A Preliminary Study On The Role Of Neutrophil And Its FcγRIIA In The Pathogenesis Of Some Vasculitides

Vasculitis is histologically defined as inflammatory cell infiltration and destruction of blood vessels. The majority of cutaneous lesions of vasculitis are likely due to immune complexes (IC) deposition/typeⅢhypersensitivity reactions. There is compelling animal and human experimental evidence that leukocytoclastic vasculitis is a hypersensitivity vasculitis, similar in nature to the experimental Arthus reaction. The immune complexes and activated neutrophils represent a key factor in this process.Neutrophils express many recognition receptors. Among these receptors, cell receptors for Fc domain of IgG antibodies (FcγR), serve as a bridge between innate and adaptive immunity, and play a major role in mediating both antibody-dependent cell-mediated cytotoxicity (ADCC) and immunophagocytosis. Recent studies using gene knockout mice have demonstrated that FcγR may also play a critical role in IC-mediated tissue injury, such as experimental Arthus reaction and vasculitis.Part one: Degranulation response of peripheral neutrophils from patients with Behcet's disease to aggregated IgG and the role of Fcgamma receptor in the degranulationBeh(?)et's disease (BD) is a recurrent multisystem vasculitis that can affect all sizes of blood vessels. Although the details of the pathogenesis of Beh(?)et's disease are not entirely understood, the immune complexes and hyperfunction of neutrophils are related to its inflammation and vascular injuries. In addition, degranulation response of neutrophils is one of the major source of inflammation.In part one, the aims of this study were several-fold. Firstly, we wanted to investigate degranulation response of neutrophils from patients with Behcet's disease upon stimulation with aggregated IgG (aIgG,stimulant for FcγR). Secondly, we sought to explore the role of Fc gamma receptor in neutrophil degranulation via these immunoglobulin aggregates. Finally, we wanted to investigate the mRNA expression of Fc gamma RⅡA (FcγRⅡA) on polymorphonuclear leukocyte (PMN) from patients with Beh(?)et's disease (BD).We developed an in vitro system, in which neutrophils in whole blood, were stimulated with either aIgG in the presence or absence of blocking anti-FcγRⅡand/or blocking anti-FcγRⅢor fMet-Leu-Phe (fMLP) supplemented with cytochalasin B(as control ), and assessed for degranulation by measuring the release of elastase with ELISA after 2h incubation. We found a highly significant difference in elastase release in response to aIgG when patients with Beh(?)et's disease were compared with healthy controls (P<0.05).In addition, blockade of FcγRⅢhad no effect both in BD patients and healthy controls (P>0.05), whereas anti-FcγRⅡfully attenuated the elastase release in response to aIgG,irrespective of patients and healthy controls (P< 0.01). Again, stimulation with fMLP (in the presence of cytochalasin B) increased the elastase release in response to aIgG both in patients and in healthy controls (P<0.01). Our results showed that the degranulation response of neutrophils from patients with Beh(?)et's disease upon stimulation with aIgG was significantly more pronounced compared with healthy controls; this degranulation was dependent on FcγRⅡ.Theseresults indicate that the involvement of neutrophils in the pathogenesis of Beh(?)et's disease through the interaction of FcγR and inmmune complexes.Part two: The mRNA expression of Fc gamma RⅡA on neutrophilsfrom patients with Behcet's disease and its significanceFcγR plays a critical role in mediating IC-mediated diseases (such as vasculitis, arthritis). In our study of part one, the results showed that degranulation response of neutrophils from patients with Beh(?)et's disease(BD) upon stimulation with aIgG was significantly more pronounced compared with healthy controls, and the degranulation was dependent on FcγRⅡ.To investigate the mRNA expression of Fc gamma RⅡA (FcγRⅡA) on polymorphonuclear leukocyte (PMN) from patients with BD, PMN were separated from the blood samples of 25 patients with active BD before treatment,15 patients with inactive BD after treatment, and 20 healthy controls. FcγRⅡA mRNA expression levels on PMN was detected by RT-PCR. Plasma myeloperoxidase (MPO) activity representing neutrophil activation, was measured spectrophotometrically. Our results showed that the expression level of FcγRⅡA on PMN and plasma MPO activity were significantly higher in patients with active BD than that in healthy controls or patients with inactive BD (P<0.01 ) , while there was no significant difference between the groups of patients with inactive BD and healthy controls (P >0.05). There were positive correlations between the expression level of FcγRⅡA on PMN and plasma MPO activity in patients with BD(r=0.391, P<0.01). These results indicate that the mRNA expression of FcγRⅡA on neutrophils increases during exacerbation of BD. It may suggest the involvement of FcγRⅡA in the activation of neutrophils in BD.Part three: Changes of adhesion molecules CD11b expression of neutrophil in peripheral and plasma activites of elastase from patients with Henoch-Sch(?)nlein purpura and its relation with diseaseactivityHenoch-Sch(?)nlein purpura (HSP) is a leukocytoclastic vasculitis with predominantly neutrophil infiltration histopathologically. Although the pathogenesis of HSP has not been clearly defined, evidences imply that neutrophil activation plays an important role in the pathogenesis of HSP. Some of the HSP patients (especially in patients with renal involvement) had 1 or more recurrences of the symptoms. There is no pathognomonic test for the diagnosis and follow-up of the disease activity in patients with HSP. The objectives in part three were to analyze the level of expression of neutrophil adhering molecules such as CD11b, and plasma levels of elastase as neutrophil activation makers in patients with HSP at the onset of the disease andduring remission. The expression of CD11b was determined by flow cytometric methods in 12 HSP at the onset of the disease and during remission in comparison with 12 healthy controls.The plasma levels of elastase were also measured by ELISA in 20 HSP at the onset of the disease and during remission in comparison with 20 healthy controls. Our results showed that patients in active stage had enhanced expression of CD11b(P< 0.01 ), and significantly higher elastase levels (P< 0.01) than patients during remission. Compared to healthy controls, patients during remission had no differences in the expression of CD11b (P > 0.05) while had higher elastase levels (P< 0.05). Significantly positive correlation was found between the expression of CD11b and elastase activity 0=0.732,P=0.007) at disease onset, no correlaton (r=0.196,P=0.541) during remission. These results indicate that neutrophils of peripheral blood in HSP are more activated at active stage than that during remission, suggesting an indicative role of the disease activity by analyzing the expression of CD11b of neutrophil and plasma levels of elastase.