Specific Granule Protein Research Articles

Perinatal granulopoiesis and risk of pediatric asthma.

There are perinatal characteristics, such as gestational age, reproducibly associated with the risk for pediatric asthma. Identification of biologic processes influenced by these characteristics could facilitate risk stratification or new therapeutic targets. We hypothesized that transcriptional changes associated with multiple epidemiologic risk factors would be mediators of pediatric asthma risk. Using publicly available transcriptomic data from cord blood mononuclear cells, transcription of genes involved in myeloid differentiation was observed to be inversely associated with a pediatric asthma risk stratification based on multiple perinatal risk factors. This gene signature was validated in an independent prospective cohort and was specifically associated with genes localizing to neutrophil-specific granules. Further validation demonstrated that umbilical cord blood serum concentration of PGLYRP-1, a specific granule protein, was inversely associated with mid-childhood current asthma and early-teen FEV1/FVCx100. Thus, neutrophil-specific granule abundance at birth predicts risk for pediatric asthma and pulmonary function in adolescence.

Analysis of expression of genes encoding neutrophil and eosinophil granule proteins during in vitro eosinophilopoiesis

Abstract Neutrophils and eosinophils, the two prominent granulocytes in circulation, are characteristically distinguished by neutrophil primary granule proteins (NPGPs) and eosinophil specific granule proteins (ESGPs). They differentiate from myeloid progenitors in the bone marrow commonly through a successive stage encompassing promyelocyte, myelocyte, metamyelocyte, and band form. The purpose of this study is to analyze bifurcation of the two granulocytes by examining expression of the NPGP- and ESGP-encoding genes during eosinophilic differentiation of cord blood (CB) CD34+ cells for 24 days. All transcripts of the NPGP-encoding genes, including PRTN3, MPO, CTSG, AZU1, and ELANE, reach peaks at day 12 and steeply decrease thereafter, while the transcripts of ESGP-encoding genes, such as MBP and ECP, reached peaks at day 18. Similarly, upon differentiation of immature eosinophilic EoL-1 cells, the NPGP transcripts are markedly downregulated accompanying by upregulated ESGP transcripts. Thus, the NPGP-encoding genes appear to be transcriptionally active even at eosinophilic promyelocyte and myelocyte stages. As these cells undergo maturation through metamyelocyte and band form stages, a transcription network come into a play that favorably shifts expression the ESGP-encoding genes.

Structural and functional analysis of CRISP3 protein in relation to diseases of exocrine pancreas

Introduction: Human cysteine rich secretory protein 3 (CRISP3), also known as specific granule protein (SGP28) is a small 28kDa protein. CRISP-3 is widely expressed in salivary glands, pancreas (particularly in pancreatic disease), lachrymal glands, prostate, myeloid precursors, human epididymis, ovary, thymus and colon. Human CRISP3 share similarity with snake venoms as well as Glioma Pathogenesis Related protein GLIPR1. Here we are reporting the extent of alignment of human CRISP3 protein with pancreatic enzymes and snake venom proteins and presence of GLIPR1 and CRISP3 genes containing conserved GHYTQVVW motifs in clinical samples of pancreatitis and pancreatic cancer. Material and Methods: Protein sequences with accession numbers for Human CRISP3, Snake Venom and enzymes present in pancreatic juices i.e. Trypsinogen, Cathepsin, Lipase, Amylase and Elastase were retrieved from swissprot, protein database. These sequences were aligned in silico for demonstrating the presence of 16 conserved cysteine residues and other motifs i.e. GENL and GHYTQVVW. The multiple sequence alignment (MSA) was performed. Presence of CRISP-3 conserved motifs in clinical samples of pancreatitis and pancreatic cancer was evaluated by PCR. DNA extracted from a small piece taken from inflamed pancreatic tail biopsy with confirmed diagnosis for pancreatitis and an excised pancreatic cancer tissue was subjected to PCR to conform presence of CRISP3 and GLIPR1 genes. Results: Our results show conservation of 9 Cysteine residues out of 16 Cysteine residues in majority of pancreatic enzymes and notable conservation of GENL and GHYTQVVW motifs across snake venom proteins. PCR yielded desired amplicon of expected size with the primers spanning conserved residues and core consensus motifs specifically designed to amplify CRISP3 gene in clinical samples of pancreatitis and pancreatic cancer. We also detected presence of GLIPR1 gene sequence in the DNA extracted from clinical samples of pancreatitis and pancreatic cancer thus confirming similarity between the pathogenesis related protein and CRISP3. Conclusion: Notable conservation of cysteine residues across pancreatic enzymes, snake venom and Human CRISP3 opens avenues of further research to evaluate the likely clinical potential of the conserved residues as putative biomarkers in exocrine pancreatic diseases. To the best of our knowledge this is the first report where primers designed around conserved GHYTQVVW motifs to amplify CRISP3 gene and its structural similarity with GLIPR1 gene sequence in clinical samples of pancreatitis and pancreatic cancer has been validated.

SWI/SNF Protein SMARCD2 Orchestrates Transcriptional Networks Controlling Hematopoiesis and Neutrophil Granulocytes in Humans, Mice and Zebrafish

Differentiation of hematopoietic stem cells follows a hierarchical program of transcriptional-regulated events. We here identify SMARCD2 (Swi/Snf-related matrix associated actin dependent regulator of chromatin, subfamily D, member 2) as critical regulator of myelopoiesis in humans, mice, and zebrafish. We studied four patients from three unrelated pedigrees presenting with a novel syndromatic phenotype comprising congenital neutropenia, specific granule deficiency, susceptibility to myelodysplasia with excess of blasts, and various skeletal anomalies. All patients had homozygous loss-of-function mutations in SMARCD2. In contrast to wildtype alleles, the variant alleles did not give rise to proteins with capacity to interact with the SWI/SNF subunits BRG1, BAF170, BAF155, and BAF47, as shown by co-immunoprecipitation experiments. In vitro, knockdown of SMARCD2 in promyelocytic NB4 cells, differentiated in the presence of ATRA, led to decreased expression of genes encoding the primary granule proteins cathelicidin (CAMP) and alpha-1-antitrypsin (AAT) as well as specific granule proteins matrix metalloproteinase-8 (MMP8), transcobalamin (TCN1) and lactoferrin (LTF). This phenotype is reminiscent of patients with specific granule deficiency, characterized by mutations in CEBPE, a known transcription factor controlling terminal neutrophil development. We therefore hypothesized that SMARCD2 may act via CEBPe and performed immunoprecipitation studies in transfected cells. Upon pull-down of SMARCD2, CEPBE could be detected, and vice versa, suggesting that both proteins physically interact to control transcriptional networks. To interrogate effects of SMARCD2 deficiency on global chromatin accessibility we made use of ATAC sequencing of undifferentiated and ATRA-differentiated NB4 cells and compared this data with comprehensive RNA-sequencing results. A specific subset of genes was found deregulated in both assays, affecting vesicular trafficking, migration and signalling pathways. To validate a role for SMARCD2 in hematopoiesis in vivo, we generated murine and zebrafish model systems. We generated Smarcd2-/- mice by injection of Smarcd2+/- murine ES cells into blastocysts, transfer into pseudo-pregnant mice and interbreeding of heterozygous Smarcd2+/- offsprings. The mutant allele was inherited in a Mendelian fashion but no viable mice were born. 14.5dpc embryos were characterized by anemia and reduced size compared to their littermates. Analysis of fetal liver hematopoiesis revealed a complete absence of CD11b+Gr1+ and CD11b+Ly6c+ cells, whereas the number of LSK stem cells was not affected. Futhermore, Smarcd2-/- embryos showed aberrations in erythroid cells such as extensive anisocytosis, multinucleated cells, and perturbed mitosis. In cytokine-driven colony forming unit assays, GM-CSF, M-CSF, and G-CSF induced myeloid cell differentiation was decreased. Transcriptional profiling of LSK stem cells revealed a striking dysbalance affecting genes involved in signaling pathways and host defence, including CEBPE-dependent genes. Among a total of 12362 detected genes, we found 4290 to be differentially expressed (DESeq2, FDR<10%). Interestingly, the majority (79%) of the 605 genes with a relatively large difference (fold-change > 1.4, FDR<1%) were up- and not downregulated. Next, we generated three Smarcd2-deficient zebrafish models using a) morpholino-mediated knockdown in Tg(mpx:EGFP)i114 and Tg(lyz:dsRed)nz50 strains of the orthologous gene or b) Crisp/Cas9-mediated genomic engineering of this locus in Tg(mpx:EGFP)i114. In all models, the numbers of neutrophil granulocytes were significantly reduced. We conclude that SMARCD2 is a critical factor orchestrating transcriptional networks controlling hematopoiesis across species, in particular regulation and maintenance of neutrophil differentation and prevention of leukemogenesis. DisclosuresAbboud:Novartis: Honoraria; MAST Therapeutics: Research Funding; Eli Lilly and Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Myeloperoxidase Stimulates Neutrophil Degranulation.

Myeloperoxidase, heme enzyme of azurophilic granules in neutrophils, is released into the extracellular space in the inflammation foci. In neutrophils, it stimulates a dose-dependent release of lactoferrin (a protein of specific granules), lysozyme (a protein of specific and azurophilic granules), and elastase (a protein of azurophilic granules). 4-Aminobenzoic acid hydrazide, a potent inhibitor of peroxidase activity of myeloperoxidase, produced no effect on neutrophil degranulation. Using signal transduction inhibitors (genistein, methoxyverapamil, wortmannin, and NiCl2), we demonstrated that myeloperoxidase-induced degranulation of neutrophils resulted from enzyme interaction with the plasma membrane and depends on activation of tyrosine kinases, phosphatidylinositol 3-kinases (PI3K), and calcium signaling. Myeloperoxidase modified by oxidative/halogenation stress (chlorinated and monomeric forms of the enzyme) lost the potency to activate neutrophil degranulation.

Role of Eosinophil Granulocytes in Allergic Airway Inflammation Endotypes.

Eosinophil granulocytes are intriguing members of the innate immunity system that have been considered important defenders during parasitic diseases as well as culprits during allergy-associated inflammatory diseases. Novel studies have, however, found new homoeostasis-maintaining roles for the cell. Recent clinical trials blocking different Th2 cytokines have uncovered that asthma is heterogeneous entity and forms different characteristic endotypes. Although eosinophils are present in allergic asthma with early onset, the cells may not be essential for the pathology. The cells are, however, likely disease causing in asthma with a late onset, which is often associated with chronic rhinosinusitis. Assessment of eosinophilia, fraction exhaled nitric oxide (FeNO) and periostin are markers that have emerged useful in assessing and monitoring asthma severity and endotype. Current scientific knowledge suggests that eosinophils are recruited by the inflammatory environment, activated by the innate interleukin (IL)-33 and prevented from apoptosis by both lymphocytes and innate immune cells such as type two innate immune cells. Eosinophils contain four specific granule proteins that exhibit an array of toxic and immune-modulatory activates. The granule proteins can be released by different mechanisms. Additionally, eosinophils contain a number of inflammatory cytokines and lipid mediators as well as radical oxygen species that might contribute to the disease both by the recruitment of other cells and the direct damage to supporting cells, leading to exacerbations and tissue fibrosis. This review aimed to outline current knowledge how eosinophils are recruited, activated and mediate damage to tissues and therapies used to control the cells.

Heterogeneous Expression of OLFM4 in Neutrophils and Plasma

Introduction: Olfactomedin 4 (OLFM4) is a glycoprotein predominantly expressed in myeloid cells and in gastrointestinal tissues. OLFM4 is stored in specific granules of human neutrophils where it defines a subset of neutrophils ranging from 5-40% OLFM4 positive neutrophils. OLFM4 has been proposed to inhibit cathepsin C, a cysteine protease essential for activation of serine proteases. Elevated levels of OLFM4 has been seen primary myelofibrosis (PMF) patients, these patients furthermore display a significantly elevated serum OLFM4.Methods: We developed monoclonal antibodies against OLFM4 and established an Enzyme Linked Immunosorbent Assay for OLFM4 for further investigation of OLFM4 in myeloid cells.Results: We observed two populations of individuals with respect to OLFM4 levels in plasma, the majority with OLFM4 in plasma between 0 and 0.1 μg/mL, mean 0.028 μg/mL while approximately 10% had OLFM4 between 4 and 60 μg/mL, mean 15 μg/mL. The levels were constant over time.The level did not relate to the size of the OLFM4 positive neutrophil subset detected in peripheral blood. We studied the biosynthesis of OLFM4 in isolated bone marrow cells from an individual with high plasma OLFM4 and an individual with low plasma OLFM4. The levels of OLFM4 mRNA were comparable and the amounts of OLFM4 synthesized and retained in cells were similar between the two individuals. We next determined whether OLFM4 might be produced by bone marrow cells and released into bone marrow plasma. Corresponding levels of OLFM4 determined in bone marrow plasma and blood plasma from two persons with high levels of OLFM4 showed lower levels in bone marrow plasma than in blood plasma, arguing against bone marrow as the direct source of OLFM4 in plasma.To estimate the amount of OLFM4 generated daily during myelopoiesis, we quantitated the amount of OLFM4 in neutrophils from 3 sets of buffy coat neutrophils, each pooled from 4 healthy donors. The amount of OLFM4 was 1.2 μg/107 neutrophils. As the production of neutrophils is about 1 x 109 cells/kg body weight/day, this would indicate production of 10 mg OLFM4/day in an adult.To rule out the liver as a production site, mRNA was determined by Affymetrix gene array in liver biopsies from 42 patients evaluated for liver steatosis. OLFM4 mRNA levels were uniformly at the border of detection in all (data not shown).OLFM4 is secreted from PMA stimulated neutrophils in parallel with other specific granule proteins. When purified OLFM4 was added to medium from neutrophils induced to degranulate with PMA, the ability to detect OLFM4 was rapidly lost, indicating that OLFM4 is highly sensitive to proteolysis. Adding a cocktail of protease inhibitors to the material secreted from PMA activated neutrophils preserved OLFM4.As we find that OLFM4 is highly sensitive to proteolysis we suggest that the differences in OLFM4 levels in plasma may be related to individual differences in the susceptibility of OLFM4 to escape degradation when neutrophils decease as part of their normal life cycle. Production of 10 mg OLFM4/day would support a plasma level of 3-4 µg/ml plasma depending of the half-life of OLFM4 in plasma. This hypothesis is not easily tested, but if proven correct, might open for novel insight into the fate of neutrophils after exiting circulation, an issue that is still a matter of debate. DisclosuresNo relevant conflicts of interest to declare.

Protein post-translational modification in host defense: the antimicrobial mechanism of action of human eosinophil cationic protein native forms.

Knowledge on the contribution of protein glycosylation in host defense antimicrobial peptides is still scarce. We have studied here how the post-translational modification pattern modulates the antimicrobial activity of one of the best characterized leukocyte granule proteins. The human eosinophil cationic protein (ECP), an eosinophil specific granule protein secreted during inflammation and infection, can target a wide variety of pathogens. Previous work in human eosinophil extracts identified several ECP native forms and glycosylation heterogeneity was found to contribute to the protein biological properties. In this study we analyze for the first time the antimicrobial activity of the distinct native proteins purified from healthy donor blood. Low and heavy molecular weight forms were tested on Escherichia coli cell cultures and compared with the recombinant non-glycosylated protein. Further analysis on model membranes provided an insight towards an understanding of the protein behavior at the cytoplasmic membrane level. The results highlight the significant reduction in protein toxicity and bacteria agglutination activity for heavy glycosylated fractions. Notwithstanding, the lower glycosylated fraction mostly retains the lipopolysaccharide binding affinity together with the cytoplasmic membrane depolarization and membrane leakage activities. From structural analysis we propose that heavy glycosylation interferes with the protein self-aggregation, hindering the cell agglutination and membrane disruption processes. The results suggest the contribution of post-translational modifications to the antimicrobial role of ECP in host defense.

NGAL Can Alternately Mediate Sunitinib Resistance in Renal Cell Carcinoma

Serum NGAL is highly expressed in patients with advanced renal cancer treated with sunitinib. We investigated the role of NGAL in sunitinib resistance in renal cell carcinoma to identify potential tactics to overcome it. NGAL expression was correlated with sunitinib sensitivity. Vascular endothelial growth factor related upstream Ras, Erk1/2 and STAT1 phosphorylation activity in Caki-1 and NGAL transfected Caki-1 cells after sunitinib treatment was analyzed using Western blot. NGAL and vascular endothelial growth factor-A interaction with sunitinib therapeutic efficacy was monitored in renal cell carcinoma tumor xenografted mice by tumor growth inhibition, serum NGAL and vascular endothelial growth factor-a levels, and microscopic examination of tumor microvascular density. Sunitinib cytotoxicity in various renal cell carcinoma cell lines was reversibly related to NGAL expression. Sunitinib showed the lowest 50% inhibitory concentration (5.53 μM) in Caki-1 cells, which had the lowest NGAL expression of these renal cell carcinoma cell lines. After sunitinib treatment adding NGAL inhibited Ras and Erk1/2 phosphorylation but activated STAT1α phosphorylation in Caki-1 cells and Caki-1 cells transfected with NGAL. In a xenograft mouse model sunitinib significantly inhibited tumor growth in Caki-1 mice. NGAL transfected Caki-1 mice had higher serum NGAL and lower vascular endothelial growth factor-A than Caki-1 mice. Microvascular density was decreased in Caki-1 mice with sunitinib treatment. NGAL in tumor cells may show crosstalk with vascular endothelial growth factor-a and alternative activation in stimulating tumor growth during sunitinib treatment. It may become a therapeutic target to reverse sunitinib resistance in renal cell carcinoma.

Azurophil granule proteins constitute the major mycobactericidal proteins in human neutrophils and enhance the killing of mycobacteria in macrophages.

Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule proteins (AZP) were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane resulting in killing of mycobacteria. Exogenous addition of AZP to murine macrophage RAW 264.7, THP-1 and peripheral blood monocyte-derived macrophages significantly reduced the intracellular survival of mycobacteria without exhibiting cytotoxic activity on macrophages. Immunofluorescence studies showed that macrophages actively endocytose neutrophil granular proteins. Treatment with AZP resulted in increase in co-localization of BCG containing phagosomes with lysosomes but not in increase of autophagy. These data demonstrate that neutrophil azurophil proteins may play an important role in controlling intracellular survival of mycobacteria in macrophages.

Olfactomedin 4 defines a subset of human neutrophils

OLFM4 was identified initially as a gene highly induced in myeloid stem cells by G-CSF treatment. A bioinformatics method using a global meta-analysis of microarray data predicted that OLFM4 would be associated with specific granules in human neutrophils. Subcellular fractionation of peripheral blood neutrophils demonstrated complete colocalization of OLFM4 with the specific granule protein NGAL, and stimulation of neutrophils with PMA resulted in corelease of NGAL and OLFM4, proving that OLFM4 is a genuine constituent of neutrophil-specific granules. In accordance with this, OLFM4 mRNA peaked at the MY/MM stage of maturation. OLFM4 was, however, present in only 20-25% of peripheral blood neutrophils, as determined by immunocytochemistry and flow cytometry, whereas mRNA for OLFM4 was present in all MY/MM, indicating post-transcriptional regulation as a basis for the heterogeneous expression of OLFM4 protein.

The SNARE protein VAMP2 but not VAMP3 mediates cAMP‐stimulated renin release from juxtaglomerular cells

Exocytosis of renin containing granules mediates renin release from juxtaglomerular (JG) cells, a process stimulated by cAMP. We found that the SNARE protein SNAP23 is involved in renin release. To mediate exocytosis, SNAP23 must interact with a vesicle associated membrane protein (VAMP). We found that VAMP2 and VAMP3 are expressed in JG cells but their role is not known. We hypothesized that VAMP2 mediates cAMP-stimulated renin release from mouse JG cells. To study the role of VAMP2 and 3 in JG cells we generated adenoviruses expressing shRNAs for these proteins. Transducing primary cultures of JG cells with either VAMP2 shRNA (n=4) or shVAMP3 (n=3) specifically decreased VAMP2 and VAMP3 respectively, without affecting other VAMPs or GAPDH. In control cells transduced with scrambled shRNA, increasing cAMP with Forskolin/IBMX stimulated renin release by 226±31% (p<0.05). However, in JG cells transduced with VAMP2-shRNA, cAMP increased renin release only by 81±27%, a 64% blockade (p<0.05). In contrast, silencing VAMP3 did not affect the stimulatory effect of cAMP on renin release (n=9). Confocal microscopy showed that VAMP2 colocalized with renin containing granules, whereas VAMP3 was expressed in JG cells but did not colocalize with renin. We concluded that VAMP2 but not VAMP3 mediates in part cAMP-stimulated renin release, identifying for the first time a specific granule protein involved in renin release. NIH/NRSA to M. Mendez.

Olfactomedin 4, An Indicator of the Existence of Neutrophil Subsets.

AbstractAbstract 3775Olfactomedin 4 (OLFM4) was initially identified as a gene highly induced in myeloid stem cells by G-CSF treatment and independently as a gene highly expressed in colon cancers. OLFM4 was predicted in a bioinformatics analysis as associated with neutrophil specific granules. We analyzed the expression of OLFM4 mRNA in myeloid cells from normal human bone marrow and demonstrated that expression of OLFM4 mRNA is similar to the expression of LCN2 which codes for the specific granule protein NGAL (Figure 1), but distinct from expression of mRNA for myeloperoxidase and gelatinase which are marker proteins for azurophil granules and gelatinase granules, respectively. Subcellular fractionation of peripheral blood neutrophils demonstrated complete co-localization of OLFM4 with NGAL, and stimulation of neutrophils with fMLP or PMA resulted in co-release of NGAL and OLFM4, indirectly proving that OLFM4 is a genuine constituent of neutrophil specific granules. [Display omitted] Interestingly, immunohistochemistry showed OLFM4 expression in only a subset of neutrophils (figure 2). We suspected that this might be dependent on the antibody, but two different commercial antibodies and an in-house antibody raised against a synthetic OLFM4 derived peptide, all polyclonal, showed similar patterns. Flow cytometry confirmed the existence of two populations of neutrophils, one expressing OLFM4 the other not. [Display omitted] Immunohistochemistry of bone marrow cells showed that OLFM4 appears in myelocytes and is maintained in the cells during further maturation of the cells to segmented neutrophils. Again, only 30% of the neutrophil precursors from bone marrow stain positive for OLFM4 indicating, that different subsets of human neutrophils may exist. Disclosures:No relevant conflicts of interest to declare.

Vesicle-mediated secretion of human eosinophil granule-derived major basic protein

Major basic protein (MBP), the predominant cationic protein of human eosinophil specific granules, is stored within crystalloid cores of these granules. Secretion of MBP contributes to the immunopathogenesis of varied diseases. Prior electron microscopy (EM) of eosinophils in sites of inflammation noted losses of granule cores in the absence of granule exocytosis and suggested that eosinophil granule proteins might be released through piecemeal degranulation (PMD), a secretory process mediated by transport vesicles. Because release of eosinophil granule-derived MBP through PMD has not been studied, we evaluated secretion of this cationic protein by human eosinophils. Intracellular localizations of MBP were studied within nonstimulated and eotaxin-stimulated human eosinophils by both immunofluorescence and a pre-embedding immunonanogold EM method that enables optimal epitope preservation and antigen access to membrane microdomains. In parallel, quantification of transport vesicles was assessed in eosinophils from a patient with hypereosinophilic syndrome (HES). Our data demonstrate vesicular trafficking of MBP within eotaxin-stimulated eosinophils. Vesicular compartments, previously implicated in transport from granules to the plasma membrane, including large vesiculotubular carriers termed eosinophil sombrero vesicles (EoSVs), were found to contain MBP. These secretory compartments were significantly increased in numbers within HES eosinophils. Moreover, in addition to granule-stored MBP, even unstimulated eosinophils contained appreciable amounts of MBP within secretory vesicles, as evidenced by immunonanogold EM and immunofluorescent colocalizations of MBP and CD63. These data suggest that eosinophil MBP, with its multiple extracellular activities, can be mobilized from granules by PMD into secretory vesicles and both granule- and secretory vesicle-stored pools of MBP are available for agonist-elicited secretion of MBP from human eosinophils. The recognition of PMD as a secretory process to release MBP is important to understand the pathological basis of allergic and other eosinophil-associated inflammatory diseases.

Evaluation of the Combined Application of Ethanol-Fixed and Formaldehyde-Fixed Neutrophil Substrates for Identifying Atypical Perinuclear Antineutrophil Cytoplasmic Antibodies in Inflammatory Bowel Disease

No clear guidelines for indirect immunofluorescence (IIF) detection and interpretation of antineutrophil cytoplasmic antibodies (ANCA) have been proposed for inflammatory bowel diseases (IBD). We evaluated the reliability of the combined use of ethanol- and formalin-fixed neutrophil substrates to identify atypical perinuclear ANCA (P-ANCA) by IIF under routine laboratory circumstances. A total of 204 IBD patients were assessed with four different fluorescent substrates in two distinct laboratories. Antibodies against myeloperoxidase, proteinase-3, and other specific granule proteins (elastase, lactoferrin, cathepsin G, lysozyme, and bactericidal permeability-increasing protein) were measured by an enzyme-linked immunosorbent assay. The combined application of ethanol- and formalin-fixed slides to detect atypical P-ANCA resulted in a lack of agreement between assays (kappa, < or =0.39) in the interassay study and moderate agreement in the interobserver study (kappa, 0.42). After atypical and typical P-ANCA patterns were combined, the consensus improved greatly. A total of 26.9% of patients were P-ANCA positive by at least two tests (44.3% of ulcerative colitis [UC] and 13.1% of Crohn's disease [CD] patients; P < 0.0001), while overall ANCA positivity was 22.5% to 34.8%. The combined application of ethanol-fixed and formaldehyde-fixed neutrophil substrates did not facilitate differentiation between P-ANCA and atypical P-ANCA, and the results were not consistent when substrates from different sources were used. Combining all P-ANCA ensures the highest sensitivity and specificity in differentiating UC from CD.

Clinical significance of eosinophilic cationic protein levels in nasal secretions of patients with nasal polyposis

Nasal polyps are characterized by eosinophilic infiltration, and frequently coexist with asthma, aspirin intolerance and allergy. Eosinophilic cationic protein (ECP) is a specific eosinophil granule protein released upon activation of eosinophils. We investigated the ECP levels in nasal secretions of patients with nasal polyposis (NP) in order to correlate them with disease severity and associated diseases and to compare ECP levels between patients with and without recurrence of NP after surgical treatment. A total of 78 patients who had surgery for NP were followed up for a minimum of 18 months. The presence of asthma, allergies or aspirin intolerance was noted. Nasal secretions were obtained 1 day before the surgery and during the follow-up period after surgery. Immunoassays were used to quantify ECP in nasal secretions and serum and interleukin (IL)-5 in nasal secretions. ECP levels in nasal secretions were higher in patients with asthma or aspirin intolerance than in patients without asthma or aspirin intolerance, while no significant differences were found between allergic and non-allergic patients. ECP levels in nasal secretions correlated significantly with IL-5 levels in nasal secretions, the degree of tissue eosinophilia and computed tomographic (CT) scores. In total, 30 patients (38%) developed recurrent NP during the follow-up period. Preoperative ECP and IL-5 levels in nasal secretions were significantly higher in patients with recurrence compared to patients without recurrence. During the follow-up period, patients without recurrence demonstrated a significant reduction in the ECP levels in nasal secretions, whereas there was no significant reduction in the ECP levels of patients with recurrence. The results of this study provide evidence that ECP levels in nasal secretions of patients with NP correlate with the presence of asthma or aspirin intolerance and severity of NP determined by CT scores.

P059 STEROIDS' EFFECTS ON ANGIOGENIC FACTORS LEVELS IN ULCERATIVE COLITIS (UC) PATIENTS

s of the 3rd ECCO Congress, Lyon, France, February 28–March 1, 2008 23 says, and the inter-observer precision based on the results of two distinct laboratories. Antibody levels to myeloperoxidase (MPO), proteinase-3 (PR3), elastase, lactoferrin, cathepsin G, lysozyme and bactericidal permeabilityincreasing protein (BPI) were measured by ELISA. Agreement was estimated by Weighted Kappa. Results: With the combined application of ethanol-fixed and formaldehydefixed substrates from four different sources, atypical P-ANCA was detected in 9.9%-39.6% of patients with UC and 9.3%-17.8% with CD. Striking differences were observed among the distributions of IIF patterns, resulting in K values ranging 0.14-0.34 for atypical P-ANCA. When atypical and typical P-ANCA patterns were combined together, the consensus greatly improved. Moderate agreement was observed in the inter-observer study regarding atypical P-ANCA (K=0.44), which also improved after grouping typical and atypical P-ANCAs together (K=0.53). Altogether, P-ANCA was detected in UC and CD patients with a frequency of 62.5% versus 20.4% (p<0.0001) and 49.0% versus 18.6% (p<0.0001) in the inter-observer study (n.s.). Antibodies against specific granule proteins occurred in low percentage of the subjects and were associated with diverse IIF patters. Multiple positivities were prevalent, and antibody specificities did not correlate with disease type or characteristics. Conclusions: The combined application of ethanol-fixed and formaldehydefixed substrates did not facilitate the differentiation between P-ANCA and atypical P-ANCA, and the results were not consistent when using substrates from different sources. Combining all P-ANCAs (typical and atypical) together ensures the highest sensitivity and specificity in differentiating UC from CD.

Production and Regulation of Eotaxin-2/CCL24 in a Differentiated Human Leukemic Cell Line, HT93

When a human leukemic cell line, HT93 was incubated with all-trans retinoic acid (ATRA), IL-5, or both, this cell line was differentiated into eosinophic lineage, in that an eosinophilic specific granule proteins, major basic protein (MBP) and eosinophil peroxidase (EPO) appeared. Both CD11b and CC chemokine receptor, CCR3 expression were upregulated, while CD71 expression was downregulated by ATRA or ATRA+IL-5. Concomitantly, marked production of eotaxin-2/CCL24 was observed, but no production of eotaxin-1/CCL11 and eotaxin-3/CCL26 was detected. Since only 20 to 30% cells incubated with ATRA became positive for CCR3, CCR3(+) population was enriched by a magnetic activated cell sorter (MACS). Enriched CCR3(+) population produced higher eotaxin-2/CCL24 than the CCR3(-) population, indicating that differentiated eosinophils are capable of producing eotaxin-2/CCL24. During the ATRA-induced differentiation, expression of a transcriptional factor, GATA-1 was significantly increased. Introduction of siRNA against GATA-1 markedly reduced the ATRA-induced differentiation markers including CD11b and CCR3, as well as reduced eotaxin-2/CCL24 production. Finally, ATRA-induced differentiation and eotaxin-2/CCL24 production were greatly enhanced in the GATA-1-overexpressed clones. These results indicate that the ability to produce eotaxin-2/CCL24 is acquired during the differentiation into eosinophilic lineage which is dependent on GATA-1 expression.

Thiocyanate-Dependent Inhibition of Spontaneous and Agonist-Induced Eosinophil Apoptosis by Eosinophil Peroxidase (EPO): A Potential Physiologic Role for Endogenously Generated HOSCN in Maintaining Eosinophil Viability.

Apoptotic eosinophils (EOs), atypically, release cytotoxic specific granule proteins that promote tissue damage in eosinophilic inflammatory states such as asthma. PMN myleloperoxidase strongly promotes PMN apoptosis by generating HOCl. We tested whether EPO plays a similarly pivotal role in EO apoptosis. In vivo, bromide (Br−), nitrite (NO2−), and thiocyanate (SCN−) compete for oxidation by EPO and H2O2 yielding, respectively, HOBr, NO2°, and HOSCN. We have shown that SCN- is the strongly preferred substrate for EPO in vivo and that HOSCN, in striking contrast to HOBr, NO2° and HOCl, is a weak, sulfhydryl-specific oxidant. Because we recently showed that HOSCN is a uniquely potent oxidant activator of endothelial cell NF-kB (Blood 107:558 2006), a powerful antagonist of apoptosis, we hypothesized that endogenously generated HOSCN would inhibit EO apoptosis. Blood EOs were isolated from mildly atopic donors by immunomagnetic separation to > 95% purity. EOs were cultured in RPMI + 10% FCS without added IL-5 and assayed for cell viability by Annexin V and Propidium Iodide (PI) staining and flow cytometry. Apoptosis was confirmed using an immunoassay of cytoplasmic histone-associated DNA fragments, caspase 3 activation and morphology. In every time course examined, EOs were first annexin+/PI-, then later annexin+/PI+. Therefore data are here reported as viability normalized to a control of EOS with 1 ng/ml IL-5, the remainder being comprised of early and late apoptotic cells. EOs cultured 2 days with 1 mM SCN− were 69% viable, a 77% relative increase (n=9, p < 0.0001) over those cultured with nothing (i.e., Cl- only), 1 mM Br-, or 1 mM NO2-, all of which had the same viability (~39%). When 0.5 nM PMA was added to activate the respiratory burst, viability with SCN− after 2 days was 63%, Cl- 5%, Br- 2%, and NO2- 14%. Surprisingly, viability with PMA and SCN− was 20% higher than that with Cl- without PMA (p < 0.05), suggesting that HOSCN not only fails to promote apoptosis but instead engenders an anti-apoptotic tone. Addition of the EPO inhibitor azide (1 mM) abrogated the protective effect of SCN− with PMA. Moreover, SCN- failed to protect EPO-devoid monocytes and lymphocytes from both spontaneous and PMA-induced apoptosis. EOs activated with the physiologic agonists C5a (33 nM) and PAF (5 μM) exhibited the same protective effect of SCN− and increased viability in activated vs. unactivated EOs. EOs treated 2 hours with and without 1mM H2O2 before adding an agonist anti-Fas antibody (1 μg/ml) had viabilities ~30% higher with SCN− than with the other halides at day 1 and at day 2, late-apoptotic cells were 42% that in Cl-. BAY 11-7085 (10μM), an inhibitor of NF-kB activation, caused rapid EO apoptosis (70% at day 1) but in this setting SCN− was not protective with or without PMA activation. Unlike PMN, Western blots for IkB-alpha showed no degradation with PMA irrespective of halide. We conclude that HOSCN generated endogenously in EOs by the EPO/H2O2/ SCN− system plays a previously unsuspected role to maintain both constitutive and agonist-stimulated EO survival. HOSCN antagonizes EO apoptosis through a mechanism that may require constitutive, but not inducible, activation of NF-kB. Because serum SCN− levels vary widely (10–300 μM) and are dietarily determined, oral supplementation with this inexpensive and innocuous pseudohalide may mitigate tissue injury in eosinophilic inflammatory states by inhibiting EO apoptosis in infiltrated organs.

Effects of Mouse and Human Lipocalin Homologues 24p3/lcn2 and Neutrophil Gelatinase–Associated Lipocalin on Gastrointestinal Mucosal Integrity and Repair

The lipocalin superfamily, including the mouse and human homologues 24p3/lcn2 and neutrophil gelatinase-associated lipocalin, show great functional diversity including roles in olfaction, transportation, and prostaglandin synthesis in mammals. Their potential role in maintaining gastrointestinal mucosal integrity and repair is, however, unclear. Changes in 24p3/lcn2 expression in the mouse gut in response to various noxious agents were examined using Northern blot, in situ hybridization, and immunohistochemistry. Effects of recombinant 24p3/lcn2 on proliferation ([3H]-thymidine uptake), and restitution (cell-wounding migration) were assessed using human colonic HT29 and HCT116 cells. In addition, the effects of recombinant 24p3/lcn2 on the amount of gastric damage were assessed in rats treated with indomethacin (20 mg/kg) and restraint. Marked up-regulation of expression of 24p3/lcn2 was seen throughout the gut in response to indomethacin or dextran sodium sulfate treatment. Expression was increased particularly in the surface epithelial cells and infiltrating inflammatory cells. Proliferation and restitution assays in the presence of recombinant wild-type sequence neutrophil gelatinase-associated lipocalin, wild-type cys(98)-24p3/lcn2, and mutant ala98-24p3/lcn2 showed that all 3 peptides caused a 3- to 4-fold increase in promigratory activity (P < .01 vs control) but did not influence proliferation. The administration of wild-type cys98-, or mutant ala98-24p3/lcn2 (25 and 50 microg/kg/h, respectively), given via the subcutaneous route, both caused similar reductions in the rat gastric damage model (60% reduction at highest dose, P < .01 vs control), although oral administration was ineffective. 24p3/lcn2 facilitates mucosal regeneration by promoting cell migration.