Edmund J. Miller

Association of IL-8 and MCP-1 With the Development of Reexpansion Pulmonary Edema in Rabbits

BACKGROUNDnThe aim of this study is to determine the relationships between the cytokines and the inflammatory response in reexpansion pulmonary edema (RPE).nnnMETHODSnWe examined the cell population, epithelial permeability measured by Evans blue dye (EB), betaglucuronidase and cytokine concentrations in bronchoalveolar lavage fluid (BALF) and/or blood using a rabbit RPE model.nnnRESULTSnWe confirmed that RPE is characterized by recruitment of polymorphonuclear leukocytes (PMNs), the release of PMN granular contents into the air spaces, and increased vascular permeability. These findings were highly correlated with increased interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) concentrations in the BALF. Growth related oncogene (GRO) was detected in the BALF from only 2 of the 7 reexpanded lungs while TNFalpha was not detected in any rabbits. A similar but less severe inflammatory response to the reexpanded lung was found in the contralateral lung.nnnCONCLUSIONSnIL-8 and MCP-1 may play important roles in the development of RPE; the inflammatory response is independent of TNFalpha and unilateral reexpansion of the lung induces an inflammatory response not only in the reexpanded lung but also in the contralateral lung.

α-Chemokine growth factors for adenocarcinomas; a synthetic peptide inhibitor for α-chemokines inhibits the growth of adenocarcinoma cell lines

Purpose: The experiments aimed to determine if α-chemokine inhibitors are effective suppressors of the growth of adenocarcinomas, a neoplasm with a high mortality rate. Methods: Expression of growth-related oncogene (GROα) and interleukin-8 (IL-8) was determined by enzyme-linked immunosorbent assay. Inhibition of α-chemokine binding to tumor cells was assessed in the presence and absence of the hexapeptide, antileukinate. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide assays were performed to determine the effect of α-chemokines, monoclonal antibodies (mAb), and antileukinate on cell proliferation. Finally, antileukinate inhibition of human, lung adenocarcinoma tumor growth, was determined in BALB/c nude mice. Results: All of the adenocarcinomas tested produced either GROα or IL-8 or both. Proliferation of lung, stomach and colon adenocarcinoma cells was inhibited by anti-GROα mAb and/or anti-IL-8 mAb while recombinant human GROα stimulated the proliferation of lung and stomach adenocarcinomas. Antileukinate inhibited GROα binding to specific receptors on adenocarcinoma cells and inhibited the proliferation of all adenocarcinomas tested. Colon-derived adenocarcinomas specifically bound IL-8 and this binding was also inhibited by antileukinate. Administration of antileukinate inu2009vivo inhibited the tumor growth of adenocarcinoma A549. Conclusions: GROα and IL-8 are necessary for the growth of lung, stomach and colon adenocarcinomas, and can be inhibited by the hexapeptide, antileukinate. The findings suggest the possibility of using α-chemokine receptor inhibitors in the treatment of adenocarcinoma.

Biological and kinetic characterization of recombinant human macrophage inflammatory peptides 2 alpha and beta and comparison with the neutrophil activating peptide 2 and interleukin 8

We examined the biological and kinetic characteristics of two new members of the intercrine family of cytokines. Human macrophage inflammatory peptides 2 alpha and beta (huMIP-2 alpha and beta) were compared to human interleukin 8 (huIL-8), neutrophil activating peptide 2 (huNAP-2), and N-formyl-L-methionyl-L-phenylalanine (fMLP). The huMIP-2 peptides were the least potent cytokine tested in triggering neutrophil degranulation. They were also less potent neutrophil chemotaxins than fMLP or huIL-8. However, they were more effective than NAP-2 in stimulating chemotaxis of neutrophils. The binding studies showed that huMIP-2 peptides could interact with specific receptors on human blood neutrophils. Moreover, huMIP-2 peptides competed for up to 60% of the huIL-8 binding sites on neutrophils whereas huIL-8 competed for almost 100% of either of the huMIP-2 peptide binding sites. These data suggest the huMIP-2 peptides have little or no affinity for 40% of the huIL-8 receptors. In addition, detectable amounts of mRNA for huMIP-2 alpha were found in samples from human alveolar macrophages stimulated with Staphylococcus aureus, toxic shock syndrome toxin-1 (TSST), but not in samples stimulated with S. aureus enterotoxin A (SEA) or Escherichia coli endotoxin (lipopolysaccharide = LPS). In conclusion, huMIP-2 alpha and beta are weak neutrophil stimulating agents, which may increase inflammation in diseases such as toxic shock syndrome.

Search for drugs that may reduce the load of neutrophil azurophilic granule enzymes in the lungs of patients with emphysema.

Neutrophil elastase and myeloperoxidase probably play an important role in the development of pulmonary emphysema. We have analyzed drugs from the major classes of agents that alter neutrophil function to determine if there are drugs in use today that can reduce the load of neutrophil elastase or myeloperoxidase in the lungs of smokers. Eleven representative drugs were tested for their ability to inhibit chemotaxis and degranulation. None of the drugs inhibited chemotaxis in a dose-response fashion at concentrations achievable in human plasma. Sulfinpyrazone, phenylbutazone, and auranofin completely inhibited the release of azurophilic granules (myeloperoxidase) and tertiary granules (beta-D-glucuronidase) when formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) was used as the stimulant, and inhibited azurophilic granule release by 69%, 19%, and 64% respectively, but not tertiary granule release when macrophage-conditioned media was used as the stimulus. In conclusion, none of the drugs tested are inhibitors of chemotaxis; however, three are excellent inhibitors of azurophilic granule enzyme release. Of these three, sulfinpyrazone, a drug that is not currently used clinically for its antiinflammatory effects, is the least toxic and should be considered as a potential drug to reduce the elastase and myeloperoxidase load in the lungs of smokers who are developing emphysema.

A synthetic peptide which specifically inhibits heat-treated interleukin-8 binding and chemotaxis for neutrophils

Interleukin-8 (IL-8) is a peptide which is secreted by stimulated human monocytes and which is chemotactic for human neutrophils. We synthesized three overlapping peptides spanning the amino-terminal region of the IL-8 sequence. None of the peptides retained the chemotactic activity of the native molecule. One of the peptides, IL-8(3–25), inhibited the neutrophil chemotactic activity of recombinant IL-8 (rIL-8) which had been preheated to 40°C but did not reduce neutrophil chemokinesis, or the chemotactic activity of unheated rIL-8, FMLP, C5a or LTB4. Interleukin-8 exhibited similar binding kinetics and chemotaxis for neutrophils regardless of whether it had been pretreated at 40°C.In addition, IL-8(3–25) was also able to decrease the binding of prehead IL-8 to neutrophils. IL-8(3–25), which can self-associate, binds directly to receptors on the neutrophil. The data suggest that heat-treated, but not untreated, IL-8 causes the IL-8(3–25) multimers to disaggregate, allowing the monomeric peptide to directly bind to the IL-8 receptor and thus inhibiting IL-8/receptor binding.

Monoclonal antibodies to rabbit α-2-macroglobulin and their use in a sensitive ELISA assay

Abstract α-2-macroglobulin (α-2-M), a plasma proteinase inhibitor, plays an important role in the pathogenesis of lung inflammation. The purpose of this study was to develop a sensitive ELISA assay for rabbit α-2-M to allow us to define the role of this protein in a rabbit model of lung inflammation/injury. Therefore, we developed hybridomas which secrete monoclonal antibodies (mAbs) against rabbit α-2-M. From the antibodies produced, two (5B6 and 3C5) were selected. Both of them were of the IgG1 subclass. 5B6 reacted with native α-2-M as well as with the “fast” form of α-2-M (native α-2-M or the “slow” form is converted to the “fast” form by reaction with a proteinase). On the other hand, 3C5 reacted only with “fast” form of α-2-M. With these antibodies, we developed two ELISA assays which were used to determine the concentration of α-2-M in the lung fluids from rabbits with lung injury.