Dysfunctional neutrophil effector organelle mobilization and microbicidal protein release in alcohol-related cirrhosis

Patients with alcohol-related cirrhosis (ALD) are prone to infection. Circulating neutrophils in ALD are dysfunctional and predict development of sepsis, organ dysfunction, and survival. Neutrophil granules are important effector organelles containing a toxic array of microbicidal proteins, whose controlled release is required to kill microorganisms while minimizing inflammation and damage to host tissue. We investigated the role of these granular responses in contributing to immune disarray in ALD. Neutrophil granular content and mobilization were measured by flow cytometric quantitation of cell-surface/intracellular markers, [secretory vesicles (CD11b), secondary granules (CD66b), and primary granules (CD63; myeloperoxidase)] before and after bacterial stimulation in 29 patients with ALD cirrhosis (15 abstinent; 14 actively drinking) compared with healthy controls (HC). ImageStream Flow Cytometry characterized localization of granule subsets within the intracellular and cell-surface compartments. The plasma cytokine environment was analyzed using ELISA/cytokine bead array. Circulating neutrophils were primed in the resting state with upregulated surface expression of CD11b ( P = 0.0001) in a cytokine milieu rich in IL-8 ( P < 0.001) and lactoferrin ( P = 0.035). Neutrophils showed exaggerated mobilization to the cell surface of primary granules at baseline ( P = 0.001) and in response to N-formyl-l-methionyl-l-leucyl-l-phenylalanine ( P = 0.009) and Escherichia coli ( P = 0.0003) in ALD. There was no deficit in granule content or mobilization to the cell membrane in any granule subset observed. Paradoxically, active alcohol consumption abrogated the hyperresponsive neutrophil granular responses compared with their abstinent counterparts. Neutrophils are preprimed at baseline with augmented effector organelle mobilization in response to bacterial stimulation; neutrophil degranulation is not a mechanism leading to innate immunoparesis in ALD.NEW & NOTEWORTHY Neutrophil granule release is dysregulated in patients with alcohol-related cirrhosis (ALD) with augmented effector organelle mobilization and microbiocidal protein release. Neutrophil granules are upregulated in ALD at baseline and demonstrate augmented responses to bacterial challenge. The granular responses in ALD did not contribute to the observed functional deficit in innate immunity but rather were dysregulated and hyperresponsive, which may induce bystander damage to host tissue. Paradoxically, active alcohol consumption abrogated the excessive neutrophil granular responses to bacterial stimulus compared with their abstinent counterparts.

The tetraspanin CD63 is involved in granule targeting of neutrophil elastase

Targeting mechanisms of neutrophil elastase (NE) and other luminal proteins stored in myeloperoxidase (MPO)–positive secretory lysosomes/primary granules of neutrophils are unknown. These granules contain an integral membrane protein, CD63, with an adaptor protein-3–dependent granule delivery system. Therefore, we hypothesized that CD63 cooperates in granule delivery of the precursor of NE (proNE). Supporting this hypothesis, an association was demonstrated between CD63 and proNE upon coexpression in COS cells. This also involved augmented cellular retention of proNE requiring intact large extracellular loop of CD63. Furthermore, depletion of CD63 in promyelocytic HL-60 cells with RNA interference or a CD63 mutant caused reduction of cellular NE. However, the proNE steady-state level was similar to wild type in CD63-depleted clones, making it feasible to examine possible effects of CD63 on NE trafficking. Thus, depletion of CD63 led to reduced processing of proNE into mature NE and reduced constitutive secretion. Furthermore, CD63-depleted cells showed a lack of morphologically normal granules, but contained MPO-positive cytoplasmic vacuoles with a lack of proNE and NE. Collectively, our data suggest that granule proteins may cooperate in targeting; CD63 can be involved in ER or Golgi export, cellular retention, and granule targeting of proNE before storage as mature NE.

Increased Unfolded Protein Response in Hematopoietic Cells from Cyclic Hematopoietic Dogs.

Canine cyclic hematopoiesis, a model of human cyclic neutropenia/congenital neutropenia (CN), is caused by a mutation in the AP3b1 gene. Human CN is caused by mutations in the Ela2 gene. The exact mechanism(s) whereby mutations in the Ela2 and AP3b1 cause CN or CH is not well understood. Elastase is directed to the primary granules by AP3 in myeloid progenitor cells, suggesting abnormal trafficking of elastase as a cause for CN/CH. Discontinuous percoll gradients on PMN cells from normal and CH dogs were performed. Fractions corresponding to the cytosol, plasma membrane, nuclei, primary, secondary and tertiary granules were collected and assayed for elastase and myeloperoxidase (MPO) enzyme activity and protein levels by Western blot using canine specific antibodies. Percoll density gradient fractionation results indicated that MPO, a primary granule protein that is not an AP3 cargo protein, is present in the primary granules in approximately equivalent amounts in both normal and CH dog PMN’s. Elastase was also localized in the primary granule fraction from both normal and CH dog PMN, but with a lower amount in CH dogs. Elastase was not present in the plasma membrane fraction in either CH or normal dog PMN’s. Quantitatively the CH dog primary granules had 10–20% of normal dog primary granule elastase. These results suggest PMN’s from CH dogs correctly sort elastase to the primary granules but at a lower level compared to PMNs from dogs without AP3b1 defect. Glucose-regulated protein (GRP78/BiP) is an indicator of ER stress. ER stress leads to activition of the unfolded protein response (UPR) and is cytoprotective. However, prolonged UPR leads to apoptosis. Analysis of GRP78 expression in PMN’s and bone marrow cell cultures from normal and CH dogs stimulated with SCF and G-CSF demonstrated 3–4 fold increase of GRP78 in CH cells compared to normal dog cells. These results indicate mistrafficking or accumulation of misfolded elastase induces the UPR in myeloid precursor cells and disrupts normal PMN production in CH dogs. These results are consistent with in vitro studies in which over-expression of mutant but not normal human elastase induces the UPR in myeloid cells and cell death. These results suggest that induction of the UPR is a common event, in myeloid progenitor cells from patients and animal models with either Ela2 or AP3b1 mutations, which ultimately results in congenital and/or cyclic neutropenia.

Neutrophil Elastase, but Not Myeloperoxidase, Depends on Serglycin Proteoglycan for Granule Localization in Neutrophils.

Granule proteins play a major role for the bactericidal activity of neutrophils. The mechanisms for sorting of proteins to granules in neutrophils remain unknown. Serglycin proteoglycan with glycosaminoglycan side chains is the major intracellular proteoglycan of hematopoietic cells. Neutrophil elastase, a resident of primary (azurophilic) granules, binds glycosaminoglycans. Serglycin has been proposed to play a major role in sorting and packaging of granule proteins in neutrophils and other cells. Figure Figure We have investigated the expression and localization of granule proteins in neutrophils from a serglycin knock out mouse. We found neutrophil elastase absent from neutrophils of serglycin knock out mice as shown by activity assay (fig. 1), Western blotting (fig. 2), and immunocytochemistry. Northern blotting (fig. 1) demonstrated the presence of neutrophil elastase mRNA both in serglycin knock out mice and in wild type mice. No other granule proteins tested (myeloperoxidase, cathepsin G, proteinase 3, bacterial permeability increasing protein (primary granules), lactoferrin, 24p3 (secondary (specific) granules), MMP-9/gelatinase B (tertiary (gelatinase) granules)) showed differences in expression or localization in neutrophils from wild type mice compared to serglycin knock out mice. Three-dimensional models of neutrophil elastase revealed a cluster of positively charged amino acids around the active site of the enzyme. This cluster may be engaged in binding the negatively charged glycosaminoglycan side chains of serglycin and thus forms the basis for serglycin mediated sorting of elastase to primary (azurophilic) granules. No counts for both bone marrow cells and peripheral blood are similar for the wild type and serglycin −/− genotypes; indicating that the lack of sorting of elastase to granules does not induce neutropenia itself. These findings disclose an important role for serglycin proteoglycan in targeting neutrophil elastase to neutrophil primary granules. This is in accordance with the recently published significance for serglycin in granule formation of mast cells. Our results also indicate that different mechanisms are responsible for sorting of different granule proteins to the same neutrophil granule subset, as none of the other tested granule proteins showed differences in localization between wild type and serglycin knock out neutrophils.