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Rationale: High-mobility group box 1 (HMGB1) is a potent inflammatory mediator elevated in sepsis and rheumatoid arthritis, although its role in cystic fibrosis (CF) lung disease is unknown.Objectives: To determine whether HMGB1 contributes to CF lung inflammation, including neutrophil chemotaxis and lung matrix degradation.Methods: We used sputum and serum from subjects with CF and a Scnn1b-transgenic (Scnn1b-Tg) mouse model that overexpresses beta-epithelial Na(+) channel in airways and mimics the CF phenotype, including lung inflammation. Human secretions and murine bronchoalveolar lavage fluid (BALF) was assayed for HMGB1 by Western blot and ELISA. Neutrophil chemotaxis was measured in vitro after incubation with human neutrophils. The collagen fragment proline-glycine-proline (PGP) was measured by tandem mass spectroscopy.Measurements and Main Results: HMGB1 was detected in CF sputum at higher levels than secretions from normal individuals. Scnn1b-Tg mice had elevated levels of HMGB1 by Western blot and ELISA. We demonstrated that dose-dependent chemotaxis of human neutrophils stimulated by purified HMGB1 was partially dependent on CXC chemokine receptors and that this could be duplicated in CF sputum and BALF from Scnn1b-Tg mice. Neutralization by anti-HMGB1 antibody, in both the sputum and BALF-reduced chemotaxis, which suggested that HMGB1 contributed to the chemotactic properties of these samples. Intratracheal administration of purified HMGB1 induced neutrophil influx into the airways of mice and promoted the release of PGP. PGP was also elevated in Scnn1b-Tg mice and CF serum.Conclusions: HMGB1 expression contributes to pulmonary inflammation and lung matrix degradation in CF airway disease and deserves further investigation as a biomarker and potential therapeutic target. [ABSTRACT FROM AUTHOR]

Neutrophil influx and activation contributes to organ damage in several major lung diseases. This inflammatory influx is initiated and propagated by both classical chemokines such as interleukin-8 and by downstream mediators such as the collagen fragment cum neutrophil chemokine Pro-Gly-Pro (PGP), which share use of the ELR + CXC receptor family. Benzyloxycarbonyl-proline-prolinal (ZPP) is known to suppress the PGP pathway via inhibition of prolyl endopeptidase (PE), the terminal enzyme in the generation of PGP from collagen. However, the structural homology of ZPP and PGP suggests that ZPP might also directly affect classical glutamate-leucine-arginine positive (ELR+) CXC chemokine signaling. In this investigation, we confirm that ZPP inhibits PE in vitro, demonstrate that ZPP inhibits both ELR + CXC and PGP-mediated chemotaxis in human and murine neutrophils, abrogates neutrophil influx induced by murine intratracheal challenge with LPS, and attenuates human neutrophil chemotaxis to sputum samples of human subjects with cystic fibrosis. Cumulatively, these data demonstrate that ZPP has dual, complementary inhibitory effects upon neutrophil chemokine/matrikine signaling which make it an attractive compound for clinical study of neutrophil inhibition in conditions (such as cystic fibrosis and chronic obstructive pulmonary disease) which evidence concurrent harmful increases of both chemokine and matrikine signaling., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Here, we describe a novel pathogenic entity, the activated PMN (polymorphonuclear leukocyte, i.e., neutrophil)-derived exosome. These CD63 + /CD66b + nanovesicles acquire surface-bound neutrophil elastase (NE) during PMN degranulation, NE being oriented in a configuration resistant to α1-antitrypsin (α1AT). These exosomes bind and degrade extracellular matrix (ECM) via the integrin Mac-1 and NE, respectively, causing the hallmarks of chronic obstructive pulmonary disease (COPD). Due to both ECM targeting and α1AT resistance, exosomal NE is far more potent than free NE. Importantly, such PMN-derived exosomes exist in clinical specimens from subjects with COPD but not healthy controls and are capable of transferring a COPD-like phenotype from humans to mice in an NE-driven manner. Similar findings were observed for another neutrophil-driven disease of ECM remodeling (bronchopulmonary dysplasia [BPD]). These findings reveal an unappreciated role for exosomes in the pathogenesis of disorders of ECM homeostasis such as COPD and BPD, providing a critical mechanism for proteolytic damage., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Neutrophils (PMNs) are key mediators of inflammatory processes throughout the body. In this study, we investigated the role of acrolein, a highly reactive aldehyde that is ubiquitously present in the environment and produced endogenously at sites of inflammation, in mediating PMN-mediated degradation of collagen facilitating proline-glycine-proline (PGP) production. We treated peripheral blood neutrophils with acrolein and analyzed cell supernatants and lysates for matrix metalloproteinase-9 (MMP-9) and prolyl endopeptidase (PE), assessed their ability to break down collagen and release PGP, and assayed for the presence of leukotriene A4 hydrolase (LTA4H) and its ability to degrade PGP. Acrolein treatment induced elevated production and functionality of collagen-degrading enzymes and generation of PGP fragments. Meanwhile, LTA4H levels and triaminopeptidase activity declined with increasing concentrations of acrolein thereby sparing PGP from enzymatic destruction. These findings suggest that acrolein exacerbates the acute inflammatory response mediated by neutrophils and sets the stage for chronic pulmonary and systemic inflammation.

A role for the collagen-derived tripeptide, N-acetyl proline-glycine-proline (NAc-PGP), in neutrophil recruitment in chronic airway inflammatory diseases, including COPD and cystic fibrosis, has recently been delineated. Due to structural similarity to an important motif for interleukin-8 (CXCL8) binding to its receptor, NAc-PGP binds to CXCR1/2 receptors, leading to neutrophil activation and chemotaxis. In an effort to develop novel CXCL8 antagonists, we describe the synthesis of four chiral isomers of NAc-PGP (NAc-L-Pro-Gly-L-Pro (LL-NAc-PGP), NAc-L-Pro-Gly-D-Pro (LD-NAc-PGP), NAc-D-Pro-Gly-L-Pro (DL-NAc-PGP), and NAc-D-Pro-Gly-D-Pro (DD-NAc-PGP)), characterize them by circular dichroism and NMR spectroscopy, compare their structures to the equivalent region of CXCL8, and test them as potential antagonists of ll-NAc-PGP and CXCL8. We find that LL-NAc-PGP superimposes onto the CXCR1/2 contacting E(29)S(30)G(31)P(32) region of CXCL8 (0.59A rmsd for heavy atoms). In contrast, DD-NAc-PGP has an opposing orientation of key functional groups as compared to the G(31)P(32) region of CXCL8. As a consequence, DD-NAc-PGP binds CXCR1/2, as demonstrated by competition with radiolabeled CXCL8 binding in a radioreceptor assay, yet acts as a receptor antagonist as evidenced by inhibition of CXCL8 and LL-NAc-PGP mediated neutrophil chemotaxis. The ability of DD-NAc-PGP to prevent the activation of CXC receptors indicates that DD-NAc-PGP may serve as a lead compound for the development of CXCR1/2 inhibitors. In addition, this study further proves that using a different technical approach, namely preincubation of NAc-PGP instead of simultaneous addition of NAc-PGP with radiolabeled CXCL8, the direct binding of NAc-PGP to the CXCL8 receptor is evident., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Leukotriene A(4) hydrolase (LTA(4)H) is a proinflammatory enzyme that generates the inflammatory mediator leukotriene B(4) (LTB(4)). LTA(4)H also possesses aminopeptidase activity with unknown substrate and physiological importance; we identified the neutrophil chemoattractant proline-glycine-proline (PGP) as this physiological substrate. PGP is a biomarker for chronic obstructive pulmonary disease (COPD) and is implicated in neutrophil persistence in the lung. In acute neutrophil-driven inflammation, PGP was degraded by LTA(4)H, which facilitated the resolution of inflammation. In contrast, cigarette smoke, a major risk factor for the development of COPD, selectively inhibited LTA(4)H aminopeptidase activity, which led to the accumulation of PGP and neutrophils. These studies imply that therapeutic strategies inhibiting LTA(4)H to prevent LTB(4) generation may not reduce neutrophil recruitment because of elevated levels of PGP.

Chronic neutrophilic inflammation is a manifestation of a variety of lung diseases including cystic fibrosis (CF). There is increasing evidence that fragments of extracellular matrix proteins, such as collagen and elastin, play an important role in inflammatory cell recruitment to the lung in animal models of airway inflammation. Unfortunately, the association of these peptides with human disease and the identification of therapeutic targets directed toward these inflammatory pathways have remained elusive. In this study, we demonstrate that a novel extracellular matrix-derived neutrophil chemoattractant, proline-glycine-proline (PGP), acts through CXC receptors 1 and 2 on neutrophils, similar to N-acetylated proline-glycine-proline (N-alpha-PGP). We describe the specific multistep proteolytic pathway involved in PGP generation from collagen, involving matrix metalloproteases 8 and 9 and prolyl endopeptidase, a serine protease for which we identify a novel role in inflammation. PGP generation correlates closely with airway neutrophil counts after administration of proteases in vivo. Using CF as a model, we show that CF sputum has elevated levels of PGP peptides and that PGP levels decline during the course of CF inpatient therapy for acute pulmonary exacerbation, pointing to its role as a novel biomarker for this disease. Finally, we demonstrate that CF secretions are capable of generating PGP from collagen ex vivo and that this generation is significantly attenuated by the use of inhibitors directed toward matrix metalloprotease 8, matrix metalloprotease 9, or prolyl endopeptidase. These experiments highlight unique protease interactions with structural proteins regulating innate immunity and support a role for these peptides as novel biomarkers and therapeutic targets for chronic, neutrophilic lung diseases.

Myasthenia gravis (MG) is caused by T cell-dependent antibodies reactive with acetylcholine receptors. These autoreactive antibodies cause muscle weakness by interfering with neuromuscular transmission via removal of acetylcholine receptors from the neuromuscular junction as well as changing the architecture of the junction itself. Consequently, muscle fatigue is a debilitating aspect of MG often leading to more general feelings of tiredness not directly due to muscle weakness. We have previously described two peptides that are mimetics of antigen receptors on certain autoreactive T and B cells that are involved in MG. When used as vaccines in the rat model of MG, these peptides prevented and ameliorated disease and muscle fatigue by blunting acetylcholine receptor antibody responses. Such disease protection resulted from vaccine-induced anergizing antibodies against acetylcholine receptor-specific T and B cell antigen receptors. The present study prospectively evaluated the efficacy of these two vaccines in spontaneous acquired MG in pet dogs. When compared to historical controls that were prospectively studied, the vaccines increased the proportion of remitted dogs from 17 to 75%. In comparison to retrospectively studied historical controls that spontaneously remitted from MG, the vaccines accelerated the rate of decline in acetylcholine receptor antibody titers which resulted in a 3-fold decrease in the mean time to remission. These results are suggestive of a new type of targeted therapy that can drive autoimmune responses into long-term remission and possibly afford a means of determining whether correction of a physical cause of muscle weakness also corrects the perception of chronic, generalized fatigue.

We describe the tripeptide neutrophil chemoattractant N-acetyl Pro-Gly-Pro (PGP), derived from the breakdown of extracellular matrix (ECM), which shares sequence and structural homology with an important domain on alpha chemokines. PGP caused chemotaxis and production of superoxide through CXC receptors, and administration of peptide caused recruitment of neutrophils (PMNs) into lungs of control, but not CXCR2-deficient mice. PGP was generated in mouse lung after exposure to lipopolysaccharide, and in vivo and in vitro blockade of PGP with monoclonal antibody suppressed PMN responses as much as chemokine-specific monoclonal antibody. Extended PGP treatment caused alveolar enlargement and right ventricular hypertrophy in mice. PGP was detectable in substantial concentrations in a majority of bronchoalveolar lavage samples from individuals with chronic obstructive pulmonary disease, but not control individuals. Thus, PGP's activity links degradation of ECM with neutrophil recruitment in airway inflammation, and PGP may be a biomarker and therapeutic target for neutrophilic inflammatory diseases.

In a previously described case of Waldenstrom's Macroglobulinemia, complicated by polyneuropathy, the IgM/lambda monoclonal antibody (mAb) was highly reactive with myelin basic protein (MBP). Given our demonstration that V lambda x, a recently described murine lambda variable region gene product, can itself bind MBP as well as confer MBP reactivity to an Ab, the possibility of a shared idiotypy between murine V lambda x and this human IgM/lambda anti-MBP was investigated. We characterized the epitope specificity of the macroglobulinemia patient's MBP-reactive IgM/lambda using indirect ELISA procedures with MBP, a citrullinated isomer of MBP termed C8, or peptide fragments of MBP as the coating antigens and monospecific Ab to V lambda x as the secondary Ab. The patient's MBP-reactive IgM/lambda was recognized by Ab specific for V lambda x and, like murine mAb containing V lambda x bound human MBP but not MBP-C8 nor other common autoantigens such as DNA, thyroglobulin, or actin. The anti-MBP reactivity was selective for MBP peptide 90-170 and preferentially recognized MBP peptide 84-96. Thus, the patient's macroglobulin and perhaps certain other human Ab with a 'V lambda x idiotype' bind to MBP peptide residues 84-96, an immunodominant peptide in multiple sclerosis patients. Such binding may be involved in the pathogenesis of neural damage in patients with neuroimmunologic disorders related to plasma cell dyscrasias or autoimmunity.

Signals relayed through the magnocellular layers of the LGN travel on axons with faster conduction speeds than those relayed through the parvocellular layers. As a result, magnocellular signals might reach cerebral cortex appreciably before parvocellular signals. The relative speed of these two channels cannot be accurately predicted based solely on axon conduction speeds, however. Other factors, such as different degrees of convergence in the magnocellular and parvocellular channels and the retinal circuits that feed them, can affect the time it takes for magnocellular and parvocellular signals to activate cortical neurons. We have investigated the relative timing of visual responses mediated by the magnocellular and parvocellular channels. We recorded individually from 78 magnocellular and 80 parvocellular neurons in the LGN of two anesthetized monkeys. Visual response latencies were measured for small spots of light of various intensities. Over a wide range of stimulus intensities the fastest magnocellular response latencies preceded the fastest parvocellular response latencies by about 10 ms. Because parvocellular neurons are far more numerous than magnocellular neurons, convergence in cortex could reduce the magnocellular advantage by allowing parvocellular signals to generate detectable responses sooner than expected based on the responses of individual parvocellular neurons. An analysis based on a simple model using neurophysiological data collected from the LGN shows that convergence in cortex could eliminate or reverse the magnocellular advantage. This observation calls into question inferences that have been made about ordinal relationships of neurons based on timing of responses.