C3d Elicits Neutrophil Degranulation and Decreases Endothelial Cell Migration, with Implications for Patients with Alpha-1 Antitrypsin Deficiency.

Tomás P Carroll,Noel G Mcelvaney,Michelle Casey,Laura T Fee,Emer P Reeves,Emer Mchugh,Michael E O’Brien,Ciara Gough,Ann M Hopkins,Mark Murphy,Debananda Gogoi

doi:10.3390/biomedicines9121925

Abstract

Alpha-1 antitrypsin (AAT) deficiency (AATD) is characterized by increased risk for emphysema, chronic obstructive pulmonary disease (COPD), vasculitis, and wound-healing impairment. Neutrophils play a central role in the pathogenesis of AATD. Dysregulated complement activation in AATD results in increased plasma levels of C3d. The current study investigated the impact of C3d on circulating neutrophils. Blood was collected from AATD (n = 88) or non-AATD COPD patients (n = 10) and healthy controls (HC) (n = 40). Neutrophils were challenged with C3d, and degranulation was assessed by Western blotting, ELISA, or fluorescence resonance energy transfer (FRET) substrate assays. Ex vivo, C3d levels were increased in plasma (p < 0.0001) and on neutrophil plasma membranes (p = 0.038) in AATD compared to HC. C3d binding to CR3 receptors triggered primary (p = 0.01), secondary (p = 0.004), and tertiary (p = 0.018) granule release and increased CXCL8 secretion (p = 0.02). Ex vivo plasma levels of bactericidal-permeability-increasing-protein (p = 0.02), myeloperoxidase (p < 0.0001), and lactoferrin (p < 0.0001) were significantly increased in AATD patients. In endothelial cell scratch wound assays, C3d significantly decreased cell migration (p < 0.0001), an effect potentiated by neutrophil degranulated proteins (p < 0.0001). In summary, AATD patients had increased C3d in plasma and on neutrophil membranes and, together with neutrophil-released granule enzymes, reduced endothelial cell migration and wound healing, with potential implications for AATD-related vasculitis.

Highlights

Alpha-1 antitrypsin (AAT) is an abundant plasma protein that plays a key role in the innate immune response
The mean C3d concentration was significantly increased in ZZ-AAT deficiency (AATD) (~2 μg/mL) compared to MM-healthy controls (HC) (p < 0.0001), confirming that complement activation and C3d production are increased in ZZ-AATD individuals (Figure 1A)
MM-chronic obstructive pulmonary disease (COPD) patients compared to ZZ-AATD (p < 0.0001), indicating that the increase in complement activation is related to a lack of AAT, as previously reported [19]

Summary

Introduction

Alpha-1 antitrypsin (AAT) is an abundant plasma protein that plays a key role in the innate immune response. The majority of circulating AAT is produced by hepatocytes and is generated as a single polypeptide chain that is post-translationally modified by glycosylation through the addition of N–glycosidically linked oligosaccharides [1,2]. AAT deficiency (AATD) is an autosomal codominant disorder resulting in an increased risk for the development of emphysema, panacinar in pathology, often by the third or fourth decade. AATD is caused by mutation of the SERPINA1 gene [3]. The most common mutations known to cause AATD are the Z (Glu342Lys) and S (Glu264Val) mutations. There are over 100 disease-causing mutations reported to date [4–6]. The Z mutation occurs in approximately 95% of individuals with severe AATD [7].

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