Neutrophils are the first line of defense at the site of an infection. They encounter and kill microbes intracellularly upon phagocytosis or extracellularly by degranulation of antimicrobial proteins and the release of Neutrophil Extracellular Traps (NETs). NETs were shown to ensnare and kill microbes. However, their complete protein composition and the antimicrobial mechanism are not well understood. Using a proteomic approach, we identified 24 NET-associated proteins. Quantitative analysis of these proteins and high resolution electron microscopy showed that NETs consist of modified nucleosomes and a stringent selection of other proteins. In contrast to previous results, we found several NET proteins that are cytoplasmic in unstimulated neutrophils. We demonstrated that of those proteins, the antimicrobial heterodimer calprotectin is released in NETs as the major antifungal component. Absence of calprotectin in NETs resulted in complete loss of antifungal activity in vitro. Analysis of three different Candida albicans in vivo infection models indicated that NET formation is a hitherto unrecognized route of calprotectin release. By comparing wild-type and calprotectin-deficient animals we found that calprotectin is crucial for the clearance of infection. Taken together, the present investigations confirmed the antifungal activity of calprotectin in vitro and, moreover, demonstrated that it contributes to effective host defense against C. albicans in vivo. We showed for the first time that a proportion of calprotectin is bound to NETs in vitro and in vivo.

/pdf/neutrophil-extracellular-traps-contain-calprotectin-a-3pp8q0svix.pdf

Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans

Engagement of the receptor for advanced glycation end products (RAGE) by products of nonenzymatic glycation/oxidation triggers the generation of reactive oxygen species (ROS), thereby altering gene expression. Because dissection of the precise events by which ROS are generated via RAGE is relevant to the pathogenesis of complications in AGE-related disorders, such as diabetes and renal failure, we tested the hypothesis that activation of NADPH oxidase contributed, at least in part, to enhancing oxidant stress via RAGE. Here we show that incubation of human endothelial cells with AGEs on the surface of diabetic red blood cells, or specific AGEs, (carboxymethyl)lysine (CML)-modified adducts, prompted intracellular generation of hydrogen peroxide, cell surface expression of vascular cell adhesion molecule-1, and generation of tissue factor in a manner suppressed by treatment with diphenyliodonium, but not by inhibitors of nitric oxide. Consistent with an important role for NADPH oxidase, although macrophages derived from wild-type mice expressed enhanced levels of tissue factor upon stimulation with AGE, macrophages derived from mice deficient in a central subunit of NADPH oxidase, gp91phox, failed to display enhanced tissue factor in the presence of AGE. These findings underscore a central role of NADPH oxidase in AGE-RAGE-mediated generation of ROS and provide a mechanism for altered gene expression in AGE-related disorders.

https://www.physiology.org/doi/pdf/10.1152/ajpendo.2001.280.5.E685

Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE.

The S100 protein family: History, function, and expression

BAFF (BLyS, TALL-1, THANK, zTNF4) is a member of the TNF superfamily that specifically regulates B lymphocyte proliferation and survival. Mice transgenic (Tg) for BAFF develop an autoimmune condition similar to systemic lupus erythematosus. We now demonstrate that BAFF Tg mice, as they age, develop a secondary pathology reminiscent of Sjogren's syndrome (SS), which is manifested by severe sialadenitis, decreased saliva production, and destruction of submaxillary glands. In humans, SS also correlates with elevated levels of circulating BAFF, as well as a dramatic upregulation of BAFF expression in inflamed salivary glands. A likely explanation for disease in BAFF Tg mice is excessive survival signals to autoreactive B cells, possibly as they pass through a critical tolerance checkpoint while maturing in the spleen. The marginal zone (MZ) B cell compartment, one of the enlarged B cell subsets in the spleen of BAFF Tg mice, is a potential reservoir of autoreactive B cells. Interestingly, B cells with an MZ-like phenotype infiltrate the salivary glands of BAFF Tg mice, suggesting that cells of this compartment potentially participate in tissue damage in SS and possibly other autoimmune diseases. We conclude that altered B cell differentiation and tolerance induced by excess BAFF may be central to SS pathogenesis.

/pdf/association-of-baff-blys-overexpression-and-altered-b-cell-4db4dm5ot8.pdf

Association of BAFF/BLyS overexpression and altered B cell differentiation with Sjögren’s syndrome

S100A8 and S100A9 are small calcium-binding proteins that are highly expressed in neutrophil and monocyte cytosol and are found at high levels in the extracellular milieu during inflammatory conditions. Although reports have proposed a proinflammatory role for these proteins, their extracellular activity remains controversial. In this study, we report that S100A8, S100A9, and S100A8/A9 caused neutrophil chemotaxis at concentrations of 10(-12)-10(-9) M. S100A8, S100A9, and S100A8/A9 stimulated shedding of L-selectin, up-regulated and activated Mac-1, and induced neutrophil adhesion to fibrinogen in vitro. Neutralization with Ab showed that this adhesion was mediated by Mac-1. Neutrophil adhesion was also associated with an increase in intracellular calcium levels. However, neutrophil activation by S100A8, S100A9, and S100A8/A9 did not induce actin polymerization. Finally, injection of S100A8, S100A9, or S100A8/A9 into a murine air pouch model led to rapid, transient accumulation of neutrophils confirming their activities in vivo. These studies 1) show that S100A8, S100A9, and S100A8/A9 are potent stimulators of neutrophils and 2) strongly suggest that these proteins are involved in neutrophil migration to inflammatory sites.

Proinflammatory Activities of S100: Proteins S100A8, S100A9, and S100A8/A9 Induce Neutrophil Chemotaxis and Adhesion

https://mp.bmj.com/content/molpath/50/3/113.full.pdf

Functional and clinical aspects of the myelomonocyte protein calprotectin.

L1 is a major and highly immunogenic protein component of human granulocytes. It was purified from random samples of blood-donor leukocytes. An antiserum to L1 was initially raised in rabbits by immunization with protein fractions from preparative agarose gel electrophoresis. The main problem in purifying L1 was its poor stability when carried through multiple steps. Preparative isoelectric focussing was therefore adopted as an efficient one-step method. The isoelectric focussing pattern of L1 was greatly influence by the presence of EDTA or calcium ions in the sample buffer. With low EDTA concentrations or calcium excess, L1 focussed as seven protein bands in two regions, pH 6.1-6.5 and pH 7.6-8.4. Conversely, L1 was found only in the pH-6.1-6.5 region when excess EDTA was added to the sample. Irrespective of conditions, the bulk of L1 focussed at pH 6.3 and pH 6.5. Also the electrophoretic mobility of L1 was strongly influenced by calcium ions, suggesting uptake of calcium by the protein. In the presence of calcium, L1 adhered to glass surfaces and filters used for concentration of protein solutions. The latter problem could be prevented by addition of EDTA. The molecular mass of L1 was determined to be about 36.5 kDa. The molecule was shown to consist of three non-covalently linked 12.5-kDa subunits.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1983.tb07522.x

Purification and Partial Characterization of a Highly Immunogenic Human Leukocyte Protein, the L1 Antigen

The LI antigen is a highly immunogenic protein of about 36,500 daltons that can be purified from granulocytes with good yield. Immunocytochemistry with a rabbit anti-serum raised against LI showed it to be present in the cytoplasm of vjrtually all resting peripheral neutrophils and monocytes. Moreover, immunofluorescence staining demonstrated variable expression of LI on the plasma membrane of both these cell types, usually along with lysozyme. This indicated that LI represents a secretory product like lysozyme as their coexpression on the surface of vital cells was contrasted by the absence of lactoferrin. Cytoplasmic LI was well preserved by both precipitating and cross-linking fixatives, the latter being preferable to avoid leaching out of antigenic material and to obtain good cellular morphology. Thus, fixation for 3 minutes at room temperature in glutaraldehyde (l%)–formaldehyde (3%) afforded excellent immunoperoxidase staining, particularly when a calcium-containing buffer was used. LI was not found in eosinophilic granulocytes or in resting B- and T-lymphocytes. Neither did blast transformation of lymphocytes seem to induce LI expression.

Distribution of a new myelomonocytic antigen (L1) in human peripheral blood leukocytes. Immunofluorescence and immunoperoxidase staining features in comparison with lysozyme and lactoferrin.

https://www.jaad.org/article/S0190-9622(86)70152-7/pdf

Clinical and laboratory studies

Immunological methods have been developed for quantitation of a protein, provisionally called L1, which constitutes about 5% of the total protein in granulocytes. The protein has a mol wt of about 51000 daltons and an isoelectric point of about 6.3–6.5. It is readily released from leucocytes during storage and following immunological injury to the cell membrane. The concentration of L1 in normal plasma is about 300 ng/ml, but levels above the normal mean + 5 SD were found in 23 out of 30 patients with various malignant diseases and in 4 patients with septicaemia. It is suggested that plasma levels of L1 may reflect the rate of leucocyte turnover in vivo and that the L1 release phenomenon may be used for studies on leucocyte reactions in vitro.

Inge Dale

Papers

Functional and clinical aspects of the myelomonocyte protein calprotectin.

Purification and Partial Characterization of a Highly Immunogenic Human Leukocyte Protein, the L1 Antigen

Distribution of a new myelomonocytic antigen (L1) in human peripheral blood leukocytes. Immunofluorescence and immunoperoxidase staining features in comparison with lysozyme and lactoferrin.

Clinical and laboratory studies

Release and Quantitation of a Leucocyte Derived Protein (L1)