BAL neutrophilia in azithromycin-treated lung transplant recipients: Clinical significance.

BACKGROUND: Azithromycin decreases airway neutrophilia and can prevent chronic lung allograft dysfunction (CLAD) after lung transplantation (LTx). It also can be used to treat lymphocytic bronchiolitis, as it decreases the submucosal infiltrating IL-17 positive lymphocytes. Some patients, while receiving azithromycin, (re)develop increased airway neutrophilia, which we hypothesize to result in worse outcome and to be regulated by an IL-17-independent mechanism. METHODS: LTx recipients, transplanted between 2001 and 2012, were investigated and categorized in a study group of patients with increased broncho-alveolar lavage (BAL) neutrophilia (≥15%) and a matched control group with low BAL neutrophilia (<15%), both groups while already being on azithromycin treatment. CLAD-free and overall survival were compared between groups. Cell differentials and 33 proteins in BAL were analyzed to identify underlying mechanisms. RESULTS: The study group (n=72) demonstrated a significantly lower CLAD-free (p=0.015) and overall survival (p=0.041) compared to the control group (n=37). Absolute BAL neutrophils and eosinophils were increased in the study group, which was paralleled by elevated inflammatory cytokines (IL-1ß/IL-1Ra, IL-4 and IL-6) and chemokines (CXCL8/IL-8, CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1ß, CCL11/eotaxin) concentrations compared to the control group (all p<0.05). CONCLUSION: Patients with elevated airway neutrophilia despite azithromycin, experience worse CLAD-free and overall survival. In these patients, IL-1ß might play a central role giving rise to neutrophils, eosinophils, macrophages and B-cells. This provides an opportunity to further investigate the modulation of this pathway.

Differential cytokine, chemokine and growth factor expression in phenotypes of chronic lung allograft dysfunction.

BACKGROUND: Chronic lung allograft dysfunction is a heterogeneous entity limiting long-term survival after lung transplantation. Different clinical phenotypes (bronchiolitis obliterans syndrome [BOS]-neutrophilic BOS-restrictive allograft syndrome [RAS]) have been identified but the mechanisms remain elusive. METHODS: In this study, we measured 34 different cytokines, chemokines, and growth factors in bronchoalveolar lavage fluid of 20 stable patients, 20 patients suffering from non-neutrophilic BOS, 17 from neutrophilic BOS, and 20 from RAS using classic enzyme-linked immunosorbent assay and multiplex technology. RESULTS: Total cell count and % neutrophils were elevated in neutrophilic BOS and RAS compared to stable and non-neutrophilic BOS patients, whereas also the % eosinophils was elevated at diagnosis of RAS. Levels of interleukin (IL)-1ß (P<0.01), IL-1Rα (P<0.001), IL-6 (P<0.001), IL-8/CXCL8 (P<0.001), IP-10/CXCL10 (P<0.05), MCP-1/CCL2 (P<0.05), macrophage inflammatory protein (MIP)-1α/CCL3 (P<0.001), MIP-1ß/CCL4, and vascular endothelial growth factor (VEGF; P<0.05) were differentially regulated in RAS compared to stable, whereas in neutrophilic BOS IL-1ß (P<0.001), IL-1Rα (P<0.01), IL-7 (P<0.05), IL-8/CXCL8 (P<0.001), MCP-3/CXCCL7 (P<0.05) and MIP-1α/CCL-3 (P<0.05) were significantly upregulated compared to stable patients. We could not detect any differences between non-neutrophilic BOS and stable patients. Interestingly, bronchoalveolar lavage IL-6, interferon gamma-induced protein (IP)-10/CXCL10 and interferon-inducible T-cell alpha chemoattractant/chemokine (C-X-C motif) ligand 11 (ITAC/CXCL10) were associated with survival after diagnosis in RAS patients. CONCLUSION: There were major differences in cytokine and chemokine expression in our different study groups. Especially IL-6, but also IP-10/CXCL10, and VEGF may be interesting mediators in RAS.

Molecular origin of the hydrolytic activity and fixed regioselectivity of a Zr(IV) -substituted polyoxotungstate as artificial protease.

A multitechnique approach has been applied in order to identify the thermodynamic and kinetic parameters related to the regioselective hydrolysis of human serum albumin (HSA) promoted by the Wells-Dawson polyoxometalate (POM), K15 H[Zr(α2 -P2 W17 O61 )2 ]. Isothermal titration calorimetry (ITC) studies indicate that up to four POM molecules interact with HSA. While the first interaction site is characterized by a 1:1 binding and an affinity constant of 2×10(8) M(-1) , the three remaining sites are characterized by a lower global affinity constant of 7×10(5) M(-1) . The higher affinity constant at the first site is in accordance with a high quenching constant of 2.2×10(8) M(-1) obtained for fluorescence quenching of the Trp214 residue located in the only positively charged cleft of HSA, in the presence of K15 H[Zr(α2 -P2 W17 O61 )2 ]. In addition, Eu(III) luminescence experiments with an Eu(III) -substituted POM analogue have shown the replacement of water molecules in the first coordination sphere of Eu(III) due to binding of the metal ion to amino acid side chain residues of HSA. All three interaction studies are in accordance with a stronger POM dominated binding at the positive cleft on the one hand, and interaction mainly governed by metal anchoring at the three remaining positions, on the other hand. Hydrolysis experiments in the presence of K15 H[Zr(α2 -P2 W17 O61 )2 ] have demonstrated regioselective cleavage of HSA at the Arg114Leu115, Ala257Asp258, Lys313Asp314 or Cys392Glu393 peptide bonds. This is in agreement with the interaction studies as the Arg114Leu115 peptide bond is located in the positive cleft of HSA and the three remaining peptide bonds are each located near an upstream acidic residue, which can be expected to coordinate to the metal ion. A detailed kinetic study has evidenced the formation of additional fragments upon prolonged reaction times. Edman degradation of the additional reaction products has shown that these fragments result from further hydrolysis at the initially observed cleavage positions, indicating a fixed selectivity for K15 H[Zr(α2 -P2 W17 O61 )2 ].

Selective hydrolysis of hen egg white lysozyme at Asp-X peptide bonds promoted by oxomolybdate.

The activity of oxomolybdate(VI) towards hen egg white lysozyme (HEWL) was examined under physiological and slightly acidic pH conditions. Purely hydrolytic cleavage of HEWL in the presence of 10 to 100 mM of oxomolybdate(VI) after incubation at pH 5.0 and 60 °C for 2 to 7 days was observed in SDS-PAGE experiments. Four cleavage sites, which all occurred at Asp-X sequences and included the Asp18-Asn19, Asp48-Gly49, Asp52-Trp53 and Asp101-Gly102 peptide bonds, were identified with Edman degradation. The molecular interaction between [MoO4](2-) and HEWL was studied by circular dichroism (CD) and (1)H-(15)N heteronuclear single quantum correlation (HSQC) NMR spectroscopy. CD spectroscopy revealed a significant decrease in the α-helical content of HEWL upon addition of oxomolybdate, while (1)H-(15)N HSQC NMR spectroscopy identified the residues which were most affected upon interaction with [MoO4](2-). (95)Mo NMR measurements, performed on oxomolybdate solutions containing HEWL, identified the monomeric [MoO4](2-) form as active species in the hydrolytic reaction. The hydrolysis of the Asp-Gly model peptide in the presence of oxomolybdate(VI) was studied by (1)H NMR, further supporting a hydrolytic mechanism where polarisation of the carbonyl is followed by internal nucleophilic attack on the Asp residue.

Citrullination and proteolytic processing of chemokines by Porphyromonas gingivalis.

The outgrowth of Porphyromonas gingivalis within the inflammatory subgingival plaque is associated with periodontitis characterized by periodontal tissue destruction, loss of alveolar bone, periodontal pocket formation, and eventually, tooth loss. Potential virulence factors of P. gingivalis are peptidylarginine deiminase (PPAD), an enzyme modifying free or peptide-bound arginine to citrulline, and the bacterial proteases referred to as gingipains (Rgp and Kgp). Chemokines attract leukocytes during inflammation. However, posttranslational modification (PTM) of chemokines by proteases or human peptidylarginine deiminases may alter their biological activities. Since chemokine processing may be important in microbial defense mechanisms, we investigated whether PTM of chemokines by P. gingivalis enzymes occurs. Upon incubation of interleukin-8 (IL-8; CXCL8) with PPAD, only minor enzymatic citrullination was detected. In contrast, Rgp rapidly cleaved CXCL8 in vitro. Subsequently, different P. gingivalis strains were incubated with the chemokine CXCL8 or CXCL10 and their PTMs were investigated. No significant CXCL8 citrullination was detected for the tested strains. Interestingly, although considerable differences in the efficiency of CXCL8 degradation were observed with full cultures of various strains, similar rates of chemokine proteolysis were exerted by cell-free culture supernatants. Sequencing of CXCL8 incubated with supernatant or bacteria showed that CXCL8 is processed into its more potent forms consisting of amino acids 6 to 77 and amino acids 9 to 77 (the 6-77 and 9-77 forms, respectively). In contrast, CXCL10 was entirely and rapidly degraded by P. gingivalis, with no transient chemokine forms being observed. In conclusion, this study demonstrates PTM of CXCL8 and CXCL10 by gingipains of P. gingivalis and that strain differences may particularly affect the activity of these bacterial membrane-associated proteases.

Regioselective hydrolysis of human serum albumin by Zr(IV)-substituted polyoxotungstates at the interface of positively charged protein surface patches and negatively charged amino acid residues.

Complexes comprising the Lewis acidic Zr(IV) metal and protein binding polyoxotungstate ligands of Lindqvist-, Keggin- and Wells-Dawson-type were found to region selectively hydrolyze human serum albumin at four distinct positions. Higher reactivities were found for structures with higher polyoxometalate charges and the cleavage positions were found in protein regions of mixed charge. Both findings suggest an electrostatic nature of the observed reactivity.

The Sputum Colour Chart as a predictor of lung inflammation, proteolysis and damage in non-cystic fibrosis bronchiectasis: a case-control analysis.

BACKGROUND AND OBJECTIVE: Non-cystic fibrosis bronchiectasis (NCFB) is characterized by a vicious cycle of airway infection, inflammation and structural damage with inappropriate mucus clearance. Our aim was to relate the value of proteolytic enzymes, proteolytic enzyme activity and inflammatory markers to disease severity and symptoms in patients with NCFB. METHODS: Sputum induction in NCFB patients and healthy controls was performed. Sputum was analysed for total and differential cell count, markers of inflammation (CXCL8 (also known as interleukin-8) and tumour necrosis factor-α (TNF-α)) and proteolytic enzymes (neutrophil elastase (NE), gelatin zymography and total gelatinolytic activity (TGA)). Each patient was evaluated by spirometry, Leicester Cough Questionnaire (LCQ) and Sputum Colour Chart (SCC). Patient files were analysed to determine Pseudomonas aeruginosa colonization status. The computed tomography (CT) closest to the date sputum induction was scored by a radiologist. RESULTS: NCFB patients showed significantly higher neutrophils, CXCL8, TNF-α, NE and TGA than healthy controls. TGA subanalysis showed that the majority of the activity was NE (82 ± 6.4%). Residual activity was mainly zinc ion-dependent matrix metalloproteinase (MMP) activity (18 ± 6.4%). Subanalysis showed that patients with chronic Pseudomonas aeruginosa colonization had more activated MMP-9. Correlations were seen between proteolytic enzymes and inflammation and disease severity (spirometry and CT score), but not with the LCQ. SCC was associated with increased markers of inflammation, proteolytic enzymes and worse CT score. CONCLUSIONS: We show that sputum purulence assessment in daily clinical practice using the SCC is a quick and easy tool that reflects severity of inflammation, destruction and proteolytic enzymatic activity/presence.

Regulation of TNF-α with a focus on rheumatoid arthritis.

Cytokines and chemokines represent two important groups of proteins that control the human immune system. Dysregulation of the network in which these immunomodulators function can result in uncontrolled inflammation, leading to various diseases including rheumatoid arthritis (RA), characterized by chronic inflammation and bone erosion. Potential triggers of RA include autoantibodies, cytokines and chemokines. The tight regulation of cytokine and chemokine production, and biological activity is important. Tumor necrosis factor-α (TNF-α) is abundantly present in RA patients' serum and the arthritic synovium. This review, therefore, discusses first the role and regulation of the major proinflammatory cytokine TNF-α, in particular the regulation of TNF-α production, post-translational processing and signaling of TNF-α and its receptors. Owing to the important role of TNF-α in RA, the TNF-α-producing cells and the dynamics of its expression, the direct and indirect action of this cytokine and possible biological therapy for RA are described.

In vivo regulation of chemokine activity by post-translational modification.

Cytokines and chemokines represent two important groups of proteins that control the immune system. Dysregulation of the network in which these immunomodulators function can result in uncontrolled inflammation leading to various diseases, including rheumatoid arthritis, characterized by chronic inflammation and bone erosion. Chemokine activity is regulated at multiple levels, such as post-translational modification (PTM) of chemokines and their receptors by specific enzymes including proteases and peptidylarginine deiminases. Many in vitro experiments underscore the importance of post-translational processing of human chemokines. PTMs may enhance or reduce chemokine activity or may alter the receptor specificity of chemokine ligands. However, identification of chemokine isoforms in physiological in vivo settings forms the ultimate proof that PTM of chemokines is relevant in regulating the biological activity of these molecules. This review summarizes current knowledge on the in vivo role for PTMs in the regulation of chemokine activity.

Polyoxometalates as a novel class of artificial proteases: selective hydrolysis of lysozyme under physiological pH and temperature promoted by a cerium(IV) Keggin-type polyoxometalate.

Hen-egg-white lysozyme (HEWL) is specifically cleaved at the Trp28-Val29 and Asn44-Arg45 peptide bonds in the presence of a Keggin-type [Ce(α-PW(11)O(39))(2)](10-) polyoxometalate (POM; 1) at pH 7.4 and 37 °C. The reactivity of 1 towards a range of dipeptides was also examined and the calculated reaction rates were comparable to those observed for the hydrolysis of HEWL. Experiments with α-lactalbumin (α-LA), a protein that is structurally highly homologous to HEWL but has a different surface potential, showed no evidence of hydrolysis, which indicates the importance of electrostatic interactions between 1 and the protein surface for the hydrolytic reaction to occur. A combination of spectroscopic techniques was used to reveal the molecular interactions between HEWL and 1 that lead to hydrolysis. NMR spectroscopy titration experiments showed that on protein addition the intensity of the (31)P NMR signal of 1 gradually decreased due to the formation of a large protein/polyoxometalate complex and completely disappeared when the HEWL/1 ratio reached 1:2. Circular dichroism (CD) measurements of HEWL indicate that addition of 1 results in a clear decrease in the signal at λ=208 nm, which is attributed to changes in the α-helical content of the protein. (15)N-(1)H heteronuclear single quantum coherence (HSQC) NMR measurements of HEWL in the presence of 1 reveal that the interaction is mainly observed for residues that are located in close proximity to the first site in the α-helical part of the structure (Trp28-Val29). The less pronounced NMR spectroscopic shifts around the second cleavage site (Asn44-Arg45), which is found in the ß-strand region of the protein, might be caused by weaker metal-directed binding, compared with strong POM-directed binding at the first site.

Citrullination of TNF-α by peptidylarginine deiminases reduces its capacity to stimulate the production of inflammatory chemokines.

Citrullination, a posttranslational modification (PTM) recently discovered on inflammatory chemokines such as interleukin-8 (IL-8/CXCL8) and interferon-γ-inducible protein-10 (IP-10/CXCL10), seriously influences their biological activity. Citrullination or the deimination of arginine to citrulline is dependent on peptidylarginine deiminases (PADs) and has been linked to autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA). Chemokines are to date the first identified PAD substrates with receptor-mediated biological activity. We investigated whether cytokines that play a crucial role in RA, like interleukin-1ß (IL-1ß) and tumor necrosis factor-alpha (TNF-α), may be citrullinated by PAD and whether such a PTM influences the biological activity of these cytokines. IL-1ß and TNF-α were first incubated with PAD in vitro and the occurrence of citrullination was examined by Edman degradation and a recently developed detection method for citrullinated proteins. Both techniques confirmed that human TNF-α, but not IL-1ß, was citrullinated by PAD. Citrullination of TNF-α reduced its potency to stimulate chemokine production in vitro on human primary fibroblasts. Concentrations of the inflammatory chemokines CXCL8, CXCL10 and monocyte chemotactic protein-1 (MCP-1/CCL2) were significantly lower in supernatants of fibroblasts induced with citrullinated TNF-α compared to unmodified TNF-α. However, upon citrullination TNF-α retained its capacity to induce apoptosis/necrosis of mononuclear cells, its binding potency to Infliximab and its ability to recruit neutrophils to the peritoneal cavity of mice.

Detection and quantification of citrullinated chemokines.

BACKGROUND: Posttranslational deimination or citrullination by peptidylarginine deiminases (PAD) regulates the biological function of proteins and may be involved in the development of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. This posttranslational modification of arginine was recently discovered on inflammatory chemokines including CXCL8 and CXCL10, and significantly reduced their biological activity. To evaluate the importance of these modified chemokines in patients, methods for the detection and quantification of citrullinated chemokines are needed. Since citrullination only results in an increase of the protein mass with one mass unit and the loss of one positive charge, selective biochemical detection is difficult. Therefore, we developed an antibody-based method to specifically detect and quantify citrullination on a protein of interest. METHODOLOGY/PRINCIPAL FINDINGS: First, the citrullinated proteins were chemically modified with antipyrine and 2,3-butanedione at low pH. Such selectively modified citrullines were subsequently detected and quantified by specific antibodies raised against a modified citrulline-containing peptide. The specificity of this two-step procedure was validated for citrullinated CXCL8 ([Cit(5)]CXCL8). Specific detection of [Cit(5)]CXCL8 concentrations between 1 and 50 ng/ml was possible, also in complex samples containing an excess of contaminating proteins. This novel detection method was used to evaluate the effect of lipopolysaccharide (LPS) on the citrullination of inflammatory chemokines induced in peripheral blood mononuclear cells (PBMCs) and granulocytes. LPS had no significant effect on the induction of CXCL8 citrullination in human PBMCs and granulocytes. However, granulocytes, known to contain PAD, were essential for the production of significant amounts of [Cit(5)]CXCL8. CONCLUSION/SIGNIFICANCE: The newly developed antibody-based method to specifically detect and quantify chemically modified citrullinated proteins is proven to be effective. This study furthermore demonstrates that granulocytes were essential to obtain significant levels of [Cit(5)]CXCL8. For human PBMCs and granulocytes stimulation with LPS did not affect the citrullination of CXCL8.