We performed longitudinal in vivo imaging with [18F]-fluoride PET/CT before and after experimental arthritis onset for diseased and control DBA/1 mice and assessed arthritis progression by clinical scoring, tracer uptake studies and bone volume as well as surface roughness measurements. Arthritic animals showed significantly increased tracer uptake in the paws compared to non-diseased controls. Automated CT image analysis revealed increased bone surface roughness already in the earliest stage of the disease. Moreover, we observed clear differences between endosteal and periosteal sites of cortical bone regarding surface roughness. This study shows that in vivo PET/CT imaging is a favorable method to study arthritic processes, enabling the quantification of different aspects of the disease like pathological bone turnover and bone alteration. Especially the evaluation of bone surface roughness is sensitive to early pathological changes and can be applied to study the dynamics of bone erosion at different sites of the bones in an automated fashion.

18 F-Fluoride positron emission tomography/computed tomography for noninvasive in vivo quantification of pathophysiological bone metabolism in experimental murine arthritis.

INTRODUCTION: Evaluation of disease severity in experimental models of rheumatoid arthritis is inevitably associated with assessment of structural bone damage. A noninvasive imaging technology allowing objective quantification of pathophysiological alterations of bone structure in rodents could substantially extend the methods used to date in preclinical arthritis research for staging of autoimmune disease severity or efficacy of therapeutical intervention. Sodium 18 F-fluoride (18 F-NaF) is a bone-seeking tracer well-suited for molecular imaging. Therefore, we systematically examined the use of 18 F-NaF positron emission tomography/computed tomography (PET/CT) in mice with glucose-6-phosphate isomerase (G6PI)-induced arthritis for quantification of pathological bone metabolism. METHODS: F-fluoride was injected into mice before disease onset and at various time points of progressing experimental arthritis. Radioisotope accumulation in joints in the fore- and hindpaws was analyzed by PET measurements. For validation of bone metabolism quantified by 18 F-fluoride PET, bone surface parameters of high-resolution µCT measurements were used. RESULTS: Before clinical arthritis onset, no distinct accumulation of 18 F-fluoride was detectable in the fore- and hindlimbs of mice immunized with G6PI. In the course of experimental autoimmune disease, 18 F-fluoride bone uptake was increased at sites of enhanced bone metabolism caused by pathophysiological processes of autoimmune disease. Moreover, 18 F-fluoride signaling at different stages of G6PI-induced arthritis was significantly correlated with the degree of bone destruction. CT enabled identification of exact localization of 18 F-fluoride signaling in bone and soft tissue. CONCLUSIONS: The results of this study suggest that small-animal PET/CT using 18 F-fluoride as a tracer is a feasible method for quantitative assessment of pathophysiological bone metabolism in experimental arthritis. Furthermore, the possibility to perform repeated noninvasive measurements in vivo allows longitudinal study of therapeutical intervention monitoring.

Implementation of 89Zr production and in vivo imaging of B-cells in mice with 89Zr-labeled anti-B-cell antibodies by small animal PET/CT.

We examined the production, separation, and characterization of (89)Zr, including supplementation of a commercial Cyclone(®) 18/9 with a self-made Solid Target System (STS). Obtained [(89)Zr]Zr-oxalate was used for the labeling of anti-B cell antibodies with desferrioxamine-p-SCN as a bifunctional chelator. (89)Zr-labeled antibodies were injected in DBA/1 mice to examine usability for detection of B cells in vivo. PET measurements showed binding of (89)Zr-labeled anti-B cell antibodies in tissues with high frequencies of B cells, i.e. in spleen and lymph nodes.

In vivo molecular imaging of experimental joint inflammation by combined (18)F-FDG positron emission tomography and computed tomography.

INTRODUCTION: The purpose of this work was to establish and validate combined small animal positron emission tomography - computed tomography (PET/CT) as a new in vivo imaging method for visualisation and quantification of joint inflammation. METHODS: Signalling of radioisotope ¹8F labelled Fluorodeoxyglucose (¹8F-FDG) injected in mice with glucose-6-phosphate isomerase (G6PI)-induced arthritis was analysed by PET/CT. Accumulation of ¹8F-FDG in tissue was quantified by PET measurement, whereas high definition CT delivered anatomical information. The fusion of both images revealed in detail spatial and temporal distribution and metabolism of ¹8F-FDG. RESULTS: A distinct ¹8F-FDG signal could be measured by PET in carpal and tarsal joints, from mice with early or established arthritis. In contrast, no accumulation of ¹8F-FDG was detectable before arthritis onset. Comparison of ¹8F-FDG joint uptake with histopathological evaluation revealed a significant correlation of both methods. CONCLUSIONS: Small animal PET/CT using ¹8F-FDG is a feasible method for monitoring and, more importantly, quantitative assessment of inflammation in G6PI-arthritis. Since it is possible to perform repeated non-invasive measurements in vivo, not only numbers of animals in preclinical studies can markedly be reduced by this method, but also longitudinal studies come into reach, e. g. for individual flare-up reactions or monitoring therapy response in progressive arthritis.

Interactions of T helper cells with fibroblast-like synoviocytes: up-regulation of matrix metalloproteinases by macrophage migration inhibitory factor from both Th1 and Th2 cells.

OBJECTIVE: Interactions of immune cells, such as activated T helper cells, with fibroblast-like synoviocytes (FLS) play a crucial role in the joint destruction during human rheumatoid arthritis (RA). This study was undertaken to investigate the expression of the proinflammatory cytokine macrophage migration inhibitory factor (MIF) by T helper cells, and to assess the role of MIF in overexpression of matrix metalloproteinases (MMPs) in cocultures of FLS from arthritic mice with either Th1 or Th2 cells. METHODS: MIF expression by in vitro-polarized murine Th1 and Th2 cells was determined using 2 different generation protocols. FLS were isolated from the inflamed joints of mice with antigen-induced arthritis. MMP expression was analyzed in cocultures of the FLS with T helper cell subsets. Effects of MIF were blocked by a neutralizing anti-MIF antibody. In addition, analyses were performed on cocultures of either Th1 or Th2 cells with FLS from MIF-deficient mice. RESULTS: Both Th1 and Th2 cells expressed high quantities of MIF. MMPs were overexpressed by FLS after coculture with both Th1 and Th2 cells. Activated T helper cells were more effective than resting cells. Neutralization of MIF by an anti-MIF antibody led to a marked reduction in MMP expression in Th1- and Th2-stimulated FLS. T helper cells generated from MIF-deficient mice exhibited a T helper cell-specific cytokine profile comparable with that in wild-type cells, except in the expression of MIF, but showed an impaired ability to stimulate MMP expression in FLS. CONCLUSION: MIF is an important Th1 and Th2 cell-derived proinflammatory cytokine that stimulates MMP expression in FLS from arthritic mice, and therefore inhibition of MIF might be a promising target for novel therapeutic strategies in human RA.

Exacerbation of antigen-induced arthritis in IFN-gamma-deficient mice as a result of unrestricted IL-17 response.

Proinflammatory Th1 responses are believed to be involved in the induction and perpetuation of rheumatoid arthritis. However, the role of IFN-gamma, the major cytokine produced by Th1 cells, is still incompletely defined. In the present study, we investigated the effects of IFN-gamma deficiency (IFN-gamma(-/-)) on the course of experimental murine Ag-induced arthritis (AIA). In the acute stage of disease, IFN-gamma(-/-) AIA mice showed significantly increased inflammatory responses compared with wild-type C57BL/6 AIA mice, i.e., exacerbated joint swelling, increased delayed-type hypersensitivity reaction, and increased histopathological scores of arthritis. Intraarticular administration of exogenous IFN-gamma at induction of AIA significantly suppressed these acute aggravation effects. Stimulated cells isolated from lymph nodes and spleen of IFN-gamma(-/-) AIA mice showed increased production of IL-2, IL-4, IL-5, IL-6, but most prominently of IL-17. These elevations were paralleled by decreased humoral immune responses, with low serum levels of total and Ag-specific IgG (IgG1, IgG2a(b), IgG2b, IgG3). At immunohistology, the knee joints of IFN-gamma(-/-) AIA mice showed massive neutrophil granulocyte infiltration. Treatment with mAbs neutralizing IL-17 diminished the acute inflammation. In vitro, Th cell expansion and production of IL-17 upon restimulation were effectively and dose dependently inhibited by IFN-gamma. These results clearly demonstrate that IFN-gamma has anti-inflammatory properties during the initial phase of AIA, and indicate that IFN-gamma deficiency exerts disease-promoting effects, preferentially via IL-17-modulated pathways.

Developmental variability during early embryonic development of zebra fish, Danio rerio.

Early vertebrate embryos pass through a period of remarkable morphological similarity. Possible causes for such similarity of early embryos include modularity, developmental constraints, stabilizing selection, canalization, and exhausted genetic variability. Supposedly, each process creates different patterns of variation and covariation of embryonic traits. We study the patterns of variation of the embryonic phenotype to test ideas about possible evolutionary mechanisms shaping the early embryonic development. We use the zebra fish, Danio rerio, as a model organism and apply repeated measures of individual embryos to study temporal changes of phenotypic variability during development. In particular, we are looking at the embryonic development from 12 hours post fertilization until 27 hours post fertilization. During this time period, the development of individual embryos is documented at hourly intervals. We measured maximum diameter of the eye, length of embryo, number of somites, inclination of somites, and the yolk size (as a maternal effect). The coefficient of variation (CV) was used as a measure of variability that was independent of size. We used a principal component analysis for analysis of morphological integration. The experimental setup kept environment x genotype interactions constant. Nongenetic parental contributions had no significant effects on interindividual variability. Thus all observed phenotypic variation was based on additive genetic variance and error variance. The average CV declined from 14% to 7.7%. The decline of the CV was in particular expressed during 15-19 h post fertilization and occurred in association with multiple correlations among embryonic traits and a relatively high degree of morphological integration. We suggest that internal constraints determine the patterns of variability during early embryonic development of zebra fish.