Ziel dieser Arbeit war es herauszufinden, ob es sich bei der leukozytoklastischen Zelldesintegration der neutrophilen Granulozyten in kutaner leukozytoklastischer Vaskulitis (kLV) um Apoptose handelt. Um die zugrundeliegenden Mechanismen dieses Zellzerfalls näher zu charakterisieren, wurden Hautbiopsien von 9 Patienten mit klinisch und histologisch gesicherter kLV und 13 Hautbiopsien von Patienten mit kLV folgend auf intrakutane Histamin-Injektion im zeitlichen Verlauf untersucht. Licht und elektronenmikroskopisch wurde die Morphologie der Leukozytoklasie beurteilt. Immunhistologische Untersuchungen beinhalteten klassische pro-apoptotische Rezeptoren (CD95/CD95L), desweiteren über die Mitochondrien (Bax/Bcl-2) und auf genetischer Ebene die Apoptose regulierende Proteine (p53/p21). Es wurde zusätzlich die Expression von Zelloberflächen-Rezeptoren (FcgammaRIII/ FcgammaRII) beurteilt. Mittels der TUNEL-Methode wurde versucht die klassische DNA-Fragmentierung nachzuweisen. Obwohl strukturell und ultrastrukturell die klassischen morphologischen Merkmale der Apoptose vorlagen, konnte diese biochemisch nicht untermauert werden. DNA-Fragmentierung liess sich nur in schon von Makrophagen phagozytierten Kernfragmenten nachweisen. Neutrophile waren negativ und ihre Fragmente waren es vorwiegend. Den untersuchten Rezeptoren CD95/CD95L kommt allenfalls eine geringe und den Proteine, Bax/Bcl-2 und p53/p21 keine Rolle im Zellzerfall der Neutrophilen in dieser selbstlimitierten Entzündung zu. Interessanterweise wurde jedoch FcgammaRIII sehr stark auf der Oberfläche nicht nur intakter, sondern auch schon fragmentierter neutrophiler Granulozyten exprimiert. Dementsprechend ist anzunehmen, dass diese konstitutionell exprimierten Rezeptoren nicht nur eine Rolle in der Aktivierung, sondern auch eine Rolle in der darauf folgenden Auslösung der Apoptose in diesen Zellen spielen. Desweiteren dienen diese stark glykosylierten Rezeptoren möglicherweise als Phagozytose-Erkennungsmerkmal für Makrophagen. FcgammaRII liess sich hingegen nicht auf der Oberfläche der Neutrophilen und ihrer Fragmente nachweisen. Dies könnte einerseits durch eine Verdrängung durch FcgammaRIII bedingt sein, andrerseits könnten sie durch Immnunkomplex-Bindung eine separate oder zusätzliche Rolle in der Induktion des Zellzerfalls spielen. Zusammenfassend weist der leukozytoklastische Zellzerfall der Neutrophilen in kLV die klassischen morphologischen Merkmale der Apoptose auf, die biochemisch jedoch auf atypische Weise abläuft. In dieser Hinsicht zu erwähnen, ist vor allem die verzögerte oder durch DNasen der Makrophagen bedingte DNA-Fragmentierung. / The aim of this study was to find out whether leukocytoklastic cell desintegration of neutrophils in cutaneous leukocytoklastic vasculitis (clv) is apoptosis. In order to find out more about the underlying mechanisms leading to this form of cell desintegration and subsequent cell death skin sections of 9 patients with clinically and histologically verified clv and of 13 patients with clv after intradermal injection of histamine were examined at different timepoints. The structural and ultrastructural morphological features of leukoctyoklasia were evaluated light- and electronmicroscopically. Immunhistochemistry included well known death receptors (CD95/CD95L), mitochondrial proteins (Bax/Bcl-2), proteins interfering with the cell cycle (p53/p21) and cell surface receptors (FcgammaRIII/FcgammaRII). Furthermore DNAfragmentation was assessed by the TUNEL-method. Although the structural and ultrastructural features of leukocytoklastic cell desintegration of neutrophils in clv revealed typical apoptotic morphology, the underlying biochemistry didn`t match apoptotic means. DNA-fragmentation was only seen in a minor fraction of fragmented nuclei and prominent in already phagocytosed debris. Neutrophils at different locations (in the lumen of the postcapillary venules, within the wall of the blood vessels and in the perivascular tissue) were always negative.The examined death receptors, mitochondrial proteins and DNA-interfering proteins play no (Bcl-2/Bax, p53/p21) or only a minor (CD95/CD95L) role in the biochemical mechanism leading to the cell desintegration of these cells in this self-limited inflammation. Interestingly FcgammaRIII was highly expressed not only in intact but also extremely desintegrated neutrophils as well as on the surface of their fragments. One could therefore assume that this receptor not only plays a role in activation of these cells, but also might be involved in the following induction of the cell death programm. Maybe this highly glycosylated receptor also functions as an `eat me flag` in the recognition-mechansim by macrophages. FcgammaRII could not be shown on the cell surface of these cells in this context, which on the one hand could be due to the high expression of FcgammaRIII with no room left for FcgammaRII on the cell surface or due to a separate or additional role in the induction of the cell death programm. Finally leukocytoklastic cell desintegration shows strong resemblance to apoptosis but with non classical underlying biochmical mechanisms, most importantly to mention the delay in DNA-fragmentation which could also be due to DNAdegradation by DNases of macrophages.

No description available.

Monoklonale Antikörper (mAk) werden bereits erfolgreich zur therapeutischen Depletion verschiedener Zellpopulationen in vivo verwendet, die Mechanismen der Depletion sind jedoch unklar geblieben. In dieser Arbeit wurden im Mausmodell die molekularen Grundlagen der CD4+ T-Zelldepletion (CD4 TZD) nach einmaliger Gabe (i.p.) von 100 µg des anti-CD4-mAk YTS191.1 untersucht. Dabei konnte eine starke Korrelation zwischen Depletion und der Modulation des CD4-Moleküls von der Oberfläche beobachtet werden. Gleichzeitig zeigten sich organabhängige Unterschiede, sowohl im zeitlichen Verlauf, als auch in der Effizienz der Depletion. Im Thymus konnten weder Depletion noch Modulation detektiert werden, in Milz und Lymphknoten (Lk) war die CD4 TZD nach starker CD4-Modulation bereits nach 48 h mit 80-90 % maximal, in den Peyer-Plaques jedoch niedriger und verzögert (50-60 % nach 72 h). Anhand C3-defizienter Mäuse konnte ferner kein wesentlicher Beitrag von Komplement an der CD4 TZD beobachtet werden. Im Gegensatz dazu konnte durch die Verwendung verschiedener FcGamma-Rezeptor (FcGammaR)-defizienter Mäuse (FcGammaRI, FcGammaRII, FcGammaRIII, FcGammaRI/III und FcRGamma) wie auch durch die Blockade des FcGammaRIV eine starke, zudem organabhängige Beteiligung von FcGammaR an der CD4 TZD gezeigt werden. Während in der Milz die CD4 TZD von FcGammaRIV vermittelt wurde, waren in den Lk und Peyer-Plaques FcGammaRI/III involviert. Diese Befunde korrelierten mit der starken Expression von FcGammaRIV in Milz, Lunge, Darm, Niere und Leber, während in den Lk nur eine schwache und in Thymus und Peyer-Plaques keine Expression detektiert werden konnte. Innerhalb der Milz konnten erstmalig F4/80hoch Makrophagen als FcGammaRIV+ identifiziert und somit als potenzielle Effektorzellen der CD4 TZD bestimmt werden. Der direkte Vergleich der Depletion von CD4+ T-Zellen mit der Depletion von ICOS+ T-Zellen verdeutlichte darüber hinaus, dass die Effizienz der Zelldepletion nicht nur von den Eigenschaften des verwendeten mAk, sondern auch von denen des Zielmoleküls abhängig ist. / Monoclonal antibodies (mAb) are efficiently used for the therapeutic depletion of various cells in vivo yet the mechanisms of depletion are still unclear. In this work, the molecular principles of CD4+ T cell depletion (CD4 Tcd) by a single application of 100 µg of the anti-CD4 mAb YTS191.1.1 were investigated in the mouse. A strong correlation between the depletion and the surface modulation of the CD4 molecule could be observed. At the same time, organ-dependent differences in the kinetics as well as in the efficiency of depletion could be detected. In the thymus, neither modulation nor depletion were detectable. In the spleen and the lymph nodes (Ln), the modulation was strong and the depletion was maximal (80-90%) 48 h after mAb treatment. Interestingly, both modulation and depletion were decreased and delayed (50-60% after 72 h) in the Peyer`s patches. By using C3-deficient mice, no major contribution of complement to the CD4 Tcd was seen. On the contrary, with the help of different FcGamma-receptor (FcGammaR)-deficient mice (FcGammaRI, FcGammaRII, FcGammaRIII, FcGammaRI/III, and FcRGamma) and through the blockade of FcGammaRIV, a strong organ dependent involvement of FcGammaR could be shown. While the depletion in the spleen was clearly dependent on FcGammaRIV, in the Ln and the Peyer`s patches, FcGammaRI/III were involved. These findings correlated with the strong expression of FcGammaRIV in the spleen, the lung, the colon, the kidney, and the liver, while in the Ln the expression was weak and undetectable in the thymus and the Peyer`s patches. For the first time, F4/80high macrophages in the spleen could be identified as also being FcGammaRIV+, and are therfore considered as the potential effector cells of the CD4 Tcd. The direct comparison of the depletion of T cells via CD4 or ICOS pointed out that the target cell depletion is not only dependent on the properties of the mAb used, but also on those of the target molecule.

O lúpus eritematoso sistêmico (LES) é uma doença autoimune e a suscetibilidade às infecções está associada as suas anormalidades imunológicas e a sua terapia imunossupressora e citotóxica. Dentre as alterações que predispõem às infecções no LES estão anormalidades moleculares e funcionais dos neutrófilos: clearance e fagocitose ineficientes de imunocomplexos (IC) e bactérias, neutropenia, defeitos na quimiotaxia, redução do burst oxidativo e redução da expressão de receptores para IgG (Fc?R) e para complemento (CR). Além disso, os polimorfismos genéticos dos Fc?R têm sido associados com as disfunções imunes do LES. Os Fc?R são importantes mediadores das funções efetoras do neutrófilo e atuam em sinergismo com os CR (CR1 e CR3). O polimorfismo dos genes Fc?RIIA e Fc?RIIIB determina a expressão de variantes alélicas com diferenças funcionais, as quais podem influenciar as respostas biológicas e a suscetibilidade e o prognóstico das doenças infecciosas. Em particular, o alótipo Fc?RIIa-R131, tem menor afinidade para a IgG2, o que resulta em prejuízo na fagocitose mediada por esta imunoglobulina. A IgG2 é essencial contra bactérias encapsuladas e os pneumococos são responsáveis por 6-18% das infecções bacterianas no LES. O objetivo deste estudo foi investigar a influência dos polimorfismos genéticos dos Fc?R na fagocitose e desgranulação dos neutrófilos, associados ao polimorfismo do CR3, e à ocorrência de infecções bacterianas no LES. Os genótipos foram determinados por reações da polimerase em cadeia para os polimorfismos das variantes alélicas; a fagocitose e desgranulação foram estimuladas por IC contendo IgG (IC-IgG) e por IC-IgG e complemento (IC-IgG/SHN); a fagocitose de IC por neutrófilos e a expressão dos Fc?R e CR3 foram avaliadas por citometria de fluxo; e a desgranulação dos neutrófilos estimulada por IC foi medida pela liberação de elastase e lisozima. Os resultados mostraram: maior frequência para o genótipo R-131 no LES; associação do genótipo HNA-4a negativo para o CR3 com a suscetibilidade para o LES e associação do HNA-4a positivo com proteção; fagocitose menor em neutrófilos de pacientes com LES com genótipo HR-131 comparados aos neutrófilos do grupo controle com genótipo R-131 (IC-IgG e IC-IgG/SHN); no polimorfismo do Fc?RIIIb, a fagocitose e a lisozima foram menores em neutrófilos de pacientes com LES com genótipo HNA-1b e maior para HNA-1a/1b comparados aos controles com os respectivos genótipos (IC-IgG/SHN); a ocorrência de infecções foi mais frequentemente associada à presença do alelo R-131 e HNA-1b; nenhuma diferença foi observada para o polimorfismo HNA-4a do CR3, bem como para e elastase. Este estudo contribui para o entendimento das anormalidades nas funções dos neutrófilos no LES e para a identificação de indivíduos, cujo polimorfismo dos Fc?R e CR3 possa conferir suscetibilidade ou proteção às infecções. / Systemic lupus erythematosus (SLE) is an autoimmune disease in which disease-related and genetic factors and immunosuppressive and cytotoxic therapies all contribute to an increased susceptibility to infections. Factors predisposing to infection include defects in chemotaxis, abnormalities in neutrophil phagocytic activity, decreased immune complex (IC) and bacteria clearance, neutropenia, reduced oxidative burst, abnormalities in the expression of Fc?R (Fc?R) and complement (CR) receptors. Recent data have provided evidence that genetic polymorphism of Fc?R is associated with immune abnormalities and risk to development of SLE. Fc?R can mediate neutrophil effector functions and play a synergistic action with CR. Fc?RIIa and Fc?RIIIb display functionally relevant genetic polymorphisms, which allelic variants can influence the biological responses and the susceptibility to and course of infectious diseases. In particular, the presence of the Fc?RIIa-R131 allotype results in lower affinity binding to IgG2, a subclass of IgG specific for encapsulated bacteria. Since pneumococci accounts for 6-18% of all bacterial infections in SLE, the R131 allele can be relevant as a risk factor for infections. The aim of this study was to investigate the influence of the Fc?R polymorphisms on the phagocytosis and degranulation of neutrophils, associated with CR3 polymorphism, and bacterial infections in SLE. Genotypes were determined by polymerase chain reactions for the polymorphisms of the allelic variants; the phagocytosis and the degranulation were stimulated with IC-containing IgG (IC-IgG) and IC-IgG and complement (IC-IgG/NHS); the phagocytosis of IC by neutrophils and expression of Fc?R and CR3 were evaluated by flow cytometry, and degranulation of neutrophils stimulated with IC was measured by the release of lysozyme and elastase. The results showed a higher frequency for the genotype R-131 in SLE and association of genotype HNA-4a negative for the CR3 with susceptibility to SLE and association of HNA-4a with protection; the phagocytosis was lower in neutrophils from patients with SLE with HR-131 genotype than those in neutrophils from control group with genotype R-131 (IC-IgG and IC-IgG/NHS); in the Fc?RIIIb polymorphism, the phagocytosis and lysozyme were lower in neutrophils from patients with SLE with genotype HNA-1b and higher for HNA-1a/1b than those controls with the respective genotypes (IC-IgG/NHS); the occurrence of infections was more frequently associated with the presence of the allele R-131 and HNA-1b; no difference was observed for polymorphism HNA-4a of the CR3, neither for elastase. This study can contribute for the understanding of neutrophil abnormalities in SLE and identifying genetic markers that would predict patients Who are at high risk for infections.

O lúpus eritematoso sistêmico (LES) é uma doença autoimune e a suscetibilidade às infecções está associada as suas anormalidades imunológicas e a sua terapia imunossupressora e citotóxica. Dentre as alterações que predispõem às infecções no LES estão anormalidades moleculares e funcionais dos neutrófilos: clearance e fagocitose ineficientes de imunocomplexos (IC) e bactérias, neutropenia, defeitos na quimiotaxia, redução do burst oxidativo e redução da expressão de receptores para IgG (Fc?R) e para complemento (CR). Além disso, os polimorfismos genéticos dos Fc?R têm sido associados com as disfunções imunes do LES. Os Fc?R são importantes mediadores das funções efetoras do neutrófilo e atuam em sinergismo com os CR (CR1 e CR3). O polimorfismo dos genes Fc?RIIA e Fc?RIIIB determina a expressão de variantes alélicas com diferenças funcionais, as quais podem influenciar as respostas biológicas e a suscetibilidade e o prognóstico das doenças infecciosas. Em particular, o alótipo Fc?RIIa-R131, tem menor afinidade para a IgG2, o que resulta em prejuízo na fagocitose mediada por esta imunoglobulina. A IgG2 é essencial contra bactérias encapsuladas e os pneumococos são responsáveis por 6-18% das infecções bacterianas no LES. O objetivo deste estudo foi investigar a influência dos polimorfismos genéticos dos Fc?R na fagocitose e desgranulação dos neutrófilos, associados ao polimorfismo do CR3, e à ocorrência de infecções bacterianas no LES. Os genótipos foram determinados por reações da polimerase em cadeia para os polimorfismos das variantes alélicas; a fagocitose e desgranulação foram estimuladas por IC contendo IgG (IC-IgG) e por IC-IgG e complemento (IC-IgG/SHN); a fagocitose de IC por neutrófilos e a expressão dos Fc?R e CR3 foram avaliadas por citometria de fluxo; e a desgranulação dos neutrófilos estimulada por IC foi medida pela liberação de elastase e lisozima. Os resultados mostraram: maior frequência para o genótipo R-131 no LES; associação do genótipo HNA-4a negativo para o CR3 com a suscetibilidade para o LES e associação do HNA-4a positivo com proteção; fagocitose menor em neutrófilos de pacientes com LES com genótipo HR-131 comparados aos neutrófilos do grupo controle com genótipo R-131 (IC-IgG e IC-IgG/SHN); no polimorfismo do Fc?RIIIb, a fagocitose e a lisozima foram menores em neutrófilos de pacientes com LES com genótipo HNA-1b e maior para HNA-1a/1b comparados aos controles com os respectivos genótipos (IC-IgG/SHN); a ocorrência de infecções foi mais frequentemente associada à presença do alelo R-131 e HNA-1b; nenhuma diferença foi observada para o polimorfismo HNA-4a do CR3, bem como para e elastase. Este estudo contribui para o entendimento das anormalidades nas funções dos neutrófilos no LES e para a identificação de indivíduos, cujo polimorfismo dos Fc?R e CR3 possa conferir suscetibilidade ou proteção às infecções. / Systemic lupus erythematosus (SLE) is an autoimmune disease in which disease-related and genetic factors and immunosuppressive and cytotoxic therapies all contribute to an increased susceptibility to infections. Factors predisposing to infection include defects in chemotaxis, abnormalities in neutrophil phagocytic activity, decreased immune complex (IC) and bacteria clearance, neutropenia, reduced oxidative burst, abnormalities in the expression of Fc?R (Fc?R) and complement (CR) receptors. Recent data have provided evidence that genetic polymorphism of Fc?R is associated with immune abnormalities and risk to development of SLE. Fc?R can mediate neutrophil effector functions and play a synergistic action with CR. Fc?RIIa and Fc?RIIIb display functionally relevant genetic polymorphisms, which allelic variants can influence the biological responses and the susceptibility to and course of infectious diseases. In particular, the presence of the Fc?RIIa-R131 allotype results in lower affinity binding to IgG2, a subclass of IgG specific for encapsulated bacteria. Since pneumococci accounts for 6-18% of all bacterial infections in SLE, the R131 allele can be relevant as a risk factor for infections. The aim of this study was to investigate the influence of the Fc?R polymorphisms on the phagocytosis and degranulation of neutrophils, associated with CR3 polymorphism, and bacterial infections in SLE. Genotypes were determined by polymerase chain reactions for the polymorphisms of the allelic variants; the phagocytosis and the degranulation were stimulated with IC-containing IgG (IC-IgG) and IC-IgG and complement (IC-IgG/NHS); the phagocytosis of IC by neutrophils and expression of Fc?R and CR3 were evaluated by flow cytometry, and degranulation of neutrophils stimulated with IC was measured by the release of lysozyme and elastase. The results showed a higher frequency for the genotype R-131 in SLE and association of genotype HNA-4a negative for the CR3 with susceptibility to SLE and association of HNA-4a with protection; the phagocytosis was lower in neutrophils from patients with SLE with HR-131 genotype than those in neutrophils from control group with genotype R-131 (IC-IgG and IC-IgG/NHS); in the Fc?RIIIb polymorphism, the phagocytosis and lysozyme were lower in neutrophils from patients with SLE with genotype HNA-1b and higher for HNA-1a/1b than those controls with the respective genotypes (IC-IgG/NHS); the occurrence of infections was more frequently associated with the presence of the allele R-131 and HNA-1b; no difference was observed for polymorphism HNA-4a of the CR3, neither for elastase. This study can contribute for the understanding of neutrophil abnormalities in SLE and identifying genetic markers that would predict patients Who are at high risk for infections.

No description available.