A synthetic peptide inhibitor for alpha-chemokines inhibits the growth of melanoma cell lines.

Melanoma growth stimulatory activity (MGSA/GROalpha) is a 73 amino acid peptide sharing sequence characteristics with the alpha-chemokine superfamily. MGSA/GROalpha is produced by diverse melanoma cell lines and reported to act as an autocrine growth factor for the cells. We tested the binding of MGSA/GROalpha to melanoma cell lines, Hs 294T and RPMI7951, and found that these cells could bind to MGSA/GROalpha but not to interleukin-8. Recently, we defined a novel hexapeptide, antileukinate, which is a potent inhibitor of binding of alpha-chemokines to their receptors on neutrophils. When antileukinate was added to melanoma cells, it inhibited the binding of MGSA/ GROalpha. The growth of cells from both melanoma cell lines was suppressed completely in the presence of 100 microM peptide. The cell growth inhibition was reversed by the removal of the peptide from the culture media or by the addition of the excess amount of MGSA/GROalpha. The viability of Hs 294T cells in the presence of 100 microM peptide was > 92%. These findings suggest that MGSA/GROalpha is an essential autostimulatory growth factor for melanoma cells and antileukinate inhibits their growth by preventing MGSA/GROalpha from binding to its receptors.

Effects of dexamethasone and insulin on the acute phase response of Morris hepatoma cells and of rat hepatocytes in culture.

Dexamethasone and insulin stimulate production of several plasma proteins in primary cultures of adult rat hepatocytes but inhibit their production in primary cultures of Morris hepatoma cell line 7777W. The acute phase response elicited in cultured cells by crude cytokines from activated rat peritoneal macrophages is considerably higher in hepatocytes in the presence of hormones, and especially of dexamethasone. In hepatoma cells the hormones enhance the cytokine-induced formation of fibrinogen and cysteine proteinase inhibitor but are without significant effect on suppression of albumin and alpha-fetoprotein synthesis by macrophage supernatants.

Alpha-1-acute phase globulin in the blood of tunicamycin-injected rats. Isolation of the non-glycosylated form, its inhibitory properties and synthesis in liver slices.

Plasma of rats injected with tunicamycin (2 micrograms/g body wt) shows on crossed immunoelectrophoresis the presence of an additional, slowly migrating component of alpha 1-acute-phase globulin (alpha 1-AP-globulin). The native and modified forms of alpha 1-AP-globulin were jointly isolated on the column of immobilized antibodies and then separated by chromatofocusing on polybuffer exchanger. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate demonstrated that tunicamycin-induced form of rat alpha 1-AP-globulin has Mr of 50,000 and is devoid of carbohydrates as inferred from the lack of staining with Schiff reagent. However, during incubation with papain in vitro it is only slightly less effective than mature glycosylated alpha 1-AP-globulin (Mr 68 000) in inhibiting hydrolysis of CBZ-Lys-ONp. Incubation of liver slices from control and tunicamycin-injected rats with 14C-leucine demonstrated that tunicamycin reduces synthesis and release to the medium of alpha 1-AP-globulin and some other plasma proteins, but the proportion of aglyco-alpha 1-AP-globulin is higher than in plasma.

An anti-interleukin 8 monoclonal antibody that interferes with the binding of interleukin 8 to cellular receptors and the activation of human blood neutrophils.

Interleukin 8 (IL-8) is a proinflammatory cytokine produced by a wide variety of cells. Interleukin 8 acts as a neutrophil activator and chemotactic factor. In the current studies, we examined the properties of a monoclonal antibody against human IL-8. The estimated affinity of the antibody was 1.74 x 10(7) liters/mol. The antibody interfered with the binding of radiolabeled recombinant human IL-8 (rhIL-8) to human blood neutrophils (IC50 = 3 x 10(-7) M, at an IL-8 concentration of 2.4 nM). Neutrophil degranulation elicited by 5 x 10(-6)-4 x 10(-8) M rhIL-8 was blocked by the antibody at three-fold molar excess. However, a higher concentration of anti-IL-8 antibody was needed to suppress the chemotactic activity of rhIL-8. The inhibition of neutrophil chemotaxis triggered by 2 x 10(-7)-2 x 10(-9) M rhIL-8 required 6 x 10(-5) M antibody. Similarly, a 300-fold molar excess of anti-IL-8 antibody [10(-5) M] was necessary to abrogate the increase in cytosolic free calcium in neutrophils stimulated with 4 x 10(-8) M rhIL-8. In addition, epitope analysis using synthetic peptides corresponding to different regions of the IL-8 molecule showed that peptide consisting of residues 44-72 (corresponding to the C-terminal of the IL-8 molecule) competed with the antibody for binding to rhIL-8. Because IL-8 is an important inflammatory mediator in several human diseases, anti-IL-8 antibodies may have pharmacological potential.

The cascade of inflammatory cytokines regulating synthesis of acute phase proteins.

The acute phase cytokines: interleukin 1, tumor necrosis factor alpha (cachectin) and beta (lymphotoxin), hepatocyte stimulating factor and several interferons, all belong to the family of endotoxin-inducible, low molecular weight proteins. Their synthesis in macrophages, fibroblasts, lymphocytes, epithelial and some tumor cells is enhanced by the same cytokines, often in the autocrine manner, and suppressed by dexamethasone. The principal hepatocyte stimulating factor (HSF) regulating synthesis of acute phase proteins is probably identical with IFN-beta 2/BSF-2/IL-6, but other inflammatory cytokines (IL-1, TNF alpha, IFN-gamma) are able to induce distinct sets of acute phase proteins, or to modulate the final response pattern. The effect of hrIFN-gamma on production of acute phase proteins by human hepatoma Hep G2 cells is discussed in detail. It is concluded that the cascades of inflammatory cytokines in different tissues represent amplification and regulatory pathways controlling the development of acute phase response in vivo.

Interaction of horse plasma antithrombin III and alpha 1-proteinase inhibitor with some serine proteinases.

Antithrombin III and alpha 1-proteinase inhibitor isolated simultaneously from horse citrated plasma were tested for inhibitory activity against bovine trypsin and chymotrypsin, as well as elastase-like neutral proteinases from horse leucocytes. The stoichiometry of reaction and kinetic parameters (kass, Ko) were estimated and related to the protein pattern obtained after exposure of these proteinases to horse inhibitors as analyzed by polyacrylamide gel electrophoresis (PAGE and PAGE-SDS). As shown by fast reaction rates and low values of dissociation constants the two inhibitors effectively inactivate trypsin. On the other hand, AT III is completely inactive against chymotrypsin or leucocyte elastases with alpha 1PI only partly inhibits these enzymes.

Acute phase protein stimulation by alpha 1-antichymotrypsin-cathepsin G complexes. Evidence for the involvement of interleukin-6.

Incubation of alpha 1-antichymotrypsin-cathepsin G complexes with human lung fibroblasts caused a nearly 5-fold increase in synthesis of the cytokine interleukin-6. In turn, the fibroblast-conditioned medium induced significant synthesis of the acute phase proteins haptoglobin, fibrinogen, and alpha 1-antichymotrypsin in human Hep G2 cells, whereas a mixture of interleukin-1 and conditioned medium was considerably less stimulatory. These data indicate that proteinase-proteinase inhibitor complexes formed between plasma serpins and their target enzymes could play major roles in signaling for acute phase protein synthesis in response to injury.

Kinetics and stoichiometry of inactivation of some pancreatic and leucocyte serine proteinases by rabbit alpha 1-proteinase inhibitors F and S.

Purified rabbit alpha 1-proteinase inhibitors F and S were incubated with bovine trypsin, chymotrypsin or horse leucocyte neutral proteinases in order to determine the stoichiometry of inhibition, inactivation rates of the enzymes and dissociation constants of the complexes. Trypsin reacted with the two forms of alpha 1-PI with different velocities but in the molar ratio of 1:1 and yielding stable complexes. Chymotrypsin reacted very fast with the two forms of alpha 1-PI but required a three-fold molar excess of alpha 1-PI-S for almost complete inhibition, and again the dissociation constants were low. Leucocyte proteinases also needed certain molar excess of the inhibitor but the reaction was complicated by instability of the complexes, especially with Z-Ala-ONp as substrate. We conclude that the interaction of rabbit alpha 1-PI with leucocyte proteinases, and particularly alpha 1-PI-S with chymotrypsin, includes not only complex formation but also some inactivation of the inhibitor.

Interleukin-8 and anti-interleukin-8 autoantibodies in gingival crevicular fluid from patients with periodontitis.

Interleukin-8 (IL-8) is believed to play an important role in the pathogenesis of various forms of periodontitis. In addition, the anti-IL-8 autoantibody has been recently recognized as a potent modulator of IL-8 function. In the current study, the concentrations of IL-8 and its autoantibody in gingival crevicular fluid from patients with chronic generalized periodontitis were compared to those in gingival crevicular fluid from patients with refractory chronic periodontitis. Gingival crevicular fluids were collected from patients treated in a private periodontal clinic. Nine patients who were identified as having chronic generalized periodontitis and four with refractory chronic periodontitis were selected for the study. Patients included in the latter group had undergone supportive periodontal therapy for more than 10 years, and during that time had experienced many episodes of periodontal destruction. The gingival crevicular fluid concentrations of total protein, IL-8, free anti-IL-8 autoantibody and IL-8 bound to the autoantibody (anti-IL-8:IL-8 complexes) were examined. There were no differences in concentration of total protein, but significantly higher levels of IL-8 were detected in patients with chronic generalized periodontitis in comparison to patients with refractory chronic periodontitis (P < 0.05). In addition, anti-IL-8:IL-8 complexes were present in 90% of patients with chronic generalized periodontitis, but in only 50% of patients with refractory chronic periodontitis. The results suggest that elevated concentrations of free and complexed IL-8 can differentiate patients with chronic generalized periodontitis from patients with refractory chronic periodontitis.

The effect of azithromycin and clarithromycin on ex vivo interleukin-8 (IL-8) release from whole blood and IL-8 production by human alveolar macrophages.

We investigated the effects of azithromycin and clarithromycin, two antibiotics that possess a broad spectrum of antimicrobial activity (including antimycobacterial activity), on interleukin-8 (IL-8) release from human whole blood leucocytes and lung macrophages. Ex vivo stimulation of leukocytes with either of the antibiotics (0.04-40 mg/L) significantly increased IL-8 secretion. Incubation of alveolar macrophages with different concentrations of azithromycin or clarithromycin modified IL-8 production: it increased at a drug concentration of 4 mg/L and decreased at concentration of 400 mg/L. Our findings suggest that azithromycin and clarithromycin may alter IL-8 production, thus enhancing the clinical effectiveness of these antibiotics.

Studies on the interaction of IL-8 with human plasma alpha 2-macroglobulin: evidence for the presence of IL-8 complexed to alpha 2-macroglobulin in lung fluids of patients with adult respiratory distress syndrome.

alpha 2-Macroglobulin (alpha 2m) is a major plasma proteinase inhibitor, as well as a carrier and regulator of the function of many cytokines. IL-8 is a potent neutrophil attractant and activator, and it plays an important role in the pathogenesis of adult respiratory distress syndrome (ARDS). The concentration of both IL-8 and alpha 2m is increased in lung fluids from patients with ARDS. Therefore, interaction of IL-8 with human alpha 2m was studied. Mixtures of native and methylamine-treated alpha 2m (fast alpha 2m) with 125I-labeled IL-8 were analyzed using nonreducing gel electrophoresis. 125I-labeled IL-8 exclusively bound to fast alpha 2m, and the binding could be inhibited by unlabeled IL-8. Analysis of the IL-8-alpha 2m interaction using SDS-PAGE gels indicated that the binding was mainly noncovalent. The affinity of the binding of alpha 2m to IL-8 was measured using an equilibrium dialysis technique, and Kd was 30 nM. Bioassays revealed that fast alpha 2m did not affect IL-8-induced neutrophil degranulation or chemotaxis. However, it protected IL-8 from proteolytic degradation. In addition, IL-8 complexed to alpha 2m was detected in lung fluids from patients with ARDS. alpha 2m may therefore modulate IL-8 function in the lung.

Comparison of antiproteolytic activities of alpha-1-proteinase inhibitors from the plasma of some mammalian species.

Alpha-1-proteinase inhibitors isolated from plasmas of horse, ox, pig, rabbit and man were used for determination of some kinetic parameters of interaction with three horse leucocyte proteinases and bovine pancreatic trypsin and chymotrypsin. Effective molar ratio of enzyme-to-inhibitor, inactivation rate constant and inhibition constant were measured. In horse, ox, pig and rabbit two principal electrophoretic forms of alpha 1-PI could be distinguished. Both forms effectively inhibited trypsin but usually only one form reacted promptly and stoichiometrically with chymotrypsin and leucocyte elastases. It appears that genetic variability and functional heterogeneity of multiple forms of alpha 1-PI as well as lack of other tissue inhibitors of proteinases may be responsible for lung emphysema occurring in man and horse.

Role of cytokines and growth factors in the induced synthesis of proteinase inhibitors belonging to acute phase proteins.

The effects of various cytokines on synthesis and secretion of albumin and some proteinase inhibitors belonging to the class of macroglobulins, serpins and cysteine proteinase inhibitors were studied in the primary cultures of rat and mouse hepatocytes and established human hepatoma cell line Hep G2. In all tested systems interleukin 6 depressed the synthesis of albumin and enhanced the synthesis of antichymotrypsin (or contrapsin) and alpha-1-proteinase inhibitor, while in the rat alpha-2-macroglobulin and T-kininogen (thiostatin) were the major acute phase reactants. Smaller and variable effects were observed with interleukin-1, tumour necrosis factor, interferon-gamma, transforming growth factor-beta and epidermal growth factor. Searching for the feed-back regulatory mechanism responsible for induced synthesis of proteinase inhibitors we found that cultured human lung fibroblasts exposed to human alpha-1-antichymotrypsin or antichymotrypsin-cathepsin G complexes produce significantly more interleukin 6 which stimulates Hep G2 cells to augmented synthesis of several acute phase proteins.

Staphylococcal enterotoxin A-induced injury of human lung endothelial cells and IL-8 accumulation are mediated by TNF-alpha.

Staphylococcal enterotoxin A (SEA), a superantigen produced by some strains of Staphylococcus aureus, causes a variety of clinical manifestations ranging from food poisoning to shock. S. aureus can also be associated with the development of acute respiratory distress syndrome, and SEA has been shown to cause an inflammatory reaction in the lung. Therefore, we examined possible interactions between SEA, PBMCs, polymorphonuclear cells (PMNs), and normal human lung microvascular endothelial cells (HMVEC-L), as well as the role of these interactions on the secretion of IL-8. Injury to HMVEC-L, as measured by the release of 51Cr, increased significantly when HMVEC-L were incubated with SEA and PBMCs. IL-8 was secreted by both PBMCs and HMVEC-L. The accumulation of IL-8 in the culture medium of HMVEC-L was increased by SEA in a dose-dependent manner and was directly related to the number of PBMCs present. Although neither anti-human IL-8 nor IL-1 mAb inhibited HMVEC-L cytotoxicity, anti-human TNF-alpha mAb inhibited both the cytotoxicity and IL-8 accumulation completely. When HMVEC-L were incubated with supernatants from SEA-treated PBMCs, HMVEC-L cytotoxicity was comparable with HMVEC-L incubated with SEA and PBMCs at the same time. Although high concentrations of purified PMNs induced HMVEC-L lysis in a dose-dependent manner, the effect of PMNs was not changed in the presence of SEA. These findings suggest that TNF-alpha secreted by SEA-stimulated PBMCs plays a leading role in HMVEC-L injury.