Attenuation of interleukin 8-induced nasal inflammation by an inhibitor peptide.

Polymorphonuclear neutrophils (PMNs) infiltrate tissue in response to chemoattractants, including interleukin 8 (IL-8). Infiltrating PMNs clear microorganisms but also cause tissue damage. We previously reported the presence in human bronchial lavage of a peptide that inhibits PMN functions. The current project assessed (1) effects of a synthetic analog of this peptide (synthetic neutrophil inhibitor peptide, SNIP) on IL-8-induced nasal inflammation in humans, (2) effects of SNIP on PMN apoptosis and chemotaxis, (3) specific binding of SNIP to PMNs, and (4) evidence of larger molecules with the SNIP sequence. Results show that SNIP attenuates IL-8-induced nasal inflammation, inhibits in vitro PMN chemotaxis to IL-8, and accentuates PMNs apoptosis. PMNs contain specific SNIP-binding sites and the integrin CR3 (CD11b/CD18), or a CR3-associated molecule, is one SNIP-binding molecule. Chemotaxis to IL-8 is most potently inhibited by SNIP in the presence of fibrinogen, a CR3 ligand. Antiserum against the SNIP sequence recognizes a 70-kDa protein in bronchoalveolar lavage and an anti-SNIP immunoaffinity column binds a 70-kDa protein in U937 cell culture supernatant. U937 cell mRNA contains a 1.8-kb transcript detected with degenerate oligonucleotides designed from the SNIP sequence. These studies demonstrate that a synthetic inhibitor peptide can attenuate in vivo nasal inflammation through downregulatory effects on PMNs.

Attenuation of interleukin-8 production by inhibiting nuclear factor-kappaB translocation using decoy oligonucleotides.

Interleukin-8 (IL-8), a monocyte-derived neutrophil chemoattractant factor, is a polymorphonuclear neutrophil chemotaxin that is involved in a number of inflammatory disorders. Transcription of the IL-8 gene is controlled by regulatory proteins, including nuclear factor-kappaB (NF-kappaB), a family of proteins that is important in the transcriptional control of a number of genes. When cells are activated, NF-kappaB translocates from the cytoplasm to the nucleus, where it activates transcription by binding to a specific sequence within the 5' untranslated region of the gene. During translocation, NF-kappaB is potentially susceptible to diversion by oligonucleotides that contain the binding sequence for this protein. In the current study, we produced phosphorothioate-modified oligonucleotides containing the specific DNA sequence that NF-kappaB binds within the IL-8 gene. We then investigated the effects of transfection of monocytes with these oligonucleotides on interleukin-1beta (IL-1beta)-stimulated IL-8 production, IL-8 mRNA expression, and NF-kappaB binding activity. We found that transfection with these oligonucleotides significantly inhibited monocyte IL-8 production. A single-stranded oligonucleotide with two copies of the NF-kappaB-binding sequence was the most potent of those tested. This single-stranded oligonucleotide also inhibited IL-1beta-induced translocation of NF-kappaB to the nucleus and reduced IL-8 mRNA expression. These studies demonstrated that monocyte production of IL-8 can be attenuated using a single-stranded oligonucleotide that binds a transcriptional activating protein before it translocates to the cell nucleus. This approach ultimately may be useful in the control of inflammation involved in a number of diseases.

Modulation of monocyte chemokine production and nuclear factor kappa B activity by oxidants.

Reactive oxygen species can directly damage tissue. In this setting, amplification of tissue damage also occurs through infiltration of inflammatory cells either acutely or chronically. Several recent studies suggest that reactive oxygen species stimulate production of certain chemokines, which are potent chemoattractants for inflammatory cells. In the present study, we examined whether oxidants, generated by the combination of xanthine and xanthine oxidase (X/XO), alter chemokine production by monocytes and U937 cells. Our findings demonstrate that X/XO stimulates monocytes, but not U937 cells, to produce increased amounts of interleukin-8 (IL-8) and monocyte chemoattractant protein. This effect is attenuated by pretreatment with dimethylsulfoxide (DMSO), a scavenger of hydroxyl radicals, but is not affected by superoxide dismutase or catalase. In contrast, X/XO-induced cytotoxicity, evidenced by lactate dehydrogenase release, is mediated primarily by hydrogen peroxide, as catalase reverses this effect. Finally, exposure to X/XO causes an increase in nuclear factor kappa B (NF-kappaB), and this effect is attenuated by DMSO. These studies suggest that reactive oxygen species can induce production of molecules that amplify inflammation through attraction of inflammatory cells. It appears the hydroxyl radical is the principal oxidant species involved in stimulation of chemokine production.

Modulation of monocyte chemotactic protein-1 production by hyperoxia: importance of RNA stability in control of cytokine production.

Pulmonary oxygen toxicity occurs after prolonged administration of increased fractions of inspired oxygen. Lung damage in this setting manifests as diffuse alveolar damage. In animals exposed to hyperoxia, increased numbers of alveolar macrophages are noted 72 h after initiation of high concentrations of oxygen. Monocyte chemotactic protein-1 (MCP-1) is a cytokine released by a number of cell types that has potent chemotactic activity for monocytes, precursor cells for alveolar macrophages. In the current study, we examined whether MCP-1 production was increased in response to hyperoxia. We used the monocyte/ histiocytic U937 cell line and exposed these cells to hyperoxia for variable amounts of time, then determined MCP-1 concentrations by enzyme-linked immunosorbent assay and MCP-1 mRNA levels by Northern blot analysis. We also examined the effects of dexamethasone on the response of U937 cells to hyperoxia. Finally, as a potential mechanism for regulation of U937 MCP-1 production, we examined effects of hyperoxia on MCP-1 mRNA stability. The results demonstrate that hyperoxia stimulates MCP-1 production after 6 and 24 h of exposure. MCP-1 mRNA levels are also increased after initiation of hyperoxia in part through effects on MCP-1 transcript stability. Dexamethasone significantly reduces MCP-1 production and mRNA levels also in part through effects on transcript stability. These studies suggest monocytes may be attracted to hyperoxia-exposed lungs through enhanced MCP-1 production. MCP-1 production appears to be upregulated in part through post-transcriptional processes in this setting.

Characterization of a neutrophil inhibitor peptide harvested from human bronchial lavage: homology to influenza A nucleoprotein.

Bronchi are exposed to particulate matter, including bacteria, fungi and dusts, that should trigger release of molecules which attract polymorphonuclear neutrophils (PMN). However, normal bronchi are relatively devoid of PMN, suggesting that there exists a mechanism to dampen acute inflammation in the lung. We have previously reported that bronchial lavage from normal humans contains a nonpolar peptide that inhibits PMN chemotaxis and oxidant production. In the present study we devised preparative methods to obtain sufficient quantities of a similar inhibitor molecule for partial amino acid sequencing and allow production of truncated analogues. Amino acid sequencing demonstrated that the peptide includes a 10-amino-acid sequence that is completely homologous to a sequence of amino acids contained in the influenza A nucleoprotein. Synthesized peptides containing this 10-amino-acid sequence inhibited PMN chemotaxis and oxidant production. In addition, PMN lysates actively phosphorylated peptides containing the 10-amino-acid sequence or a partial sequence containing an apparent phosphorylation site. U937 cells were noted to be one source of this inhibitor, as a similarly sized nonpolar inhibitor peptide was purified from U937 culture supernatants. In addition, U937 and monocyte cellular lysates contained proteins recognized by an antiserum directed at the influenza A nucleoprotein. Further characterization of the molecule described in this study or related molecules may lead to significantly new antiinflammatory strategies.

Effects of influenza A nucleoprotein on polymorphonuclear neutrophil function.

Infection with influenza virus is commonly associated with polymorphonuclear neutrophil (PMNL) dysfunction and consequent secondary bacterial pneumonia. A recently isolated human-derived protein that inhibits PMNL chemotaxis and oxidant production shows a striking homology to the influenza A nucleoprotein. In the present study, the effects of purified influenza A nucleoprotein on PMNL chemotaxis, oxidant production, degranulation, and calcium homeostasis were studied. Results of the study demonstrate that purified nucleoprotein inhibits PMNL chemotaxis as well as superoxide production. In addition, purified nucleoprotein induces a rise in PMNL cytosolic calcium concentration in a manner similar to that demonstrated for crude influenza A lysates. In contrast, no difference in FMLP-stimulated PMNL elastase or beta glucuronidase release was noted after exposure to nucleoprotein. These studies suggest that the influenza A nucleoprotein may account for some of the neutrophil defect associated with cellular infection by this virus.

Hyperoxia stimulates interleukin-8 release from alveolar macrophages and U937 cells: attenuation by dexamethasone.

Pulmonary oxygen toxicity is associated with histological evidence of polymorphonuclear neutrophil (PMN) infiltration into lung parenchyma. What guides infiltration of these cells is unknown. A number of chemoattractants for PMN have been documented including interleukin-8 (IL-8), a cytokine released by alveolar macrophages (AM) and other cell types. The purposes of this study were to 1) determine whether human AM and the histiocytic U937 cell line release IL-8 in response to hyperoxia, 2) assess whether hyperoxia results in increased IL-8 steady-state mRNA levels in U937 cells and 3) establish whether dexamethasone could attenuate noted effects of hyperoxia. Our study shows that hyperoxia stimulates human AM and U937 cell release of IL-8. Hyperoxia also increases IL-8 mRNA levels in U937 cells. IL-8 released in response to hyperoxia by AM was biologically active as evidenced by ability to induce PMN chemotaxis. A polyclonal antibody to IL-8 partially attenuated this chemotactic activity. Finally, dexamethasone at concentrations of 10 microM, 1 microM, and 100 nM markedly reduced hyperoxia-induced IL-8 release and mRNA synthesis by U937 cells. We conclude that IL-8 may be important in the pathogenesis of pulmonary oxygen toxicity and that therapeutic concentrations of dexamethasone can suppress production of this cytokine.

Alteration of cellular cytosolic calcium and chemotactic peptide binding by an inhibitor of neutrophil function.

The lung is frequently exposed to particulate material that can potentially stimulate release of factors that attract polymorphonuclear neutrophils (PMN). However, few PMN are noted in the airways of normal subjects, suggesting there is some mechanism to dampen influx of these cells. We have isolated from bronchial lavage a peptide that inhibits PMN chemotaxis to formyl-methionyl-leucyl-phenylalanine (FMLP). In the present study we examined effects of this molecule on 1) chemotaxis to other agonists, 2) FMLP-stimulated PMN superoxide production, 3) PMN calcium fluxes, and 4) binding of FMLP. Our results show that purified inhibitor attenuates PMN chemotaxis to C5a and leukotriene B4. This molecule also inhibits PMN superoxide release in response to FMLP. Exposure to this inhibitor causes an abrupt rise in cytosolic calcium concentration due to a pertussis toxin-sensitive shift of intracellular calcium and attenuates subsequent influx of extracellular calcium in response to FMLP. Binding studies demonstrate the inhibitor induces increased FMLP binding at 37 degrees C but has no effects at 4 degrees C. Inhibition of chemotaxis and increased FMLP binding mediated by this molecule are attenuated by buffering PMN calcium transients. These studies suggest an inhibitor of neutrophil function present in the bronchial environment alters PMN through effects on calcium homeostasis.