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Restitution of wounds in colonic epithelium is essential in the maintenance of health. Microbial products, such as the short-chain fatty acid butyrate, can have positive effects on wound healing. We used an in vitro model of T84 colonic epithelial cells to determine if the Snail genes Slug ( SNAI2) and Snail ( SNAI1), implemented in keratinocyte monolayer healing, are involved in butyrate-enhanced colonic epithelial wound healing. Using shRNA-mediated Slug/Snail knockdown, we found that knockdown of Slug (Slug-KD), but not Snail (Snail-KD), impairs wound healing in scratch assays with and without butyrate. Slug and Snail had differential effects on T84 monolayer barrier integrity, measured by transepithelial resistance, as Snail-KD impaired the barrier (with or without butyrate), whereas Slug-KD enhanced the barrier, again with or without butyrate. Targeted transcriptional analysis demonstrated differential expression of several tight junction genes, as well as focal adhesion genes. This included altered regulation of Annexin A2 and ITGB1 in Slug-KD, which was reflected in confocal microscopy, showing increased accumulation of B1-integrin protein in Slug-KD cells, which was previously shown to impair wound healing. Transcriptional analysis also indicated altered expression of genes associated with epithelial terminal differentiation, such that Slug-KD cells skewed toward overexpression of secretory cell pathway-associated genes. This included trefoil factors TFF1 and TFF3, which were expressed at lower than control levels in Snail-KD cells. Since TFFs can enhance the barrier in epithelial cells, this points to a potential mechanism of differential modulation by Snail genes. Although Snail genes are crucial in epithelial wound restitution, butyrate responses are mediated by other pathways as well.NEW & NOTEWORTHY Although butyrate can promote colonic mucosal healing, not all of its downstream pathways are understood. We show that the Snail genes Snail and Slug are mediators of butyrate responses. Furthermore, these genes, and Slug in particular, are necessary for efficient restitution of wounds and barriers in T84 epithelial cells even in the absence of butyrate. These effects are achieved in part through effects on regulation of β1 integrin and cellular differentiation state.

N-formyl-Met-Leu-Phe (fMLF) is a model PAMP/DAMP driving human PMN to sites of injury/infection utilizing the GPCR, FPR1. We examined a microtiter plate format for measurement of FPR1 phosphorylation in adherent PMN at high densities and found that a new phosphosensitive FPR1 fragment, 25K-FPR1, accumulates in SDS-PAGE extracts. 25K-FPR1 is fully inhibited by diisopropylfluorophosphate PMN pretreatment but is not physiologic, as its formation failed to be significantly perturbed by ATP depletion, time and temperature of adherence, or adherence mechanism. 25K-FPR1 was minimized by extracting fMLF-exposed PMN in lithium dodecylsulfate at 4°C prior to reduction/alkylation. After exposure of adherent PMN to a 5 log range of PAF before or after fMLF, unlike in suspension PMN, no inhibition of fMLF-induced FPR1 phosphorylation was observed. However, PAF induced the release of 40% of PMN lactate dehydrogenase, implying significant cell lysis. We infer that PAF-induced inhibition of fMLF-dependent FPR1 phosphorylation observed in suspension PMN does not occur in the unlysed adherent PMN. We speculate that although the conditions of the assay may induce PAF-stimulated necrosis, the cell densities on the plates may approach levels observed in inflamed tissues and provide for an explanation of PAF’s divergent effects on FPR1 phosphorylation as well as PMN function.

Neutrophils are the most abundant white blood cells, with a vital role in innate immune defense against bacterial and fungal pathogens. Although mostly associated with pathological processes directly related to immune defense, they can also play a detrimental role in inflammatory conditions and have been found to have a pro-metastatic role in the spread of cancer cells. Here, we explore ways to temporarily suppress these detrimental activities. We first examined the possibility of using siRNA and antisense oligonucleotides (ASOs) for transient knockdown of the human and mouse C5a receptor, an important chemoattractant receptor involved in neutrophil-mediated injury that is associated with myocardial infarction, sepsis, and neurodegenerative diseases. We found that siRNAs and ASOs transfected into cultured cell lines can eliminate 70–90% of C5a receptor mRNA and protein within 72 h of administration, a clinically relevant time frame after a cardiovascular event. Targeted drug delivery to specific cells or tissues of interest in a mammalian host, however, remains a major challenge. Here, using phage display technology, we have identified peptides that bind specifically to CD177, a neutrophil-specific surface molecule. We have attached these peptides to fluorescent, lipid-based nanoparticles and confirmed targeting and delivery to cultured cells ectopically presenting either human or mouse CD177. In addition, we have shown peptide-nanoparticle binding specifically to neutrophils in human and mouse blood. We anticipate that these or related tagged nanoparticles may be therapeutically useful for delivery of siRNAs or ASOs to neutrophils for transient knockdown of pro-inflammatory proteins such as the C5a receptor.

Human neutrophil flavocytochrome b (Cyt b) is a heterodimeric, integral membrane protein that generates high levels of superoxide in the multisubunit NADPH oxidase complex. Since Cyt b is currently isolated in limited quantities, improved methods for purification from low levels of starting membranes (from both neutrophils and other expressing cell types) are important for the analysis of structure and catalytic mechanism. In the present study, the epitope-mapped monoclonal antibody CS9 was coupled to Sepharose beads and used as an affinity matrix for single-step immunoaffinity purification of Cyt b. Following solubilization of both human neutrophil and PLB-985 membrane fractions in the nonionic detergent octylglucoside, Cyt b was absorbed on the CS9-Sepharose affinity matrix and purified protein was eluted under non-denaturing conditions with an epitope-mimicking peptide. The high efficiency of this isolation procedure allowed Cyt b to be reproducibly purified from readily obtainable levels of starting membrane fractions (9x10(8) cell equivalents of neutrophil membranes and 2x10(9) cell equivalents of PLB-985 membranes). Since Cyt b could be affinity-purified in the detergent octylglucoside, high-level functional reconstitution was carried out directly on elution fractions by simple addition of solubilized phospholipid and subsequent dialysis for detergent removal. To our knowledge, this study describes the most efficient method for generating purified, functionally-reconstituted Cyt b and should facilitate analyses that require a highly-defined NADPH oxidase system.

The heterodimeric, integral membrane protein flavocytochrome b (Cyt b) is the catalytic core of the phagocyte NADPH oxidase and generates superoxide which plays a critical role in host defense. To better define the activation of superoxide production by this multisubunit enzyme complex, Cyt b-specific monoclonal antibodies (mAbs) and the p47phox SH3 domains (p47SH3AB) were used in the present study as probes to map surface structure and conformational dynamics in human neutrophil Cyt b. In pull-down and co-immunoprecipitation studies with detergent-solubilized Cyt b, the oxidase-inhibitory mAb CS9 was shown to share an overlapping binding site with p47SH3AB on the C-terminal region of the p22phox subunit. Similar studies demonstrated a surprising lack of overlap between the mAb 44.1 and CS9/p47SH3AB binding sites, and they indicated that the oxidase-inhibitory mAb NL7 binds a region physically separated from the p22phox C-terminal domain. Resonance energy transfer and size exclusion chromatography confirmed the above results for functionally reconstituted Cyt b and provided evidence that binding of both mAb CS9 and p47SH3AB altered the conformation of Cyt b. Further support that binding of the p47phox SH3 domains modulates the structure of Cyt b was obtained using a cell-free assay system where p47SH3AB enhanced superoxide production in the presence of a p67phox (1-212)-Rac1(Q61L) fusion protein. Taken together, this study further characterizes the structure of human neutrophil Cyt b in both detergent micelles and reconstituted membrane bilayers, and it provides evidence that the cytosolic regulatory subunit p47phox modulates the conformation of Cyt b (in addition to serving as an adapter protein) during oxidase activation.

The well-described antimicrobial and immunoregulatory properties of human cathelicidin antimicrobial protein 18 (hCAP-18) derive in part from the ability of its proteolytic fragment, LL-37 (a.k.a. CAP-37), to associate with activated immune and epithelial cells during inflammation. We now show a stable association between hCAP-18 and the cell surface of formyl-Met-Leu-Phe (fMLF)-stimulated neutrophils using two novel hCAP-18-specific mAb, H7 and N9, which recognize a single 16-kDa band, identified by N-terminal sequencing and mass spectrometry as hCAP-18. Phage display analysis of epitope-binding sites showed that both mAb probably recognize a similar five amino acid sequence near the C terminus of the prodomain. Immunoblot analysis of degranulated neutrophil supernatants resulted in mAb recognition of the 14-kDa prodomain of hCAP-18. Subcellular fractionation of unstimulated neutrophils on density gradients showed expected cosedimentation of hCAP-18 with specific granule lactoferrin (LF). fMLF stimulation resulted in an average 25% release of specific granule hCAP-18, with ∼15% of the total cellular hCAP-18 recovered from culture media, and ∼10% and ∼75%, respectively, codistributing with plasma membrane alkaline phosphatase and specific granule LF. Surface association of hCAP-18 on fMLF-stimulated neutrophils was confirmed by immunofluorescence microscopy and flow cytometry analysis, which also suggested a significant up-regulation of surface hCAP-18 on cytochalasin B-pretreated, fully degranulated neutrophils. hCAP-18 surface association was labile to 10 mM NaOH treatment but resistant to 1 M NaCl and also partitioned into the detergent phase following Triton X-114 solubilization, possibly suggesting a stable association with one or more integral membrane proteins. We conclude that fMLF stimulation promotes redistribution of hCAP-18 to the surface of human neutrophils.

Accumulation, activation, and control of neutrophils at inflammation sites is partly driven by N-formyl peptide chemoattractant receptors (FPRs). Occupancy of these G-protein-coupled receptors by formyl peptides has been shown to induce regulatory phosphorylation of cytoplasmic serine/threonine amino acid residues in heterologously expressed recombinant receptors, but the biochemistry of these modifications in primary human neutrophils remains relatively unstudied. FPR1 and FPR2 were partially immunopurified using antibodies that recognize both receptors (NFPRa) or unphosphorylated FPR1 (NFPRb) in dodecylmaltoside extracts of unstimulated and N-formyl-Met-Leu-Phe (fMLF) + cytochalasin B-stimulated neutrophils or their membrane fractions. After deglycosylation and separation by SDS-PAGE, excised Coomassie Blue-staining bands (∼34,000 Mr) were tryptically digested, and FPR1, phospho-FPR1, and FPR2 content was confirmed by peptide mass spectrometry. C-terminal FPR1 peptides (Leu(312)-Arg(322) and Arg(323)-Lys(350)) and extracellular FPR1 peptide (Ile(191)-Arg(201)) as well as three similarly placed FPR2 peptides were identified in unstimulated and fMLF + cytochalasin B-stimulated samples. LC/MS/MS identified seven isoforms of Ala(323)-Lys(350) only in the fMLF + cytochalasin B-stimulated sample. These were individually phosphorylated at Thr(325), Ser(328), Thr(329), Thr(331), Ser(332), Thr(334), and Thr(339). No phospho-FPR2 peptides were detected. Cytochalasin B treatment of neutrophils decreased the sensitivity of fMLF-dependent NFPRb recognition 2-fold, from EC50 = 33 ± 8 to 74 ± 21 nM. Our results suggest that 1) partial immunopurification, deglycosylation, and SDS-PAGE separation of FPRs is sufficient to identify C-terminal FPR1 Ser/Thr phosphorylations by LC/MS/MS; 2) kinases/phosphatases activated in fMLF/cytochalasin B-stimulated neutrophils produce multiple C-terminal tail FPR1 Ser/Thr phosphorylations but have little effect on corresponding FPR2 sites; and 3) the extent of FPR1 phosphorylation can be monitored with C-terminal tail FPR1-phosphospecific antibodies.

The catalytic core of the phagocyte NADPH oxidase is a heterodimeric integral membrane protein (flavocytochrome b (Cyt b)) that generates superoxide and initiates a cascade of reactive oxygen species critical for the host inflammatory response. In order to facilitate structural characterization, the present study reports the first direct analysis of human phagocyte Cyt b by matrix-assisted laser desorption/ionization and nanoelectrospray mass spectrometry. Mass analysis of in-gel tryptic digest samples provided 73% total sequence coverage of the gp91(phox) subunit, including three of the six proposed transmembrane domains. Similar analysis of the p22(phox) subunit provided 72% total sequence coverage, including assignment of the hydrophobic N-terminal region and residues that are polymorphic in the human population. To initiate mass analysis of Cyt b post-translational modifications, the isolated gp91(phox) subunit was subject to sequential in-gel digestion with Flavobacterium meningosepticum peptide N-glycosidase F and trypsin, with matrix-assisted laser desorption/ionization and liquid chromatography-mass spectrometry/mass spectrometry used to demonstrate that Asn-132, -149, and -240 are genuinely modified by N-linked glycans in human neutrophils. Since the PLB-985 cell line represents an important model system for analysis of the NADPH oxidase, methods were developed for the purification of Cyt b from PLB-985 membrane fractions in order to confirm the appropriate modification of N-linked glycosylation sites on the recombinant gp91(phox) subunit. This study reports extensive sequence coverage of the integral membrane protein Cyt b by mass spectrometry and provides analytical methods that will be useful for evaluating posttranslational modifications involved in the regulation of superoxide production.

A heme-bearing polypeptide core of human neutrophil flavocytochrome b(558) was isolated by applying high performance, size exclusion, liquid chromatography to partially purified Triton X-100-solubilized flavocytochrome b that had been exposed to endoproteinase Glu-C for 1 h. The fragment was composed of two polypeptides of 60-66 and 17 kDa by SDS-polyacrylamide gel electrophoresis and retained a native heme absorbance spectrum that was stable for several days when stored at 4 degrees C in detergent-containing buffer. These properties suggested that the majority of the flavocytochrome b heme environment remained intact. Continued digestion up to 4.5 h yielded several heme-associated fragments that were variable in composition between experiments. Digestion beyond 4.5 h resulted in a gradual loss of recoverable heme. N-Linked deglycosylation and reduction and alkylation of the 1-h digestion fragment did not affect the electrophoretic mobility of the 17-kDa fragment but reduced the 60-66-kDa fragment to 39 kDa. Sequence and immunoblot analyses identified the fragments as the NH(2)-terminal 320-363 amino acid residues of gp91(phox) and the NH(2)-terminal 169-171 amino acid residues of p22(phox). These findings provide direct evidence that the primarily hydrophobic NH(2)-terminal regions of flavocytochrome b are responsible for heme ligation.

Wild-type and 35 mutant formyl peptide receptors (FPRs) were stably expressed in Chinese hamster ovary cells. All cell surface-expressed mutant receptors bound N-formyl peptide with similar affinities as wild-type FPR, suggesting that the mutations did not affect the ligand-binding site. G protein coupling was examined by quantitative analysis of N-formyl-methionyl-leucyl-phenylalanine-induced increase in binding of (35)S-labeled guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) to membranes. The most prominent uncoupled FPR mutants were located in the N-terminal part of the second transmembrane domain (S63W and D71A) and the C-terminal interface of the third transmembrane domain (R123A and C124S/C126S). In addition, less pronounced uncoupling was detected with deletion mutations in the third cytoplasmic loop and in the cytoplasmic tail. Further analysis of some of the mutants that were judged to be uncoupled based on the [(35)S]GTPgammaS membrane-binding assay were found to transduce a signal, as evidenced by intracellular calcium mobilization and activation of p42/44 MAPK. Thus, these single point mutations in FPR did not completely abolish the interaction with G protein, emphasizing that the coupling site is coordinated by several different regions of the receptor. Mutations located in the putative fifth and sixth transmembrane domains near the N- and C-terminal parts of the third cytoplasmic loop did not result in uncoupling. These regions have previously been shown to be critical for G protein coupling to many other G protein-coupled receptors. Thus, FPR appears to have a G protein-interacting site distinct from the adrenergic receptors, the muscarinic receptors, and the angiotensin receptors.

Activation of the N-formyl peptide receptor (FPR) of human neutrophils by ligands such as N-formyl-methionine-leucine-phenylalanine (fMLP) induces mobilization of intracellular calcium, cell adhesion, chemotaxis, superoxide production and degranulation. Chinese hamster ovary (CHO) cells are normally devoid of FPR and unresponsive to fMLP, but when stably transfected with a human FPR cDNA, exhibited some of these same responses. Specifically, stimulation with fMLP resulted in release of intracellular calcium and chemotactic migration toward a gradient of fMLP. As in neutrophils, both processes were inhibited through receptor desensitization by prior exposure to a higher or equal concentration of ligand or by treatment with pertussis toxin. Soluble and membrane-bound fibronectin greatly increased fMLP-induced chemotaxis of CHO cells expressing FPR, but not of wild-type CHO cells, suggesting a role for FPR in activation of integrin function. Evidence for this hypothesis was obtained by demonstrating that CHO cells expressing FPR rapidly increased their adhesion to a fibronectin-coated surface after stimulation with fMLP. Both chemotaxis and adhesion were largely inhibited by RGDS peptide and a function-blocking antibody against alpha5 integrin. FPR-mediated chemotaxis of the CHO transfectants was partly inhibited by a tyrosine kinase inhibitor, herbimycin A, and blocked by a phosphoinositide 3-kinase inhibitor, wortmannin. These data suggest that stimulation of CHO FPR transfectants with a gradient of fMLP results in phosphoinositide 3-kinase-dependent chemotactic migration, which is enhanced by binding of activated alpha5beta1 to fibronectin. This non-myeloid, non-lymphoid fibroblastic cell line will thus serve as a useful model to investigate additional requirements of signal transduction molecules, adhesion molecules and cytoskeletal elements in FPR-mediated chemotaxis.

Flavocytochrome b (Cyt b) is a heterodimeric, integral membrane protein that serves as the central component of an electron transferase system employed by phagocytes for elimination of bacterial and fungal pathogens. This report describes a rapid and efficient single-step purification of Cyt b from human neutrophil plasma membranes by solubilization in the nonionic detergent dodecylmaltoside (DDM) and immunoaffinity chromatography. A similar procedure for isolation of Cyt b directly from intact neutrophils by a combination of heparin and immunoaffinity chromatography is also presented. The stability of Cyt b was enhanced in DDM relative to previously employed solubilizing agents as determined by both monitoring the heme spectrum in crude membrane extracts and assaying resistance to proteolytic degradation following purification. Gel filtration chromatography and dynamic light scattering indicated that DDM maintains a predominantly monodisperse population of Cyt b following immunoaffinity purification. The high degree of purity obtained with this isolation procedure allowed for direct determination of a 2:1 heme to protein stoichiometry, confirming previous structural models. Analysis of the isolated heterodimer by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry allowed for accurate mass determination of p22phox as indicated by the gene sequence. Affinity-purified Cyt b was functionally reconstituted into artificial bilayers and demonstrated that catalytic activity of the protein was efficiently retained throughout the purification procedure.