Miklós Kovács

The Src family kinases Hck, Fgr, and Lyn are critical for the generation of the in vivo inflammatory environment without a direct role in leukocyte recruitment

Kovács et al. examine the role of the Src family kinases Hck, Fgr, and Lyn in immune cell–mediated inflammation. Using arthritis and skin inflammation models, the authors show that mice lacking hematopoietic Hck, Fgr, and Lyn are protected from these inflammatory diseases, showing loss of myeloid cell recruitment and lack of inflammatory mediator production. Unexpectedly, the three kinases are dispensable for the intrinsic migratory ability of myeloid cells. These finding may have clinical implications in rheumatic and skin diseases.

Differential regulatory role of pituitary adenylate cyclase-activating polypeptide in the serum-transfer arthritis model.

Pituitary adenylate cyclase–activating polypeptide (PACAP) expressed in capsaicin‐sensitive sensory neurons and immune cells has divergent functions in inflammatory and pain processes. This study was undertaken to investigate the involvement of PACAP in a mouse model of rheumatoid arthritis.

Neutrophil Functions and Autoimmune Arthritis in the Absence of p190RhoGAP: Generation and Analysis of a Novel Null Mutation in Mice

β2 integrins of neutrophils play a critical role in innate immune defense, but they also participate in tissue destruction during autoimmune inflammation. p190RhoGAP (ArhGAP35), a regulator of Rho family small GTPases, is required for integrin signal transduction in fibroblasts. Prior studies have also suggested a role for p190RhoGAP in β2 integrin signaling in neutrophils. To directly test that possibility, we have generated a novel targeted mutation completely disrupting the p190RhoGAP-encoding gene in mice. p190RhoGAP deficiency led to perinatal lethality and defective neural development, precluding the analysis of neutrophil functions in adult p190RhoGAP−/− animals. This was overcome by transplantation of fetal liver cells from p190RhoGAP−/− fetuses into lethally irradiated wild-type recipients. Neutrophils from such p190RhoGAP−/− bone marrow chimeras developed normally and expressed normal levels of various cell surface receptors. Although p190RhoGAP−/− neutrophils showed moderate reduction of β2 integrin-mediated adherent activation, they showed mostly normal migration in β2 integrin-dependent in vitro and in vivo assays and normal β2 integrin-mediated killing of serum-opsonized Staphylococcus aureus and Escherichia coli. A neutrophil- and β2 integrin-dependent transgenic model of the effector phase of autoimmune arthritis also proceeded normally in p190RhoGAP−/− bone marrow chimeras. In contrast, all the above responses were completely blocked in CD18−/− neutrophils or CD18−/− bone marrow chimeras. These results suggest that p190RhoGAP likely does not play a major indispensable role in β2 integrin-mediated in vitro and in vivo neutrophil functions or the effector phase of experimental autoimmune arthritis.

Distortional effect of beam-hardening artefacts on microCT: a simulation study based on an in vitro caries model

OBJECTIVE The aim of this study was to assess quantitatively the degrading effect of artefacts caused by beam hardening on the microscopic computerized tomography (microCT) measurements of an in vitro caries model. STUDY DESIGN A simulation-based method was described, with which the degrading effect of microCT artefacts on certain parameters of the observed structure could be determined. Simulations were carried out with polychromatic and monochromatic X-ray source, and a linearization method with a second-order polynomial fit algorithm was used in specific cases to correct the beam hardening artefact. The virtual test object was a half-crown of a tooth with an artificial caries lesion. RESULTS For simulation with monochromatic X-ray source, the relative error of lesion depth and thickness measurements of the remineralized layer was found to be 1%-2%. For polychromatic X-ray source, and omitting beam hardening correction, the relative error exceeded 6%. After appropriate beam-hardening correction, the relative error of the measurement could be reduced to 1%-2%. CONCLUSION With the adjustment simulated in this study, microCT having polychromatic X-ray source resulted in the same level of error as with monochromatic source if the linearization method to correct the beam hardening was used. The presented simulation-based method is a useful way to determine artefact-caused distortions for other studies testing objects with different material and geometry.

Phosphoinositide 3-OH kinase regulates integrin-dependent processes in neutrophils by signaling through its effector ARAP3.

ARAP3, a GTPase activating protein for Rho and Arf family GTPases, is one of many phosphoinositide 3-OH kinase (PI3K) effectors. In this study, we investigate the regulatory input of PI3K upstream of ARAP3 by analyzing neutrophils from an ARAP3 pleckstrin homology (PH) domain point mutation knock-in mouse (R302, 303A), in which ARAP3 is uncoupled from activation by PI3K. ARAP3 PH domain point mutant neutrophils are characterized by disturbed responses linked to stimulation by either integrin ligands or immobilized immune complexes. These cells exhibit increased β2 integrin inside-out signaling (binding affinity and avidity), and our work suggests the disturbed responses to immobilized immune complexes are secondary to this. In vitro, neutrophil chemotaxis is affected in the mutant. In vivo, ARAP3 PH domain point mutant bone marrow chimeras exhibit reduced neutrophil recruitment to the peritoneum on induction of sterile peritonitis and also reduced inflammation in a model for rheumatoid arthritis. The current work suggests a dramatic regulatory input of PI3K into the regulation of β2 integrin activity, and processes dependent on this, by signaling through its effector ARAP3.

Urine/Plasma Neutrophil Gelatinase Associated Lipocalin Ratio Is a Sensitive and Specific Marker of Subclinical Acute Kidney Injury in Mice

Background Detection of acute kidney injury (AKI) is still a challenge if conventional markers of kidney function are within reference range. We studied the sensitivity and specificity of NGAL as an AKI marker at different degrees of renal ischemia. Methods Male C57BL/6J mice were subjected to 10-, 20- or 30-min unilateral renal ischemia, to control operation or no operation, and AKI was evaluated 1 day later by histology, immunohistochemistry, BUN, creatinine, NGAL (plasma and urine) and renal NGAL mRNA expression. Results A short (10-min) ischemia did not alter BUN or kidney histology, but elevated plasma and urinary NGAL level and renal NGAL mRNA expression although to a much smaller extent than longer ischemia. Surprisingly, control operation elevated plasma NGAL and renal NGAL mRNA expression to a similar extent as 10-min ischemia. Further, the ratio of urine to plasma NGAL was the best parameter to differentiate a 10-min ischemic injury from control operation, while it was similar in the non and control-operated groups. Conclusions These results suggest that urinary NGAL excretion and especially ratio of urine to plasma NGAL are sensitive and specific markers of subclinical acute kidney injury in mice.

Compromised bone healing following spacer removal in a rat femoral defect model

PURPOSE The clinical demand for bone grafting materials necessitated the development of animal models. Critical size defect model has been criticized recently, mainly for its inaccuracy. Our objective was to develop a dependable animal model that would provide compromised bone healing, and would allow the investigation of bone substitutes. METHODS In the first group a critical size defect was created in the femur of adult male Wistar rats, and a non-critical defect in the remaining animals (Groups II, III and IV). The defect was left empty in group II, while in groups III and IV a spacer was interposed into the gap. Osteoblast activity was evaluated by NanoSPECT/CT imaging system. New bone formation and assessment of a union or non-union was observed by μCT and histology. RESULTS The interposition model proved to be highly reproducible and provided a bone defect with compromised bone healing. Significant bone regeneration processes were observed four weeks after removal of the spacer. CONCLUSION Our results have shown that when early bone healing is inhibited by the physical interposition of a spacer, the regeneration process is compromised for a further 4 weeks and results in a bone defect during the time-course of the study.

A2.21 The role of HCK, FGR and LYN in in vivo inflammation in mice

Background and objectives Although Src family kinases participate in leukocyte function in vitro, such as integrin signal transduction, their role in inflammation in vivo is poorly understood. The role of Src family kinases in b2 integrin signalling and the requirement for b2 integrins during autoantibody-induced in vivo inflammation prompted us to test the role of Src family kinases in autoantibody-induced inflammatory disease models. Materials and methods To determine the role of Src family kinases in autoantibody-induced autoimmune diseases Hck-/-Fgr-/-Lyn-/- triple knockout and wild type mice were tested in autoantibody-induced K/B×N serum transfer arthritis, autoantibody-induced skin blistering disease and reverse passive Arthus reaction. Autoimmune disease development was carefully monitored, and inflammatory cytokine and chemokine production was determined in vivo and in vitro. Furthermore, in vivo migratory capacity of leukocytes was monitored in mixed radiation chimaeras. Results We found that Src family kinases play a critical role in myeloid cell–mediated in vivo inflammatory reactions. Mice lacking the Src family kinases Hck, Fgr, and Lyn in the hematopoietic compartment were completely protected from autoantibody-induced arthritis and skin blistering disease, as well as from the reverse passive Arthus reaction, with functional overlap between the three kinases. Though the overall phenotype resembled the leukocyte recruitment defect observed in b2 integrin–deficient (CD18-/-) mice, Hck-/-Fgr-/-Lyn-/- neutrophils and monocytes/macrophages had no cell autonomous in vivo or in vitro migration defect. Instead, Src family kinases were required for the generation of the inflammatory environment in vivo and for the release of proinflammatory mediators from neutrophils and macrophages in vitro, likely due to their role in Fcg receptor signal transduction. Conclusions Our results suggest that infiltrating myeloid cells release proinflammatory chemokine, cytokine, and lipid mediators that attract further neutrophils and monocytes from the circulation in a CD18-dependent manner. Src family kinases are required for the generation of the inflammatory environment but not for the intrinsic migratory ability of myeloid cells.

A2.22 Tyrosine Phosphorylation Pathways in Myeloid Cell-Mediated Inflammatory Diseases

Background Tyrosine kinases are major therapeutic targets in cancer but their role in immune-mediated disease pathogenesis is less understood. Methods Here we tested the role of tyrosine kinases and tyro sine kinase substrates in two autoantibody-mediated disease models, the K/BxN serum-transfer arthritis and anti-collagen VII autoantibody-induced blistering skin diseases. Results Mice genetically deficient of the Syk tyrosine kinase in the hematopoietic compartment were completely protected from autoantibody-induced arthritis and blistering skin disease. Mice lacking three myeloid-specific tyrosine kinases (Hck, Fgr and Lyn) or the tyrosine kinase substrate PLCγ 2 were also protected from autoantibody-induced diseases. In vitro, Src-family kinases were required for Syk activation by immune complexes and both were further required for activation of PLCγ 2. The Src-family–Syk–PLCγ 2 pathway mediated cytokine production by myeloid cells but not neutrophil or monocyte migration per se. Lineage-specific analyses revealed that during autoantibody-induced arhritis, this signalling pathway was required in myeloid cells (particular neutrophils) but not in mast cells or platelets. Finally, Src-family kinases were also required for activation of myeloid cells by monosodium urite crystals and their deficiency attenuated monosdium urate crystal-induced arthritis, indicating that the role of this signalling pathway is not restricted to autoantibody-mediated disease processes. Conclusions Taken together, the Src-family–Syk–PLCγ 2 pathway is an important component of both autoantibody-mediated and autoantibody-independent inflammatory disease processes.