Monocyte-derived Neutrophil Chemotactic Factor Research Articles

Ex Vivo Adenoviral Vector Gene Delivery Results in Decreased Vector-associated Inflammation Pre- and Post–lung Transplantation in the Pig

Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1β levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function.

Characterization of a 64-kd protein phosphorylated during chemotactic activation with IL-8 and fMLP of human polymorphonuclear leukocytes. I. Phosphorylation of a 64-kd protein and other proteins

Interleukin-8 (IL-8) such as LUCT (lung giant cell carcinoma-derived chemotactic protein), NAP (neutrophil activating protein) and MDNCF (monocyte-derived neutrophil chemotactic factor), and formylmethionyl-leucyl-phenylalanine (fMLP) are well-known chemoattractants for human polymorphonuclear leukocytes (PMNs) and are able to stimulate phosphorylation of 64-kd protein (p64) in these leukocytes. To elucidate the molecular mechanism of PMN activation with chemoattractants, we investigated the phosphorylation process of p64 in an intact cell. 32P-Labeled PMNs were stimulated with LUCT/IL-8, fMLP, leukotriene B4, or C5a, and phosphorylated proteins were analyzed by two-dimensional electrophoresis and autoradiography. A marked phosphorylation of p64 was observed after stimulation. A new spot of phosphorylated p64 (pp64) could be detected on the gel stained with Coomassie Brilliant Blue, indicating that the isoelectric point (pI) of p64 shifted from 5.3 to a more acidic pI by the phosphorylation forming pp64. The spot of pp64 was shown to be dephosphorylated to p64 by treatment with calf intestine alkaline phosphatase. Other proteins having molecular masses 82, 66, 58, 55 and 50 kd were also phosphorylated. The fMLP-stimulated phosphorylation was time-dependent and saturated within 5 min. Maximum stimulation was achieved with 10 nM fMLP. Phosphoamino acid analysis revealed phosphorylation of serine residues in pp64. Staurosporine (100 nM) and W-7 (100 microM) significantly inhibited the phosphorylation of p64, but H-7 slightly inhibited it. H-8 and herbimycin A did not effect phosphorylation. Phorbol myristate acetate was found to stimulate significantly. Protein kinase C did not stimulate the phosphorylation. These data suggest that protein kinase C and calmodulin-like protein are indirectly involved in the phosphorylation of p64 during chemoattractant-activation of PMN.

Recombinant Interleukin-8 Induces Changes in Cytosolic Ca2+ in Human Neutrophils

Activation of polymorphonuclear leukocytes (PMNL) by most soluble stimulants is associated with a marked increase in cytosolic free Ca2+ ([Ca2+]i). Interleukin-8 (IL-8), a monocyte-derived neutrophil chemotactic factor and potent neutrophil-activating cytokine, effectively enhanced the resting free [Ca2+]i within human PMNL in a dose-dependent manner (maximal effect with 100 ng/mL). The increase in [Ca2+]i was substantially (55%) inhibited in the absence of extracellular Ca2+. Thus, the increase was due to extra- and intracellular cooperative mobilization of Ca2+, as supported by the reduced effect of IL-8 on [Ca2+]i after quenching with Mn2+. Granulocyte-macrophage colony-stimulating factor and interferon-gamma failed to induce a change in [Ca2+]i, suggesting that they may operate through different signal pathways. Pretreatment with Bordetella pertussis toxin largely inhibited the IL-8-induced change in [Ca2+]i. Thus, IL-8-induced cooperative mobilization of intra- and extracellular Ca2+ leads to a net Ca2+ influx into the cytoplasm through a process mediated by a guanosine triphosphate-binding protein.

A macrophage LPS-inducible early gene encodes the murine homologue of IP-10

Recently, we have isolated and characterized a set of cDNA clones which encode lipopolysaccharide-inducible proteins in murine peritoneal macrophages. Here, we report the sequence and identification of one of these cDNAs previously termed C7. Nucleotide sequence analysis revealed an open reading frame encoding a predicted polypeptide composed of 98 amino acids, which contained a 21 amino acid residue signal peptide, indicating approximately 9 kDa of mature protein. The deduced protein sequence showed homology (67% identity, 77% considering conservative amino acid changes) with the human INFγ-inducible gene IP-10, a member of the recently described superfamily of chemotactic and mitogenic proteins which includes platelet factor 4, monocyte-derived neutrophil chemotactic factor (NAF, NAP-1, IL-8), and MGSA/gro/KC. Thus C7 would appear to represent the murine homologue of the human IP-10 gene or a very closely related gene.

Monocyte-derived neutrophil chemotactic factor (MDNCF/IL-8) resides in a gene cluster along with several other members of the platelet factor 4 gene superfamily

Monocyte-derived neutrophil chemotactic factor (MDNCF/IL-8, suggested gene symbol IL8) is a cytokine that chemoattracts and activates neutrophils. Using a panel of human-rodent cell hybrids that preferentially segregate human chromosomes and in situ hybridization, the MDNCF/IL-8 gene was placed on the human gene map at position 4q12-q21. This is the same location where at least three other members (platelet factor 4, melanoma growth stimulatory activity, and interferon-gamma induced factor) of the platelet factor 4 gene super-family reside. In addition, a restriction fragment length polymorphism was identified using MDNCF as a probe in screening genomic DNA digested with HindIII from unrelated individuals.

Interleukin 8 (monocyte-derived neutrophil chemotactic factor) dynamically regulates its own receptor expression on human neutrophils.

The regulation of monocyte-derived neutrophil chemotactic factor (MDNCF)/interleukin 8 (IL 8) receptor expression by the MDNCF/IL 8 ligand was examined using freshly isolated human peripheral blood neutrophils. MDNCF/IL 8 down-regulated greater than 90% of its own receptor expression within 10 min at 37 degrees C. This down-regulation was associated with internalization of the ligand. The radiolabeled MDNCF/IL 8 molecules after internalization were proteolytically degraded, and trichloroacetic acid-soluble molecules were released into the culture medium starting at 60 min. Lysosomotropic agents could inhibit this degradation of ligand suggesting the involvement of lysosomal enzymes in this proteolytic digestion. MDNCF/IL 8 receptors reappeared on the cell surface within 10 min after removal of free ligands from the culture medium. Cycloheximide did not alter the reappearance of the receptor suggesting that de novo protein synthesis of MDNCF/Il 8 receptors is not involved in this event and that receptors probably recycled. The addition of lysosomotropic agents partially inhibited the reappearance/recycling of the receptors, although none of these agents inhibited the binding of ligand to the surface receptors or ligand internalization. Ammonium chloride reduced the MDNCF/IL 8-induced neutrophil chemotactic response in a dose-dependent fashion. These data suggest that MDNCF/IL 8 receptor expression is dynamically regulated by MDNCF/IL 8 and that the rapid recycling of MDNCF/IL 8 receptors may be essential for the chemotactic response of neutrophils.

Genomic structure of the human monocyte-derived neutrophil chemotactic factor IL-8.

To identify possible regulatory sites of the gene expression, we cloned the genomic DNA of human monocyte-derived neutrophil chemotactic factor (MDNCF/IL-8), whose mRNA is induced by IL-1 or TNF and determined its entire nucleotide sequence. The results show that the MDNCF/IL-8 gene consists of 4 exons and 3 introns with a single "CAT"- and "TATA"-like structure. The 5'-flanking region of MDNCF/IL-8 gene shows no overall sequence similarity with that of other cytokine and acute phase reactant genes whose production is also affected by IL-1 and TNF. The 5' flanking region, however, contains potential binding sites for several nuclear factors including activation factor-1, activation factor-2, IFN regulatory factor-1, and hepatocyte nuclear factor-1. In addition, the glucocorticoid responsive element and heat shock element were located in the 5'-flanking region. Inasmuch as PMA induces MDNCF/IL-8 mRNA accumulation in human PBMC and a glucocorticoid inhibits the induction of MDNCF/IL-8 mRNA by LPS, the expression of this gene is probably regulated by interaction of these nuclear factors with the 5'-flanking DNA.

Purification and characterization of cytokine-induced neutrophil chemoattractant produced by epithelioid cell line of normal rat kidney (NRK-52E cell)

Interleukin-1β, tumor necrosis factor-α and lipopolysaccharide stimulated normal rat kidney cell line (NRK-52E) to produce a chemotactic factor for rat neutrophils. This cytokine-induced neutrophil chemoattractant (CINC) was purified to obtain a single band with a M.W. of 63,000 on SDS-PAGE. The purified CINC induced detectable migration and strong chemotaxis of neutrophils at concentrations of 10 −9M and 10 −7–10 −8M, respectively. Checkerboard analysis indicated that CINC was a real chemotactic factor. Amino acid composition of CINC showed that CINC has a resemblance to human monocyte-derived neutrophil chemotactic factor (MDNCF) rather than human complement fragment, C5a.

Identification and characterization of specific receptors for monocyte-derived neutrophil chemotactic factor (MDNCF) on human neutrophils.

Specific receptors for a recently purified and cloned monocyte-derived neutrophil chemotactic factor (MDNCF) have been identified on the surface of normal human peripheral blood neutrophils using 125I-labeled recombinant human MDNCF (125I-MDNCF). Competitive binding of 125I-MDNCF to human neutrophils reached a maximal level at 1-3 h at 4 degrees C. The Scatchard analysis showed that there are approximately 20,000 receptors per cell with a single type of high affinity binding (Kd, 8 x 10(-10) M). The receptors for MDNCF are clearly distinct from the receptors for other cytokines and chemotactic agents, e.g., IL-1 alpha, TNF-alpha, and FMLP, C5a, leukotriene B4, and platelet activating factor. Based on the SDS-PAGE analysis of chemically crosslinked 125I-MDNCF receptor complex, there are two polypeptides that bind MDNCF; the molecular weight of these two MDNCF receptors were estimated to be 67,000 and 59,000. Treatment of a promyelocytic cell line, HL60, with 1.25% DMSO for 5 d in vitro increased the number of receptors up to 7,000 receptors/cell with a Kd of 1.2 x 10(-9) M.

Mitogenic activation of human T cells induces two closely related genes which share structural similarities with a new family of secreted factors.

Previously we have isolated about 60 novel cDNA clones whose corresponding mRNAs are induced by mitogenic activation in human peripheral blood T cells. Here we describe the primary structure and regulation of two such cloned genes, pAT 464 and pAT 744, which may encode new lymphokines/cytokines. Similar to IL-2, both genes require the synergy of agents such as PHA and PMA for optimal expression, and, in addition, the induction of both is sensitive to the immunosuppressive drug cyclosporin A. The two genes can be expressed in T cells, B cells, and the promyelocytic cell line HL60, but they are not expressed in human fibroblasts, suggesting that their expression is restricted to hematopoietic lineages. The predicted peptides encoded by these two clones feature hydrophobic N-terminal leaders characteristic of secreted proteins. The predicted size of both proteins is about 8 kDa upon cleavage of the putative leader peptide. pAT 464 and pAT 744 are very similar to each other and also share some critical amino acid similarity with a newly emerging family of secreted factors including connective tissue activating factor III, platelet factor 4, an IFN-gamma-induced factor, macrophage inflammatory protein, and a factor chemotactic to neutrophils (3-10C, monocyte-derived neutrophil chemotactic factor, neutrophil-activating factor). Some of these factors have been shown to display functions associated with an inflammatory response and/or have mitogenic activities. Collectively, the data presented here suggest that pAT 464 and pAT 744 encode novel lymphokines/cytokines which may play roles during an immune response similar to those enacted by these structurally related factors.

Three forms of monocyte-derived neutrophil chemotactic factor (MDNCF) distinguished by different lengths of the amino-terminal sequence

Human monocyte-derived neutrophil chemotactic factor (MDNCF) was purified from culture supernatant of lipopolysaccharide-stimulated human peripheral blood mononuclear leukocytes on a column of Sepharose-bound murine monoclonal anti-MDNCF. About 65% of the culture fluid chemotactic activity was bound to the column. The unbound 35% probably represents chemotactic activity of other cytokines in the culture fluid. More than 85% of the bound activity was eluted by pH 2.5 glycine buffer. When this material was applied to an HPLC-CM column, gradient elution produced four well-separated A 280 peaks, each of which had chemotactic activity. N-terminal amino acid analysis of the four peaks revealed three different sequences. One (MDNCF-c) was identical to the sequence that we reported previously. The other two (MDNCF-a and -b) had seven and five additional amino acids, respectively, at the N-terminus. MDNCF-a, -b and -c accounted for 8, 47 and 45% of the total MDNCF peptide. Alignment with the MDNCF cDNA sequence shows that MDNCF-a results from cleavage of a 20 residue signal peptide. MDNCF-c results from culture fluid proteolytic cleavage of the N -terminal sequences of MDNCF-a and -b at an R-S bond. The three peptides occurred in the four HPLC-CM peaks in different ratios. The bulk of any one peptide was distributed in two adjacent HPLC-CM peaks. This suggests that each peptide exists in a minimum of two states. In contrast to our previous multi-step purification, the immunoaffinity and HPLC-CM column sequence resulted in complete purification of MDNCF in two steps and led to identification of two additional MDNCF peptides, one of which has not heretofore been detected.

Synthesis and biological characterization of monocyte-derived neutrophil chemotactic factor

MDNCF is a human monocyte-derived, 72-residue chemotactic peptide, which has sequence similarity with members of a family of pro-inflammatory cytokines. The peptide was synthesized by the solid-phase method, and is identical to the natural peptide in amino acid composition, sequence and chemotactic potency. MDNCF forms two loops via a neighboring pair of disulfide bridges, the probable locations of which are residues 7–34 and 9–50. Reduction and alkylation eliminated chemotactic activity. MDNCF fragments 7–37, 30–72 and 17–72 were all biologically inactive. The data suggest that the region of the clustered pair of disulfide bridges is important for biological activity.

Purification of a human monocyte-derived neutrophil chemotactic factor that has peptide sequence similarity to other host defense cytokines.

Stimulated human monocytes release several proteins thought to play a role in inflammation, including interleukin 1, tumor necrosis factor, and plasminogen activator. We have purified another proinflammatory protein that is chemotactic for human neutrophils from conditioned medium of lipopolysaccharide-stimulated monocytes. After a series of steps that included anion-exchange chromatography, gel filtration, and HPLC on cation-exchange and reverse-phase columns, an apparently pure protein was obtained that migrated as a single 7-kDa band on NaDodSO4/polyacrylamide gels under reducing or nonreducing conditions. The amino acid composition of this monocyte-derived neutrophil chemotactic factor was different from that of interleukin 1 and tumor necrosis factor. N-terminal amino acid sequence of the first 42 residues was determined. This portion of the molecule has up to 56% sequence similarity with several proteins that may be involved in host responses to infection or tissue injury. It is identical to a portion of a sequence deduced from an mRNA induced by staphylococcal enterotoxin treatment of human leukocytes. At the optimal concentration of 10 nM, 50% of neutrophils added to chemotaxis assay wells migrated toward the pure attractant. Potency and efficacy are comparable to that of fMet-Leu-Phe, which is often used as a reference. In contrast to many attractants, the protein was not chemotactic for human monocytes.