Neutrophils are indispensable in the innate immune defense against invading microorganisms. Neutrophils contain SVs and several subsets of granules that are essential for their function. Proteins present in neutrophil SVs and granules are synthesized during terminal granulopoiesis in the bone marrow. The heterogeneity of granules, as determined by marker proteins characteristic of each granule subset, is thought to result from differences in the biosynthetic windows of major classes of granule proteins, a process referred to as targeting by timing. Qualitative proteomic analysis of neutrophil granules, SVs, and plasma membrane has been performed before. Here, we performed subcellular fractionation on freshly isolated human neutrophils by nitrogen cavitation and density centrifugation on a four-layer Percoll gradient. Granule subsets were pooled and subjected to SDS-PAGE, and gel pieces were in-gel-digested with trypsin. The resulting peptides were analyzed using LTQ Orbitrap XL tandem MS. A total of 1292 unique proteins were identified and grouped, according to the neutrophil fraction, in which they displayed maximal expression. In addition to various known neutrophil proteins, several uncharacterized proteins were found, as well as proteins not described previously in neutrophils. To study the correlation between mRNA expression in neutrophil precursors and the localization of their cognate proteins, the distribution of 126 identified proteins was compared with their mRNA expression profiles. The neutrophil subcellular proteome profiles presented here may be used as a database in combination with the mRNA array database to predict and test the presence and localization of proteins in neutrophil granules and membranes.

Papillon-Lefèvre syndrome (PLS) results from mutations that inactivate cysteine protease cathepsin C (CTSC), which processes a variety of serine proteases considered essential for antimicrobial defense. Despite serine protease-deficient immune cell populations, PLS patients do not exhibit marked immunodeficiency. Here, we characterized a 24-year-old woman who had suffered from severe juvenile periodontal disease, but was otherwise healthy, and identified a homozygous missense mutation in CTSC indicative of PLS. Proteome analysis of patient neutrophil granules revealed that several proteins that normally localize to azurophil granules, including the major serine proteases, elastase, cathepsin G, and proteinase 3, were absent. Accordingly, neutrophils from this patient were incapable of producing neutrophil extracellular traps (NETs) in response to ROS and were unable to process endogenous cathelicidin hCAP-18 into the antibacterial peptide LL-37 in response to ionomycin. In immature myeloid cells from patient bone marrow, biosynthesis of CTSC and neutrophil serine proteases appeared normal along with initial processing and sorting to cellular storage. In contrast, these proteins were completely absent in mature neutrophils, indicating that CTSC mutation promotes protease degradation in more mature hematopoietic subsets, but does not affect protease production in progenitor cells. Together, these data indicate CTSC protects serine proteases from degradation in mature immune cells and suggest that neutrophil serine proteases are dispensable for human immunoprotection.

Objective. To quantify immunoglobulin and C1q on circulating cell-derived microparticles (MPs) in patients with systemic lupus erythematosus (SLE) and to determine whether immunoglobulin and C1q levels are correlated with clinical and serologic parameters.Methods. Sixty-eight clinically well-characterized SLE patients, 38 healthy controls, 6 patients with systemic sclerosis (SSc), and 6 patients with rheumatoid arthritis (RA) were included. The numbers of annexin V-binding MPs displaying IgG, IgM, or C1q were enumerated by flow cytometry. MP protein levels were determined by mass spectrometry in clinically defined subsets of SLE patients and controls. The MP IgG load was determined by flow cytometric analysis of all samples from SLE patients and healthy controls.Results. SLE patients had significantly increased total and relative numbers of IgG-positive MPs (P ‫؍‬ 0.0004), with a much higher average IgG load per MP (P < 0.0001) than healthy controls. Quantitative mass spectrometry of purified MPs verified significantly increased IgG, IgM, and C1q levels in SLE patients. In RA and SSc patients, the average IgG load per MP was significantly lower than in SLE patients (P ‫؍‬ 0.006 and P ‫؍‬ 0.05, respectively). Also, the IgM load and C1q load per MP were significantly higher in SLE patients than in the control groups (P < 0.05), except for IgM in the RA group. IgG-positive MPs were significantly associated with the presence of anti-double-stranded DNA, anti-extractable nuclear antigen, and antihistone antibodies, with total IgG, and with decreased leukocyte counts. Average IgG load per MP was associated with lower concentrations of MPs, the presence of anti-C1q antibodies, and complement consumption. Systemic lupus erythematosus (SLE) is an autoimmune disease with a wide range of clinical manifestations (1). One of the serologic hallmarks of SLE is the presence of circulating, high-affinity autoantibodies against nuclear constituents (2). These autoantibodies may form circulating proinflammatory immune complexes (ICs) that directly trigger plasmacytoid dendritic cells (PDCs) and the complement system, e.g., in the kidneys (3). Also, IC and autoantibody activation of SLE neutrophils prone to NETosis, i.e., specialized neutrophil cell death, contributes to the sustained PDC activation that is typical of SLE (4,5). The etiology of antinuclear autoimmunity in SLE remains unclear, but persistent, poorly cleared circulating cellular remnants, including microparticles (MPs) and neutrophil extracellular traps, are likely sources of immunogenic autoantigens (6-9). Conclusion. Our findings indicate that circulating cell-derived MPs in SLE patients

Objective. Characterization of the abundance, origin, and annexin V (AnxV)-binding capabilities of circulating microparticles (MPs) in SLE patients and healthy controls and to determine any associations with clinical parameters.Methods. Seventy unselected SLE patients and 29 sex-and age-matched healthy control subjects were included in the study. MPs were isolated from citratetreated plasma and characterized by flow cytometry using AnxV or antibodies to platelet, leukocyte, or endothelial cell surface markers.Results. SLE patients had significantly increased concentrations of AnxV-nonbinding (AnxV؊) MPs (P < 0.0001), while the concentrations of total MPs (P ‫؍‬ 0.011) and AnxV-binding (AnxV؉) MPs (P < 0.0001) were decreased, as compared with controls. Based on flow cytometric characteristics, 2 subgroups of AnxV؊ MPs could be discerned: AnxV؊ cell-derived MPs (CDMPs) and AnxV؊ MPs of unknown nature (UNMPs). Both fractions were significantly increased in SLE patients (P ‫؍‬ 0.007 and P ‫؍‬ 0.0018, respectively). Platelet-and leukocyte-derived MPs were decreased in the SLE patients (P < 0.0001), whereas no difference was observed for endothelial cell-derived MPs (P ‫؍‬ 0.14). The concentrations of AnxV؊ CDMPs correlated with the concentrations of endothelial cell-derived MPs, the disease activity score, active nephritis, hypertension, history of arterial thrombosis, and triglyceride levels (P < 0.05 for all comparisons).Conclusion. The concentrations and composition of MPs in SLE patients differ markedly from those in healthy subjects. Overall MP numbers were significantly decreased, but two distinct subpopulations of AnxV؊ MPs were significantly increased. These findings call for further characterization of MPs in SLE patients to elucidate their role in disease pathogenesis.