Reduced Neutrophil Extracellular Trap (NET) Formation During Systemic Inflammation in Mice With Menkes Disease and Wilson Disease: Copper Requirement for NET Release.

Neutrophil extracellular traps (NETs) contribute to pathological disorders, and their release was directly linked to numerous diseases. With intravital microscopy (IVM), we showed previously that NETs also contribute to the pathology of systemic inflammation and are strongly deposited in liver sinusoids. Over a decade since NET discovery, still not much is known about the metabolic or microenvironmental aspects of their formation. Copper is a vital trace element essential for many biological processes, albeit its excess is potentially cytotoxic; thus, copper levels are tightly controlled by factors such as copper transporting ATPases, ATP7A, and ATP7B. By employing IVM, we studied the impact of copper on NET formation during endotoxemia in liver vasculature on two mice models of copper excess or deficiency, Wilson (ATP7B mutants) and Menkes (ATP7A mutants) diseases, respectively. Here, we show that respective ATP7 mutations lead to diminished NET release during systemic inflammation despite unaltered intrinsic capacity of neutrophils to cast NETs as tested ex vivo. In Menkes disease mice, the in vivo effect is mostly due to diminished neutrophil infiltration of the liver as unmutated mice with a subchronic copper deficiency release even more NETs than their controls during endotoxemia, whereas in Wilson disease mice, excess copper directly diminishes the capacity to release NETs, and this was further confirmed by ex vivo studies on isolated neutrophils co-cultured with exogenous copper and a copper-chelating agent. Taken together, the study extends our understanding on how microenvironmental factors affect NET release by showing that copper is not a prerequisite for NET release but its excess affects the trap casting by neutrophils.

Cerebellar superoxide dismutase expression in Menkes' kinky hair disease: an immunohistochemical investigation.

This comparative immunohistochemical study deals with the expression of the cytosolic Cu/Zn-binding and mitochondrial Mn-dependent superoxide dismutases (SODs) in the cerebella of five patients with Menkes' kinky hair disease (MKHD) and five age-matched controls. Several cell types, including Purkinje cells and reactive astrocytes, of all MKHD patients examined were intensely stained by an antibody to Mn SOD, but not by an anti-Cu/Zn SOD antibody. By contrast, the cells of the five controls reacted very weakly or not at all with the anti-Mn SOD antibody, but were strongly reactive with the antibody to Cu/Zn SOD. These results suggest that the increased Mn SOD immunoreactivity in MKHD reflects enzyme induction as a protective mechanism against the highly toxic superoxide anion generated under the disease conditions.

Evidence for serum factors modulating antibody production in normal and macular mice.

The macular mutant mouse is a model of Menkes' kinky hair disease, which is characterized by a deficiency of ceruloplasmin in the serum. In hemizygotic mice (ml/y), the antibody production against sheep red blood cells (SRBC) was decreased. Treatment with normal (+/y) mouse serum increased in a dose-dependent manner the antibody response of ml/y mouse spleen cells in vitro. However, +/y mouse serum had no effect on +/y mouse spleen cells, even at high doses up to 5%. In contrast, ml/y mouse serum decreased antibody production in +/y mouse spleen cells in a dose-dependent manner. Antibody production in +/y mouse spleen cells decreased with time, after pretreatment with ml/y mouse serum. However, serum from ml/y mice injected with ceruloplasmin did not decrease antibody production in +/y mouse spleen cells but rather increased it in ml/y mouse spleen cells. Ceruloplasmin had no effect on the production of antibodies against SRBC in ml/y and +/y mouse spleen cells in vitro. These findings suggest that one or more enhancers of antibody production exist in normal mouse serum and that one or more suppressors of normal antibody production exist in ml/y mouse serum. It is proposed that the activities of these factors in serum may be regulated by ceruloplasmin.

The decreased immune responses in macular mouse, a model of Menkes' kinky hair disease.

The macular mutant mouse is a murine model of the Menkes' kinky hair disease, characterized by a copper deficiency in serum. The immune response of its hemizygote (ml/y) was examined, herein. Ml/y mice which were not treated with Cu were atrophy of lymphoid tissues on day 14. However, kidney, brain, heart and lung weights were higher in ml/y mice without Cu treatment than in normal (+/y) mice. When compared to cells from +/y mice, spleen cells from ml/y mice exhibited similar proliferation-curves stimulated by Con A or LPS. Lymph node cells from ml/y mice showed a significantly decreased mixed lymphocyte reaction (MLR) response when stimulated by spleen cells from Balb/c mice. Spleen cells from ml/y mice demonstrated similar stimulation against lymph node cells from Balb/c mice. Antibody production against sheep red blood cells (SRBC) in vivo, a T cell dependent response, was suppressed in ml/y mice. By contrast, the antibody production against dinitrophenyl-ficoll, a T cell independent response was similar in +/y and ml/y. The antibody production against SRBC in vitro was also suppressed in ml/y mice. However, when the T cell-enriched fraction of ml/y mouse spleen cells was replaced by the T cell-enriched fraction of +/y mouse spleen cells, the antibody production against SRBC was recovered. The percentage of Ly-5-positive cells (B cell) from ml/y mice was greater than those from +/y mice. The percentage of Thy-1.2-positive cells (T cell) was decreased, and the decrease was most prominent with the L3/T4-positive T cell (helper T cell) subset.

Immunodeficiency and syndromes: a nosographic approach.

Immunodeficiency disorders can be classified on clinical grounds into two broad groups according to whether all features are the result of the immune defect (immunodeficiency syndromes) or whether many, even prominent, features cannot be explained by the immune defect (syndromes with immunodeficiency). X-linked agammaglobulinemia and X-linked chronic granulomatous disease are paradigmatic examples of immunodeficiency syndromes. Despite some overlap (for instance extra-immune symptoms, although minor, are present in several variants of severe combined immunodeficiency and chronic granulomatous disease) immunodeficiency syndromes and syndromes with immunodeficiency are easily distinguishable. Together with the pathogenetic classification of the WHO, the present approach to a clinical classification amplifies the operational concept of immunodeficiency also from a therapeutic point of view.